WO2019217243A1 - Methods and compositions related to the next generation vaccine - Google Patents

Abstract

Disclosed are methods and compositions related to polypeptides comprising a fusion of the needle tip protein and translocator protein of a type III secretion apparatus (T3SA) from a type III secretion system (T3SS) of a Gram negative bacteria. Disclosed herein are fusion polypeptides comprising a fusion of a needle tip protein, such as, Bsp22, LcrV, BipD, PcrV, CT053, or CT668, or an antigenic fragment thereof; and a translocator protein, such as, BopB, YopB, BipB, PopB, CopB, or CopB2, or an antigenic fragment thereof from a Type III secretion system (T3SS) of a Gram negative bacteria, such as, Bordetella, Burkholderia, Chlamvdia, Pseudononas, Vibrio, or Yersinia.

Description

METHODS AN D COMPOSITIONS RELATED TO THE NEXT

GENERATION VACCINE

I. CROSS-REFERENCE TO RELATED APPLICATIONS

1. The present application claims the priority benefit of U.S. Provisional Patent Application Serial No. 62/667,599, filed May 6, 2018, which is incorporated by reference in its entity herein.

II. BACKGROUND

2. Bordetella pertussis is a Gram-negative bacterial pathogen that causes pertussis, or whooping cough, a highly contagious, severe respiratory disease that is life threatening for infants and young children. This pathogen colonizes the trachea and secretes toxins that paralyze the cilia, which prevents clearance of mucous. Severe (paroxysmal), non productive coughing fits are a result and attempts to acquire oxygen are manifested by the characteristic“whoop” upon gasps for air. The majority of deaths associated with pertussis are actually caused by secondary respiratory infections resulting from the inability to clear pulmonary secretions. In the 1940s a whole-cell pertussis (wP) vaccine was introduced that dramatically reduced the mortality caused by pertussis. Due to side effects attributed to the wP vaccine, a new' acellular pertussis (aP) vaccine was developed and introduced in the US and other parts of the world in the 1990s. Although the aP vaccine has few side effects, its protective efficacy is lower than that of the wP vaccine. In 2012, which is considered the most recent major epidemic, 48,277 cases of pertussis w'ere reported and, in 2015, 20,762 cases ere reported. During the last 15 years, in addition to a greater overall incidence of pertussis, there is growing concern over the increase in the peak number of reported cases for each ensuing epidemic. In 2015, 45% of the 0.5 to 6-year-old children that contracted pertussis had been vaccinated with DTaP at least three times (with five vaccinations being optimal: 2, 4, 6, 15 months and one at 4-6 years). Additionally, there is evidence that selective pressure is causing B. pertussis to eliminate virulence factors that are components of the aP vaccine, further compromising the vaccine’s efficacy. Taken together, a better vaccine is needed.

DL SUMMARY

3. Disclosed are methods and compositions related to polypeptides comprising a fusion of the needle tip protein and translocator protein of a type III secreti on apparatus (T3SA) from a type III secretion system (T3SS) of a Gram negative bacteria. 4. Disclosed herein are fusion polypeptides comprising a fusion of a needle tip protein (such as, for example, Bsp22, LcrV, BipD, PcrV, CT053, or CT668) or an antigenic fragment thereof and a translocator protein (such as, for example, BopB, YopB, BipB, PopB, CopB, or CopB2 ) or an antigenic fragment thereof from a Type III secretion system (T3SS) of a Gram negative bacteria (such as, for example, Bordetella spp., Burkholderia spp., Chlamydia spp., Pseudomonas spp., Vibrio spp. or Yersinia spp.); wherein the gram negative bacteria is not a Salmonella enterica or Shigella spp.

5. In one aspect, disclosed herein are fusion polypeptides, wherein the fusion polypeptide is arranged such that the needle tip protein is 5’ of the translocator protein.

6. Also disclosed herein are fusion polypeptides of any preceding aspect, wherein the fusion further comprises an adjuvant such as, for example, Cholera Toxin or antigenic fragment thereof (such as, for example, CTA1) or double mutant labile toxin (dmLT) or an antigenic fragment thereof labile toxin (such as, for example, LTA1) from Enterotoxigenic Escherichia coll In some aspect, the dmLT or fragment thereof can also be fused to the needle tip protein-translocator protein fusion at the 5’ end

7. In one aspect, disclosed herein are fusion polypeptides of any preceding aspect, wherein the fusion polypeptide further comprises pertussis toxoid (PTd).

8. Also disclosed herein are compositions comprising a T3SA needle tip protein (such as, for example, Bsp22, LcrV, BipD, PcrV, or CdsF) or an antigenic fragment thereof from a Gram negative bacteria (such as, for example, Bordetella spp., Burkholderia spp., Chlamydia spp., Pseudomonas spp., Vibrio spp., or Yersinia spp.) and a T3SA first translocator protein (such as, for example, BopB, YopB, BipB, PopB, or CopB/CopB2 ) or an antigenic fragment thereof from a Gram negati ve bacteri a; wherein the gra negative bacteria is not a Salmonella enterica or Shigella spp. In one aspect, the composition can comprise the needle tip protein or fragment thereof and the translocator protein or fragment thereof as separate components or as a fusion polypeptide. Also disclosed herein are compositions of any preceding aspect, wherein the composition comprises an adjuvant (such as, for example, dmLT, LTA1, cholera toxin, or CTA1) and/or bacterial toxin protein such as a pertussis toxoid (PTd).

9. In one aspect, disclosed herein are vaccines comprising the fusion

polypeptides or compositions of any preceding aspect. In some embodiments, the vaccine can further comprise an acellular gram negative vaccine or active components thereof. In one aspect, the vaccine can comprise pertussis toxoid (PTd). 10. Also disclosed herein are methods of treating, inhibiting, or preventing an infection of a Gram negative bacteria (such as, for example, Bordeteta spp., Burkholderia spp., Chlamydia spp., Pseudomonas spp., Vibrio spp., or Yersinia spp.) in a subject comprising administering to the subject the fusion polypeptide, composition, or vaccine of any preceding aspect.

11. In one aspect, disclosed herein are methods of treating, inhibiting, or preventing an infection of a Gram negative bacteria of any preceding aspect, wherein the method further inhibits or prevents colony formation of the bacteria and/or transmission of the bacteria to another subject.

12. Also disclosed herein are methods of eliciting an immune response in a subject to a Gram negative bacteria (such as, for example, Bordeteta spp., Burkholderia spp., Chlamydia spp., Pseudomonas spp., Vibrio spp., or Yersinia spp.) comprising administering to the subject the fusion polypeptide, composition, or vaccine of any preceding aspect. For example, disclosed herein are methods of eliciting an immune response against at least one Gram negative bacteria serovar in a subject in need thereof, comprising administering to the subject a composition comprising at least one needle tip protein or an antigenic fragment thereof and/or at least one translocator protein or an antigenic fragment thereof; wherein said composition is administered in an amount sufficient to elicit an immune response to said at least one Gram negative bacteria serovar in said subject; and wherein the Gram negative bacteria is not a Shigella spp. or Salmonella enterica.

13. In one aspect, disclosed herein are methods of eliciting an immune response in a subject to a Gram negative bacteria of any preceding aspect, wherein the immune response provides sterilizing immunity.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

14. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

15. Figure 1 shows the protective efficacy of intranasally administered 22BF against B. bronchiseptica challenge. Mice (n=T0) were vaccinated intranasally biweekly three times with the indicated formulation which contained 10 gg protein ± dmLT. Zoetis vaccine was delivered subcutaneously on day 1 and 21 as per manufacturer’s directions. Mice were challenged with 1.3xl0⁷ B. bronchiseptica on day 56. BopB was not available at day 0. #

P< 0.05 compared to survival of mice vaccinated with PBS.

..... 3 ..... 16. Figure 2 shows the weight gain/loss of vaccinated mice during B. bronchiseptica challenge. Mice (same as above) were weighed daily in p.m. Note that the 22BF+dmLT mice gain weight and have small error bars.

17. Figures 3 A and FIG. 3B show the protective efficacy of 22BF+dmLT. Mice were vaccinated on days 0, 14, 28 and challenged on day 56 with a sublethal dose of if bronchiseptica. Figure 3A shows that on day 7 of the challenge, the CFU/lung were determined. * = P < 0.05, ** = P < 0.01 when compared to dmLT. Figure 3B shows the decrease in CFU compared to the 22BF average. * =^: P < 0.05, ** ==^: P < 0.01 when compared to 22BF.

18. Figures 4A, 4B, and 4C show the kinetics of IgG response. Blood was collected on days -1, 13, 27, 41, and 55. The kinetics of anti-Bsp22, -BopB and -dmLT IgG were assessed in all sera and shown in (4A) BopB, (4B) Bsp22, and (4C) dmLT Typical logarithmic increases were seen. * = P value of < 0.05 when comparing to PBS controls.

19. Figures 5 A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, and 51 show stimulation of antibody secreting cells from bone marrow, spleen and lungs. Bone marrow, spleens and lungs were collected on day 56. Single cell suspensions from 5 mice per group were stimulated in vitro Bsp22, BopB or dmLT IgG (black) and IgA (white) ASC were measured by ELISpot. Bars represent mean ASC per 10⁶ cells +SD from replicate wells. Data for bone marrow is shown in (4 A), (5D), and (5G) for Bsp22, BopB, and dmLT, respectively. Data for spleen is shown in (5B), (5E), and (5H) for Bsp22, BopB, and dmLT, respectively. Data for lungs is shown in (5C), (5F), and (51) for Bsp22, BopB, and dmLT, respectively.

20. Figures 6 A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, and 61 show Thl cytokine secretion. Splenocytes were extracted from 5 mice of each group and incubated with Bsp22, BopB or dmLT. After 48 h, supernatants were collected and levels of cytokine secretion in response to specified antigen were then measured (in pg/mi) using an MSD cytokine detection plate.

Each bar represents mean of triplicate wells ±S.D. Asterisk specified a P < 0.05 when comparing specified groups. Data for IFN-y is shown in (6A), (6B), and (6C) for Bsp22, BopB, and dmLT, respectively. Data for TNF-a is shown in (6D), (6E), and (6F) for Bsp22, BopB, and dmLT, respectively. Data for IL-Ib is shown in (6G), (6H), and (61) for Bsp22, BopB, and dmLT, respectively.

21. Figures 7 A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, and 71 show Thl cytokine secretion. Splenocytes were extracted from 5 mice of each group and incubated with Bsp22, BopB or dmLT. After 48 h, supernatants were collected and levels of cytokine secretion in response to specified antigen were then measured (in pg/mi) using an MSD cytokine detection plate.

4 Each bar represents mean of triplicate wells ±S.D. Asterisk specified a P < 0 05 when comparing specified groups. Data for IL-2 is shown in (7A), (7B), and (7C) for Bsp22,

BopB, and dmLT, respectively. Data for IL-10 is shown in (7D), (7E), and (7F) for Bsp22, BopB, and dmLT, respectively. Data for IL-6 is shown in (7G), (7H), and (71) for Bsp22, BopB, and dmLT, respectively.

22 Figures 8 A, 8B, and 8C shows IL-17 secretion Splenocytes were extracted from 5 mice of each group and incubated with Bsp22, BopB or dmLT. After 48 h, supernatants were collected and levels of IL-17 secretion in response to labeled antigen w'ere then measured by the MSD® U-Plex Platform Multiplex Assay and the data is shown in (8A) BopB, (8B) Bsp22, and (8C) dmLT Each bar represents the mean of triplicate wells ± S.D. Significance (Asterisk = P < 0.05) was calculated for the comparison between labeled groups

23. Figures 9 A, 9B, 9C, 9D, 9E, and 9F show' Th2 cytokine secretion. Splenocytes w'ere extracted from 5 mice of each group and incubated with Bsp22, BopB or dmLT. After 48 h, supernatants were collected and level s of cytokine secretion in response to specified antigen were then measured (in pg/ml) using an MSD cytokine detection plate. Each bar represents mean of triplicate wells ±S.D. Asterisk specified a P < 0.05 when comparing specified groups. Data for IL-4 is shown in (9 A), (9B), and (9C) for Bsp22, BopB, and dmLT, respectively. Data for IL-5 is shown in (9D), (9E), and (9F) for Bsp22, BopB, and dmLT, respectively.

24 Figure 10 shows the change in weight in percentage after infection with sub!etha! dosage of B. pertussis intranasally. There was an observable difference in weight loss between mice vaccinated with the 22BF+dmLT+PTd formulation and those that only received PBS. By Day 7 all mice aside from PBS treated mice had recovered to within 3% of pre-infection weight.

25. Figures 1 1 A, 1 IB, 11C, and 1 ID show serum antibody responses to BopB, Bsp22, Pertussis Toxin Mutant, and dmLT Mice were immunized on days 0, 14, and 28 with 22BF+PTd admixed with dmLT. Serum IgG antibodies specific for BopB, Bsp22, PTd, and dmLT w'ere measured by ELISA and the data is shown in (1 1 A) BopB, (1 IB) Bsp22, (11C) PTd, and (1 ID) dmLT. Data are the mean titers (EU ml^A-l) from group pools of animal samples. An asteri sk indicates a P value of 0 05 when comparing vaccinated mice and the PBS controls. No responses w'ere seen in the control mice that received PBS. Mice vaccinated with Infanrix only displayed a response against pertussis toxin mutant, which is part of its formulation. 26. Figure 12 shows the Lung Colony forming units (CFU) from mice 3 days post intranasal infection. Mice vaccinated intranasaily with 22BF+PTd and dmLT showed statistical (P < 0.05) decreases in lung CPUs when compared to PBS treated mice. The mice vaccinated intradermally and mice vaccinated intramuscularly with 22BF+PTd and dmLT either showed sterilizing immunity, or no statistical decrease in lung CPUs. Infanrix appeared to show a decrease in lung CFU, but this was not statistically significant (P > 0.05). (* = P < 0.05, KW p-value = 0.0003).

27. Figure 13 shows the Lung Colony forming units (CFU) from mice 7 days post intranasal infection. Mice vaccinated intranasaily with 22BF+PTd and dmLT showed statistical (P < 0.05) decreases in lung CPUs when compared to PBS treated mice, with 60% of the mice showing sterilizing immunity. The mice vaccinated intramuscularly or intradermally with 22BF+PTd and dmLT showed no statistical decrease in lung CPUs.

Infanrix appeared to display sterilizing immunity with CFU measuring below the limit of detection. (* ^::: P < 0.05, ** P < 0.01, KW p-value = 0.0001).

28. Figure 14 shows the protective efficacy of LTA1 -DBF vs DBF+dmLT. Mice were vaccinated intramuscularly on days 0, 14 and 28 with the indicated pg of DBF+ 0.1 pg dmLT or DBF equivalent of LTA1 -DBF. The positive control was DBF+dmLT delivered intranasaily. On day 56 the mice were challenged with Shigella flexneri. Figure 14 indicates the percent survival of mice post infection with Shigella flexneri.

29. Figures 15 A, 15B, and 15C show the kinetics of IgG response. Mice from Figure 14 were bled prior to vaccination and on day 42. Sera were assessed for anti-IpaD, - IpaB and -dmLT IgG, and the data is shown in (15 A) IpaD, (15Bb) IpaB, and (15C) dmLT. Differences in the IgG levels in mice vaccinated with dmLT vs. LTA1 are attributed to the recognition of the entire dmLT on the well.

30. Figures 16A, 16B, and 16C show the stimulation of antibody secreting cells from bone marrow. Bone marrow⁷ was collected on day 56. Single cell suspensions from 5 mice per group were stimulated in vitro IpaD, IpaB or dmLT. IgG (black) and IgA (white) ASC were measured by ELISpot, and the data is shown in (16A) IpaD, (16B) IpaB, and (16C) dmLT. Bars represent mean ASC per 10⁶ cells +SD from replicate welis.

31. Figures 17 A, 17B, and 17C show the stimulation of antibody secreting cells from spleen. Spleens w^?ere collected on day 56. Single cell suspensions from 5 mice per group were stimulated in vitro IpaD, IpaB or dmLT. IgG (black) and IgA (white) ASC were measured by ELISpot. Bars represent mean ASC per I 0⁶ cells +SD from replicate wells. 32. Figures 18 A, 18B, and 18C show the stimulation of antibody secreting cells from lungs. Lungs were collected on day 56. Single cell suspensions from 5 mice per group were stimulated in vitro IpaD, IpaB or dmLT. IgG (black) and IgA (white) ASC were measured by ELISpot, and the data is shown in (18A) IpaD, (18B) IpaB, and (18C) dmLT. Bars represent mean ASC per 10⁶ cells +SD from replicate wells.

33. Figures 19A, 19B, 19C, and 19D show the protective efficacy and IgG response kinetics of LTA1-22BF. Mice were vaccinated on days 0, 14, 28 and challenged on day 56 with a sublethal dose of B. pertussis. On day 7 of the challenge, the CFU/lung were determined. Figure 19A shows the CFU/lung while Figure 19B shows the decrease in CFU compared to the PBS average. * = P < 0 05 when compared to PBS. Figures 19C and 19D how the kinetics of the response of the anti-Bsp22 and anti-BopB IgG, respectively. No difference is seen between the mice vaccinated with 22BF+dmLT and LTA1-22BF.

34. Figure 20 shows the ADPr activity of L-antigens. LTA1 was fused to DBF, 22BF or SseB LTAi, however, must retain its ADP-ribosylation activity to maintain adjuvant activity. The ADPr of NAD+ was biotin conjugated and LTAI transferred the biotin- ADPr moiety to ARF4. The biotin was then detected with Streptavidin-IRBOO. Lane 1 : LTAI -DBF; 2: LTA1-22BF; 3 : LTAI -SseB.

V. DETAILED DESCRIPTION

35. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary-.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting

A. Definitions

36. As used in the specification and the appended claims, the singular forms“a,” “an” and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a pharmaceuti cal carrier” includes mixtures of two or more such carriers, and the like.

37. Ranges can be expressed herein as from“about” one particular value, and/or to“about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another embodiment. It will be further understood

7 that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. For example, if the value“10” is disclosed, then“about 10” is also disclosed. It is also understood that when a value is disclosed that“less than or equal to” the value,“greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value“10” is disclosed the“less than or equal to 10” as well as“greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point“10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

38. The needle tip protein and/or translocator proteins or antigenic portions thereof disclosed herein are used to elicit an immune response in subjects to whom they are administered. By "elicit an immune response",“induces or enhances an immune response”, or“stimulates an immune response” which are used interchangeably herein, is meant that the subject mounts one or both of an innate and/or an adaptive immune reaction against antigenic determinants of the proteins or antigenic portions thereof that are administered. Preferably a statistically measurable induction or increase in an immune response over a control sample to which the needle tip protein and/or translocator proteins or antigenic portions thereof disclosed herein has not been administered. Preferably the induction or enhancement of the immune response results in a prophylactic or therapeutic response in a subject. In particular, the adaptive immune reaction entails production of e.g. B and T cell lymphocytes and antibodies specific for binding and forming complexes with the antigenic determinants. In some embodiments, the proteins and/or antigenic fragments thereof elicit a protective immune response in the subject, i.e. administration of one or more of the proteins and/or antigenic portions thereof results in an immune response that is protective against later challenge by the disease causing organism itself, either preventing infection altogether, or lessening the impact of infection by decreasing disease symptoms that would otherwise occur, had the subject not been vaccinated as described herein. 39. “Vaccine” as used herein is a preparation that stimulates an immune response that produces immunity against particular antigens, e.g. Gram negative bacteria. Vaccines may be administered prophylactically (for example, to prevent or inhibit the establi shment of an infection) or therapeutically to inhibit, reduce, or treat an established infection, or to ameliorate the effects or symptoms of an infection. Vaccines may contain, but are not limited to, live, attenuated infectious material such as viruses or bacteria, and dead or inactivated organisms or purified products derived therefrom. A vaccine can be administered by injection, orally, or by inhalation. Injections may be, but are not limited to, subcutaneous (sc), intramuscular (im), intraperitonea! (ip), intradermai (id) or intravenous (iv).

40. “Optional” or“optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

41. The term“subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term“patient” refers to a subject under the treatment of a. clinician, e.g., physician or veterinarian.

42. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

B. Compositions

43. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein.

These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular needle tip protein (such as, for example, IpaD, SipD, SseB, Bsp22,

LcrV, BipD, PcrV, CT053, or CT668), translocator protein (such as, for example, IpaB,

SipB, SseC, BopB, YopB, BipB, PopB, CopB, or CopB2), or fusion polypeptide thereof

9 (such as, for example, 22BF, BurkF, PaF, YerF, CT053~CopB, CT053-CopB2, CT668-CopB, or CT668-CopB2) is disclosed and discussed and a number of modifications that can be made to a number of molecules including the needle tip protein (such as, for exampl e, IpaD, SipD, SseB, Bsp22, LcrV, BipD, PcrV, CT053, or CT668), translocator protein (such as, for example, IpaB, SipB, SseC, BopB, YopB, BipB, PopB, CopB, or CopB2), or fusion polypeptide thereof (such as, for example, 22BF, BurkF, PaF, YerF, CT053-CopB, CT053- CopB2, CT668~CopB, or CT668-CopB2) are discussed, specifically contemplated is each and every combination and permutation of needle tip protein such as, for example, IpaD, SipD, SseB, Bsp22, LcrV, BipD, PcrV, CT053, or CT668), translocator protein (such as, for example, IpaB, SipB, SseC, BopB, YopB, BipB, PopB, CopB, or CopB2), or fusion polypeptide thereof (such as, for example, , DBF, SI, S2, 22BF, BurkF, PaF, YerF, CT053- CopB, CT053-CopB2, CT668-CopB, or CT668-CopB2) and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combinati on molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety_' of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

44. To infect a host, B. pertussis uses an arsenal of well-characterized virulence factors. These factors include pertussis toxin (PT), adenylate cyclase toxin (ACT), the type

III secretion system (T3 SS), tracheal cytotoxin (TCT), dermonecrotic toxin (DNT), filamentous hemagglutinin (FHA), pertactin (PRN), and iipooligosaccharide (LOS). Current aP vaccines are comprised of PT, FHA, PRN, and the fimbria! proteins in varying

proportions, but not necessarily all four proteins. Though the aP vaccine causes fewer adverse reactions than the wP vaccine, it is not as efficacious. This same situation exists for other pathogenic Gram negative bacteria. Accordingly, disclosed herein are fusion polypeptides from a Type III secretion system (T3 SS) of a Gram negative bacteria (such as, for example,

Shigella spp.. Salmonella enterica , Bordetella spp. (such as, for example B. pertussis and/or

B. bronchiseptica), Burkholderia spp. (such as, for example, B. cepacian, B mallei, and/or B. pseudomallei), Chlamydia spp. (such as, for example, C. trachomatis), Pseudomonas spp., Vibrio spp. or Yersinia spp.) comprising a polypeptide of needle tip protein (such as, for example, IpaD, SipD, SseB, Bsp22, LcrV, BipD, PcrV, CT053, or CT668) or an antigenic fragment thereof and polypeptides of a translocator protein (such as, for example, IpaB, SipB, SseC, BopB, YopB, BipB, PopB, CopB, or CopB2) or an antigenic fragment thereof. In some aspect, the fusion polypeptide does not comprise a needle tip protein polypeptide or translocator polypeptide from a Shigella spp. (IpaD and IpaB) or a Salmonella spp. (such as, for example, S. enterica) (SipD, SseB, SipB, and SseC) It is recognized and herein contemplated that the disclosed polypeptides can be separate components of a composition or more preferably a fusion construct. By a "fusion polypeptide" is meant a peptide,

polypeptide, or protein that is translated from a single, contiguous nucleic acid molecule, and which comprises sequences from at least two different proteins or antigenic regions thereof. Typically, the individual sequences are joined via a linker or spacer sequence of e.g. from about 2 to about 20 amino acids, usually from about 2 to about 10 amino acids. The amino acids in linking sequences are typically uncharged and the linker sequence usually does not exhibit secondary or tertiary' structure, but does allow the fused protein/peptide segments to adopt functional secondary, tertiary, etc. conformations. One such exemplary fusion polypeptide includes Bsp22 (as set forth in SEQ ID NO: 4 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 3) and BopB (as set forth in SEQ ID NO: 6 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 5). The amino acid sequence of this chimera (i.e., 22BF) is set forth in SEQ ID NO: 2. The chimera may be encoded by any suitable nucleic acid sequence, e.g. the exemplary nucleic acid sequence depicted in SEQ ID NO: 1.

45. Thus, in one aspect, disclosed herein are fusion polypeptides comprising a fusion of a needle tip protein (such as, for example, IpaD, SipD, SseB, Bsp22, LcrV, BipD,

PcrV, CT053, or CT668) or an antigenic fragment thereof and a translocator protein (such as, for example, IpaB, SipB, SseC, BopB, YopB, BipB, PopB, CopB, or CopB2 ) or an antigenic fragment thereof from a Type III secretion system (T3SS) of a Gram negative bacteria (such as, for example, Bordetella spp., Burkholderia spp., Chlamydia spp., Pseudomonas spp.,

Vibrio spp. or Yersinia spp. For example, the fusion polypeptide can comprise a fusion of the

Shigella spp. needle tip protein (IpaD) and first translocator protein (IpaB) or fragments thereof (the fusion referred to as DBF), Salmonella spp. (such as, for example, S. enterica)

SPI-1 needle tip protein (SipD) (as set forth in SEQ ID NO: 52 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 51) and translocator protein (SipB) (as set forth in

..... 11 ...... SEQ ID NO: 54 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 53) or fragments thereof (the fusion referred to as SI) (as set forth in SEQ ID NO: 56 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 55), Salmonella spp.(such as, for example, S. enterica) SPI-2 needle tip protein (SseB) (as set forth in SEQ ID NO: 62 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 61) and translocator protein

(SseC) (as set forth in SEQ ID NO: 64 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 63) or fragments thereof (the fusion referred to as S2) (as set forth in SEQ ID

NO: 66 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 65), Bordetella spp. needle-tip protein (Bsp22) and translocator protein (BopB), or fragments thereof (the fusion referred to as 22BF); a fusion of the Yersinia spp. needle-tip protein (LcrV) (as set forth in SEQ ID NO: 42 and encoded by the nucleic acid sequence as set forth in SEQ ID

NO: 41) and translocator protein (YopB) (as set forth in SEQ ID NO: 44 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 43), or fragments thereof (the fusion referred to as YerF) (as set forth in SEQ ID NO: 46 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 45), a fusion of th Q Burkholderia spp. needle-tip protein (BipD)

(as set forth in SEQ ID NO: 22 and encoded by the nucleic acid sequence as set forth in SEQ

ID NO: 21) and translocator protein (BipB) (as set forth in SEQ ID NO: 24 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 23), or fragments thereof (the fusion referred to as BurkF) (as set forth in SEQ ID NO: 26 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 25); a fusion of the Pseudomonas spp. needle-tip protein (PcrV) (as set forth in SEQ ID NO: 32 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 31) and translocator protein (PopB) (as set forth in SEQ ID NO: 34 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 33), or fragments thereof (the fusion referred to as PaF) (as set forth in SEQ ID NO: 36 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 35); and/or a fusion of the Chlamydia spp. needle-tip protein CT053 (as set forth in SEQ ID NO: 74 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 73) or CT668 (as set forth in SEQ ID NO: 84 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 83) and translocator protein

(CopB (as set forth in SEQ ID NO: 76 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 75) or CopB2 (as set forth in SEQ ID NO: 92 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 91)), or fragments thereof such fusions including but not limited to CT053-CopB (the CT053-CopB fusion as set forth in SEQ ID NO: 78 and encoded by the nucleic acid sequence as set forth in SEQ) ID NO: 77), CT668-CopB (the

CT668-CopB fusion as set forth in SEQ ID NO: 86 and encoded by the nucleic acid sequence

..... 12 -- as set forth in SEQ ID NO: 85), CTQ53-CopB2 (the CTQ53-CopB2 fusion as set forth in SEQ ID NO: 94 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 93), and CT053~CopB2 (the CT668~CopB2 fusion as set forth in SEQ ID NO: 100 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 99) and collectively each fusion being referred to as ChlamF. In one aspect, the fusion polypeptide does not comprise any needle tip protein or transiocator protein or fragment thereof from a Salmonella spp. or a Shigella spp. Accordingly, disclosed herein are fusion polypeptides comprising a fusion of a needle tip protein (such as, for example, Bsp22, LcrV, BipD, PcrV, CT053, or CT668) or an antigenic fragment thereof and a transiocator protein (such as, for example, BopB, YopB, BipB, PopB, CopB, or CopB2 ) or an antigenic fragment thereof from a Type 01 secretion system (T3SS) of a Gram negative bacteria (such as, for example, Bordetella spp.,

Burkholderia spp., Chlamydia spp., Pseudomonas spp., Vibrio spp. or Yersinia spp. wherein the gram negative bacteria is not a Salmonella enter! ca or Shigella spp.

46. It is understood and herein contemplated that the arrangement of the polypeptides in a fusion construct can have significant impact on the antigenicity of the fusion construct. Accordingly, in one aspect, disclosed herein are fusion polypeptides, wherein the fusion polypeptide is arranged such that the needle tip protein is 5’ of the transiocator protein.

47. The present invention provides compositions for use in eliciting an immune response and/or vaccinating an individual against Gram negative bacterial infection, and/or against disease symptoms caused by Gram negative bacterial infection. The compositions include one or more substantially purified proteins, polypeptides or antigenic regions thereof as described herein, or substantially purified nucleic acid sequences (e.g. DNA cDNA, RNA, etc.) encoding such proteins, polypeptides or antigenic regions thereof, and a

pharmacologically suitable/compatible carrier. By "substantially purified" is meant that the molecule is largely free of other organic molecules, cellular debris, solvents, etc. when tested using standard techniques known to those of skill in the art (e.g. gel electrophoresis, column chromatography, sequencing, mass spectroscopy, etc.). For example, the molecule is generally at least about 50, 55, 60, 65, 70, or 75% pure by wt%, and preferably is at least about 80, 85, 90, 95% or more preferably pure (e.g. 96, 97, 98, 99 or even 100% pure). The preparation of proteins, polypeptides, and peptides as described herein is well-known to those in the art, and includes, for example, recombinant preparation; isolation from a natural source; chemical synthesis; etc. The purification of proteinaceous materials is also known. However, specific exemplary methods for preparing the vaccinating agents utilized in the practice of the invention are described in detail in the Examples section below.

48. In addition, the composition may contain adjuvants, many of which are known in the art. For example, adjuvants suitable for use in the invention include but are not limited to: bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopoly saccharide (EPS), Lipid A derivatives, immunostimuiatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof. Non-toxic derivatives of LPS include monophosphory! lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of three de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred non-toxic derivative of LPS is 3 De-O-acylated monophosphoryl lipid A. Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoaikyi glucosaminide phosphate derivatives, e.g. RC-529.

49. Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. Immunostimuiatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double- stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimuiatory. The CpGs can include nucleotide

modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded, e.g. replacement of guanosine with 2'-deoxy-7-deazaguanosine. The CpG sequence may include, for example, the motif GTCGTT or TTCGTT. The CpG sequence may be specific for inducing a Thl immune response, such as a CpG- A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN, CpG-A and CpG-B ODNs Preferably, the CpG is a CpG-A ODN Preferably, the CpG oligonucleotide is constructed so that the 5^! end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers".

50. Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention. Preferably, the protein is derived from E. coli (e.g. E coli heat labile enterotoxin "LT"), cholera ("CT")(Table 1), or pertussis ("PT").

Table 1 Cholera Toxin (CTA 1) subunits and sequences

51. The toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits. Preferably, the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated. More preferably, the adjuvant is a detoxified LT mutant such as LT- K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins and detoxified derivatives thereof, particularly LT-K63 and LT-R72, is known. Such adjuvants are described, for example, in issued US Patent No. 8,039,007 (the complete contents of which is hereby incorporated by reference in entirety). Various interleukins may also be used as adjuvants to increase the immune response in a subject. In preferred embodiments, the adjuvant is a mucosal adjuvant such as, for example, the double mutant heat-labile toxin (dmLT) as set forth in SEQ ID NOs: 113 and 114) from enterotoxigenic E. coli or the active moiety thereof known as LTA1 (as set forth in SEQ ID NO: 13 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 12) and encoded by nor cholera toxin or the active moiety thereof known as CTA1. Accordingly, disclosed herein are fusion polypeptides of any preceding aspect, wherein the fusion further comprises an adjuvant such as, for example, double mutant labile toxin (dmLT) or an antigenic fragment thereof (such as, for example, LTA1 or CTA1) from Enterotoxigenic Escherichia coli. In some aspect, the dmLT or fragment thereof can also be fused to the needle tip protein-transioeator protein fusion at the 5’ end. For example, specifically disclosed herein are LTA1-DBF, LTA1-S1 (as set forth in SEQ ID NO: 57 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 58), LTA1-S2 (as set forth in SEQ ID NO: 68 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 67), LTAl -SseB (as set forth in SEQ) ID NO: 70 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 69), LTA1-22BF (as set forth in SEQ ID NO: 18 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 17), LTAl-BurkF (as set forth in SEQ ID NO: 28 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 27), LT.A1- CT668~CopB (as set forth in SEQ ID NO: 88 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 87), LTAl-CT668-CopB2 (as set forth in SEQ ID NO: 102 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 101), LTAI-CT053~CopB (as set forth in SEQ ID NO: 80 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 79), LTAl-CT053-CopB2 (as set forth in SEQ ID NO: 96 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 95), LTAI -PaF (as set forth in SEQ ID NO: 38 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 37), and LTA1- YerF (as set forth in SEQ ID NO: 48 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 47).

52. Whooping cough still causes significant mortality and morbidity in children ail over the world. It also continues to be a problem in adults whose immunity has waned. Herein is disclosed a strong candidate for a new protective vaccine based on research on the T3SS proteins and resulting subunit vaccines, including the vaccine against shigellosis. It is demonstrated herein that the vaccine has 100% protective efficacy against B. hronchiseptica using 22BF+dmI,T. While this is a remarkable step forward, examined herein is the immune response and the protective efficacy of 22BF+dmLT ± PTd against B. pertussis. The vaccine can also be taken a step further by eliciting sterilizing immunity so that the B. pertussis transmission chain can be broken.

53. Originally, the mechanism of protection against B. pertussis, an extracellular organism, was thought to be the humoral immune response, however, cell-mediated immunity has been found to also be important for protection with bacterial clearance mediated by Th l and ThI 7 cells. By measuring cytokines corresponding to specific immune pathways, Ross et al. concluded that the wP vaccine promotes Thl and Thl 7 responses while the aP vaccine elicits a mix of Thl and Th2 responses. These differences likely account for the increased protection seen for the wP vaccine. A study in a baboon model compared wP vaccines with an aP vaccine and confirmed that the wP elicits a Thl /Thl 7 response while the aP vaccine elicits a Thl/Th2 response. Moreover, these studies found that aP does not prevent colonization or transmission of B. pertussis, even in asymptomatic subjects. Thus, the current pertussis resurgence could be due, in part, to the ability of the aP vaccine to protect the host against the overt symptoms of the disease while not preventing colonization and the resulting transmission of B. pertussis to susceptible children. Furthermore, protection of newborns against pertussis via aP or wP is problematic due not only to possible side effects but also because newborns lack the ability to mount a vaccine-induced Thl response elicited through the requisite antigen presentation and T-cell activation. Although it has been shown, in some cases, that neonatal immunization can prime the immune system for subsequent booster vaccinations, the development of a protective pertussis vaccine for infants remains a need.

54. As noted above, the current aP vaccine does not provide sterilizing immunity. That is, the aP vaccine protects the immunized host, but does not stop colonization and transmission of the Bordetella spp. In one aspect, disclosed herein are fusion polypeptides of any preceding aspect, wherein the composition or fusion polypeptide further comprises an acellular Gram negative vaccine component (such as, for example, the acellular pertussis vaccine (aP) component pertussis toxoid (PTd)).

55. Pertussis toxin (PTX) is produced by Bordetella pertussis, the bacterium responsible for whooping cough. Pertussis toxin is a multi-component protein composed of six non-covalently bound subunits ranging in molecular weight from approximately about 9 kDa to about 28 kDa. These subunits are designated as SI, S2, S3, S4 and S5 and occur in native pertussis toxin in a ratio of 1 : 1 : 1 :2: 1, where the subunit S4 is present in two copies The largest subunit SI, also called the A protom er, is responsible for the ADP- ribosy!transferase activity. List Labs produces Pertussis Toxin Mutant R9K, E129A (both in the SI subunit), a genetically inactivated mutant of pertussis toxin, which has a modified sequence encoding the enzyme subunit (Table 2). Virulence of this pertussis mutant is reduced relative to that found with the wild type.

Table 2 Pertussis Toxic Mutant R9K, E129A

Subunit [ AA sequence

^"s^hunkT

! AAGAACAGGCTGGCTGACGTGGCTGGCGATT j AILAVTAPVTSPAWADDPPA CTTGCCGTCACGGCGCCCGTGACTTCGCCGGC TVYRY D SRPPED V F QN GFTA

ATGGGCCGACGATCCTCCCGCCACCGTATACC WGNNDNVLDHLTGRSCQV

GCTATGACTCCCGCCCGCCGGAGGACGTTTTC GS SN S AF V STS S SRRYTE VYL

CAGAACGGATTCACGGCGTGGGGAAACAACG EHRMQEAVEAERAGRGTGH

ACAATGTGCTCGACCATCTGACCGGACGTTCC FIGYIYEVRADNNFY GAAS S

TGCCAGGTCGGCAGCAGCAACAGCGCTTTCGT YFEY VD rY GDNAGRILAGA

CTCCACCAGCAGCAGCCGGCGCTATACCGAG LATYQ SEY LAHRRIPPENIRR

GTCTATCTCGAACATCGCATGCAGGAAGCGGT VTRWHNGITGETTTTEYSN

CGAGGCCGAACGCGCCGGCAGGGGCACCGGC ARYVSQQTRANPNPYTSRRS

CA CTTC A TCGGCTA C ATCTA CG A A GT C CGCGC VASIVGTLVRMAPVIGACM

CGACAACAATTTCTACGGCGCCGCCAGCTCGT ARQAESSEAMAAWSERAGE

ACTTCGAATACGTCGACACTTATGGCGACAAT AMVLVYYESIAYSF (SEQ ID

GCCGGCCGTATCCTCGCCGGCGCGCTGGCCAC NO: 104)

CTACCAGAGCGAATATCTGGCACACCGGCGC

ATTCCGCCCGAAAACATCCGCAGGGTAACGC

GGGTCTATCACAACGGCATCACCGGCGAGAC

CACGACCACGGAGTATTCCAACGCTCGCTACG

TCAGCCAGCAGACTCGCGCCAATCCCAACCCC

TACACATCGCGAAGGTCCGTAGCGTCGATCGT

CGGCACATTGGTGCGCATGGCGCCGGTGATA

GGCGCTTGCATGGCGCGGCAGGCCGAAAGCT

CCGAGGCCATGGCAGCCTGGTCCGAA CGCGC

CGGCGAGGCGATGGTTCTCGTGTACTACGAA

AGCATCGCGTATTCGTTCTAG (SEQ ID NO: 103)

Subunit 2 ATGCCGATCGACCGCAAGACGCTCTGCCATCT MPIDRKTLCHLLSVLPLALL

CCTGTCCGTTCTGCCGTTGGCCCTCCTCGGAT GSHVAIIASTPGIVIPPQEQIT

CTCACGTGGCGCGGGCCTCCACGCCAGGCATC QHGGPYGRCANKTRALTVA

GTCATTCCGCCGCAGGAACAGATTACCCAGC ELRGSGDLQEYLRHVTRGW

ATGGCAGCCCCTATGGACGCTGCGCGAACAA STEAL YDGTYLGGEY GGVIK

GACCCGTGCCCTGACCGTGGCGGAATTGCGC DGTPGGAFDLKTTFCIMTTR

GGCAGCGGCGATCTGCAGGAGTACCTGCGTC NTGQPATDHYYSNVTATRL

ATGTGACGCGCGGCTGGTCAATATTTGCGCTC LSSTNSRLCAVFVRSGQPV1G

TACGATGGCACCTATCTCGGCGGCGAATATGG ACTSPYDGKYWSMYSRLRK

CGGCGTGATCAAGGACGGAACACCCGGCGGC MLYLIYVAGISVRVHVSKEE

GCATTCGACCTGAAAACGACGTTCTGCATCAT QYYDYEDATFETYALTGISI

GACCACGCGCAATACGGGTCAACCCGCAACG CNPGSSLC (SEQ ID NO: 106)

GATCACTACTACAGCAACGTCACCGCCACTCG

CCTGCTCTCCAGCACCAACAGCAGGCTATGCG

CGGTCTTCGTCAGAAGCGGGCAACCGGTCATT

GGCGCCTGCACCAGCCCGTATGACGGCAAGT

ACTGGAGCATGTACAGCCGGCTGCGGAAAAT

GCTTTACCTGATCTACGTGGCCGGCATCTCCG

TACGCGTCCATGTCAGCAAGGAAGAACAGTA

TTACGACTATGAGGACGCAACGTTCGAGACTT

ACGCCCTTACCGGCATCTCCATCTGCAATCCT

GGATCATCCTTATGCTGA (SEQ ID NO: 105)

Subunit 3 ! ATGCTGATCAACAACAAGAAGCTGCTTCATCA MLINNKKLLHHILPILVLALL

! CATTCTGCCCATCCTGGTGCTCGCCCTGCTGG GMRTAQ A V APGT VTPP KALF | GCATGCGCACGGCCCAGGCCGTTGCGCCAGG TQQGGAYGRCPNGTRALTV ! CATCGTCATCCCGCCGAAGGCACTGTTCACCC AELRGNAELQTYLRQITPGW ! AACAGGGCGGCGCCTATGGACGCTGCCCGAA SI Y GLY DGIY LGQ AY GGIIK ! CGGAACCCGCGCCTTGACCGTGGCCGAACTG DAPPGAGFIYRETFCITTIYK ! CGCGGCAACGCCGAATTGCAGACGTATTTGC TGQPAADHYYSKVTATRLL ! GCCAGATAACGCCCGGCTGGTCCATATACGGT A STN SRLC A VFVRDG QS VIG CTCTATGACGGTACGTACCTGGGCCAGGCGTA ACASPYEGRYRDMYDALRR

CGGCGGCATCATCAAGGACGCGCCGCCAGGC LLYMIYMSGLAVRVHVSKE

GCGGGGTTCATTTATCGCGAAACTTTCTGCAT EQYYDYED ATF QTY ALTGI S

CACGACCATATACAAGACCGGGCAACCGGCT LCNPAASIC (SEQ ID NO: 108)

GCGGATCACTACTACAGCAAGGTCACGGCCA

CGCGCCTGCTCGCCAGCACCAACAGCAGGCT

GTGCGCGGTATTCGTCAGGGACGGGCAATCG

GTCATCGGAGCCTGCGCCAGCCCGTATGAAG

GCAGGTACAGAGACATGTACGACGCGCTGCG

GCGCCTGCTGTACATGATCTATATGTCCGGCC

TTGCCGTACGCGTCCACGTCAGCAAGGAAGA

GCAGTATTACGACTACGAGGACGCCACATTCC

AGACCTATGCCCTCACCGGCATTTCCCTCTGC

AACCCGGCAGCGTCGATATGCTGA (SEQ ID

NO: 107)

ATGCTGAGACGCTTCCCCACTCGAACCACCGC MLRRFPTRTTAPGQGGARRS

CCCGGGACAGGGCGGCGCCCGGCGGTCGCGC RVRALAWLLASGAMTHLSP

GTGCGCGCCCTGGCGTGGTTGCTGGCATCCGG ALADVPYVLVKTNMVVTSV

CGCGATGACGCATCTTTCCCCCGCCCTGGCCG AMKPYEVTPTRMLVCGIAA

ACGTTCCTTATGTGCTGGTGAAGACCAATATG KLGAAASSPDAHVPFCFGKD

GTGGTCACCAGCGTAGCCATGAAGCCGTATG LKRPGSSPMEVMLRAVFMQ

AAGTCACCCCGACGCGCATGCTGGTCTGCGGC QRPLRMFLGPKQLTFEGKPA

ATCGCCGCCAAACTGGGCGCCGCGGCCAGCA LELIRMVECSGKQDCP

GCCCGGACGCGCACGTGCCGTTCTGCTTCGGC ID NO: 1 10)

AAGGATCTCAAGCGTCCCGGCAGCAGTCCCA

TGGAAGTCATG1TGCGCGCCGTCTTCATGCAA

CAACGGCCGCTGCGCATGTTTCTGGGTCCCAA

GCAACTCACTTTCGAAGGCAAGCCCGCGCTCG

AACTGATCCGGATGGTCGAATGCAGCGGCAA

GCAGGATTGCCCCTGA (SEQ ID NO: 109)

ATGCAGCGGCAAGCAGGATTGCCCCTGAAGG MQRQAGLPLKANPMHTIASI CGAACCCCATGCATACCATCGCATCCATCCTG LLS LGIYSPADVAGLP^IL TTGICCG FGCTCGGCA IATACAGCCCGGC EGA YKNFTVQELALKLKGKNQE CGTCGCCGGCTTGCCGACCCATCTGTACAAGA FCLTAFMSGRSLVRACLSDA ACTTCACTGTCCAGGAGCTGGCCTTGAAACTG GHEHDTWFDTMLGFAISAY AAGGGCAAGAATCAGGAGTTCTGCCTGACCG ALKSRIALTVEDSPYPGTPG CCTTCATGTCGGGCAGAAGCCTGGTCCGGGCG DLLELQTCPLNGY CE (SEQ ID TGCCTGTCCGACGCGGGACACGAGCACGACA NO: 1 12)

CGTGGTTCGACACCATGCTTGGCTTTGCCATA TCCGCGTATGCGCTCAAGAGCCGGATCGCGCT GACGGTGGAAGACTCGCCGTATCCGGGCACT CCCGGCGATCTGCTCGAACTGCAGATCTGCCC GCTCAACGGATATTGCGAATGA (SEQ ID NO:

56. It is understood and herein contemplated that the disclosed polypeptides, adjuvants, and acellular vaccine components for use in eliciting an immune response or for treating, inhibiting, or preventing a Gram negative bacterial infection can be administered in compositions such as vaccines as individual polypeptides or as a fusion construct or a combination thereof. Thus, in one aspect, disclosed herein are compositions comprising a T3SA needle tip protein (such as, for example, Bsp22, LerV, BipD, PcrV, CT053, or CT668) ip or an antigenic fragment thereof from a Gram negative bacteria (such as, for example, Bordetella spp., Burkholderia spp., Chlamydia spp., Pseudomonas spp., Vibrio spp. or Yersinia spp.) and a T3SA translocator protein (such as, for example, BopB, YopB, BipB, PopB, CopB, or CopB 2) or an antigenic fragment thereof from a Gram negative bacteria; wherein the gram negative bacteria is not a Salmonella enierica or Shigella spp. In one aspect, the composition can comprise the needle tip protein or fragment thereof and the translocator protein or fragment thereof as separate components or as a fusion polypeptide. Also disclosed herein are compositions of any preceding aspect, wherein the composition comprises an adjuvant (such as, for example, cholera toxin, CTA1, dmLT, or LTA1) and/or bacterial toxin protein, such as a pertussis toxoid (PTd). Thus, in one aspect, disclosed herein are vaccines comprising any of the peptides, polypeptides, proteins, fusion peptides, fusion polypeptides, fusion proteins, or compositions disclosed herein. In some embodiments, the vaccine can further comprise an acellular gram negative vaccine or active components thereof.

h Sequence similarities

57. It is understood that as discussed herein the use of the terms homology and identity mean the same thing as similarity. Thus, for example, if the use of the word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences. Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not.

58. In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences.

This identity of particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of genes and proteins herein disclosed (such as, for example, Bsp22, LcrV, BipD, PcrV, CT053, CT668, BopB, YopB, BipB, PopB, CopB, CopB2, 22BF, BurkF, PaF, YerF, CT053~CopB, CT053-CopB2, CT668-CopB, or CT668-CopB2) typically have at least bout 70, 71, 72, 73, 74, 75, 76, 77. 78, 79, 80, 81, 82, 83, 84, 85. 86, 87, 88, 89, 90, 91, 92,

93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence. Those of skill in the art. readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can he calculated after aligning the two sequences so that the homology is at its highest level.

59. Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (G AP, BESTFIT,

FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

60. It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.

61. For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above. For example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods. As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods. As yet another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculati on methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).

2. Nucleic adds

62. There are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example Bsp22, LcrV, BipD, PcrV,

21 _ CTOS 3, CT668, BopB, YopB, BipB, PopB, CopB, CopB2, 22BF, BurkF, PaF, YerF, CT053- CopB, CT053-CopB2, CT668-CopB, or CT668-CopB2 or antigenic fragments thereof, as well as various functional nucleic acids. The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, that the expressed mRNA will typically be made up of A, C, G, and U. Likewise, it is understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantageous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment.

a) Nucleotides and related molecules

63. A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenin-9-yl (A), cytosin-l-yl (C), guanin-9-yl (G), uracii-l-yl (U), and thymin-l-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety'^· of a nucleotide is pentavalent phosphate. An non-limiting example of a nucleotide would be 3'- AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). There are many varieties of these types of molecules available in the art. and available herein.

64. A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine, as well as modifications at the sugar or phosphate moieties. There are many varieties of these types of molecules available in the art and available herein.

65. Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson- Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid. There are many varieties of these types of molecules available in the art and available herein.

66. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically

..... 22— linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety. (Letsinger et at., Proc Natl Acad. Sci. USA ,

1989, 86, 6553-6556). There are many varieties of these types of molecules available in the art and available herein.

67. A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.

68. A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.

b) Sequences

69. There are a variety of sequences related to the protein molecules involved in the signaling pathways disclosed herein, for example Bsp22, LcrV, BipD, PcrV, CT053, CT668, BopB, YopB, BipB, PopB, CopB, CopB2, 22BF, BurkF, PaF, YerF, CT053-CopB, CT053-CopB2, CT668-CopB, or CT668-CopB2, or any of the nucleic acids disclosed herein for making Bsp22, LcrV, BipD, PcrV, CT053, CT668, BopB, YopB, BipB, PopB, CopB, CopB2, 22BF, BurkF, PaF, YerF, CT053-CopB, CT053-CopB2, CT668-CopB, or CT668- CopB2, all of which are encoded by nucleic acids or are nucleic acids. The sequences for the human analogs of these genes, as well as other analogs, and alleles of these genes, and splice variants and other types of vari ants, are avai lable in a vari ety of protein and gene databases, including GENBANK*'. Those of skill in the art understand how to resolve sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences. Primers and/or probes can be designed for any given sequence given the information disclosed herein and known in the art.

3. Nucleic Add Delivery

70. In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the disclosed nucleic acids can be in the form of naked DNA or RNA, or the nucleic acids can be in a vector for delivering the nucleic acids to the cells, whereby the antibody-encoding DNA fragment is under the transcriptional regulation of a promoter, as would be well understood by one of ordinary skill in the art. The vector can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTTN, LIPOFECT AMINE (GIBCO-BRL, Inc., Gaithersburg, MD),

SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRAN SFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by

electroporation, the technology for which is available from Genetronics, Inc. (San Diego,

CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, A Z).

71. As one example, vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g , Pastan et ah, Proc. Natl. Acad. Sci. U.S.A. 85:4486, 1988; Miller et al , Mol Cell. Biol. 6:2895, 1986). The recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding a broadly neutralizing antibody (or active fragment thereof). The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994), adeno-associated viral (AAV) vectors (Goodman et al, Blood 84: 1492-1500, 1994), lenti viral vectors (Naidini et al., Science 272:263-267, 1996), pseudotyped retroviral vectors (Agrawal et al, Exper. Hemalol. 24:738-747, 1996). Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al, Blood 87:472-478, 1996). This disclosed compositions and methods can be used in conjunction with any of these or other commonly used gene transfer methods.

72. As one example, if the antibody-encoding nucleic acid is delivered to the cells of a subject in an adenovirus vector, the dosage for administration of adenovirus to humans can range from about 10⁷ to about 10⁹ plaque forming units (pfu) per injection but can be as high as about 10^l2 pfu per injection (Crystal, Hum. Gem Her. 8:985-1001, 1997; Alvarez and Curiel, Hum. Gene Ther. 8:597-613, 1997). A subject can receive a single injection, or, if additional injections are necessary, they can be repeated at six month intervals (or other appropriate time intervals, as determined by the skilled practitioner) for an indefinite period and/or until the efficacy of the treatment has been established. 73. Parenteral administration of the nucleic acid or vector, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. For additional discussion of suitable formulations and various routes of administration of therapeutic compounds, see, e.g., Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.

4. Delivery of the compositions to cells

74. There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery' systems and non-vira! based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants, or physi co-mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff, J. A , et ah, Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991). Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, the methods will be modified to specifically function with large DNA molecules. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier.

a) Nucleic add based delivery systems

75. Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53 :83-88, (1993)).

76. As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as 22BF into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. Viral vectors are, for example,

Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio vims, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses

25— which make them suitable for use as vectors. Retroviruses include Murine Maloney

Leukemia vims, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector.

However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Preferred vectors of this type will carry coding regions for Interleukin 8 or 10.

77. Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene/promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material . The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.

(1) Retroviral Vectors

78. A retrovirus is an animal virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms. Retroviral vectors, in general, are described by Verma, I.M., Retroviral vectors for gene transfer.

79. A retrovirus is essentially a package which has packed into it nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat. In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus. Typically a retroviral genome, contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell. Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag

..... 26— transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome. The removal of the gag, pol, and env genes allows for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.

80. Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery , but lacks any packaging signal. When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in eis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals

(2) Adenoviral Vectors

81. The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61 : 1213-1220 (1987); Massie et al., Mol Cell Biol. 6:2872-2883 (1986); Haj -Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al, J. Virology 61 : 1226-1239 (1987); Zhang "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery⁷ to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92: 1580-1586 (1993); Kirshenbaum, ,/. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92: 1085-1092 (1993); Moul!ier, Nature Genetics 4: 154-159 (1993); La. Salle, Science 259:988-990 (1993); Gomez-Foix, ./. Biol.

( hem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner,

_ 7 _ Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73 : 1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Cail!aud, Eur. J. Neuroscience 5: 1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)).

Recombinant adenoviaises achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J Virology 55:442-449 (1985); Seth, et a! , J. Virol. 51 :650-655 (1984); Seth, et al , Mol. Cell Biol. 4: 1528-1533 (1984); Varga et al., J. I iro!ogy 65:6061 -6070 ( 1991 }; Wickham et al ., Cell 73 :309-319 (1993)).

82. A viral vector can be one based on an adenovirus which has had the El gene removed and these virons are generated in a cell line such as the human 293 cell line. In another preferred embodiment both the El and E3 genes are removed from the adenovirus genome.

5. Adeno-assoeiaied viral vectors

83. Another type of viral vector is based on an adeno-associated virus (AAV).

This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19 Vectors which contain this site specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and/or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

84. In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell- specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.

85. Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. US Patent No. 6,261,834 is herein incorporated by reference for material related to the AAV vector.

86. The disclosed vectors thus provide DMA molecules which are capable of integration into a mammalian chromosome without substantial toxicity.

28— 87. The inserted genes in viral and retroviral usually contain promoters, and/or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

a) Large payload viral vectors

88. Molecular genetic experiments with large human herpesviruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpesviruses (Sun et a!., Nature genetics 8: 33-41, 1994; Cotter and Robertson,. Carr OpinMol Ther 5: 633-644, 1999). These large DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have the potential to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV

recombinants can maintain large pieces of DNA in the infected B-cells as episomal DNA. Individual clones carried human genomic inserts up to 330 kb appeared genetically stable The maintenance of these episomes requires a specific EBV nuclear protein, EBNA1, const! tutively expressed during infection with EBV Additionally, these vectors can be used for transfection, w'here large amounts of protein can be generated transiently in vitro.

Herpesvirus ampiicon systems are also being used to package pieces of DNA > 220 kb and to infect cells that can stably maintain DNA as episomes.

89. Other useful systems include, for example, replicating and host-restricted non- repiicating vaccinia vims vectors.

b) Nora-midek add based systems

90. The disclosed compositions can be delivered to the target cells in a variety of ways. For example, the compositions can be delivered through electroporation, or through iipofection, or through calcium phosphate precipitation. The delivery' mechanism chosen will depend in part on the type of ceil targeted and whether the delivery' is occurring for example in vivo or in vitro.

91. Thus, the compositions can comprise, in addition to the disclosed needle tip protein-translocator protein fusion (such as, for example, 22BF) or vectors for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a. compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into

..... 29— the respiratory tract to target cells of the respiratory' tract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1 :95-100 (1989); Feigner et al. Proc. Nail. Acad. Sci USA 84:7413-7417 (1987); U. S. Pat. No. 4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

92. In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), delivery of the compositions to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as

LIPOFECTIN, LIPOFECTAMINE (GIBCG-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRAN SFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

93. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate ^'hem., 2:447-451, (1991); Bagshawe, K.D., Br. .]. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. ,/. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother ., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews , 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol 42:2062-2065, (1991)). These techniques can be used for a variety of other specific cell types. Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through ceil specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta,

1 104: 179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in ciathrin-coated pits, enter the cell via ciathrin-coated vesicles, pass through an acidified endosome in which the receptors are

30 -- sorted, and then either recycle to the cell surface, become stored intracellular!y, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

94. Nucleic acids that are delivered to cells which are to be integrated into the host cell genome, typically contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral integration systems can also be incorporated into nucleic acids which are to be delivered using a non-nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained in the delivery system can become integrated into the host genome.

95. Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods necessary to promote homologous recombination are known to those of skill in the art.

c) In vivo/ex vivo

96. As described above, the compositions can be administered in a

pharmaceutically acceptable carrier and can be delivered to the subject’s cells in vivo and/or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).

97. If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocol s well known in the art. The compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopicaliy transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject. 6. Expression systems

98. The nucleic acids that are delivered to cells typically contain expression controlling systems. For example, the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

a) Viral Promoters and Enhancers

99. Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B vims and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter. The early and late promoters of the SV40 vims are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Tiers et ah, Nature, 273 : 113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HmdWl E restriction fragment (Greenway, P.J. et at , Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species also are useful herein.

100. Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Set. 78: 993 (1981)) or 3' (Lusky, M.L., et al., Mol. Cell Bio. 3 : 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al, Cell 33 : 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al. Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell vims for general expression.

Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

32 -- 101. The promotor and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

102. In certain embodiments the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV4G promoters, cytomegalovirus (full length promoter), and retroviral vector LTR.

103. It has been shown that all specific regulatory_' elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

104. Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary' for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3’ untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct

b) Markers

105. The viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Preferred marker genes are the E. Coli lac Z gene, which encodes b-galactosidase, and green fluorescent protein.

..... 33 ...... 106 In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow

independent of a supplemented media. Two examples are: CHO DHFR- cells and mouse LTK- cells. These ceils lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary' for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non -supple ented media.

107 The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection.

Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet 1 : 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P.

Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et ah, Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puromycin.

7. Peptides

a) Protein variants

108 As discussed herein there are numerous variants of the needle tip protein- translocator protein fusion (such as, for example, Bsp22, LcrV, BipD, PcrV, CT053, CT668, BopB, YopB, BipB, PopB, CopB, CopB2, 22BF, BurkF, PaF, YerF, CT053-CopB, CT053- CopB2, CT668-CopB, or CT668-CopB2) that are known and herein contemplated. In addition, to the known functional strain variants there are derivatives of the needle tip protein and translocator protein which also function in the disclosed methods and compositions. Protein variants and derivatives are well understood to those of skill in the art and can involve amino acid sequence modifications. For example, antino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogen! city to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than from about 2 to about 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of from about 1 to about 10 amino acid residues; and deletions will range from about I to about 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place.

Such substitutions generally are made in accordance with the following Tables 3 and 4 and are referred to as conservative substitutions.

TABLE 3: Amino Acid Abbreviations

Amino Add Abbreviations

Alanine Ala A

Allosoleucine Alie

Arginine Arg R

Asparagine Asn N

Aspartic acid Asp D

Cysteine Cys C

Glutamic acid Glu E

Glutamine G3n 0

Glycine Gly G Histidine His H

Isolelucme 11e I

Leucine Leu L

Lysine Lys K

Phenylalanine Phe F

Proline Pro P

Pyroglutamic acid pGlu

Serine Ser S

Threonine Thr T

Tyrosine Tyr Y

Tryptophan Trp W

Valine Val V

TABLE 4: Amino Acid Substitutions

Original Residue Exemplary Conservative

Substitutions, others are known in the art.

Ala Ser

Arg Lys; Gin

Asn Gin; His

Asp Glu

Cvs Ser

Gin Asn, Lys

Glu

Gly Pro

His Asn;Gln

He Leu; Val

Leu He: Val

Lys Arg; Gin

Met Leu; lie

Phe Met: Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

Val lie; Leu

109 Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 4, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. send or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g , glycine, in this case, or (e) by increasing the number of sites for sulfation and/or glycosylation.

1 10. For example, the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.

1 11 Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also may be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg, is accomplished for example by deleting one of the basic residues or substituting one by g!utaminyl or histidyl residues.

1 12. Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E Creighton, Proteins: Structure and Molecular Properties, W. FI. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N- terrninal amine and, in some instances, amidation of the C-terminal carboxyl.

1 13. It is understood that one way to define the variants and derivatives of the disclosed proteins herein is through defining the variants and derivatives in terms of homology /identity to specific known sequences. For example, SEQ ID NO: 1 sets forth a particular sequence of Bordetella needle tip protein-translocator protein fusion (22BF) and SEQ ID NO: 2 sets forth a particular sequence of a 22BF fusion protein. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level. 1 14 Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection

115 The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, i Science 244:48-52, 1989, Jaeger et al. Proc. Natl Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989.

116 It is understood that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations.

1 17 As this specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i e. all nucleic acids having a sequence that encodes one particular protein sequence as well as ail nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and even,_' sequence is in fact disclosed and described herein through the disclosed protein sequence. For example, one of the many nucleic acid sequences that can encode the protein sequence set forth in SEQ ID NO: 2 is set forth in SEQ ID NO: 1 It is understood that for this mutation all of the nucleic acid sequences that encode this particular derivative of the 22BF are also disclosed. It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein in the particular needle tip protein-translocator protein fusion (such as, for example, 22BF) from which that protein arises is also known and herein disclosed and described.

118 It is understood that there are numerous amino acid and peptide analogs which can be incorporated into the disclosed compositions. For example, there are numerous D amino acids or amino acids which have a different functional substituent then the amino acids shown in Table 3 and Table 4. The opposite stereo isomers of naturally occurring peptides

..... 38 -- are disclosed, as well as the stereo isomers of peptide analogs. These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site specific way.

119 Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include— CH?.NH— ,— CH₂S— ,— CH2— CH2— ,— CH=CH— (cis and trans),— COCH2— , ~CH(OH)CH2~, and— CHH2SO— (These and others can be found in Spatola, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p 267 (1983); Spatola, A. F , Vega Data (March 1983), Vol. 1 , Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D et al, Int J Pept Prot Res 14: 177-185 (1979) (-CH2NFI-, - CH2CH2--); Spatola et al. Life Sci 38:1243-1249 (1986) (— CH2--S); Hann J. Chem. Soc Perkin Trans. I 307-314 (1982) (— CH=CH— , cis and trans); Almquist et al. J Med. Chem. 23: 1392-1398 (1980) (-COCH2-); Jennings-White et al. Tetrahedron Lett 23:2533 (1982) (- -COCH2-); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (-- CH(OH)CH_2~); Ho!!aday et al. Tetrahedron. Lett 24:4401-4404 (1983) (~C(OH)CH_2~); and Hruby Life Sci 31 :189-199 (1982) (— CH2— S— ); each of which is incorporated herein by reference A particularly preferred non-peptide linkage is— CH2NH— . It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g- aminobutyric acid, and the like.

120 Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.

121 D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. 8. Pharmaceutical carriers/Delivery of pharmaceutical products

122. As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

123. The compositions may be administered orally, parenterally (e.g.,

intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used herein, "topical intranasal administration” means deli very of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition.

However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

124. Parenteral administration of the composition, if used, is generally

characterized by injection Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.

125. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et ah, Bioconjugate

..... 40 ...... Chern., 2:447-451, (1991 ); Bagshawe, K.D., Br. J. Cancer , 60:275-281 , (1989), Bagshawe, et ah, Br. J. Cancer, 58:700-703, (1988); S enter, et ah, Bioconjugate Ghent., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immnnother ., 35:421-425, (1992), Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062- 2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes

(including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biophysica Acta, 1104: 179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-eoated pits, enter the cell via clathrin-eoated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored

intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of

macromolecuies, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

126 The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

127. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the

pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.6, and most preferably about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of

administration and concentration of composition being admini stered.

128. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

129. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

130. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmicaUy, vaginally, rectally, intranasa!!y), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous,

intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or

transderm al ly.

131. Preparations for parenteral administration include sterile aqueous or n on- aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte

replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

132. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. 133. Compositions for oral administration include powders or granules,

suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets.

Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.

134. Some of the compositions may potentially be administered as a

pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di- , tri-alkyl and aryl amines and substituted ethanolamines.

b) Therapeutic Uses

135. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of

Monoclonal Antibodies, Ferrone et af, eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et a!.. Antibodies in Human Diagnosis and Therapy, Haber et ah, eds., Raven Press, New' York (1977) pp 365-389 A typical daily dosage of the antibody used alone might range from about 1 pg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.

136. In a preferred embodiment, the amount of protein that is administered per dose of vaccine is in the range of from about 0.0001 to about 1000 pg/kg. In one embodiment, the amount is in the range of from about 0 001 to about 1000 pg/kg of body weight of the recipient. In one embodiment, the amount is in the range of from about 0.01 to about 1000

..... 43 ...... pg/kg of body weight of the recipient. In one embodiment, the amount is in the range of from about 0.01 to about 100 pg/kg of body weight of the recipient. Those of skill in the art will recognize that the precise dosage may vary from situation to situation and from patient to patient, depending on e.g. age, gender, overall health, various genetic factors, and other variables known to those of skill in the art. Dosages are typically determined e.g. in the course of animal and/or human clinical trials as conducted by skilled medical personnel, e.g. physicians or veterinarians.

C. Methods of using the compositions

137. Herein, the protective efficacy of the Bordetella spp. tip/translocator fusion, 22BF, is examined against lethal lung challenge and with complete (sterilizing) clearance of colonizing bacteria. Unlike some components of the current aP vaccine, Bsp22 and BopB are required for infection and are not mutable since they must be retained structurally and functionally within the context of a large nanomachine residing within the Bordetella cell envelope. Furthermore, targeting the Bordetella T3SA renders the pathogen less able to tight off the host innate and adaptive immune responses. Regardless of whether 22BF is protective alone or when used with components of the current aP vaccine, the innovation of this high risk, high reward investigation lies in whether this subunit vaccine can elicit sterilizing immunity and thereby prevent the colonization that results in host to host transmission. It has been reported that Bsp22 (a component of the 22BF fusion vaccine) does not elicit a serum antibody response in humans during the course of natural infection and is not a protective antigen in mice. Nevertheless, as shown herein, protective and sterilizing immunity can be obtained with the compositions disclosed herein.

138. Thus, in one aspect, disclosed herein are methods of eliciting an immune response in a subject to a Gram negative bacteria (such as, for example, Bordetella spp., Burkholderia spp., Chlamydia spp., Pseudomonas spp., Vibrio spp. Enteropathogenic or Enterohemorrhagic E. coli or Yersinia spp.) comprising administering to the subject the fusion polypeptides, compositions, or vaccines disclosed herein. Accordingly, in one aspect, disclosed herein are methods of eliciting an immune response against at least one Gram negative bacteria serovar in a subject in need thereof, comprising administering to the subject a composition comprising at least one needle tip protein or a fragment thereof and/or at least one translocator protein or a fragment thereof; wherein said composition is administered in an amount sufficient to elicit an immune response to said at least one Gram negative bacteria serovar in said subject; and wherein the Gram negative bacteria is not a Shigella spp. or Salmonella spp. In one aspect, the immune response elicited provides sterilizing immunity to the infectious bacterium.

139. As can be appreciated by the skilled artisan, the methods of eliciting an immune response can be used for the purpose of treating, inhibiting, or preventing an infection of a Gram negati ve bacteria (such as, for example, Bordeiella spp., Burkholderia spp., Chlamydia spp. Pseudomonas spp. Vibrio spp. Enteropathogenic or

Enterohemorrhagic E. coli or Yersinia spp.y. Thus, in one aspect, disclosed herein are methods of treating, inhibiting, or preventing an infection of a Gram negative bacteria in a subject comprising administering to the subject a therapeutic amount of any of the fusion polypeptides, compositions, or vaccines disclosed herein. As one goal of any vaccine is not only to prevent infection or reducing the severity of disease in the individual receiving the vaccine, but also to prevent further transmission of the infectious agent (sterilizing immunity), it is understood and herein contemplated that the disclosed methods of treatment, inhibition, or preventing an infection can further comprise inhibiting and/or preventing colony formation of the bacteria and/or transmission of the bacteria to another subject.

140. The term“therapeutically effective” refers to the amount of the composition used that is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

141. The term“treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

142. The term“inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, response, condition, or disease. This can also include, for example, a

4_{5 .....} 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

143. By“reduce” or other forms of the word, such as“reducing” or“reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary' for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

144. By“prevent” or other forms of the word, such as“preventing” or

“prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a parti cular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

IX Examples

145. The following examples are put forth so as to provide those of ordinary_' skill in the art with a complete disclosure and descripti on of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

1. Example 1

a) Use of a T3SS needle tip /translocator protein fusion as a protective antigen against B. pertussis :

146. The dominant antigen eliciting protection against Gram-negative pathogens is LPS, which confers O-antigen serotype specificity. The initial project focused on Shigella , however, there are at least 58 distinct Shigella serotypes. This reduces broad-spectrum efficacy for live, attenuated and whole, killed vaccines, which tend to be somatic antigen driven. IpaD and IpaB are the surface-localized needle tip and first translocator proteins of the T3SA, respectively. They are essential for virulence in all Shigella, are >98% conserved across ail Shigella species, and provide serotype-independent protection. When

IpaD+IpaB+dmLT was given IN to mice, the formulation was about 80-90% protective against lethal challenge by homologous and heterologous Shigella spp. (Table 5). To reduce the production cost, IpaD and IpaB were genetically fused to make DBF. Not only did the DBF provide protection against lethal challenge, it also unexpectedly increased the cell- mediated immunity, most notably the IL-17 and IFN-g responses. When the Salmonella enterica tip and first translocator proteins of the T3SSs of SPI-l and SPI-2 {Salmonella Pathogenicity Islands 1 and 2) were fused, about 70% protection against lethal challenge by two S. enterica serovars was observed when both fusion proteins were administered simultaneously (Table 5).

Table 5. Protective efficacy of tip/translocator fusions against challenge by appropriate pathogen.

Mice (n=10) were vaccinated 3 times biweekly with the vaccine and then challenged with indicated pathogen at day 56. Protection is indicated as percent survival after 21 days.

147. B. bronchi septica and B. pertussis infections have been shown to also require a T3SS for virulence. To determine if a subunit of the T3SS could be used to confer protection, B. pertussis Bsp22 and BopB, the T3SS needle tip and first translocator proteins, which are 98% conserved with those of B. bronchiseptica were genetically fused. Mice were vaccinated biweekly, three times with 22BF, 22BF+dmLT, Bsp22+dmLT, or PBS. One group was also vaccinated twice with the Zoetis canine vaccine that is a killed cellular extract of B. bronchiseptica. The 22BF+dmLT exhibited 100% protection against ft bronchiseptica in a mouse lethal pulmonary model, while the commercial vaccine provided 80% protection (Fig. 1). Moreover, the mice vaccinated with 22BF+dmLT did not show signs of illness and they actually gained weight throughout the challenge (Fig. 2). In contrast, the mice vaccinated with the Zoetis vaccine lost weight and then regained it, however, they continued to display low'er health scores than the mice vaccinated with 22BF+dmLT for the remainder of the experiment (Fig. 2). 148. Next, the protective efficacy of the 22BF fusion with and without dmLT and in comparison to the needle tip protein Bsp22 and translocator protein BopB and the effect of fusion of dmLT relative to concurrent administration was examined. Mice were vaccinated IN on days 0, 14, 28 and challenged with Bordeteta bronchiseptica on day 56 On day 7 mice were sacrificed and the colony forming units (CFU)/lung were determined (Figure 3). 22BF+dmLT reduced the B. bronchiseptica CFU lung burden by 96.5%. Blood and fecal samples were collected on days -1, 13, 27, 41, and 55 The kinetics of IgG were assessed. Typical logarithmic increases were seen (Figure 4) regardless of the antigen on the well. No IgA was detected in fecal samples of any of the mice. Antibody secreting cells in the bone marrow, spleen, and lungs (Figure 5) were stimulated with Bsp22, BopB and dmLT. All organs from mice that had been vaccinated with the single proteins or double proteins or fusion, as long as the dmLT was present, exhibited some level of IgG or IgA ASCs

149. While protection is important, the key to a next generation subunit vaccine against Bordetella spp. must be that it exhibits sterilizing immunity (to prevent carriage). The lungs of the mice that survived the challenge of Fig. 2 w'ere examined to look for sterilizing immunity. Ail of the remaining 22BF and PBS vaccinated mice exhibited CFU/lung. In contrast, the 22BF+dmLT vaccinated mice demonstrated 38% sterilizing immunity while only 12% of the mice vaccinated with the Zoetis vaccine demonstrated sterilizing immunity. Thus, the 22BF represents an innovative new antigen to protect against B. bronchiseptica and B. pertussis due to the conservation of these antigens.

150. It is shown herein that the use of a broad, serotype-independent subunit vaccine against Shigella spp. and S. enterica serotypes. These vaccines are based on the fusion of the T3SA tip and first translocator proteins, which are highly conserved within a given bacterial genus. The DBF has also been shown to protect monkeys from severe diarrhea and S. enterica S1S2 fusions are protective in a bovine calf model. A genetic fusion of the B. pertussis T3SA tip/translocator system, 22BF, was generated which protects 100% of the mice challenged with a lethal dose of the heterologous B bronchiseptica. Furthermore,

22BF elicited 38% sterilizing immunity while the commercial vaccine provided 12%. Herein, not only was the B. bronchiseptica experiment repeated, but also it demonstrated that the

22BF+dmLT protects against if pertussis. B. bronchiseptica was chosen as the first challenge pathogen since it lacks pertussis toxin (PT), which could conceivably compromise adequate assessment of the 22BF. PT is an especially important toxin secreted by B. pertussis that can significantly affect the health of the host Shiga toxin was also considered when DBF vaccinated mice were challenged with S. dysenteriae , however, DBF protected 40% of those qg -- vaccinated mice while the individual proteins did not. Furthermore, due to the disease progression of S. dysenteriae, mice killed by the Shiga toxin versus those succumbing to the bacterial infection (based on health score immediately preceding death) were distinguished. 22BF provides some level of protection agai nst B. pertussis , which can be boosted by the addition of pertussis toxoid (PTd). At the end of this study, it was demonstrated that 22BF protects against both B. bronchiseptica and B. pertussis . Results for two vaccination routes and doses are obtained. PTd is required in the protection against B. pertussis was shown herein. Finally, it is demonstrated that 22BF+dmLT elicits >90, if not 100%, sterilizing immunity.

b) Cytokine Assay

151. Cytokines w^ere collected from splenocytes collected on Day 55. 100 pL of homogenized splenocytes were seeded at a concentration of 5xl0⁶ cells / rnL onto a flat- bottom 96-well plate. 100 pL of BopB, Bsp22, or dmLT at a concentration of 20 pg/mL was added onto cells (bringing final protein concentration to 10 pg/mL) Plates were incubated at 37°C and 5% CO2 for 48 hours. After incubation, plates were centrifuged at 1600 rpm at 4°C for seven minutes. Supernatants were collected and stored at -20°C until analysis. Cytokines were analyzed using the MSD® U-Plex Platform Multiplex Assay for the following cytokines for the following immune responses: Thl : IFN-y, TNF-a, EL-Ib, IL-2, IL-6, IL-10; (Figures 6 and 7) Thl 7: IL-17A; (Figure 8) Th2: IL-4, 1L-5 (Figure 9)

2. Example 2: Assess the respective humoral and cell-mediated immune responses elicited by 22BF+dmLT delivered IN and IM.

152. IN delivery of 20 pg 22BF+2.5 pg dmLT protects 100% of vaccinated mice from death and provide 38% sterilizing immunity in the lungs following B. bronchiseptica challenge. 50 pg or 15 pg 22BF IN is delivered and then in parallel deliver 100 pg or 20 pg 22BF IM or ID to assess the humoral and cell mediated responses in each case. Based on previous work, it is known that some of these dose/route combinations do not protect mice while others offer protection.

153. First the effect of the route of administrati on of 22BF fol lowing challenge was assessed. Group l is a 22BF+ 2.5 pg dmLT IN, group 2 is 22BF+ 2.5 pg dmLT IM, and group 3 is a 22BF+ 2.5 pg dmLT IN. For each administrative route, PBS vaccinated controls ere included (Groups 4, 5, and 6). Group 7 (n=T0) is vaccinated subcutaneously on day 1 and 21 with the Zoetis vaccine. After day 56 following initial vaccination, mice were challenged with a suhiethai dosage of B. pertussis intranasal iy. There was an observable difference in weight loss between mice vaccinated with the 22BF+dmLT+PTd formulation

..... 49 ...... and those that only received PBS. By Day 7 all mice aside from PBS treated mice had recovered to within 3% of pre infection weight.

154. The immune responses can be compared with the protective efficacy and potentially define a protective correlate in the mouse model. The correlate is useful in the development of the 22BF vaccine, making the necessary adjustments when it is translated to humans. The systemic immune response can be assessed by measuring serum IgG against BopB, Bsp22, and dmLT, as well as the mucosal immune response by assessing IgA in fecal pellets. Cytokine secretion is assessed in splenocytes from vaccinated mice.

155. This experiment can be performed in two parts. The IN route can use a high dose of 50 pg and low dose of 15 pg. The IM route uses a high dose of 100 pg and a low dose of 40 pg. For each route, three groups of female C57BL/6 mice (10/group) are vaccinated on days 0, 14 and 28. Group 1 is a 22BF+ 2.5 pg dmLT IN, group 2 is 22BF+ 2.5 pg dmLT IM, and group 3 is a 22BF+ 2.5 pg dmLT IN. For each administrative route, PBS vaccinated controls were included (Groups 4, 5, and 6) Group 7 (n=10) is vaccinated subcutaneously on day 1 and 21 with the Zoetis vaccine. Blood and fecal pellets are collected on days -l, 13, 27, 41 and 55 to assess mucosal and systemic humoral responses. Individual samples are tested for the presence of anti-Bsp22, -BopB, -dmLT, -PTd IgG and IgA antibodies by ELISA. Mice were immunized on days 0, 14, and 28 with 22BF+PTd admixed with dmLT,. Serum IgG antibodies specific for BopB, Bsp22, PTd, and dmLT w'ere measured by ELISA and EFN-y/IL-17A secreting cells by ELISpot and cytokine secretion using Multi array assays. BAL is collected to measure IgG and IgA responses. GraphPad Prism 5.04 can be used for graphics and statistical comparisons. Differences were analyzed using t test or ANOVA where appropriate. A P value of less than 0.05 is considered significant for all compari sons.

156. For these experiments, serum IgG levels are >10³ EU/m!, antibody secreting cells at >50 IgG ASC/10⁶ cells or >20 IgA ASC/10⁶ cells, and cytokine secreting cells at >50 IFN-g/Ί O⁶ cells and IL-17/10⁶ cells. There can also be unique systemic and mucosal humoral immune responses from mice immunized via the IN and IM routes. 50 pg IN was chosen to facilitate an increase in sterilizing immunity. The 100 pg IM dose was based on prior findings. Antibody secreting cells specific for both proteins are detected in both mucosal and memory compartments. Finally, the full profile of cytokine secretion elicited by the vaccine can demonstrate a dose and administration route dependence. Thus, these two routes (each with a high and low dose) are expected to give rise to unique immune response profiles. 3. Example 3: Determine the protective efficacy of 22BF+dmLT against B. bronchiseptica and B. pertussis challenge.

157. As discussed above, it is demonstrated herein that initial protective efficacy of 22BF+dmLT against B. bronchiseptica challenge. Insight is gained into the immune responses elicited by two doses of 22BF delivered IN and IM. Here, mice can be vaccinated and challenged with B. bronchiseptica and B. pertussis. In addition to assessing protective efficacy and sterilizing immunity of the 22BF+dmLT as well as the requirement for PTd, a protective correlate can be established for use. This method was used to identify a protective correlate associated with DBF protection of mice against Shigella. This, however, prior to the present disclosure has never been determined for such a vaccine type against an extracellular pathogen.

a) Assess protective efficacy of the 22BF+dmLT delivered IN and ΪM against B. bronshiseptica challenge using the mouse lung model with two challenge doses - a lethal dose to assess survival and a sublethal dose to assess sterilizing immunity.

158. Protective efficacy of 22BF+dmLT delivered IN, IM, and ID against a B. bronchiseptica challenge can be assessed in the mouse lung model. A high dose of B. bronchiseptica can be administered initially to assess protection via the lethal dose. In a second trial, a lower dose can be used to assess sterilizing immunity

159. Experimental Details: Mice (20/group) are vaccinated IN on days 0, 14 and 28. Serum, and stool samples are collected as described above to measure specific antibody- responses to confirm that immune responses are comparable to those obtained above. For bacterial challenges, 10 mice are challenged on day 56 with 1 x 10⁷ B. bronchiseptica (lethal dose) and 10 animals are challenged with 1 x 1()⁶ B. bronchiseptica (sub-lethal dose). The mouse experiment can be repeated with vaccination occurring by the IM route. Survival can be ploted and a Log-rank test used to evaluate the differences. A P value of less than 0.05 is considered significant for all comparisons. Association of protective efficacy and markers of humoral and cellular immunity can be assessed with logistic regression models (see Figure 1).

160. With respect to the IN vaccinated mice, at both doses, some level of protection is shown in the lethal lung model. The mice vaccinated with 50 pg are protected with complete sterilizing immunity. The 15 pg dose gives >90% protection with a moderate level of sterilizing immunity. Similarly, 100 pg 22BF+dmLT delivered IM has a high level of protection as well as sterilizing immunity, but perhaps not 100%, but greater than 70% protection. The 40 pg dose shows minimal protection. With these results, a protective correlate for B. bronchiseptica can be predicted, as long as the immune responses were above the levels anticipated.

b) Assess protective efficacy of the 22BF+dmLT ± PTd delivered IN, IM, and ID against B. pertussis challenge using the mouse lung model with two challenge doses - a lethal dose to assess survival and a subletbal dose to assess sterilizing immunity.

161. The ultimate test of the 22BF formulation is the protective efficacy against B. pertussis. Here, the protective efficacy of 22BF+dmLT is tested with a focus on a B. pertussis challenge using the mouse lung model. Vaccinations occur IN with PTd. Furthermore, a high dose of B. pertussis is used initially to assess protection via the lethal lung model.

162. Mice (10/group) are vaccinated IN on days 0, 14 and 28. Serum, and stool samples are collected as described above to measure specific antibody responses to confirm a comparable immune response. For bacterial challenges, all mice can be challenged on day 56 with 1 x 10 ' B. pertussis (lethal dose). The experiment can be repeated using IM route and ID route again with the most protective vaccine and challenge with a lethal dose and a sub- lethal dose to assess protection and sterilizing immunity. Survival can be plotted and a Log- rank test used to evaluate the differences A P value of less than 0 05 is considered significant for all comparisons.

163. PTd can additionally be administered for protecti on against B. pertussis and to prevent the cellular damage associated with PT as well as increase sterilizing immunity. Mice can be vaccinated IN, IM, or ID with 22BF+PTd and dmLT and challenged with B. pertussis. Lung CPU were measured at day 3 (Figure 12) and day 7 (Figure 13) post challenge. As with the predicted B. bronchiseptica results, 100% protection and sterilizing immunity is obtained with 50 pg 22BF+dmLT+PTd delivered IN with reduced protection for the 15 pg dose delivered IN. Similarly, the 100 pg 22BF+dmLT+PTd delivered IM and ID achieves some significant level of protection, but sterilizing immunity is limited though could be greater at higher dosage by day 7 post challenge. Antibodies are important, but the impact of cytokines cannot be ignored.

4. Example 4: L’TA-1 fusion

164. LTAI is the active moiety of lethal toxin from Enterotoxigenic E. coii (ELECT). The activity of the LTAI is required for the adjuvant activity of dmLT. The double mutants are in the region usually targeted by a protease to allow A I to traffic to the cy toplasm of intestinal cells to cause the secretory diarrhea. Without the protease the LT still has some activation of cAMP. Likewise, LTAI remains active.

..... 52 -- a) LTA1 -Fusions:

165. The LTAl-fusions were expressed in a manner similar to the fusion alone. The LTA1 sequence was inserted 5’ to the start of the each fusion. Some of the LTAl-fusions required a small linker between the LTA1 and fusion in order for protein production to occur. LTA1-DBF, LTA1-S1, LTA1-S2, LTAl-SseB, LTA1-22BF, LTAl-BurkF, and LTAl-PaF were produced. One of the assays that appear to be required for adjuvant activity is the ability to ADP ribosy!ate ARF4. The ADP ribosy!ation assay was performed with the LTAl-fusions. In the assay, ADPr was biotin conjugated and when mixed with LTA1 and rARF4, the LTA1 transferred the biot-ADPr to rARF4. The biotin was then detected with Streptavidin-IR800 (Figure 20).

b) LTAl-Fusion protective efficacy:

166. Mice were vaccinated parenterally with LTA1-DBF or DBF+dmLT (Figure 14). Although the DBF+dmLT delivered IN elicited complete protection, this formulation cannot be given to humans since the dmLT can travel the olfactory nerve to the brain causing Bell’s palsy. Thus, parenteral routes or other mucosal routes are required DBF (100 gg) + dmLT (0. lgg) delivered IM was only 50% protective while LTA1-DBF at a concentration of 100 gg DBF equivalent was 70% protective. The lower dilutions, regardless of formulation, exhibited less protective efficacy. Similarly, LTAl-22BF+PTd elicited significant protective efficacy at a concentration of 80 gg of 22BF equivalents (Figure 19A-D)

c) LTA1-DBF immune response:

167. When the kinetics of the IgG titer was examined, responses agai nst IpaD and IpaB were essentially the same. Mice from Figure 14 were bled prior to vaccination and on day 42. Sera were assessed for anti -IpaD, -IpaB and -dmLT IgG. The lower IgG titers of the LTA1 samples can be attributed to the lower recognition of the dmLT on the well for the LTA1 samples vs the samples from mice vaccinated with dmLT (Figure 15). No IgA was detected in the fecal samples of the mice vaccinated IM, but was detectable in mice vaccinated IN with DBF+dmLT. Antibody-secreting ceils (ASCs) present in the bone marrow (Figure 16), spleen (Figure 17), and lungs (Figure 18) were also stimulated with IpaD, IpaB and dmLT. In each compartment, anti-IpaD, anti-IpaB, and anti -dmLT IgG ASCs could be detected. Interestingly, IgA ASCs could also be detected in the bone marrow against all dilutions of LTA1-DBF and resemble a curve similar to the dose escalation. A similar phenomenon was seen in the IgA ASCs from the lungs, but less pronounced. d) LTA1-DBF Purification.

168. The yield of LTA1-DBF was very low. Therefore, a linker was inserted in the DNA sequence between LTA1 and DBF to encode GSAAS (Seq. ID No. 14). The mother plasmid was Novagen’s pACYCDuet-l . The translocator for each fusion cannot be made without its cognate chaperone. Therefore, the complex of LT A 1 -DBF/Hi stag- IpgC (IPG chaperone comprises the nucleic acid sequence as set forth in SEQ ID NO: 10 which encodes the amino acid sequence as set forth in SEQ ID NO: 1 l)was produced from the plasmid pACYC-His-IpgC-LTAl -GSAAS-DBF where the ipgC gene was inserted into the

BamHI/Hindlll sites allowing for expression of His-tag IpgC and LTA1-GSAAS-DBF (nucleic acid sequence as set forth in SEQ ID NO: 15 and amino acid sequence as set forth in SEQ ID NO: 16)) was inserted at the Ndel-Xhol site. The DBF sequence had a 3’ stop codon prior to the Xhol restriction site.

169. pACYC-His-IpgC-LTAl-GSAAS-DBF was transformed into Tuner cells. A small overnight culture of LB+ Chloramphenicol (Cm) that had been inoculated with the freezer stock of the cells was transferred to 81, TB, and grown at 37 °C until OD= 1 -1 .5, add 0.5mM IPTG with 20ug/!iter AEBSF, 16C overnight, harvested at 4000rpm for 15min at 4 °C, and resuspended in IMAC binding buffer. The cells were frozenat -80 °C until ready for purification. After thawing the suspension was sonicated at 70% amplitude for 3-4min, 15s on, 30s off, clarified by centrifugation at 13000rpm for 3()min at 4 °C and decanted to obtain supernatant.

170. IMAC purification with 5ml NiNTA FF crude column on AKTA was as follows: (1 ) equilibrate column with 5CV binding buffer (20mM Tris, SOOniM NaCl, 5mM Imidazole pH 7.9), (2) load supernatant on column, collect FT in outlet!, (3) wash with binding buffer for 30CV, (4) elute with linear 0-60% elution buffer (20raM Tris, SOOniM NaCl, 500mM Imidazole pH 7.9) for 10CV, (5) elute with 60% elution buffer for 2CV, (6) w'ash column with 100% elution buffer for 3CV, (7) re-equilibrate column with 5CV binding buffer for 5CV.

171. HIC purification of the protein was as follows: Dilute pooled fraction into equal volume of 2X HIC binding buffer (50mM Sodium Phosphate (dibasic), 1M

Ammonium Sulfate, pH 7.0). Purify with 5ml HIC Phenyl HP column: (!) equilibrate column with 5CV binding buffer, (2) load diluted sample on column, collect FT in outlet 1, (3) wash with binding buffer for 5CV, (4) elute with linear 0-100% elution buffer (5mM Sodium Phosphate (dibasic), pH 7.0) for 40CV, (6) elute with 100% elution buffer for 6CV, (7) wash column with 100% elution buffer for 3CV, (8) reequilibrate column with binding buffer for 5CV.

172. Pooled fractions were dialyzed in 4L Q binding buffer for 2hrs, exchanged buffer, and then dialyzed overnight.

173. Purification using a 5mL Q FT columns on AKTA was as follows: (1) equilibrate column with 5CV binding buffer (50niM Tris, pH 8.0), (2) load dialyzed sample on column, collect FT in outlet!, (3) wash with binding buffer for 5CV, (4) elute with linear 0-30% elution buffer (50mM Tris, 1M NaCl, pH 8.0) for 20CV, (6) elute with 100% elution buffer for 5CV, (7) wash column with 100% elution buffer for 3CV, (8) re-equilibrate column with binding buffer for 5CV.

174. To facilitate final IMAC purification 8x IM.AC binding buffer (NO Imidazole) was added to pooled fractions to obtain lx and then LDAO to 0.05% was added

175. Purification by LDAO IMAC using 5ml NiNTA FF w¾s as follows: (1) equilibrate column with 5CV LDAO (20mM Tris, SOOrnM NaCl, 0.05%LDAO pH 7.9) binding buffer, (2) load supernatant on column, fractionate FT, (3) wash with binding buffer for 5CV, fractionate, (4) wash with 3% LDAO elution buffer (20mM Tris, 500mM NaCl, 500mM Imidazole, 0 005% LDAO pH 7 9) for 5CV, fractionate (5) elute with 6% LDAO elution buffer for 6.65CV, fractionate (6) elute with 100% LDAO elution buffer for 5CV, (8) re-equilibrate column with 5CV binding buffer for 5CV.

176. Pooled samples were dialyzed in 4L PBS+0.005%LDAO, exchanged buffer after 2hrs, and then dialyzed overnight.

e) Protective efficacy of LTA1-22BF

177. The initial assessment of the protective efficacy of the LTA1-22BF is presented here and demonstrated that LTA1 -22BF+rPT reduced the CPU lung burden by 99.8% while the 22BF+dmLT+rPT reduced it to 99.98% (Figure 19 (a)). Non-toxic pertussis toxin was added to the groups that prior to challenge by B. pertussis to offset the damage that pertussis toxin would cause and negatively impact the challenge. The kinetics of the IgG response is also shown where no differences are seen between the two groups (Figure 19 (b))

178. The mother plasmid was Novagerr s pACYCDuet- 1. The translocator for each fusion cannot be made without its cognate chaperone. Therefore, the complex of LTA1-

22BF/Histag-BcrHI is produced from the plasmid pACYC-His-BcrHl-LTAl-22BF where the brcHI gene (as set forth in SEQ ID NO: 7 with a histidine tag and encodes the amino acid sequence as set forth in SEQ ID NO: 8, the sequence minus the his-tag set forth in SEQ ID

..... 55 ...... NO: 9) is inserted into the BamHI/Hindlli sites allowing for expression of His-tag BcrHI and 22BF is inserted at the Ndel-Xhol site. The 22BF sequence has a 3 stop codon prior to the Xhol restriction site.

179. pACYC-His-BcrHI- 22BF was transformed into Tuner cells. A small overnight culture of LB+ Chloramphenicol (Cm) that had been inoculated with the freezer stock of the cells was transferred to 8L TB, grown at 37 °C until QD= 1-1.5, added 0.5mM IPTG with 20ug/liter AEBSF, 16 °C overnight, harvested at 4000rpm for 15min at 4C, and resuspended in IMAC binding buffer. The cells were frozen at -80 until ready for

purification. After thawing, the suspension was sonicated at 70% amplitude for 3-4min, 15s on, 30s off, clarified by centrifugation at 130()0rpm for 30min at 4 °C, and decanted to obtain supernatant.

180. IMAC purification with 5 ml NINTA FF crude column on AKTA was as follows: (!) equilibrate column with 5CV binding buffer (IMAC elution buffer: 20mM Tris, 500mM NaCl, 500mM Imidazole pH 7.9), (2) load supernatant on column, collect FT in outlet!, (3) wash with binding buffer for 30CV, (4) elute with linear 0-60% elution buffer for 10CV, (5) elute with 60% elution buffer for 2CV, (6) wash column with 100% elution buffer for 3CV, (7) re-equilibrate column with 5CV binding buffer (IMAC binding buffer: 20raM Tris, 500mM NaCl, 5mM Imidazole pH 7.9) for 5CV.

181. Diluted pooled fractions 20X into Q binding buffer Q binding buffer: 50mM Tris, pH 8.0). Purification with 3x 5mL Q FF columns on AKTA w¾s as follows: (!) equilibrate column with 6CV binding buffer, (2) load dialyzed sample on column, collect FT in outlet 1, (3) wash with binding buffer for 12CV, (4) elute with 15% elution buffer (Q elution buffer: 50mM Tris, 1M NaCl, pH 8.0) for 6CV, (5) elute with linear 15-40% elution buffer for 34CV, (6) elute with 100% elution buffer for 6CV, (7) wash column with 100% elution buffer for 3CV, (S)re-equilibrate column with binding buffer for 6CV.

182. To facilitate final IMAC purification 8x IMAC binding buffer (NO Imidazole) was added to pooled fractions to obtain lx and then LDAO to 0.05%> was added. Purification by LDAO IMAC using 5ml NiNTA FF w¾s as follows: (!) equilibrate column with 5CV LDAO binding buffer (LDAO IMAC binding buffer: 20mM Tris, 5Q0mM NaCl,

0.05%LDAQ pH 7.9), (2) load supernatant on column, fractionate FT, (3) wash with binding buffer for 5CV, fractionate, (4) wash with 3% LDAO elution buffer (LDAO IMAC elution buffer: 20mM Tris, 500mM NaCl, 500mM Imidazole, 0.005% LDAO pH 7.9) for 5CV, fractionate (5) elute with 6% LDAO elution buffer for 6.65CV, fractionate (6) elute with 100% LDAO elution buffer for 5CV, (8) re-equilibrate column with 5CV binding buffer for 5 CV.

183. Pooled samples were dialyzed in 4L PB S+0.005%LD AO, exchanged buffer after 2hrs, and then dialyzed overnight.

g) LTAl-BurkF purification

184. The mother plasmid was Novagen’s pACYCDuet-1. The translocator for each fusion cannot be made without its cognate chaperone. Therefore, the complex of LTAl- BurkF/Histag-BicA (SEQ ID NOs: 28 and 20) was produced from the plasmid pACYC-His- BicA-LTAl-BurkF (as set forth in SEQ ID NO: 20 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 19) wherein the bicA gene was inserted into the BamHI/TIindlll sites allowing for expression of His-tag BicA and LTAl-BurkF was inserted at the Ndel- Xhol site. The BurkF sequence had a 3’ stop codon prior to the Xhol restriction site.

185. pACYC-His-BicA-LTAl -BurkF was transformed into Tuner cells. A small overnight culture of LB+ Chloramphenicol (Cm) that had been inoculated with the freezer stock of the cells was transferred to 8L TB, grown at 37 °C until OD= 1-1.5, added 0.5mM IPTG with 20ug/liter AEBSF, 16 °C overnight, harvested at 4000rpm for 15min at 4C, and resuspended in IMAC binding buffer. The cells were frozen at -80 until ready for

purification. After thawing the suspension was sonicated at 70% amplitude for 3-4min, 15s on, 30s off, Clarified by centrifugation at 130Q0rpm for 30min at 4 °C, and decanted to obtain supernatant.

186. IMAC purification with 5 ml NiNTA FF crude column on AKTA was as follows: (1 ) equilibrate column with 5CV binding buffer (20mM Tris, 500mM NaCl, 5mM Imidazole pH 7.9), (2) load supernatant on column, collect FT in outlet!, (3) wash with binding buffer for 30CV, (4) elute with linear 0-60% elution buffer (20raM Tris, 500mM NaCl, 500mM Imidazole pH 7.9) for 10CV, (5) elute with 60% elution buffer for 2CV, (6) w'ash column with 100% elution buffer for 3CV, (7) re-equilibrate column with 5CV binding buffer for 5CV.

187. The pooled fractions were diluted 20X into Q binding buffer (50mM Tris, pH 8.0). Purification using 3x 5mL Q FF columns on AKTA was as follows: (1) equilibrate column with 6CV binding buffer, (2) load dialyzed sample on column, collect FT in out!etl , (3) wash with binding buffer for 12C V, (4) elute with 15% elution buffer (50mM Tris, 1M NaCl, pH 8.0) for 6CV, (5) elute with linear 15-40% elution buffer for 34CV, (6) elute with 100% elution buffer for 6CV, (7) wash column with 100% elution buffer for 3CV, ^re equilibrate column with binding buffer for 6CV. 188 To facilitate final IMAC purification add 8x IMAC binding buffer (NO Imidazole) to pooled fractions to obtain lx and then LDAO to 0.05% was added:

189. Purification by LDAO IMAC using 5ml NiNTA FF was as follows: (1) equilibrate column with 5CV LDAO binding buffer (20mM Tris, 500niM NaCl,

0.05%LDAO pH 7.9), (2) load supernatant on column, fractionate FT, (3) wash with binding buffer for 5CV, fractionate, (4) wash with 3% LDAO elution buffer (20mM Tris, 500mM NaCl, 500mM Imidazole, 0.005% LDAO pH 7.9) for 5CV, fractionate (5) elute with 6% LDAO elution buffer for 6.65CV, fractionate (6) elute with 100% LDAO elution buffer for 5CV, (8) re-equilibrate column with 5CV binding buffer for 5CV.

190 Pooled samples were dialyzed in 4L PBS+() 005%LDAO, exchanged buffer after 2hrs, and then dialyzed overnight.

h) LTAl-PaF

191. The PaF+dmLT vaccinated mice exhibited 100% survival with 44% sterilizing immunity against Pa challenge in a mouse lethal pulmonary model, while the PBS vaccinated mice exhibited 60% survival but all had >10^{8 9} CFU/luns.

192. The mother plasmid is Novagen’s pACYCDuet-l . The translocator for each fusion cannot be made without its cognate chaperone. Therefore, the complex of LTA1- PaF/Histag-PcrHI was produced from the plasmid pACYC-His-PcrHl-LTAl-PaF where the brcHI gene was inserted into the Band 11/1 lindll l sites allowing for expression of His-tag Perl 11 (as set forth in SEQ ID NO: 30 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 29) and LTAl-PaF (as set forth in SEQ ID NO: 38 and encoded by the nucleic acid sequence as set forth in SEQ ID NO: 37) which was inserted at the Ndel-Xhol site. The PaF sequence had a 3’ stop codon prior to the Xhol restriction site. The purification of LTAl-PaF was the same of for LTA1-22BF.

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Norton EB, Lawson LB, Freytag LC, Clements JD Characterization of a mutant Escherichia coli heat-labile toxin, LT(R192G/L2l lA), as a safe and effective oral adjuvant. Clinical and vaccine immunology : CVL 201 1; 18(4):546~51. doi: 10.1128/CVI.00538-10. PubMed PMID: 21288994; PMCID: 3122563.

Norton EB, Lawson LB, Mahdi Z, Freytag LC, Clements JD. The A subunit of Escherichia coli heat-labile enterotoxin functions as a mucosal adjuvant and promotes IgG2a, IgA, and Thl 7 responses to vaccine antigens. Infect Immun. 2012;80(7):2426-35. doi:

10.1128/IAI.00181-12. PubMed PMID: 22526674; PMCID: 3416479.

Qin L, Gilbert PB, Corey L, McElrath MJ, Self SG. A framework for assessing

immunological correlates of protection in vaccine trials. J Infect Dis. 2007; 196(9): 1304-12. doi: 10.1086/522428. PubMed PMID: 17922394. Ross PJ, Sutton CE, Higgins S, Allen AC, Walsh K, Misiak A, Lavelle EC, McLoughlin RM, Mills KH. Relative contribution of Thl and Thl7 cells in adaptive immunity to Bordetella pertussis: towards the rational design of an improved acellular pertussis vaccine PLoS Pathog. 2Q13;9(4):el003264. doi: lQ.137I/journai.ppat. l003264. PubMed PMID: 23592988; PMCID: PMC3617212.

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10.1 128/C VI.00449- 15. PubMed PMID: 26561389, PMCID: PMC471 1092

F. Sequences

SEQ O) NO: 1 22BF Nucleotide Sequence

CATatgaccatigatctcggagtttcactcacgtcgcaggccggcggcctgcaaggcatcgacctcaagagcatggata tccagactctcatggtgtatgtgcagggtcgtcgcgccgaactcctcacggctcaaatgcagacccaggccgaagtggtgcagaagg ccaatgaacgcatggcgcagctcaacgaggtcctgtccgcgctgtcccgggccaaggccgagtttccgcccaatccgaagccggg cgacaccatcccgggctgggacaaccagaaggtcagccggatcgaggttcctctcaatgatgcgctgcgcgctgccggcctgacg ggcatgttcgaagcgcgcgatggccaagtgaccgcccccggcggccggggtacgcaggtcgtgaacggcacgggcgtcatggcc ggttccacgacctataaggaacicgaaagigcctacaccaccgtaaaggggatgctggatacggcgtccaatacgcaacagatggac atgatcaggctgcaggccgccagcaacaagcgcaacgaggctttcgaggtcatgaccaacaccgagaagcggcgcagcgacctg aacagttccatcaccaacaacatgcgcaagcttatgaccgtcatgagtacgaccatatccacagccccgagcggcgccgcgcttgcg ccgtcicgcatagatatgcgggcaccggagcccgggagigccggcgaaggcgccggcatcctggcgccggtgacgacgctggct ctggcggcgggccggccggcttttccagcgtcaccgtcgctgcgcaccgcgcccgtcctggatccgccagtgcgcgatctcagccc cgccgacttggccgacctgctgcgcgtcttgcgatccagggcggtggacgggcagttggccacggcgcgcgagaacctgcagga cgcgcaagtcaaggcgaagcagaacacccaggcccagctcgacaagctggacgcatggtttcggaaggccgaagaggccgaga gcaagggatggctgagcaaggtgttcggctggatcggcaaggtgctggcggtcgtggcatcggccctggcggtgggctttgccgcc gtcgccagcgtggccaccggcgcggcggccacacccatgctgctgctcagcggcatggcactggtcagcgccgtgacatcgctgg ccgaccagatatcgcaagaggcgggaggcccgcctatcagcctgggcgggtttctctccgggctggccggacgtctgctgacagc gttgggggtggatcagtegeaggccgaeeaaattgccaagatcgtegecggcctggccgtgcccgtcgtcttgctgatcgaacccca gatgctgggcgaaatggcgcaaggcgtggccaggctggctggcgccagcgatgccaccgcggggtacatagccatggcgatgtc catcgtggcggcgatcgcggtcgccgcgatcaatgccgccggtacagccggcgcgggtagcgcttcggcgatcaagggggcctg ggatcgggccgccgcggtagccacccaggtccttcaagggggtacggcagtggcgcaaggcggcgtcggcgtgtcgatggcagt cgatcgcaaacaggccgatctcctggtcgccgacaaggcggatctggcggcgagcctgacaaaactgcgggcggccatggagcg tgaggcggacgatatcaagaagatcctggctcaattcgacgaggcctatcacatgatcgcgaagatgatcagcgatatggcgagtac gcacagccaggtcagcgccaacctcgggcggcgccaggcggtgtagCTCGAG

SEQ ID NO: 2 22BF Amino Acid Sequence

MTIDLGVSLTSQAGGLQGIDLKSMDIQTLMVYVQGRRAELLTAQMQTQAEV

VQKANERMAQLNEVLSALSRAKAEFPPNPKPGDTIPGWDNQKVSRIEVPLNDALRA

AGLTGMFEARDGQVTAPGGRGTQVVNGTGVMAGSTTYKELESAYTTVKGMLDTA

SNTQQMDMIRLQAASNKRNEAFEVMTNTEKRRSDLNSSITNNMRKLMTYMSTTIST

APSGAALAPSRIDMRAPEPGSAGEGAGILAPVTTLALAAGRPAFPASPSLRTAPVL PVRDLSPADLADLLRVLRSRAVDGQLATARENLQDAQVKAKQNTQAQLDKLDAWF RKAEEAESKGWLSKVFGWIGKVLAWASALAVGFAAVASVATGAAATPMLLLSGM ALVSAVTSLADQISQEAGGPPISLGGFLSGLAGRLLTALGVDQSQADQIAKIVAGLAV PVVLLIEPQMLGEMAQGVARLAGASDATAGYIAMAMSIVAAIAVAAINAAGTAGAG SASAIKGAWDRAAAVATQVLQGGTAVAQGGVGVSMAVDRKQADLLVADKADLA ASLTKLRAAMEREADDIKKILAQFDEAYHMIAKMISDMASTFISQVSANLGRRQAV

SEQ ID NO: 3 Bsp22 Nucleotide Sequence

CATatgaccattgatctcggagtttcactcacgtcgcaggccggcggcctgcaaggcatcgacctcaagagcatggata tccagactctcatggtgtatgtgcagggtcgtcgcgccgaactcctcacggctcaaatgcagacccaggccgaagtggtgcagaagg ccaatgaacgcatggcgcagctcaacgaggtcctgtccgcgctgtcccgggccaaggccgagtttccgcccaatccgaagccggg cgacaccatcccgggctgggacaaccagaaggtcagccggatcgaggttcctctcaatgatgcgctgcgcgctgccggcctgacg ggcatgttcgaagcgcgcgatggccaagtgaccgcccccggcggccggggtacgcaggtcgtgaacggcacgggcgtcatggcc ggttccacgacctataaggaactcgaaagtgcctacaccaccgtaaaggggatgctggatacggcgtccaatacgcaacagatggac aacagttccatcaccaacaacatgcgc

SEQ ID NO: 4 Bsp22 Amino Add Sequence

MTIDLGVSLTSQAGGLQGIDLKSMDIQTLMVYVQGRRAELLTAQMQTQAEV

VQKANERMAQLNEVLSALSRAKAEFPPNPKPGDTIPGWDNQKVSRIEVPLNDALRA

AGLTGMFEARDGQVTAPGGRGTQVVNGTGVMAGSTTYKELESAYTTVKGMLDTA

SNTQQMDMIRLQAASNKRNEAFEVMTNTEKRRSDLNSSITNNMR

SEQ ID NO: 5 BopB Nucleotide Sequence

Atgaccgtcatgagtacgaccatatccacagccccgagcggcgccgcgcttgcgccgtctcgcatagatatgcgggcac cggagcccgggagtgccggcgaaggcgccggcatcctggcgccggtgacgacgctggctctggcggcgggccggccggcttttc cagcgtcaccgtcgctgcgcaccgcgcccgtcctggatccgccagtgcgcgatctcagccccgccgacttggccgacctgctgcgc gtcttgcgatccagggcggtggacgggcagttggccacggcgcgcgagaacctgcaggacgcgcaagtcaaggcgaagcagaa cacccaggcccagctcgacaagctggacgcatggtttcggaaggccgaagaggccgagagcaagggatggctgagcaaggtgtt cggctggatcggcaaggtgctggcggtcgtggcatcggccctggcggtgggctttgccgccgtcgccagcgtggccaccggcgcg gcggccacacccatgctgctgctcagcggcatggcactggtcagcgccgtgacatcgctggccgaccagatatcgcaagaggcgg gaggcccgcctatcagcctgggcgggtttctctccgggctggccggacgtctgctgacagcgttgggggtggatcagtcgcaggcc gaccaaattgccaagatcgtcgccggcctggccgtgcccgtcgtcttgctgatcgaaccccagatgctgggcgaaatggcgcaagg cgtggccaggctggctggcgccagegatgccaccgcggggtacatagccatggcgatgtccatcgtggeggegatcgcggtcgec gcgatcaatgccgccggtacagccggcgcgggtagcgcttcggcgatcaagggggcctgggatcgggccgccgcggtagccacc caggtccttcaagggggtacggcagtggcgcaaggcggcgtcggcgtgtcgatggcagtcgatcgcaaacaggccgatctcctggt cgccgacaaggcggatctggcggcgagcctgacaaaactgcgggcggccatggagcgtgaggcggacgatatcaagaagatcct ggctcaattcgacgaggcctatcacatgatcgcgaagatgatcagcgatatggcgagtacgcacagccaggtcagcgccaacctcg ggcggcgccaggcggtgtagCTCGAG

SEQ ID NO: 6 BopB Amino Acid Seqnence

MTVMSTTISTAPSGAALAPSRIDMRAPEPGSAGEGAGILAPVTTLALAAGRPA

FP ASP SLRT AP VLDPP VRDL SP ADLADLLRVLRSRAVDGQL AT ARENLQD AQ VK AK

QNTQAQLDKLDAWFRKAEEAESKGWLSKVFGWIGKVLAWASALAVGFAAVASV

ATGAAATPMLLLSGMALVSAVTSLADQISQEAGGPPISLGGFLSGLAGRLLTALGVD

QSQADQIAKIVAGLAVPVVLLIEPQMLGEMAQGV RLAGASDATAGYIAMAMSIVA

AIAVAAINAAGTAGAGSASAIKGAWDRAAAVATQVLQGGTAVAQGGVGVSMAVD

RKQADLLVADKADLAASLTKLRAAMEREADDIKKILAQFDEAYHMIAKMISDMAST

H S Q V S ANLGRRQ A V

SEQ ID NO: 7 His-BcrHl chaperone with histidine tag nndeotide seqnence ATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGATGCCAAA GTCAGCCGAGCAGGGCGGCTCCCCGGCGTCAGCTTCGCATGAGGCGTTGCGCCA TATTCTCGACGCAGGCGCTTCGATGGGCAGCTTGCAGGGGTTGGACGAGGTGCA ACAGCAGGCGTTGTACGCGATCGCTCATGGCGCCTACGAACAGGGCCGCTATGC CGACGCGTTGAAAATGTTCTGCCTGCTGGTCGCGTGCGATCCGCTGGAAGCCCGT TATCTGCTGGCCCTGGGCGCCGCGGCCCAGGAGCTGGGGCTGTACGAGCATGCC TTGCAGCAATACGCGGCCGCGGCGGCTTTGCAGTTGGACTCCCCCAGGCCCCTGT TGCATGGCGCCGAGTGCCTGTATGCGTTGGGTCGTCGCCGCGACGCCCTGGATAC GCTCGACATGGTGCTTGAGTTGTGCGGGTCGCCGGAGCATGCGGCCCTGCGCGA ACGGGCCGAGTCGCTGCGCAGGAGCTATGCACGTGCCGACTGAAAGCTT

SEQ ID NO: 8 His-BcrHl with histidine tag chaperone amino acid sequence MGSSHHHHHHSQDPMPKSAEQGGSPASASHEALRHILDAGASMGSLQGLDE VQQQALYAIAHGAYEQGRYADALKMFCLLVACDPLEARYLLALGAAAQELGLYEH ALQQ Y A A A A ALQLDSPRPLLHGAECL YALGRRRD ALDTLDM VLELCGS PEH AALRE RAESLRRSYARAD

SEQ ID NO: 9 BcrHl amino add sequence

MPKSAEQGGSPASASHEALRHILDAGASMGSLQGLDEVQQQALYAIAHGAY

EQGRYADALKMFCLLVACDPLEARYLLALGAAAQELGLYEHALQQYAAAAALQLD

SPRPLLHGAECLYALGRRRDALDTLDMVLELCGSPEHAALRERAESLRRSYARAD

SEQ ID NO: 10 IpgC Chaperone of DBF nucleic acidsequence

CCatgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggcagccatatgctcgagatgtcttta aatatcaccgaaaatgaaagcatctctactgcagtaattgatgcaattaactctggcgctacactgaaagatattaatgcaattcctgatga tatgatggatgacatttattcatatgcttatgacttttacaacaaaggaagaatagaggaagctgaagttttcttcaggtttttatgtatatacg acttttacaatatagactacattatgggactcgcagctatttatcagataaaagaacagttccaacaagcagcagacctttatgctgtcgct tttgcattaggaaaaaatgactatacaccagtattccatactggacaatgccagcttcggttgaaagcccccttaaaagctaaagagtgct tcgaactcgtaattcaacacagcaatgatgaaaaattaaaaataaaagcacaatcatacttggacgcaattcaggatatcaaggagtag GATCC

SEQ ID NO: 11 IpgC Chaperone of DBF Amino Add sequence

MSLNITENESIST AVID AIN SGATLKDINAIPDDMMDDIYSYAYDFYNKGRIEE AEVFFRFLCIYDF YNVD YIMGL AAI Y QIKEQF QQ AADL YAVAF ALGKND YTPVFHT G QCQLRLKAPLKAKECFELVIQHSNDEK LKIK AQSYLDA IQDIKE

SEQ ID NO: 12 LTA1 nucleic acid seqnence

CATAtggacaatggcgatcgtttataccgtgccgactcgcgtcccccagatgagattaaacgtagcggtgggttaatgc cacgtgggcacaatgagtattttgaccgtggaacacagatgaacattaacctttacgatcatgcccgtgggacccagaccgggtttgtc cgttatgatgacgggtatgttagtacgagtttgtccttacgctccgcacaccttgcgggacaaagtattttatcaggctacagcacatatta catttatgtgatcgccactgccccaaacatgttcaatgtgaacgatgtgttgggggtttacagcccccatccatatgaacaagaagtctcg gcccttggggggatcccatatagccagatttatggttggtaccgcgtaaattttggtgtgattgatgaacgtttgcatcgtaaccgtgaata ccgcgatcgctactaccgtaacttgaacattgcacctgccgaggacggctatcgttagcgggattcccacccgatcatcaggcgtgg cgtgaggaaccgtggatccatcacgcccctcaggggtgcgggaacagtagtcgc

SEQ ID NO: 13 LTA1 amino add sequence

MDN GDRL YRAD SRPPDEIKRS GGLMPRGHNE YFDRGT QMNINL YDHARGT Q TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSP HPYEQEVSALGGIPYSQIYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYR L AGFPPDHQ AWREEPWIHHAPQGCGNS SR

SEQ ID NO: 14 GSAAS Linker amino acid sequence

GSAAS

SEQ ID NO: 15 L T A 1 - GSAAS-DBF (IpaD-LE-IpaB) nucleic add seqnence

CATAtggacaatggcgatcgtttataccgtgccgactcgcgtcccccagatgagattaaacgtagcggtgggttaatgc cacgtgggcacaatgagtattttgaccgtggaacacagatgaacattaacctttacgatcatgcccgtgggacccagaccgggtttgtc cgttatgatgacgggtatgttagtacgagttgtccttacgctccgcacaccttgcgggacaaagtattttatcaggctacagcacatatta catttatgtgatcgccactgccccaaacatgttcaatgtgaacgatgtgttgggggtttacagcccccatccatatgaacaagaagtctcg gcccttggggggatcccatatagccagatttatggttggtaccgcgtaaattttggtgtgattgatgaacgtttgcatcgtaaccgtgaata ccgcgatcgctactaccgtaacttgaacattgcacctgccgaggacggctatcgttagcgggattcccacccgatcatcaggcgtgg cgtgaggaaccgtggatccatcacgcccctcaggggtgcgggaacagtagtcgcgggtccgcggcatccatgaatat aacaactctg actaatagta tttccacctc atcattcagt ccaaacaata ccaacggttc atcaaccgaa acagttaatt ctgatataaa aacaacgacc agttctcatc ctgtaagttc ccttactatg ctcaacgaca cccttcataa tatcagaaca acaaatcagg cattaaagaa agagctttca caaaaaacgt tgactaaaac atcgctagaa gaaatagcat tacattcatc tcagattagc atggatgtaa ataaatccgc tcaactattg gatattcttt ccaggaacga atatccaatt aataaagacg caagagaatt attacattca gccccgaaag aagccgagct tgatggagat caaatgatat ctcatagaga actgtgggct aaaattgcaa actccatcaa tgatattaat gaacagtatc tgaaagtata tgaacatgcc gttagttcat atactcaaat gtatcaagat tttagcgctg ttctttccag tcttgccggc tggatctctc ccggaggtaa cgacggaaac tccgtgaaat tacaagtcaa ctcgcttaaa aaggcattgg aagaactcaa ggaaaaatat aaagataaac cgctatatcc agcaaataat actgttagtc aggaacaagc aaataaatgg cttacagaat taggtggaac aatcggcaag gtatctcaaa aaaaeggggg atatgtgtc agtataaaca tgaccccaat agacaatatg ttaaaaagct tagataatct aggtggaaat ggcgaggttg tgctagataa tgcaaaatat caggcatgga atgccggatt ctctgccgaa gatgaaacaa tgaaaaataa tcttcaaact ttagttcaaa aatacagtaa tgccaatagt attttgata atttagtaaa ggttttgagt agtacaataa gctcatgtac agatacagat aaactttttc tccatttc CTCGAG atgcataatgta agcaccacaa ccactggttt tcctcttgcc aaaatattga cttccactgagcttggagac aatactatcc aagctgcaaa tgatgcagct aacaaattat tttctcttacaattgctgat ettactgcta accaaaatat taatacaaet aatgcacact caacttcaaatatattaatc cctgaactta aagcaccaaa gtcattaaat gcaagttccc aactaacgcttttaattgga aaccttattc aaatactcgg tgaaaaatct ttaactgcat taacaaataaaattactgct tggaagtccc agcaacaggc aagacagcaa aaaaacctag aattctccgataaaattaac actcttctat ctgaaactga aggactaacc agagactatg aaaaacaaattaataaacta aaaaacgcag attctaaaat aaaagaccta gaaaataaaa ttaaccaaattcaaacaaga ttatcgaacc tcgatccaga gtcaccagaa aagaaaaaat taagccgggaagaaatacaa ctcactatca aaaaagacgc agcagttaaa gacaggacat tgattgagcagaaaaccctg tcaattcata gcaaacttac agataaatca atgcaaetcg aaaaagaaatagactctttt tctgcatttt caaaeacagc atctgctgaa cagctatcaa cccagcagaaatcattaacc ggacttgcca gtgttactca attgatggca acctttattc aactagttggaaaaaataat gaagaatctt taaaaaatga tctggctcta ttccagtctc tccaagaatcaagaaaaact gaaatggaga gaaaatctga tgagtatgct gctgaagtac gtaaagcagaagaactcaac agagtaatgg gttgtgttgg gaaaatactt ggggcacttt taactatcgttagtgttgtt gcagcagctt tttctggagg agcctctcta gcactggcag ctgttggtttagctcttatg gttacggatg ctatagtaca agcagcgacc ggcaattcct tcatggaacaagccctgaat ccgatcatga aagcagtcat tgaaccctta atcaaactcc tttcagatgcatttacaaaa atgctcgaag gcttgggcgt cgactcgaaa aaagccaaaa tgattggctctattctgggg gcaatcgcag gcgctcttgt cctagttgca gcagtcgttc tcgtagccactgttggtaaa caggcagcag caaaactgc agaaaatatt ggcaaaataa taggtaaaaccctcacagac cttataccaa agtttctcaa gaatttttct tctcaactgg acgatttaatcactaatgct gttgccagat taaataaatt tcttggtgca gcgggtgatg aagtaatatccaaacaaatt atttccaccc atttaaacca agcagtttta ttaggagaaa

gtgttaactctgccacacaa gcgggaggaa gtgtcgcttc tgctgttttc cagaacagcg cgtcgacaaatctagcagac ctgacattat cgaaatatca agttgaacaa ctgtcaaaat atatcagtgaagcaatagaa aaattcggcc aattgcagga agtaattgca gatctattag cctcaatgtccaactctcag gctaatagaa ctgatgttgc aaaagcaatt ttgcaacaaa ctactgcttga GGATCC

SEQ ID NO: 16 L TA l - GSAAS-IpaD-LE-IpaB (DBF) Amino Add sequence

MDN GDRL YRAD SRPPDEIKRS GGLMPRGHNE YFDRGT QMNINL YDHARGT Q TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSP HPYEQEVSALGGIPYSQIYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYR LAGFPPDHQAWREEPWIHHAPQGCGNSSRGSAASMNITTLTNSISTSSFSPNNTNGSS TETVNSDIKTTTSSHPSSLTMLNDTLHNIRTTNQALKKELSQKTLRNEYPINKDAREL LHS APKEAELDGDQMISHRELW AKIANSINDINEQ YLKVYEH AYS S YTQMYQDF S A VLSSLAGWISPGGNDGNSYKLQVNSLKKALEELKEKYKDKPLYPANNTVSQEQANK WLTELGGTIGKVSQKNGGYVVSINMTPIDNMLKSLDNLGGNGEVVLDNAKYQAWN GFSAEDETMKNNLQTLVQKYSNANSIFDNLVKVLSSTISSCTDTD1 .ELHFLEMHNV STTTTGFPLAKILTSTELGDNTIQAANDAAN LFSLTLADLTANQNINTTNAHSTSNILI PELKAPKSLNASSQLTLLIGNLIQILGEKSLTALTNKITAWKSQQQARQQKNLEFSDKI NTLLSETEGLTRDYEKQINKLKNADSKIKDLENKINQIQTRLSNLDPESPEKKKLSREE IQLTIKKDAAVKDRTLIEQKTLSIHSKLTDKSMQLEKEIDSFSAFSNTASAEQLSTQQK SLTGL A S VTQLM ATF IQL V GKNNEE SLKNDL ALF Q SLQE SRKTEMERK SDE Y AAE VR KAEELNRVMGCVGKILGALLTIVSVVAAAFSGGASLALAAVGLALMVTDAIVQAAT GNSFMEQALNPIMKAVIEPLIKLLSDAFTKMLEGLGVDSKKAKMIGSILGAIAGALVL

VAAVVLVATVGKQ AAAKLAENIGKIIGKTLTDLIPKFLKNF S SQLDDLITNAVARLN

KFLGAAGDEVISKQIISTHLNQAVLLGESVNSATQAGGSVASAVFQNSASTNLADLT

LSKYQVEQLSKYISEAIEKFGQLQEVIADLLASMSNSQANRTDVAKAILQQTTA

SEQ ID NO: 17 LTA 1-22BF nucleic acid sequence

CATAtggacaatggcgatcgtttataccgtgccgactcgcgtcccccagatgagattaaacgtagcggtgggttaatgc cacgtgggcacaatgagtatttgaccgtggaacacagatgaacattaacctttacgatcatgcccgtgggacccagaccgggtttgtc cgttatgatgacgggtatgtagtacgagtttgtcctacgctccgcacacctgcgggacaaagtatttatcaggctacagcacatatta catttatgtgatcgccactgccccaaacatgttcaatgtgaacgatgtgttgggggttacagcccccatccatatgaacaagaagtctcg gcccttggggggatcccatatagccagatttatggttggtaccgcgtaaattttggtgtgattgatgaacgtttgcatcgtaaccgtgaata ccgcgatcgctactaccgtaacttgaacattgcacctgccgaggacggctatcgtttagcgggattcccacccgatcatcaggcgtgg cgtgaggaaccgtggatccatcacgcccctcaggggtgcgggaacagtagtcgcatgaccattgatctcggagtttcactcacgtcgc aggccggcggcctgcaaggcatcgacctcaagagcatggatatccagactctcatggtgtatgtgcagggtcgtcgcgccgaactcc tcacggctcaaatgcagacccaggccgaagtggtgcagaaggccaatgaacgcatggcgcagctcaacgaggtcctgtccgcgct gtcccgggccaaggccgagtttccgcccaatccgaagccgggcgacaccatcccgggctgggacaaccagaaggtcagccggat cgaggttcctctcaatgatgcgctgcgcgctgccggcctgacgggcatgttcgaagcgcgcgatggccaagtgaccgcccccggcg gccggggtacgcaggtcgtgaacggcacgggcgtcatggccggttccacgacctataaggaactcgaaagtgcctacaccaccgta aaggggatgetggataeggegtccaatacgeaacagatggaeatgatcaggctgcaggccgccagcaacaagcgcaacgaggctt tcgaggtcatgaccaacaccgagaagcggcgcagcgacctgaacagttccatcaccaacaacatgcgcaagcttatgaccgtcatga gtacgaccatatccacagccccgagcggcgccgcgcttgcgccgtctcgcatagatatgcgggcaccggagcccgggagtgccgg cgaaggcgccggcatcctggcgccggtgacgacgctggctctggcggcgggccggccggcttttccagcgtcaccgtcgctgcgc accgcgcccgtcctggatccgccagtgcgcgatctcagccccgccgacttggccgacctgctgcgcgtcttgcgatccagggcggt ggacgggcagttggccacggcgcgcgagaacctgcaggacgcgcaagtcaaggcgaagcagaacacccaggcccagctcgac aagctggacgcatggtttcggaaggccgaagaggccgagagcaagggatggctgagcaaggtgttcggctggatcggcaaggtg ctggcggtcgtggcatcggccctggcggtgggctttgccgccgtcgccagcgtggccaccggcgcggcggccacacccatgctgc tgctcagcggcatggcactggtcagcgccgtgacatcgctggccgaccagatatcgcaagaggcgggaggcccgcctatcagcct gggcgggtttctctccgggctggccggacgtctgctgacagcgttgggggtggatcagtcgcaggccgaccaaattgccaagatcgt cgccggcctggccgtgcccgtcgtcttgctgatcgaaccccagatgctgggcgaaatggcgcaaggcgtggccaggctggctggc gccagcgatgccaccgcggggtacatagccatggcgatgtccatcgtggcggcgatcgcggtcgccgcgatcaatgccgccggta cagccggcgcgggtagcgcttcggcgatcaagggggcctgggatcgggccgccgcggtagccacccaggtccttcaagggggta cggcagtggcgcaaggcggcgtcggcgtgtcgatggcagtcgatcgcaaacaggccgatctcctggtcgccgacaaggcggatct ggcggcgagcctgacaaaactgcgggcggccatggagcgtgaggcggacgatatcaagaagatcctggctcaattcgacgaggc ctatcacatgatcgcgaagatgatcagcgatatggcgagtacgcacagccaggtcagcgccaacctcgggcggcgccaggcggtg tagCTCGAG^"

SEQ ID NO: 18 LTA1-22BF Amino add sequence

MDNGDRLYRADSRPPDEIKRSGGLMPRGHNEYFDRGTQMNINLYDHARGTQ

TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSP

HPYEQEVSALGGIPYSQIYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYR

X.AGFPPDHQAWREEPWIFIHAPQGCGNSSRMTIDLGVSLTSQAGGLQGIDLKSMDIQT

LMVYVQGRRAELLTAQMQTQAEWQKANERMAQLNEVLSALSRAKAEFPPNPKPG

DTIPGWDNQKVSRIEVPLNDALRAAGLTGMFEARDGQVTAPGGRGTQVVNGTGVM

AGSTTYKELESAYTTVKGMLDTASNTQQMDMIRLQAASNKKNEAFEVMTNTEKRR

SDLNSSITNNMRKLMTVMSTTISTAPSGAALAPSRIDMRAPEPGSAGEGAGILAPVTT

LALAAGRPAFPASPSLRTAPVLDPPVRDLSPADLADLLRVLRSRAVDGQLATARENL

QDAQVKAKQNTQAQLDKLDAWFRKAEEAESKGWLSKVFGWIGKVLAWASALAV

GFAAVASVATGAAATPMLLLSGMALVSAVTSLADQISQEAGGPPISLGGFLSGLAGR

LLTALGVDQSQADQIAKIVAGLAVPVVLLIEPQMLGEMAQGVARLAGASDATAGYI

AMAMSIVAALAVAAINAAGTAGAGSASAIKGAWDRAAAVATQVLQGGTAVAQGG

VGVSMAVDRKQADLLVADKADLAASLTKLRAAMEREADDIKKILAQFDEAYHMIA

KMISDMASTHSQVSANLGRRQAV*

SEQ ID NO: 19 His-BicA (Chaperone of Burk f ) nucleic acid sequence

ATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGATGACGCA ACGCGACGTGAACATAGACGACATCGAGGCGCAGGAAATGGCGGCGGCGCTGCT

GGACGCGGTCCAGAACGGCGCGACGCTGAAGGACCTGCATCAGGTGCCGCAGGA

CCTGATGGACGGCATCTATGCGTTCGCGTACCGCTTCTACCAGCAGGGGCGGCTC

G.ACGACGCGGAGGTGTTCTTCCGCTTTCTGCGCATCTACGACTTCTACAACGCCG

AATACGCGATGGGGCTCGCGGCGGTGTGCCAGTTGAAGAAGGAGTACGCGCGGG

CGATCGATCTGTATGCACTCGCGTATTCGCTGTCGAAGGACGACCACCGGCCGAT

GTTCCACACCGGCCAATGCCATCTGCTGATGGGCAAGGCGGCGCTCGCGCGGCG

CTGCTTCGGCATCGTCGTCGAGCGCTCGCGCGACGAGCGCCTCGCGCAGAAGGC

GCAGTCCTATCTCGACGGGCTCGACGAAGTGGGCGCCGACGCGGCGCCCGCATC

CGCCGGGAACGACCACTGAGCGGCCGC SEQ ID NO: 20 His-BicA ( Chaperone ofBurkF) Amino add sequence

MGSSHHHHHHSQDPMTQRDVNIDDIEAQEMAAALLDAVQNGATLKDLHQV PQDLMDGIYAFAYRFYQQGRLDDAEVFFRFLRIYDFYNAEYAMGLAAVCQLKKEY ARAIDLYALAYSLSKDDHRPMFHTGQCHLLMGKAALARRCFGIWERSRDERLAQK

AQ S Y L D GL DEV GAD A AP A S A GND H -

SEQ ID NO: 21 BipD nucleic acid sequence

CATATGAACATGCACGTGGACATGGGTCGTGCGCTGACCGTTCGTGATTG

GCCGGCGCTGGAGGCGCTGGCGAAAACCATGCCGGCGGATGCGGGTGCGCGTGC

GATGACCGATGATGACCTGCGTGCGGCGGGTGTGGACCGTCGTGTTCCGGAGCA

GAAGCTGGGTGCGGCGATTGATGAATTCGCGAGCCTGCGTCTGCCGGATCGTATC

GACGGTCGTTTCGTGGATGGCCGTCGTGCGAACCTGACCGTTTTTGATGATGCGC

GTGTTGCGGTTCGTGGTCATGCGCGTGCGCAACGTAACCTGCTGGAGCGTCTGGA

GACCGAACTGCTGGGTGGCACCCTGGATACCGCGGGTGACGAAGGTGGCATTCA

GCCGGACCCGATCCTGCAAGGCCTGGTGGATGTTATCGGTCAGGGCAAAAGCGA

TATTGACGCGTACGCGACCATCGTGGAAGGTCTGACCAAGTATTTTCAAAGCGTG

GCGGACGTTATGAGCAAACTGCAGGATTACATTAGCGCGAAGGATGACAAAAAC

ATGAAGATCGACGGTGGCAAGATCAAAGCGCTGATTCAGCAAGTGATCGACCAC

CTGCCGACCATGCAGCTGCCGAAGGGTGCGGATATTGCGCGTTGGCGTAAAGAG

CTGGGCGACGCGGTTAGCATCAGCGATAGCGGTGTGGTTACCATTAACCCGGAC

AAACTGATCAAGATGCGTGATAGCCTGCCGCCGGATGGCACCGTTTGGGATACC

GCGCGTTACCAAGCGTGGAACACCGCGTTCAGCGGTCAGAAAGGCCAGCATCCG

GAACGTCGTGCGGATGCGCGTCGTAAATATAGCCACCAGAACAGCAACTTTGAT

AACCTGGTGAAGGTTCTGAGCGGTGCGATTAGCACCCTGACCGACACCCAGAGC

TATCTGCAAATC

SEQ ID NO: 22 BipD amino acid sequence

MNMHVDMGRALTVRDWPALEALAKTMPADAGARAMTDDDLRAAGVDRR VPEQKLGA AIDEF A SLRLPDRIDGRF VDGRRANLTVFDD ARV AVRGHAR AQRNLLE RLETELLGGTLDTAGDEGGIQPDPILQGLVDVIGQGKSDIDAYATIVEGLTKYFQSVA DVMSKLQDYISAKDDKMviKIDGGKIKALIQQVIDHLPTMQLPKGADIARWRKELGD

AVSISDSGVVTtNPDKLIKMRDSLPPDGTVWDTARYQAWNTAFSGQKGQHPERRAD

ARRKYSHQNSNFDNLVKVLSGAISTLTDTQSYLQI

SEQ ID NO: 23 BipB nucleic acid sequence

ATGAGCAGCGGTGTTCAAGGTGGCCCGGCGGCGAACGCGAACGCGTACC

AGACCCACCCGCTGCGTGATGCGGCGAGCGCGCTGGGCACCCTGAGCCCGCAGG

CGTATGTGGATGTGGTTAGCGCGGCGCAACGTAACTTCCTGGAGCGTATGAGCC

A AC T GGC G AGC G A AC AGT GC GAT GC GCA AC C GGC GGC GC AT GAT GC GC GT C T GG

ATGATCGTCCGGCGCTGCGTGCGCCGCAGGAACGTGACGCGCCGCCGCTGGGTG

CGAGCGATACCGGTAGCCGTGCGAGCGGTGCGGCGAAACTGACCGAGCTGCTGG

GTGTGCTGATGAGCGTTATTAGCGCGAGCAGCCTGGACGAACTGAAGCAACGTA

GCGATATCTGGAACCAGATGAGCAAAGCGGCGCAAGACAACCTGAGCCGTCTGA

GC GAT GCGTTT C AGCGT GCGACC GACGAGGCG AAAGCGGCGGCGGAT GCGGC GG

AACAGGCGGCGGCGGCGGCGAAGCAAGCGGGTGCGGACGCGAAAGCGGCGGAT

GCGGCGGTGGATGCGGCGCAAAAACGTTACGATGACGCGGTTAAGCAGGGCCTG

C C GG AT G ACC GT C T GCA A AGC C T GA A AGC GGC GC T GGAGC AGGC GC GT C AGC A A

GCGGGTGATGCGCATGGTCGTGCGGATGCGCTGCAGGCGGATGCGACCAAGAAA

CTGGACGCGGCGAGCGCGCTGGCGACCCAAGCGCGTGCGTGCGAACAGCAAGTG GATGACGCGGTTAACCAGGCGACCCAGCAATATGGTGCGAGCGCGAGCCTGCGT

ACCCCGCAAAGCCCGCGTCTGAGCGGTGCGGCGGAGCTGACCGCGGTGCTGGGC

AAGCTGCAGGAACTGATTAGCAGCGGCAACGTTAAAGAGCTGGAAAGCAAGCA

G A A AC T GTT C ACC GAG AT G C A AGC G A AG CGT G AG GC GG A AC T GC A A A AG A A A A

GCGACG AAT AT C AGGC GC AAGT GAAGAA AGCGGAGG AAAT GC AG AAAACGAT G

GGTTGC ATCGGCAAGATTGTGGGTTGGGTT ATT ACCGCGGTTAGCTTTGCGGCGG

CGGCGTTTACCGGTGGCGCGAGCCTGGCGCTGGCGGCGGTGGGCCTGGCGCTGG

CGCJTTGGTGACGAGATTAGCCGTGCGACCACCGGTGTGAGCTTCATGGACAAGC

TGATGCAGCCGGTTATGGATGCGATCCTGAAACCGCTGATGGAGATGATTAGCA

GCCTGATCACCAAGGCGCTGGTTGCGTGCGGCGTTGATCAGCAAAAAGCGGAAC

TGGCGGGTGCGATTCTGGGTGCGGTTGTTACCGGTGTGGCGCTGGTTGCCKJCGGC

GTTTGTTGGTGCGAGCGCGGTGAAAGCGGTTGCGAGCAAGGTTATCGACGCGAT

GGCGGGTCAGCTGACCAAGCTGATGGATAGCGCGATTGGCAAAATGCTGGTGCA

ACTGATCGAGAAATTCAGCGAAAAGAGCGGTCTGCAGGCGCTGGGTAGCCGTAC

CGCGACCGCGATGACCCGTATGCGTCGTGCGATTGGCGTTGAGGCGAAGGAAGA

CGGTATGCTGCTGGCGAACCGTTTTGAAAAAGCGGGCACCGTGATGAACGTTGG

TAACCAAGTGAGCCAAGCGGCGGGTGGCATTGTGGTTGGCGTTGAGCGTGCGAA

AGCGATGGGTCTGCTGGCGGATGTGAAAGAAGCGATGTATGACATCAAGCTGCT

GGGTGATCTGCTGAAACAGGCGGTGGACGCGTTTGCGGAGCACAACCGTGTTCT

GGCGCAACTGATGCAGCAAATGAGCGATGCGGGCGAAATGCAGACCAGCACCG

GCAAGCTGATCCTGCGTAACGCGCGTGCGGTTTAAGGATCC

SEQ ID NO: 24 BipB amino acid sequence

MSSGVQGGPAANANAYQTHPLRDAASALGTLSPQAYVDWSAAQRNFLER MSQLASEQCDAQPAAHDARLDDRPALRAPQERDAPPLGASDTGSRASGAAKLTELL G VLM S VI S A S SLDELKQR SDI WN QM SK A AQDNL SRL SD AF QRATDEAK AAAD AAEQ AAAAAKQAGADAKAADAAVDAAQKRYDDAVKQGLPDDRLQSLKAALEQARQQA GDAHGRADALQADATKKLDAASALATQARACEQQVDDAVNQATQQYGASASLRT PQSPRLSGAAELTAVLGKLQELISSGNVKELESKQKLFTEMQAKREAELQKKSDEYQ AQVKKAEEMQKTMGCIGKIVGWVITAVSFAAAAFTGGASLALAAVGLALAVGDEIS RATTGVSFMDKLMQPVMDAILKPLMEMISSLITKALVACGVDQQKAELAGAILGAV VT GVAL VAAAF V GAS AVK AV ASK VID AMAGQLTKLMD S AIGKML VQLIEKF SEKS GLQ ALGSRT AT AMTRMRRAI GVE AKEDGMLL ANRF EK AGT VMN VGN Q V SQ A AGG IWGVERAKAMGLLADVKEAMYDIKLLGDLLKQAVDAFAEHNRVLAQLMQQMSD A GEMQ T S T GKLILRN ARA V

CATATGAACATGCACGTGGACATGGGTCGTGCGCTGACCGTTCGTGATTG

GCCGGCGCTGGAGGCGCTGGCGAAAACCATGCCGGCGGATGCGGGTGCGCGTGC

GATGACCGATGATGACCTGCGTGCGGCGGGTGTGGACCGTCGTGTTCCGGAGCA

GAAGCTGGGTGCGGCGATTGATGAATTCGCGAGCCTGCGTCTGCCGGATCGTATC

GACGGTCGTTTCGTGGATGGCCGTCGTGCGAACCTGACCGTTTTTGATGATGCGC

GT GTTGC GGTTCGTGGTC ATGCGC GTGC GC AACGT A ACCTGC TGGAGC GTCTGGA

GACCGAACTGCTGGGTGGCACCCTGGATACCGCGGGTGACGAAGGTGGCATTCA

GCCGGACCCGATCCTGCAAGGCCTGGTGGATGTTATCGGTCAGGGCAAAAGCGA

TATTGACGCGTACGCGACCATCGTGGAAGGTCTGACCAAGTATTTTCAAAGCGTG

GCGGACGTTATGAGCAAACTGCAGGATTACATTAGCGCGAAGGATGACAAAAAC

ATGAAGATCGACGGTGGCAAGATCAAAGCGCTGATTCAGCAAGTGATCGACCAC

CTGCCGACCATGCAGCTGCCGAAGGGTGCGGATATTGCGCGTTGGCGTAAAGAG

CTGGGCGACGCGGTTAGCATCAGCGATAGCGGTGTGGTTACCATTAACCCGGAC AAACTGATCAAGATGCGTGATAGCCTGCCGCCGGATGGCACCGTTTGGGATACC

GCGCGTTACCAAGCGTGGAACACCGCGTTCAGCGGTCAGAAAGGCCAGCATCCG

GAACGTCGTGCGGATGCGCGTCGTAAATATAGCCACCAGAACAGCAACTTTGAT

AACCTGGTGAAGGTTCTGAGCGGTGCGATTAGCACCCTGACCGACACCCAGAGC

TATCTGCAAATCAAGCTTATGAGCAGCGGTGTTCAAGGTGGCCCGGCGGCGAAC

GCGAACGCGTACCAGACCCACCCGCTGCGTGATGCGGCGAGCGCGCTGGGCACC

CTGAGCCCGCAGGCGTATGTGGATGTGGTTAGCGCGGCGCAACGTAACTTCCTG

GAGCGTATGAGCCAACTGGCGAGCGAACAGTGCGATGCGCAACCGGCGGCGCAT

GATGCGCGTCTGGATGATCGTCCGGCGCTGCGTGCGCCGCAGGAACGTGACGCG

CCGCCGCTGGGTGCGAGCGATACCGGTAGCCGTGCGAGCGGTGCGGCGAAACTG

ACCGAGCTGCTGGGTGTGCTGATGAGCGTTATTAGCGCGAGCAGCCTGGACGAA

CTGAAGCAACGTAGCGATATCTGGAACCAGATGAGCAAAGCGGCGCAAGACAA

CCTGAGCCGTCTGAGCGATGCGTTTCAGCGTGCGACCGACGAGGCGAAAGCGGC

GGCGGATGCGGCGGAACAGGCGGCGGCGGCGGCGAAGCAAGCGGGTGCGGACG

CGAAAGCGGCGGATGCGGCGGTGGATGCGGCGCAAAAACGTTACGATGACGCG

GTTAAGCAGGGCCTGCCGGATGACCGTCTGCAAAGCCTGAAAGCGGCGCTGGAG

CAGGCGCGTCAGCAAGCGGGTGATGCGCATGGTCGTGCGGATGCGCTGCAGGCG

GATGCGACCAAGAAACTGGACGCGGCGAGCGCGCTGGCGACCCAAGCGCGTGC

GTGCGAACAGCAAGTGGATGACGCGGTTAACCAGGCGACCCAGCAATATGGTGC

GAGCGCGAGCCTGCGTACCCCGCAAAGCCCGCGTCTGAGCGGTGCGGCGGAGCT

GACCGCGGTGCTGGGCAAGCTGCAGGAACTGATTAGCAGCGGCAACGTTAAAGA

GCTGGAAAGCAAGCAGAAACTGTTCACCGAGATGCAAGCGAAGCGTGAGGCGG

AACTGCAAAAGAAAAGCGACGAATATCAGGCGCAAGTGAAGAAAGCGGAGGAA

ATGCAGAAAACGATGGGTTGCATCGGCAAGATTGTGGGTTGGGTTATTACCGCG

GTTAGCTTTGCGGCGGCGGCGTTTACCGGTGGCGCGAGCCTGGCGCTGGCGGCG

GTGGGCCTGGCGCTGGCGGTTGGTGACGAGATTAGCCGTGCGACCACCGGTGTG

AGCTTCATGGACAAGCTGATGCAGCCGGTTATGGATGCGATCCTGAAACCGCTG

ATGGAGATGATTAGCAGCCTGATCACCAAGGCGCTGGTTGCGTGCGGCGTTGAT

CAGCAAAAAGCGGAACTGGCGGGTGCGATTCTGGGTGCGGTTGTTACCGGTGTG

GCGCTGGTTGCGGCGGCGTTTGTTGGTGCGAGCGCGGTGAAAGCGGTTGCGAGC

AAGGTTATCGACGCGATGGCGGGTCAGCTGACCAAGCTGATGGATAGCGCGATT

GGC A A A AT GC T GGT GC A AC T GAT C GAG A A ATT C AGC G A A A AG AGC GGT C T GC AG

GCGCTGGGTAGCCGTACCGCGACCGCGATGACCCGTATGCGTCGTGCGATTGGC

GTTG AGGCG AAGG A AGACGGT ATGCTGCTGGCG A ACCGTTTT G AAAAAGC GGGC

ACCGTGATGAACGTTGGTAACCAAGTGAGCCAAGCGGCGGGTGGCATTGTGGTT

GGCGTTGAGCGTGCGAAAGCGATGGGTCTGCTGGCGGATGTGAAAGAAGCGATG

TATGACATCAAGCTGCTGGGTGATCTGCTGAAACAGGCGGTGGACGCGTTTGCG

GAGCACAACCGTGTTCTGGCGCAACTGATGCAGCAAATGAGCGATGCGGGCGAA

ATGCAGACCAGCACCGGCAAGCTGATCCTGCGTAACGCGCGTGCGGTTTAAGGA

ICC

SEQ ID NO: 26 BurkF amino acid sequence

MNMHVDMGRALTVRDWPALEALAKTMPADAGARAMTDDDLRAAGVDRR VPEQKLGAAJDEFASLRLPDRIDGRFVDGRRANLTVFDDARVAVRGHARAQRNLLE RLETELLGGTLDTAGDEGGIQPDPILQGLVDVIGQGKSDIDAYATIVEGLTKYFQSVA DVMSKLQDYISAKDDKNMKIDGGKIKALIQQVIDHLPTMQLPKGADIARWRKELGD AVSISDSGVVTINPDKLIKMRDSLPPDGTVWDTARYQAWNTAFSGQKGQHPERRAD ARRK Y SHQN SNFDNL VKVL SGAISTLTDTQ S YLQIKLMS SGVQGGP AANANAY QTH PLRDAASALGTLSPQAYVDVVSAAQRNFLERMSQLASEQCDAQPAAHDARLDDRP ALRAPQERDAPPLGASDTGSRASGAAKLTELLGVLMSVISASSLDELKQRSDIWNQM SKAAQDNLSRLSDAFQRATDEAKAAADAAEQAAAAAKQAGADAKAADAAVDAA

QKRYDDAVKQGLPDDRLQSLKAALEQARQQAGDAHGRADALQADATKKLDAASA

LATQARACEQQVDDAVNQATQQYGASASLRTPQSPRLSGAAELTAVLGKLQELISS

GNVKELESKQKLFTEMQAKREAELQKKSDEYQAQVKKAEEMQKTMGCIGKIVGW

VITAVSFAAAAFTGGASLALAAVGLALAVGDEISRATTGVSFMDKLMQPVMDAILK

PLMEMISSLITKALVACGVDQQKAELAGAILGAWTGVALVAAAFVGASAVKAVAS

KVIDAMAGQLTKLMDSAIGKMLVQLIEKFSEKSGLQALGSRTATAMTRMRRAIGVE

AKEDGMLL ANRFEK AGTVMN VGNQ V SQ A AGGIVY GVERAKAMGLL AD VKEAMY

DIKLLGDLLKQAVDAFAEHNRVLAQLMQQMSDAGEMQTSTGKLILKNARAV

SEQ ID NO: 27 LTAl-BurkF nucleic add sequence

CATatggacaatggcgatcgtttataccgtgccgactcgcgtcccccagatgagattaaacgtagcggtgggttaatgcc acgtgggcacaatgagtatttgaccgtggaacacagatgaacattaaccttacgatcatgcccgtgggacccagaccgggtttgtcc gttatgatgacgggtatgttagtacgagtttgtcctacgctccgcacacctgcgggacaaagtattttatcaggctacagcacatattac atttatgtgatcgccactgccccaaacatgttcaatgtgaacgatgtgttgggggtttacagcccccatccatatgaacaagaagtctcg gcccttggggggatcccatatagecagatttatggttggtaccgcgtaaattttggtgtgattgatgaacgtttgcategtaacegtgaata ccgcgatcgctactaccgtaacttgaacatgcacctgccgaggacggctatcgtttagcgggatcccacccgatcatcaggcgtgg cgtgaggaaccgtggatccatcacgcccctcaggggtgcgggaacagtagtcgcCATATGAACATGCACGTGGA CATGGGTCGTGCGCTGACCGTTCGTGATTGGCCGGCGCTGGAGGCGCTGGCGAA AACCATGCCGGCGGATGCGGGTGCGCGTGCGATGACCGATGATGACCTGCGTGC GGCGGGTGTGGACCGTCGTGTTCCGGAGCAGAAGCTGGGTGCGGCGATTGATGA ATTCGCGAGCCTGCGTCTGCCGGATCGTATCGACGGTCGTTTCGTGGATGGCCGT CGTGCGAACCTGACCGTTTTTGATGATGCGCGTGTTGCGGTTCGTGGTCATGCGC GTGCGCAACGTAACCTGCTGGAGCGTCTGGAGACCGAACTGCTGGGTGGCACCC TGGATACCGCGGGTGACGAAGGTGGCATTCAGCCGGACCCGATCCTGCAAGGCC TGGTGGATGTTATCGGTCAGGGCAAAAGCGATATTGACGCGTACGCGACCATCG TGGAAGGTCTGACCAAGTATTTTCAAAGCGTGGCGGACGTTATGAGCAAACTGC AGGATTACATTAGCGCGAAGGATGACAAAAACATGAAGATCGACGGTGGCAAG ATCAAAGCGCTGATTCAGCAAGTGATCGACCACCTGCCGACCATGCAGCTGCCG AAGGGTGCGGATATTGCGCGTTGGCGTAAAGAGCTGGGCGACGCGGTTAGCATC AGCGAT AGCGGTGT GGTT ACC ATT AACCCGGAC AAACTG ATC AAG AT GCGT GAT AGCCTGCCGCCGGATGGCACCGTTTGGGATACCGCGCGTTACCAAGCGTGGAAC ACCGCGTTCAGCGGTCAGAAAGGCCAGCATCCGGAACGTCGTGCGGATGCGCGT CGTAAATATAGCCACCAGAACAGCAACTTTGATAACCTGGTGAAGGTTCTGAGC GGTGCGATTAGCACCCTGACCGACACCCAGAGCTATCTGCAAATCAAGCTTATG AGCAGCGGTGTTCAAGGTGGCCCGGCGGCGAACGCGAACGCGTACCAGACCCAC CCGCTGCGTGATGCGGCGAGCGCGCTGGGCACCCTGAGCCCGCAGGCGTATGTG GATGTGGTTAGCGCGGCGCAACGTAACTTCCTGGAGCGTATGAGCCAACTGGCG AGCGAACAGTGCGATGCGCAACCGGCGGCGCATGATGCGCGTCTGGATGATCGT CCGGCGCTGCGTGCGCCGCAGGAACGTGACGCGCCGCCGCTGGGTGCGAGCGAT ACCGGTAGCCGTGCGAGCGGTGCGGCGAAACTGACCGAGCTGCTGGGTGTGCTG ATGAGCGTT ATT AGCGCGAGCAGCCTGGACGAACTGAAGCAACGT AGCGAT ATC TGGAACCAGATGAGCAAAGCGGCGCAAGACAACCTGAGCCGTCTGAGCGATGCG TTTCAGCGTGCGACCGACGAGGCGAAAGCGGCGGCGGATGCGGCGGAACAGGC GGC GGC GGC GGC G A AGC A AGC GGGT GC GG AC GC G A A AGC GGC GG AT GC GGC GG TGGATGCGGCGCAAAAACGTTACGATGACGCGGTTAAGCAGGGCCTGCCGGATG ACCGTCTGCAAAGCCTGAAAGCGGCGCTGGAGCAGGCGCGTCAGCAAGCGGGTG ATGCGCATGGTCGTGCGGATGCGCTGCAGGCGGATGCGACCAAGAAACTGGACG CGGCGAGCGCGCTGGCGACCCAAGCGCGTGCGTGCGAACAGCAAGTGGATGACG CGGTTAACCAGGCGACCCAGCAATATGGTGCGAGCGCGAGCCTGCGTACCCCGC AAAGCCCGCGTCTGAGCGGTGCGGCGGAGCTGACCGCGGTGCTGGGCAAGCTGC

AGGAAC T GATT AGC AGCGGC AAC GIT AA AGAGCT GGAAAGC AAGC AGAAACTG

TTCACCGAGATGCAAGCGAAGCGTGAGGCGGAACTGCAAAAGAAAAGCGACGA

ATATCAGGCGCAAGTGAAGAAAGCGGAGGAAATGCAGAAAACGATGGGTTGCA

TCGGCAAGATTGTGGGTTGGGTTATTACCGCGGTTAGCTTTGCGGCGGCGGCGTT

TACCGGTGGCGCGAGCCTGGCGCTGGCGGCGGTGGGCCTGGCGCTGGCGGTTGG

TGACGAGATTAGCCGTGCGACCACCGGTGTGAGCTTCATGGACAAGCTGATGCA

GCCGGTTATGGATGCGATCCTGAAACCGCTGATGGAGATGATTAGCAGCCTGAT

CACCAAGGCGCTGGTTGCGTGCGGCGTTGATCAGCAAAAAGCGGAACTGGCGGG

TGCGATTCTGGGTGCGGTTGTTACCGGTGTGGCGCTGGTTGCGGCGGCGTTTGTT

GGTGCGAGCGCGGTGAAAGCGGTTGCGAGCAAGGTTATCGACGCGATGGCGGGT

C AGCT G ACC A AGCT GAT GG AT AGCGCG ATT GGC A A A ATGCTGGT GC AACTGAT C

GAGAAATTC AGCGAAAAGAGCGGT CT GC AGGCGCT GGGT AGCCGTACCGCGACC

GCGATGACCCGTATGCGTCGTGCGATTGGCGTTGAGGCGAAGGAAGACGGTATG

CTGCTGGCGAACCGTTTTGAAAAAGCGGGCACCGTGATGAACGTTGGTAACCAA

GTGAGCCAAGCGGCGGGTGGCATTGTGGTTGGCGTTGAGCGTGCGAAAGCGATG

GGTCTGCTGGCGGATGTGAAAGAAGCGATGTATGACATCAAGCTGCTGGGTGAT

CTGCTGAAACAGGCGGTGGACGCGTTTGCGGAGCACAACCGTGTTCTGGCGCAA

CTGATGCAGCAAATGAGCGATGCGGGCGAAATGCAGACCAGCACCGGCAAGCTG

ATCCTGCGTAACGCGCGTGCGGTTTAAGGATCC

SEQ ID NO: 28 LTAl-BurkF Amino add sequence

MDNGDRLYRADSRPPDEIKRSGGLMPRGHNEYFDRGTQMNINLYDHARGTQ TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSP HPYEQEVSALGGIPYSQIYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYR LAGFPPDHQAWREEPWIHHAPQGCGNSSRMNMHVDMGRALTVRDWPALEALAKT MPADAGARAMTDDDLRAAGVDRRVPEQKLGAAIDEFASLRLPDREDGRFVDGRRA NLTVFDDARVAVRGHARAQRNLLERLETELLGGTLDTAGDEGGIQPDPILQGLVDVI GQGKSDIDAYATIVEGLTKYFQSVADVMSKLQDYISAKDDKNMKIDGGKIKALIQQ VIDHLPTMQLPKGADLARWRKELGDAVSISDSGVVTINPDKLIKMRDSLPPDGTYWD TARYQAWNTAFSGQKGQHPERRADARRKYSHQNSNFDNLVKVLSGAISTLTDTQSY LQIKLMSSGVQGGPAANANAYQTHPLRDAASALGTLSPQAYVDWSAAQRNFLER MSQLASEQCDAQPAAHDARLDDRPALRAPQERDAPPLGASDTGSRASGAAKLTELL GVLMSVISASSLDELKQRSDIWNQMSKAAQDNLSRLSDAFQRATDEAKAAADAAEQ AAAAAKQAGADAKAADAAVDAAQKRYDDAVKQGLPDDRLQSLKAALEQARQQA GDAHGRADALQADATKKLDAASALATQARACEQQVDDAVNQATQQYGASASLRT PQSPRLSGAAELTAVLGKLQELISSGNVKELESKQKLFTEMQAKREAELQKKSDEYQ AQVKKAEEMQKTMGCIGKIVGWVITAVSFAAAAFTGGASLALAAVGLALAVGDEIS RATTGVSFMDKLMQPVMDAILKPLMEMISSLITKALVACGVDQQKAELAGAILGAV VTGVALVAAAFVGASAVKAVASKVIDAMAGQLTKLMDSAIGKMLVQLIEKFSEKS GLQALGSRTATAMTRMRRAIGVEAKEDGMLLANRFEKAGTVMNVGNQVSQAAGG IWGVERAKAMGLLADVKEAMYDIKLLGDLLKQAVDAFAEHNRVLAQLMQQMSD AGEMQ T S TGKLILRN ARA V-

SEQ ID NO: 29 His-PcrH { Chaperone ofPaF ) nucleic add sequence

ATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGATGAACCA GCCGACCCCTTCCGACACCGACCAGCAACAGGCGCTGGAGGCCTTCCTGCGCGA CGGCGGCACCCTGGCGATGCTTCGCGGACTCAGCGAGGACACCCTGGAGCAGCT

CTATGCGCTGGGCTTCAACCAGTACCAGGCGGGCAAGTGGGACGACGCGCAGAA

GATCTTCCAGGCACTGTGCATGCTCGACCACTACGACGCCCGCTACTTTCTCGGC CTGGGCGCCTGCCGCCAGTCCCTCGGTCTCTATGAACAGGCCCTGCAGAGCTACA

GCTACGGCGCGCTGATGGACATCAACGAGCCGCGCTTTCCCTTCCATGCCGCCGA

GTGCCACCTGCAACTGGGTGATCTCGACGGAGCCGAGAGTGGCTTCTACTCGGCC

CGGGCCCTGGCCGCGGCACAGCCGGCGCACGAGGCCCTGGCCGCGCGTGCCGGC

G C CAT GT T GG A AGC C GT A AC C G C G AG A A AGG AT C G AGC C T AT G A AT C C GAT A AC

GCTTGAAAGCTT

SEQ ID NO: 30 His- Per H (Chaperone of PaF) Amino acid sequence

QL YALGFNQ Y Q AGKWDD AQKIF Q ALCMLDHYD ARYFLGLGACRQ SLGLYEQ ALQ S YSYGALMDINEPRFPFHAAECHLQLGDLDGAESGFYSARALAAAQPAHEALAARAG AMLEAVTARKDRAYESDNA-

SEQ II ) NO: 31 PcrV nucleic acid sequence

CATATGGAAGTCAGAAACCTTAATGCCGCTCGCGAGCTGTTCCTGGACGA

GCTCCTGGCCGCGTCGGCGGCGCCTGCCAGTGCCGAGCAGGAGGAACTGCTGGC

CCTGTTGCGCAGCGAGCGGATCGTGCTGGCCCACGCCGGCCAGCCGCTGAGCGA

GGCGCAAGTGCTCAAGGCGCTCGCCTGGTTGCTCGCGGCCAATCCGTCCGCGCCT

CCGGGGCAGGGCCTCGAGGTACTCCGCGAAGTCCTGCAGGCACGTCGGCAGCCC

GGTGCGCAGTGGGATCTGCGTGAGTTCCTGGTGTCGGCCTATTTCAGCCTGCACG

GGCGTCTCGACGAGGATGTCATCGGTGTCTACAAGGATGTCCTGCAGACCCAGG

ACGGCAAGCGCAAGGCGCTGCTCGACGAGCTCAAGGCGCTGACCGCGGAGTTGA

AGGTCTACAGCGTGATCCAGTCGCAGATCAACGCCGCGCTGTCGGCCAGGCAGG

GCATCAGGATCGACGCTGGCGGTATCGATCTGGTCGACCCCACGCTATATGGCTA

TGCCGTCGGCGATCCCAGGTGGAAGGACAGCCCCGAGTATGCGCTGCTGAGCAA

TCTGGATACCTTCAGCGGCAAGCTGTCGATCAAGGATTTTCTCAGCGGCTCGCCG

AAGCAGAGCGGGGAACTCAAGGGCCTCAGCGATGAGTACCCCTTCGAGAAGGAC

AACAACCCGGTCGGCAATTTCGCCACCACGGTGAGCGACCGCTCGCGTCCGCTG

AACGACAAGGTCAACGAGAAGACCACCCTGCTCAACGACACCAGCTCCCGCTAC

AACTCGGCGGTCGAGGCGCTCAACCGCTTCATCCAGAAATACGACAGCGTCCTG

AGCGACATTCTCAGCGCGATC

SEQ ID NO: 32 Pcr V amino acid sequence

MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEA QVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRL DEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSARQGIRIDA GGIDL VDPTL Y GY A VGDPRWKD SPEY ALL SNLDTF S GKL SIKDFL S GSPKQ S GELKG LSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFI QKYDSVLSDILSAI

SEQ ID NO: 33 PopB nucleic acid sequence

ATGAACCCGATTACGCTGGAACGTGCTGGTCTGCCGTATGGTGTTGCCGA

TGCTGGTGACATCCCGGCTCTGGGTCGCCCGGTCGCACGTGATGTGGAAAGTCTG

CGTGTTGAACGTCTGGCAGCACCGGCAGCTGCAAGCGCATCTGGCACCGGTGTC

GCTCTGACGCCGCCGTCTGCAGCAAGTCAGCAACGTCTGGAAGTTGCTAACCGC

GCGGAAATTGCCTCACTGGTCCAGGCAGTGGGTGAAGACGTGGGTCTGGCACGT

CAAGTGGTTCTGGCAGGTGCATCGACCCTGCTGAGCGCAGGTCTGATGTCGCCGC

AGGCGTTCGAAATTGAACTGGCCAAAATCACCGGCGAAGTTGAAAATCAGCAGA

AAAAACTGAAACTGACGGAAATCGAACAGGCCCGTAAACAGAACCTGCAAAAA

ATGGAAGATAACCAGCAAAAAATCCGCGAATCGGAAGAAGCTGCGAAAGAAGC GCAGAAAAGCGGCCTGGCCGCAAAAATTTTTGGTTGGATTTCTGCTATCGCGAGT

ATTATCGTGGGTGCAATCATGGTTGCAACCGGTGTCGGTGCTGCAGCAGGTGCAC

TGATGATTGCTGGCGGTGTCATGGGTGTCGTGAGTCAGTCCGTGCAGCAAGCAGC

TGCGGATGGTCTGATCTCAAAAGAAGTGATGGAAAAACTGGGCCCGGCCCTGAT

GGGTATTGAAATGGCCGTGGCACTGCTGGCCGCAGTTGTCTCCTTTGGTGGTTCA

GCAGTTGGTGGTCTGGCACGTCTGGGTGCAAAAATCGGCGGTAAAGCTGCGGAA

ATGACGGCATCCCTGGCTTCAAAAGTGGCAGACCTGGGCGGTAAATTCGGCTCTC

TGGCGGGCCAGTCACTGTCGCATAGCCTGAAACTGGGTGTGCAAGTTTCTGATCT

GACCCTGGACGTTGCAAACGGCGCCGCACAGGCTACGCACAGTGGTTTTCAAGC

GAAAGCTGCGAATCGTCAGGCCGATGTTCAAGAATCCCGTGCAGACCTGACCAC

GCTGCAGGGTGTCATTGAACGTCTGAAAGAAGAACTGAGCCGCATGCTGGAAGC

CTTTCAGGAAATTATGGAACGCATCTTCGCAATGCTGCAAGCGAAAGGCGAAAC

CCTGCACAATCTGTCTTCCCGTCCGGCGGCTATCTGAGGATCC

SEQ ID NO: 34 PopB amino acid sequence

MNPITLERAGLPYGVADAGDIPALGRI^>VARDVESLRVERLAA]⁵AAASASGTG

VALTPPSAASQQRLEVANRAEIASLVQAVGEDVGLARQVVLAGASTLLSAGLMSPQ

LAAKIFGWISAIASIIVGAIMVATGVGAAAGALMIAGGVMGVVSQSVQQAAADGLIS

KEVMEKLGPALMGIEMAVALLAAWSFGGSAVGGLARLGAKIGGKAAEMTASLAS

KVADLGGKFGSLAGQSLSHSLKLGVQVSDLTLDVANGAAQATHSGFQAKAANRQA

D V Q ESR ADLTTLQG VIERLKEEL SRMLE AF QEIMERIF AMLQ AKGETLHNL S SRP A AI

SEQ ID NO: 35 PaF micleic acid sequence

CATATGGAAGTCAGAAACCTTAATGCCGCTCGCGAGCTGTTCCTGGACGA GCTCCTGGCCGCGTCGGCGGCGCCTGCCAGTGCCGAGCAGGAGGAACTGCTGGC CCTGTTGCGCAGCGAGCGGATCGTGCTGGCCCACGCCGGCCAGCCGCTGAGCGA GGCGCAAGTGCTCAAGGCGCTCGCCTGGTTGCTCGCGGCCAATCCGTCCGCGCCT CCGGGGCAGGGCCTCGAGGTACTCCGCGAAGTCCTGCAGGCACGTCGGCAGCCC GGTGCGCAGTGGGAT^'CTGCGTGAGTTCCTGGTGTCGGCCTATTTCAGCCTGCACG GGCGT CT CG ACG AGGATGT CAT CGGT GT CT AC AAGGAT GTCCTGC AG AC CC AGG ACGGCAAGCGCAAGGCGCTGCTCGACGAGCTCAAGGCGCTGACCGCGGAGTTGA AGGTCTACAGCGTGATCCAGTCGCAGATCAACGCCGCGCTGTCGGCCAGGCAGG GC AT C AGG AT CG ACGCT GGCGGT ATCG AT CTGGT CG ACCC C ACGCT AT ATGGCT A TGCCGTCGGCGATCCCAGGTGGAAGGACAGCCCCGAGTATGCGCTGCTGAGCAA TCTGGATACCTTCAGCGGCAAGCTGTCGATCAAGGATTTTCTCAGCGGCTCGCCG AAGCAGAGCGGGGAACTCAAGGGCCTCAGCGATGAGTACCCCTTCGAGAAGGAC AACAACCCGGTCGGCAATTTCGCCACCACGGTGAGCGACCGCTCGCGTCCGCTG AACGACAAGGTCAACGAGAAGACCACCCTGCTCAACGACACCAGCTCCCGCTAC AACTCGGCGGTCGAGGCGCTCAACCGCTTCATCCAGAAATACGACAGCGTCCTG AGCGACATTCTCAGCGCGATCGGATCCATGAACCCGATTACGCTGGAACGTGCT GGTCTGCCGTATGGTGTTGCCGATGCTGGTGACATCCCGGCTCTGGGTCGCCCGG TCGCACGTGATGTGGAAAGTCTGCGTGTTGAACGTCTGGCAGCACCGGCAGCTG CAAGCGCATCTGGCACCGGTGTCGCTCTGACGCCGCCGTCTGCAGCAAGTCAGC AACGTCTGGAAGTTGCTAACCGCGCGGAAATTGCCTCACTGGTCCAGGCAGTGG GTGAAGACGTGGGTCTGGCACGTCAAGTGGTTCTGGCAGGTGCATCGACCCTGCT GAGCGCAGGTCTGATGTCGCCGCAGGCGTTCGAAATTGAACTGGCCAAAATCAC C GGC GA AGTT GA A A ATC AGC AGA A A A A AC T G A A AC T G AC GG A A AT CG A AC AGG CCCGTAAACAGAACCTGCAAAAAATGGAAGATAACCAGCAAAAAATCCGCGAA TCGGAAGAAGCTGCGAAAGAAGCGCAGAAAAGCGGCCTGGCCGCAAAAATTTTT GGTTGGATTTCTGCTATCGCGAGTATTATCGTGGGTGCAATCATGGTTGCAACCG

GT GTC GGT GC T GC AGC AGGT GC AC T GAT G ATTGC T GGC GGT GTC AT GGGT GT CGT

GAGTCAGTCCGTGCAGCAAGCAGCTGCGGATGGTCTGATCTCAAAAGAAGTGAT

GGAAAAACTGGGCCCGGCCCTGATGGGTATTGAAATGGCCGTGGCACTGCTGGC

CGCAGTTGTCTCCTTTGGTGGTTCAGCAGTTGGTGGTCTGGCACGTCTGGGTGCA

AAAATCGGCGGTAAAGCTGCGGAAATGACGGCATCCCTGGCTTCAAAAGTGGCA

GACCTGGGCGGTAAATTCGGCTCTCTGGCGGGCCAGTCACTGTCGCATAGCCTGA

AACTGGGTGTGCAAGTTTCTGATCTGACCCTGGACGTTGCAAACGGCGCCGCACA

GGCTACGCACAGTGGTTTTCAAGCGAAAGCTGCGAATCGTCAGGCCGATGTTCA

AG A AT C C C GT GCAGACCT GAC C AC GC TGC AGGGT GT C ATT G A AC GT C T G A A AG A

AGAACTGAGCCGCATGCTGGAAGCCTTTCAGGAAATTATGGAACGCATCTTCGC

AATGCTGCAAGCGAAAGGCGAAACCCTGCACAATCTGTCTTCCCGTCCGGCGGC

T AT C T GAGGAT C C

SEQ ID NO: 36 PaF amino aekl sequence

MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIYLAHAGQPLSEA

QVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRL

GGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKG

LSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFI

QK YD S VL SOIL S AIG SMNPITLERAGLP Y G VAD AGDIP ALGRP V ARD VE SLRVERL A A

PAAASASGTGVALTPPSAASQQRLEVANRAEIASLVQAVGEDVGLARQVVLAGAST

LLSAGLMSPQAFEIELAKITGEVENQQKKLKLTEIEQARKQNLQKMEDNQQKIRESE

EAAKEAQKSGLAAKIFGWISAIASIIVGAIMVATGVGAAAGALMIAGGVMGVVSQS

VQQAAADGLISKEVMEKLGPALMGIEMAVALLAAVVSFGGSAVGGLARLGAKIGG

KAAEMTASLASKVADLGGKFGSLAGQSLSHSLKLGVQVSDLTLDVANGAAQATHS

GF Q AK A ANRQ AD VQE SRADL TTLQG VIERLKEEL SRMLE AF QEIMERIF AML Q AKG

ETLHNLSSRPAAI

SEQ ID NO: 37 LTAl-PaF nucleic acid sequence

CATatggacaatggcgatcgtttataccgtgccgactcgcgtcccccagatgagattaaacgtagcggtgggttaatgcc acgtgggcacaatgagtattttgaccgtggaacacagatgaacattaacctttacgatcatgcccgtgggacccagaccgggtttgtcc gttatgatgacgggtatgtagtacgagtttgtccttacgctccgcacaccttgcgggacaaagtatttatcaggctacagcacatattac atttatgtgatcgccactgccccaaacatgttcaatgtgaacgatgtgttgggggtttacagcccccatccatatgaacaagaagtctcg gcccttggggggatcccatatagccagatttatggttggtaccgcgtaaattttggtgtgattgatgaacgtttgcatcgtaaccgtgaata ccgcgatcgctactaccgtaacttgaacattgcacctgccgaggacggctatcgttagcgggattcccacccgatcatcaggcgtgg

TAATGCCGCTCGCGAGCTGTTCCTGGACGAGCTCCTGGCCGCGTCGGCGGCGCCT

GCCAGTGCCGAGCAGGAGGAACTGCTGGCCCTGTTGCGCAGCGAGCGGATCGTG

CTGGCCCACGCCGGCCAGCCGCTGAGCGAGGCGCAAGTGCTCAAGGCGCTCGCC

TGGTTGCTCGCGGCCAATCCGTCCGCGCCTCCGGGGCAGGGCCTCGAGGTACTCC

GCGAAGTCCTGCAGGCACGTCGGCAGCCCGGTGCGCAGTGGGATCTGCGTGAGT

TCCTGGTGTCGGCCTATTTCAGCCTGCACGGGCGTCTCGACGAGGATGTCATCGG

TGTCTACAAGGATGTCCTGCAGACCCAGGACGGCAAGCGCAAGGCGCTGCTCGA

C G AGC TC A AGGC GC T GAC CGC GGAGTT G A AGGT C T AC AGC GT GAT C C AGT CGC A

GATCAACGCCGCGCTGTCGGCCAGGCAGGGCATCAGGATCGACGCTGGCGGTAT

CGATCTGGTCGACCCCACGCTATATGGCTATGCCGTCGGCGATCCCAGGTGGAAG

GAC AGC CCCGAGT AT GC GCTGCTGAGC AATCTGGAT ACCTTC AGCGGC AAGCT G

TCGATCAAGGATTTTCTCAGCGGCTCGCCGAAGCAGAGCGGGGAACTCAAGGGC

CTCAGCGATGAGTACCCCTTCGAGAAGGACAACAACCCGGTCGGCAATTTCGCC ACCACGGTGAGCGACCGCTCGCGTCCGCTGAACGACAAGGTCAACGAGAAGACC

ACCCTGCTCAACGACACCAGCTCCCGCTACAACTCGGCGGTCGAGGCGCTCAAC

CGCTTCATCCAGAAATACGACAGCGTCCTGAGCGACATTCTCAGCGCGATCGGAT

CCATGAACCCGATTACGCTGGAACGTGCTGGTCTGCCGTATGGTGTTGCCGATGC

TGGTGACATCCCGGCTCTGGGTCGCCCGGTCGCACGTGATGTGGAAAGTCTGCGT

GTTGAACGTCTGGCAGCACCGGCAGCTGCAAGCGCATCTGGCACCGGTGTCGCT

CTGACGCCGCCGTCTGCAGCAAGTCAGCAACGTCTGGAAGTTGCTAACCGCGCG

GAAATTGCCTCACTGGTCCAGGCAGTGGGTGAAGACGTGGGTCTGGCACGTCAA

GTGGTTCTGGC AGGTGC ATCGACCCTGCTGAGCGC AGGTCTG ATGTCGCCGC AGG

C GTTCGAAATTGAACT GGCC AAA ATC ACCGGCGAAGTT G AAAATC AGC AGAAAA

AACTGAAACTGACGGAAATCGAACAGGCCCGTAAACAGAACCTGCAAAAAATG

GAAGATAACCAGCAAAAAATCCGCGAATCGGAAGAAGCTGCGAAAGAAGCGCA

GAAAAGCGGCCTGGCCGCAAAAATTTTTGGTTGGATTTCTGCTATCGCGAGTATT

ATCGTGGGTGCAATCATGGTTGCAACCGGTGTCGGTGCTGCAGCAGGTGCACTG

ATGATTGCTGGCGGTGTCATGGGTGTCGTGAGTCAGTCCGTGCAGCAAGCAGCTG

CGGATGGTCTGATCTCAAAAGAAGTGATGGAAAAACTGGGCCCGGCCCTGATGG

GTATTGAAATGGCCGTGGCACTGCTGGCCGCAGTTGTCTCCTTTGGTGGTTCAGC

AGTTGGTGGTCTGGCACGTCTGGGTGCAAAAATCGGCGGTAAAGCTGCGGAAAT

GACGGCATCCCTGGCTTCAAAAGTGGCAGACCTGGGCGGTAAATTCGGCTCTCTG

GCGGGCCAGTCACTGTCGCATAGCCTGAAACTGGGTGTGCAAGTTTCTGATCTGA

CCCTGGACGTTGCAAACGGCGCCGCACAGGCTACGCACAGTGGTTTTCAAGCGA

AAGCTGCGAATCGTCAGGCCGATGTTCAAGAATCCCGTGCAGACCTGACCACGC

TGCAGGGTGTCATTGAACGTCTGAAAGAAGAACTGAGCCGCATGCTGGAAGCCT

TTCAGGAAATTATGGAACGCATCTTCGCAATGCTGCAAGCGAA.AGGCGAAACCC

TGCAC A ATCTGTCTTCCCGTCCGGCGGCTATCTGAGGATCC

SEQ ID NO: 38 LTAl-PaF Amino add sequence

TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSP

HPYEQEVSALGGIPYSQIYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYR

LAGFPPDHQAWREEPWIHHAPQGCGNSSRMEVRNLNAARELFLDELLAASAAPASA

EQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQA

RJ QPG AQ WDLREF L V S A YF S LHGRLDED VKJV YKD VLQTQDG KRK ALLDELK ALT

AELKVYSVIQSQINAALSARQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLS

NLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLTSIDK

VNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLSDILSAIGSMNPITLERAGLPYGVA

DAGDIPALGRPVARDVESLRVERLAAPAAASASGTGVALTPPSAASQQRLEVANRAE

IASLVQAVGEDVGLARQWLAGASTLLSAGLMSPQAFEIELAKITGEVENQQKKLKL

TEIEQARKQNLQKMEDNQQKIRESEEAAKEAQKSGLAAKIFGWISAIASIIVGAIMVA

TGVGAAAGALMIAGGVMGVVSQSVQQAAADGLISKEVMEKLGPALMGIEMAVAL

LAAVVSFGGSAVGGLARLGAKIGGKAAEMTASLASKVADLGGKFGSLAGQSLSHSL

KLGVQVSDLTLDVANGAAQATHSGFQAKAANRQADVQESRADLTTLQGVIERLKE

EL SRMLE AF QEIMERIF AMLQ AKGETLHNL S SRP AAI -

SEQ ID NO: 39 SycD ( Chaperone for YerF) nucleic acid sequence

ATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGatgcaacaagag acgacagacactcaagaataccagctggcaatggaatccttcctaaaaggagggggaactatcgccatgctcaacgaaatttcaagtg acactttagagcaactctactctcttgcgtttaaccaataccagtcaggaaaatacgaggatgctcacaaggtctttcaagctctctgtgtg ctagaccactatgattcacgtttctttttagggctaggcgcttgtcgtcaagccatggggcaatacgacttagcgattcatagctacagcta tggcgccataatggatataaaagaacctcgttttccgtttcatgctgccgaatgtttactgcaaaagggagagcttgctgaagcagaaag

7_{5 .....} tggcttgttcttggctcaagagctatcgcagacaaacctgagtttaaggagctttccacccgagtagctcaatgttagaagcaattaaat tgaaaaaggagatggaacatgagtgcgttgataacccatgaAAGCTT

SEQ ID NO: 40 SycD (Chaperone for YerF) Amino add sequence

SDTLEQLYSLAFNQYQSGKYEDAHKVFQALCVLDHYDSRFFLGLGACRQAMGQYD

LAIHSYSYGAIMDiKEPRFPFHAAECLLQKGELAEAESGLFLAQELIADKPEFKELSTR V S SMLEAIKLKKEMEHEC VDNP-

SEQ ID NO: 41 LcrV amino acid sequence

CATATGATTAGAGCCTACGAACAAAACCCACAACATTTTATTGAGGATCT AG AAAAAGTT AGGGT GGAAC AACTT ACTGGTC ATGGTTCTT C AGTTTT AGAAGA ATTGGTTCAGTTAGTCAAAGATAAAAATATAGATATTTCCATTAAATATGATCCC AGAAAAGATTCGGAGGTTTTTGCCAATAGAGTAATTACTGATGATATCGAATTGC

TCAAGAAAATCCTAGCTTATTTTCTACCCGAGGATGCCATTCTTAAAGGCGGTCA

TTATGACAACCAACTGCAAAATGGCATCAAGCGAGTAAAAGAGTTCCTTGAATC

ATCGCCGAATACACAATGGGAATTGCGGGCGTTCATGGCAGTAATGCATTTCTCT

TTAACCGCCGATCGTATCGATGATGATATTTTGAAAGTGATTGTTGATTCAATGA

ATCATCATGGTGATGCCCGTAGCAAGTTGCGTGAAGAATTAGCTGAGCTTACCGC

C GAATT AAAG ATTT ATT C AGTT ATT C AAGCCGAA ATT AAT A AGC ATCTGTCT AGT

AGTGGCACCATAAATATCCATGATAAATCCATTAATCTCATGGATAAAAATTTAT

ATGGTT AT AC AG AT G AAGAGATTTTT AAAGCC AGCGC AGAGT AC AA AATTCTCG

AGAAAATGCCTCAAACCACCATTCAGGTGGATGGGAGCGAGAAAAAAATAGTCT

CGATAAAGGACTTTCTTGGAAGTGAGAATAAAAGAACCGGGGCGTTGGGTAATC

TGAAAAACTCATACTCTTATAATAAAGATAATAATGAATTATCTCACTTTGCCAC

CACCTGCTCGGATAAGTCCAGGCCGCTCAACGACTTGGTTAGCCAAAAAACAAC

TCAGCTGTCTGATATTACATCACGTTTTAATTCAGCTATTGAAGCACTGAACCGTT

TCATTCAGAAATATGATTCAGTGATGCAACGTCTGCTAGATGACACGTCTGGTAA

A

SEQ ID NO: 42 LcrV amino acid sequence

MIRAYEQNPQHFIEDLEKVRVEQLTGHGSSVLEELVQLVKDKNIDISIKYDPR

KDSEVFANRVITDDIELLKKrLAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNT

QWELRAFMAVMHFSLTADRIDDDILKVTVDSMNHHGDARSKLREELAELTAELKIYS

VIQAEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKASAEYKILEKMPQTTIQVD

GSEKKIVSIKDFLGSENKRTGALGNLKNSYSYNKDNNELSHFATTCSDKSRPLNDLV

SQKTTQLSDITSRFNSAIEALNRFIQKYDSVMQRLLDDTSGK

SEQ ID NO: 43 YopB nucleic acid sequence

ATGAGTGCGTTGATAACCCATGATCGCTCAACGCCAGTAACTGGAAGTCT ACTTCCCTACGTCGAGACACCAGCGCCCGCCCCCCTTCAGACTCAACAAGTCGCG GGAGAACTGAAGGATAAAAATGGTGGGGTGAGTTCTCAGGGCGTACAGCTCCCT GCACCACTAGCAGTGGTTGCCAGCCAAGTCACTGAAGGACAACAGCAAGAAATC ACTAAATTATTGGAGTCGGTCACCCGCGGCACGGCAGGATCTCAACTGATATCA AATTATGTTTCAGTGCTAACGAATTTTACGCTCGCTTCACCTGATACATTTGAGAT TGAGTTAGGTAAGCTAGTTTCTAATTTAGAAGAAGTACGCAAAGACATAAAAAT C GCT GAT ATT C AGCGTCTTC AT GAAC AAAAC AT GAAGAAAATTGAAGAGAATC A AGAGAAAATCAAAGAAACAGAAGAGAATGCCAAGCAAGTCAAGAAATCCGGCA TGGC ATC A AAGATTTTT GGCT GGCT C AGCGCC AT AGCCTC AGT GGTT ATCGGT GC CATCATGGTGGCCTCAGGGGTAGGAGCCGTTGCCGGTGCAATGATGATTGCCTCA

GGC GT A ATT GGG AT GGCG A AT AT GGCT GT G A A AC A AGC GGCGGA AG AT GGC C TG

ATATCCCAAGAGGCAATGCAAGTATTAGGGCCGATACTCACTGCGATTGAAGTC

GCATTGACTGTAGTTTCAACCGTAATGACCTTTGGCGGTTCGGCACTAAAATGCC

T GGCTG AT ATT GGCGC AAA ACT CGGT GC T A AC ACCGC AAGT C TT GCT GCT AAAGG

AGCCGAGTTTTCGGCCAAAGTTGCCCAAATTTCGACAGGCATATCAAACACTGTC

GGGAATGCAGTGACTAAATTAGGGGGCAGTTTTGGTAGTTTAACAATGAGCCAT

GTAATCCGTACAGGATCACAGGCAACACAAGTCGCCGTTGGTGTGGGCAGCGGA

ATAACTCAGACCATCAATAATAAAAAACAAGCTGATTTACAACATAATAACGCT

GATTT GGCCTT GAAC AAGGC AG AC AT GGC AGCGTT AC AAAGT ATTATTGACCGA

C TC A AAGAAGAGTT ATCC C ATTTGTC AGAGTC AC ATC A AC AAGT GAT GGA ACT G

ATTTTCCAGATGATTAATGCAAAAGGTGACATGCTGCATAATTTGGCCGGCAGAC

CCCATACTGTTTAAGGTACC

SEQ ID NO: 44 YopB amino acid sequence

MSALITHDRSTPVTGSLLPYVETPAPAPLQTQQVAGELKDKNGGVSSQGVQL PAPLAVVASQVTEGQQQEITKLLESVTRGTAGSQLISNYVSVLTNFTLASPDTFEIELG KLVSNLEEVRKDIKIADIQRLHEQNMKKIEENQEKIKETEENAKQVKKSGMASKIFG WLS AIAS VVIGAIM VA SGVGAVAGAMMIASGVIGM ANMAVKQ A AEDGLISQEAMQ VLGPILT AIEVALT VYSTVMTF GGS ALKCL ADIGAKLGANT ASL AAKGAEF S AK VAQ I S TGI SNT VGN A VTKLGGSF GSLTMSH VIRT GS Q AT Q V A VGVGS GIT QTINNKKQ ADL QHNNADLALNKADMAALQSIIDRLKEELSHLSESHQQVMELIFQMINAKGDMLHNL AGRPHTV

SEQ ID NO: 45 YerF nucleic acid sequence

CATATGATTAGAGCCTACGAACAAAACCCACAACATTTTATTGAGGATCT AGAAAAAGTTAGGGTGGAACAACTTACTGGTCATGGTTCTTCAGTTTTAGAAGA ATTGGTTCAGTTAGTCAAAGATAAAAATATAGATATTTCCATTAAATATGATCCC AG A AAAGATTCGG AGGTTTTTGCC AAT AGAGT AATT ACTG AT GAT ATCGAATT GC TCAAGAAAATCCTAGCTTATTTTCTACCCGAGGATGCCATTCTTAAAGGCGGTCA TTATGACAACCAACTGCAAAATGGCATCAAGCGAGTAAAAGAGTTCCTTGAATC ATCGC CGAAT AC AC AATGGG AATT GCGGGC GTT CAT GGC AGT AAT GC ATTTCT CT TTAACCGCCGATCGTATCGATGATGATATTTTGAAAGTGATTGTTGATTCAATGA ATCATCATGGTGATGCCCGTAGCAAGTTGCGTGAAGAATTAGCTGAGCTTACCGC CGAATTAAAGATTTATTCAGTTATTCAAGCCGAAATTAATAAGCATCTGTCTAGT AGTGGCACCATAAATATCCATGATAAATCCATTAATCTCATGGATAAAAATTTAT AT GGTT AT AC AG AT GAAG AGATTTTT AAAGCC AGCGC AG AGT AC AAAATT CTCG AGAAAATGCCTCAAACCACCATTCAGGTGGATGGGAGCGAGAAAAAAATAGTCT CGATAAAGGACTTTCTTGGAAGTGAGAATAAAAGAACCGGGGCGTTGGGTAATC T GAAAAACTC AT ACTCTT AT AAT AAAG AT AAT AAT GAATT ATCTC ACTTTGCC AC CACCTGCTCGGATAAGTCCAGGCCGCTCAACGACTTGGTTAGCCAAAAAACAAC TCAGCTGTCTGATATTACATCACGTTTTAATTCAGCTATTGAAGCACTGAACCGTT TCATTCAGAAATATGATTCAGTGATGCAACGTCTGCTAGATGACACGTCTGGTAA AGGATCCATGAGTGCGTTGATAACCCATGATCGCTCAACGCCAGTAACTGGAAG TCTACTTCCCTACGTCGAGACACCAGCGCCCGCCCCCCTTCAGACTCAACAAGTC GCGGGAGAACTGAAGGATAAAAATGGTGGGGTGAGTTCTCAGGGCGTACAGCTC CCTGCACCACTAGCAGTGGTTGCCAGCCAAGTCACTGAAGGACAACAGCAAGAA AT C AC T A A ATT ATT GG AGT C GGTC AC C C GC GGC AC GGC AGG AT C TC A AC T GAT AT CAAATTATGTTTCAGTGCTAACGAATTTTACGCTCGCTTCACCTGATACATTTGAG ATTGAGTTAGGTAAGCTAGTTTCTAATTTAGAAGAAGTACGCAAAGACATAAAA ATCGCTGATATTCAGCGTCTTCATGAACAAAACATGAAGAAAATTGAAGAGAAT

C AAGAGAAAATC AA AG AAAC AGAAGAGA AT GC C AAGC AAGTC AAGAA ATCCGG

CATGGCATCAAAGATTTTTGGCTGGCTCAGCGCCATAGCCTCAGTGGTTATCGGT

GCCATCATGGTGGCCTCAGGGGTAGGAGCCGTTGCCGGTGCAATGATGATTGCCT

C AGGCGT A ATTGGG AT GGC GAAT AT GGC T GT G A A AC A AGCGGC GGA AGAT GGC C

TGATATCCCAAGAGGCAATGCAAGTATTAGGGCCGATACTCACTGCGATTGAAG

TCGCATTGACTGTAGTTTCAACCGTAATGACCTTTGGCGGTTCGGCACTAAAATG

CCTGGCTGATATTGGCGCAAAACTCGGTGCTAACACCGCAAGTCTTGCTGCTAAA

GGAGCCGAGTTTTCGGCCAAAGTTGCCCAAATTTCGACAGGCATATCAAACACT

GTCGGGAATGC AGT GACT AAATT AGGGGGC AGTTTTGGT AGTTT AAC AAT GAGC

CATGTAATCCGTACAGGATCACAGGCAACACAAGTCGCCGTTGGTGTGGGCAGC

GGAATAACTCAGACCATCAATAATAAAAAACAAGCTGATTTACAACATAATAAC

GCTGATTTGGCCTTGAACAAGGCAGACATGGCAGCGTTACAAAGTATTATTGACC

GACTCAAAGAAGAGTTATCCCATTTGTCAGAGTCACATCAACAAGTGATGGAAC

T G ATTTTCC AG AT G ATT A ATGC A AAAGGT G AC AT GCTGC AT A ATTTGGCCGGC AG

ACCCCATACTGTTTAAGGTACC

SEQ ID NO: 46 YerF amino acid sequence

MIRAYEQNPQHF^'IEDLEKVRVEQLTGHGSSVLEELVQLVKDKNIDISIKYDPR KDSE ANRVITDDIELLKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNT QWELRAFMAVMHF SET ADRIDDDILK VIYT⁾ SMNHHGD ARSKLREEL AELT AELKI Y S VIQAEINKHLSSSGTINIHDKSrNLMDKNLYGYTDEEIFKASAEYKILEKMPQTTIQVD GSEKKI V SIKDF LGSENKRT GALGNLKN S Y S YNKDNNEL SHF ATT C SDK SRPLNDL V SQKTTQLSDITSRFNSAIEALNRFIQKYDSYMQRLLDDTSGKGSMSALITHDRSTPVT GSLLPYVETPAPAPLQTQQVAGELKDKNGGVSSQGVQLPAPLAVVASQVTEGQQQE ITKLLESVTRGTAGSQLISNYVSVLTNFTLASPDTFEIELGKLVSNLEEVRKDIKIADIQ RLHEQNMKKIEENQEKIKETEENAKQVKKSGMASKIFGWLSAIASVVIGAIMVASGV GAVAGAMMIASGVIGMANMAVKQAAEDGLISQEAMQVLGPILTAIEVALTVVSTV MTFGGSALKCLADIGAKLGANTASLAAKGAEFSAKVAQISTGISNTVGNAVTKLGGS FGSLTMSHVIRTGSQATQVAVGVGSGITQTINNKKQADLQHNNADLALNKADMAA LQSIIDRLKEELSHLSESHQQVMELIFQMINAKGDMLHNLAGRPF1TV

SEQ ID NO: 47 LTAl-YerF nucleic add sequence

CATatggacaatggcgatcgtttataccgtgccgactcgcgtcccccagatgagattaaacgtagcggtgggttaatgcc acgtgggcacaatgagtattttgaccgtggaacacagatgaacattaacctttacgatcatgcccgtgggacccagaccgggtttgtcc gttatgatgacgggtatgttagiacgagtitgtccttacgctccgcac accttgcgggacaaagtatttatcaggctacagcacataltac atttatgtgatcgccactgccccaaacatgttcaatgtgaacgatgtgttgggggtttacagcccccatccatatgaacaagaagtctcg gcccttggggggatcccatatagccagatttatggttggtaccgcgtaaattttggtgtgatgatgaacgtttgcatcgtaaccgtgaata ccgcgatcgctactaccgtaacttgaacatgcacctgccgaggacggctatcgtttagcgggattcccacccgatcatcaggcgtgg cgtgaggaaccgtggatccatcacgcccctc aggggtgcgggaacagtagtcgcC AT ATGATT AG AGC C T AC GA ACAAAACCCACAACATTTTATTGAGGATCTAGAAAAAGTTAGGGTGGAACAACT TACTGGTCATGGTTCTTCAGTTTTAGAAGAATTGGTTCAGTTAGTCAAAGATAAA AATATAGATATTTCCATTAAATATGATCCCAGAAAAGATTCGGAGGTTTTTGCCA ATAGAGTAATTACTGATGATATCGAATTGCTCAAGAAAATCCTAGCTTATTTTCT ACCCGAGGATGCCATTCTTAAAGGCGGTCATTATGACAACCAACTGCAAAATGG CATCAAGCGAGTAAAAGAGTTCCTTGAATCATCGCCGAATACACAATGGGAATT GCGGGCGTTCATGGCAGTAATGCATTTCTCTTTAACCGCCGATCGTATCGATGAT G AT ATTTTGAAAGTG ATT GTT G ATTC AAT GAAT CAT CAT GGT GAT GC CCGT AGC A AGTTGCGTGAAGAATTAGCTGAGCTTACCGCCGAATTAAAGATTTATTCAGTTAT TCAAGCCGAAATTAATAAGCATCTGTCTAGTAGTGGCACCATAAATATCCATGAT AAATCCATTAATCTCATGGATAAAAATTTATATGGTTATACAGATGAAGAGATTT

TTAAAGC C AGCGC AGAGT AC AAAATT CTCGAGAAAATGCCTC AAACC ACC ATTC

AGGTGGATGGGAGCGAGAAAAAAATAGTCTCGATAAAGGACTTTCTTGGAAGTG

AGAATAAAAGAACCGGGGCGTTGGGTAATCTGAAAAACTCATACTCTTATAATA

AAGATAATAATGAATTATCTCACTTTGCCACCACCTGCTCGGATAAGTCCAGGCC

GCTCAACGACTTGGTTAGCCAAAAAACAACTCAGCTGTCTGATATTACATCACGT

TTTAATTCAGCTATTGAAGCACTGAACCGTTTCATTCAGAAATATGATTCAGTGA

TGCAACGTCTGCTAGATGACACGTCTGGTAAAGGATCCATGAGTGCGTTGATAAC

CCATGATCGCTCAACGCCAGTAACTGGAAGTCTACTTCCCTACGTCGAGACACCA

GCGCCCGCCCCCCTTCAGACTCAACAAGTCGCGGGAGAACTGAAGGATAAAAAT

GGTGGGGTGAGTTCTCAGGGCGTACAGCTCCCTGCACCACTAGCAGTGGTTGCCA

GCCAAGTCACTGAAGGACAACAGCAAGAAATCACTAAATTATTGGAGTCGGTCA

CCCGCGGC ACGGC AGGAT CT C AACTGAT ATC AAATTAT GTTT C AGT GCT AACGAA

TTTTACGCTCGCTTCACCTGATACATTTGAGATTGAGTTAGGTAAGCTAGTTTCTA

ATTTAGAAGAAGTACGCAAAGACATAAAAATCGCTGATATTCAGCGTCTTCATG

AACAAAACATGAAGAAAATTGAAGAGAATCAAGAGAAAATCAAAGAAACAGAA

GAGAATGCCAAGCAAGTCAAGAAATCCGGCATGGCATCAAAGATTTTTGGCTGG

CTCAGCGCCATAGCCTCAGTGGTTATCGGTGCCATCATGGTGGCCTCAGGGGTAG

GAGCCGI I GCCGGTGCAA I'GAT GATTGCCTC AGGCGTAAT I'GGGA IGGCGAAT A

TGGCTGTGAAACAAGCGGCGGAAGATGGCCTGATATCCCAAGAGGCAATGCAAG

T ATT AGGGCC GAT ACT C ACTGCG ATT GAAGTCGC ATTGACTGT AGTTT C A ACCGT

AATGACCTTTGGCGGTTCGGCACTAAAATGCCTGGCTGATATTGGCGCAAAACTC

GGTGCTAACACCGCAAGTCTTGCTGCTAAAGGAGCCGAGTTTTCGGCCAAAGTTG

CCCAAATTTCGACAGGCATATCAAACACTGTCGGGAATGCAGTGACTAAATTAG

GGGGCAGTTTTGGT AGTTT AACAATGAGCCATGTAATCCGTACAGGATCACAGG

CAACACAAGTCGCCGTTGGTGTGGGCAGCGGAATAACTCAGACCATCAATAATA

AAAAACAAGCTGATTTACAACATAATAACGCTGATTTGGCCTTGAACAAGGCAG

ACATGGCAGCGTTACAAAGTATTATTGACCGACTCAAAGAAGAGTTATCCCATTT

GTCAGAGTCACATCAACAAGTGATGGAACTGATTTTCCAGATGATTAATGCAAA

AGGTGACATGCTGCATAATTTGGCCGGCAGACCCCATACTGTTTAAGGTACC

SEQ ID NO: 48 LTAl-YerF Ammo add sequence

MDNGDRLYRADSRPPDEIKRSGGLMPRGHNEYFDRGTQMNINLYDHARGTQ TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSP HPYEQEVSALGGIPYSQIYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYR L AGFPPDHQ A WREEPWIHH APQGC GNS SRMIRA YEQNPQHFIED LEK VRVEQLTGH GSSVLEELVQLVKDKNIDISIKYDPRKDSEVFANRVITDDIELLKKILAYFLPEDAILK GGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKVIVDS MNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTrNIHDKSINLMDKNLYG YTDEEIFK AS AEYKILEKMPQTTIQ VDGSEKKIVSIKDFLGSENKRT GALGNLKN SYS

YNKDNNELSHFATTCSDKSRPLNDLVSQKTTQLSDITSRFNSAIEALNRFIQKYDSVM

QRLLDDTSGKGSMSALITHDRSTPVTGSLLPYVETPAPAPLQTQQVAGELKDKNGGV

SSQGVQLPAPLAVYASQVTEGQQQEITKLLESVTRGTAGSQLISNYVSVLTNFTLASP

DTFEIELGKLVSNLEEVRKDIKIADIQRLHEQNMKKIEENQEKIKETEENAKQVKKSG

MASKIFGWLSAIASVVIGAIMVASGVGAVAGAMMIASGVIGMANMAVKQAAEDGL

ISQEAMQVLGPILTAIEVALTWSTVMTFGGSALKCLADIGAKLGANTASLAAKGAE

FSAKVAQISTGISNTVGNAVTKLGGSFGSLTMSHVIR.TGSQATQVAVGVGSGITQTIN

GDMLHNLAGRPHTV- SEQ ID NO: 49 His-SicA chaperone for Si nucleic sequence

ATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGatggactaccaga acaacgtcagcgaagaacgtgttgcggaaatgatttgggatgccgttagtgaaggcgccacgctaaaagacgttcatggaatccctca agatatgatggacggtttatatgctcatgcttatgagttttataaccagggacgactggatgaagctgagacgttctttcgtttctatgcatt tatgatttttacaatcccgattacaccatgggactggcggcagtatgccaactgaaaaaacaatttcagaaagcatgtgacctttatgcag tagcgtttacgttacttaaaaatgattatcgccccgtttttttaccgggcagtgtcaattataatgcgtaaggcagcaaaagccagacagt gttttgaacttgtcaatgaacgtactgaagatgagtctctgcgggcaaaagcgttggtctatctggaggcgctaaaaacggcggagaca gagcagcacagegagcaggagaaggagtaaAAGCTT

SEQ ID NO: 50 His-SicA chaperone for SI Amino add sequence

MGSSHHHHHHSQDPMDYQNNVSEERVAEMIWDAVSEGATLKDVHGIPQDM MDGLYAHAYEFYNQGRLDEAETFFRFLCIYDFYNPDYTMGLAAVCQLKKQFQKAC DL Y A V AF TLLKND YRP VFF T GQCQLLMRK AAK ARQCFEL VNERTEDE SLRAK AL VY LEALKTAETEQHSEQE E*

SEQ ID NO: 51 SipD nucleic acid sequence

atgctaatattcaaaattattccgcttctcctcatccggggatcgttgccgaacggccgcagactccctcggcgagcgagc acgtcgagactgccgtggtaccgtctaccacagaacatcgcggtacagatatcatttcattatcgcaggcggctactaaaatccaccag gcacagcagacgctgcagtcaacgccaccgatctctgaagagaataatgacgagcgcacgctggcgcgccagcagttgaccagca gcctgaatgcgctggcgaagtccggcgtgtcattatccgcagaacaaaatgagaacctgcggagcgcgttttctgcgccgacgtcgg ccttatttagcgcttcgcctatggcgcagccgagaacaaccatttctgatgctgagatttgggatatggtttcccaaaatatatcggcgata ggtgacagctatctgggcgtttatgaaaacgtgtcgcagtctataccgatttttatcaggccttcagtgatattctttccaaaatgggagg ctggttataccaggtaaggacggtaataccgttaagctagatgttacctcactcaaaaatgatttaaacagttagtcaataaatataatca aataaacagtaataccgttttatttccagcgcagtcaggcagcggcgttaaagtagccactgaagcggaagcgagacagtggctcagt gaatgaatttaccgaatagctgcctgaaatcttatggatccggttatgtcgtcaccgttgatctgacgccattacaaaaaatggttcagga tattgatggtttaggcgcgccgggaaaagactcaaaactcgaaatggataacgccaaatatcaagcctggcagtcgggttttaaagcg caggaagaaaatatgaaaaccacattacagacgctgacgcaaaaatatagcaatgccaattcattgtacgacaacctggtaaaagtgct gagcagtacgataagtagcagcctggaaaccgccaaaagcttcctgcaagga

SEQ ID NO: 52 SipD amino acid sequence

MLNIQNYSASPHPGIVAERPQTPSASEHVETAVVPSTTEHRGTDIISLSQAATKI

HQ AQQTLQ STPPISEENNDERTL ARQQLT S SLNAL AKSGV SL S AEQNENLRS AF S APT

SALFSASPMAQPRTTISDAEiWDMVSQNiSAIGDSYLGVYENVVAVYTDFYQAFSDIL

SKMGGWLLPGKDGNTVKLDVTSLKNDLNSLVNKYNQINSNTVLFPAQSGSGVKVA

TEAEARQWLSELNLPNSCLKSYGSGYVVTVDLTPLQKMVQDIDGLGAPGKDSKLEM

DNAKYQAWQSGFKAQEENMKTTLQTLTQKYSNANSLYDNLVKVLSSTISSSLETAK

SFLQG

SEQ ID NO: 53 SipB nucleic acid sequence

atggtaaatgacgcaagtagcattagccgtagcggatatacccaaaatccgcgcctcgctgaggcggcttttgaaggcgtt cgtaagaacacggactttttaaaagcggcggataaagcttttaaagatgtggtggcaacgaaagcgggcgacctaaagccggaaca aagtccggcgagagcgctattaatacggtgggtctaaagccgcctacggacgccgcccgggaaaaactctccagcgaagggcaatt gacattactgcttggcaagttaatgaccctactgggcgatgtttcgctgtctcaactggagtctcgtctggcggtatggcaggcgatgatt gagtcacaaaaagagatggggattcaggtatcgaaagaattccagacggctctgggagaggctcaggaggcgacggatctctatga agccagtatcaaaaagacggataccgccaagagtgtttatgacgctgcgaccaaaaaactgacgcaggcgcaaaataaattgcaatc gctggacccggctgaccccggctatgcacaagctgaagccgcggtagaacaggccggaaaagaagcgacagaggcgaaagagg ccttagataaggccacggatgcgacggttaaagcaggcacagacgccaaagcgaaagccgagaaagcggataacattctgaccaa attccagggaacggctaatgccgcctctcagaatcaggtttcccagggtgagcaggataatctgtcaaatgtcgcccgcctcactatgc tcatggccatgttattgagattgtgggcaaaaatacggaagaaagcctgcaaaacgatctgcgcttttcaacgcctgcaggaaggg cgtcaggcggagatggaaaagaaatcggctgaattccaggaagagacgcgcaaagccgaggaaacgaaccgcattatgggatgta tcgggaaagtcctcggcgcgctgctaaccattgtcagcgtgtggccgctgtttttaccggtggggcgagtctggcgctggctgcggt gggacttgcggtaatggtggccgatgaaattgtgaaggcggcgacgggagtgtcgtttattcagcaggcgctaaacccgattatgga gcatgtgctgaagccgttaatggagctgattggcaaggcgattaccaaagcgctggaaggattaggcgtcgataagaaaacggcag agatggccggcagcattgttggtgcgattgtcgccgctattgccatggtggcggtcattgtggtggtcgcagttgtcgggaaaggcgc ggcggcgaaactgggtaacgcgctgagcaaaatgatgggcgaaacgataagaagttggtgcctaacgtgctgaaacagttggcgc aaaacggcagcaaactctttacccaggggatgcaacgtattactagcggtctgggtaatgtgggtagcaagatgggcctgcaaacga atgccttaagtaaagagctggtaggtaataccctaaataaagtggcgttgggcatggaagtcacgaataccgcagcccagtcagccg gtggtgttgccgagggcgtatttattaaaaatgccagcgaggcgcttgctgattttatgctcgcccgttttgccatggatcagattcagca gtggcttaaacaatccgtagaaatatttggtgaaaaccagaaggtaacggcggaactgcaaaaagccatgtcttctgcggtacagcaa aatgcggatgcttcgcgttttattctgcgccagagtcgcgcataa

SEQ ID NO: 54 SipB amino add sequence

MVNDASSISRSGYTQNPRLAEAAFEGVRKNTDFLKAADKAFKDVVATKAGD

LKAGTKSGESAINTVGLKPPTDAAREKLSSEGQLTLLLGKLMTLLGDVSLSQLESRL

AVWQAMIESQKEMGIQVSKEFQTALGEAQEATDLYEASIKKTDTAKSVYDAATKKL

TQAQNKLQSLDPADPGYAQAEAAVEQAGKEATEAKEALDKATDATVKAGTDAKA

KAEKADNILTKFQGTANAASQNQVSQGEQDNLSNVARLTMLMAMFIEIVGKNTEES

LQNDLALFNALQEGRQAEMEKKSAEFQEETRKAEETNRIMGCIGKVLGALLTIVSW

AAVFTGGASLALAAVGLAYMVADEIVKAATGVSFIQQALNPIMEHVLKPLMELIGK

AITKALEGLGVDKKTAEMAGSIVGATVAAIAMyAVIVWAWGKGAAAKLGNALS

KMMGETIKKLVPNVLKQLAQNGSKLFTQGMQRITSGLGNVGSKMGLQTNALSKEL

VGNTLNKVALGMEVTNTAAQSAGGVAEGVFIKNASEALADFMLARFAMDQIQQWL

KQ S VEIF GENQKVT AELQKAMS S AVQQNAD ASRFILRQSRA

SEQ ID NO: 55 SI nucleic acid sequence

atgcttaatattcaaaattattccgcttctcctcatccggggatcgttgccgaacggccgcagactccctcggcgagcgagc acgtcgagactgccgtggtaccgtctaccacagaacatcgcggtacagatatcatttcattatcgcaggcggctactaaaatccaccag gcacagcagacgctgcagtcaacgccaccgatctctgaagagaataatgacgagcgcacgctggcgcgccagcagttgaccagca gcctgaatgcgctggcgaagtccggcgtgtcattatccgcagaacaaaatgagaacctgcggagcgcgttttctgcgccgacgtcgg ccttatttagcgcttcgcctatggcgcagccgagaacaaccatttctgatgctgagatttgggatatggtttcccaaaatatatcggcgata ggtgacagctatctgggcgtttatgaaaacgttgtcgcagtctataccgatttttatcaggccttcagtgatattctttccaaaatgggagg ctggttattaccaggtaaggacggtaataccgttaagctagatgttacctcactcaaaaatgatttaaacagttagtcaataaatataatca aataaacagtaataccgtttatttccagcgcagtcaggcagcggcgttaaagtagccactgaagcggaagcgagacagtggctcagt gaattgaatttaccgaatagctgcctgaaatcttatggatccggttatgtcgtcaccgttgatctgacgccattacaaaaaatggttcagga tattgatggtttaggcgcgccgggaaaagactcaaaactcgaaatggataacgccaaatatcaagcctggcagtcgggttttaaagcg caggaagaaaatatgaaaaccacattacagacgctgacgcaaaaatatagcaatgccaattcattgtacgacaacctggtaaaagtgct gagcagtacgataagtagcagcctggaaaccgccaaaagcttcctgcaaggagtcgacatggtaaatgacgcaagtagcattagcc agcttttaaagatgtggtggcaacgaaagcgggcgaccttaaagccggaacaaagtccggcgagagcgctattaatacggtgggtct aaagccgcctacggacgccgcccgggaaaaactctccagcgaagggcaattgacattactgcttggcaagttaatgaccctactggg cgatgtttcgctgtctcaactggagtctcgtctggcggtatggcaggcgatgattgagtcacaaaaagagatggggattcaggtatcga aagaattccagacggctctgggagaggctcaggaggcgacggatctctatgaagccagtatcaaaaagacggataccgccaagagt gtttatgacgctgcgaccaaaaaactgacgcaggcgcaaaataaattgcaatcgctggacccggctgaccccggctatgcacaagct gaagccgcggtagaacaggccggaaaagaagcgacagaggcgaaagaggccttagataaggccacggatgcgacggttaaagc aggcacagacgccaaagcgaaagccgagaaagcggataacattctgaccaaattccagggaacggctaatgccgcctctcagaatc aggtttcccagggtgagcaggataatctgtcaaatgtcgcccgcctcactatgctcatggccatgtttatgagattgtgggcaaaaatac ggaagaaagcctgcaaaacgatcttgcgcttttcaacgccttgcaggaagggcgtcaggcggagatggaaaagaaatcggctgaatt ccaggaagagacgcgcaaagccgaggaaacgaaccgcattatgggatgtatcgggaaagtcctcggcgcgctgctaaccattgtca gcgttgtggccgctgtttttaccggtggggcgagtctggcgctggctgcggtgggacttgcggtaatggtggccgatgaaatgtgaa ggcggcgacgggagtgtcgtttattcagcaggcgctaaacccgattatggagcatgtgctgaagccgttaatggagctgattggcaag si gcgattaccaaagcgctggaaggattaggcgtcgataagaaaacggcagagatggccggcagcatgttggtgcgattgtcgccgct attgccatggtggcggtcattgtggtggtcgcagttgtcgggaaaggcgcggcggcgaaactgggtaacgcgctgagcaaaatgat gggcgaaacgattaagaagttggtgcctaacgtgctgaaacagttggcgcaaaacggcagcaaactctttacccaggggatgcaac gtattactagcggtctgggtaatgtgggtagcaagatgggcctgcaaacgaatgccttaagtaaagagctggtaggtaataccctaaat aaagtggcgttgggcatggaagtcacgaataccgcagcccagtcagccggtggtgttgccgagggcgtatttattaaaaatgccagc gaggcgcttgctgattttatgctcgcccgttttgccatggatcagattcagcagtggctaaacaatccgtagaaatattggtgaaaacc agaaggtaacggcggaactgcaaaaagccatgtcttctgcggtacagcaaaatgcggatgcttcgcgttttattctgcgccagagtcg cgcataa

SEQ ID NO: 56 SI amino acid sequence

MLNIQNYSASPHPGIVAERPQTPSASEHVETAVVPSTTEHRGTDIISLSQAATKI HQAQQTLQSTPPISEENNDERTLARQQLTSSLNALAKSGVSLSAEQNENLRSAFSAPT SALFSASPMAQPRTTISDAEIWDMVSQ ISAIGDSYLGVYENWAVYTDFYQAFSDIL SKMGGWLLPGKDGNTVKLDVTSLKNDLNSLVNKYNQINSNTVLFPAQSGSGVKVA TEAEARQWLSELNLPNSCLKSYGSGYVVTVDLTPLQKMVQDIDGLGAPGKDSKLEM DN AK Y Q AW Q SGFK AQEENMKTTLQTLTQK YSNANSL YDNLVKVL S STI S S SLET AK SFLQGVDMVNDASSISRSGYTQNPRLAEAAFEGVRKNTDFLKAADKAFKDVVATKA GDLK AGTK S GE SAINT VGLKPPTDAAREKL S SEGQLTLLLGKLMTLLGD VSL S QLE S RLAVWQAMIESQKEMGIQVSKEFQTALGEAQEATDLYEASIKKTDTAKSVYDAATK KLTQAQNKLQSLDPADPGYAQAEAAVEQAGKEATEAKEALDKATDATVKAGTDA KAKAEKADNILTKFQGTANAASQNQVSQGEQDNLSNVARLTMLMAMFIEIVGKNTE E SLQ ND L ALFN AL Q EGRQ AEMEKK S AEFQEETRK AEETNRIMGC IGK VLGAL LTI V S VVAAVFTGGASLALAAVGLAVMVADEIVKAATGVSFIQQALNPIMEHVLKPLMELI GKAITKALEGLGVDKKTAEMAGSIVGAIVAAIAMVAVIVVVAVVGKGAAAKLGNA LSKMMGETIKKLVPNVLKQLAQNGSKLFTQGMQRITSGLGNVGSKMGLQTNALSKE LVGNTLNKVALGMEVTNTAAQSAGGVAEGVFIKNASEALADFMLARFAMDQIQQW LKQSVEIFGENQKVTAEEQKAMSSAVQQNADASRFILRQSRA

SEQ ID NO: 57 LTA1-GSAAS-S1 Nucleic add sequence

CATatggacaatggcgatcgtttataecgtgccgactcgcgteccceagatgagattaaacgtagcggtgggttaatgcc aegtgggeacaatgagtattttgaeegtggaacaeagatgaaeattaacetttaegatcatgcccgtgggacccagaccgggtttgtcc gttatgatgacgggtatgttagtacgagtttgtccttacgctccgcacaccttgcgggacaaagtatttatcaggctacagcacatattac atttatgtgatcgccactgccccaaacatgttcaatgtgaacgatgtgttgggggttacagcccccatccatatgaacaagaagtctcg gcccttggggggatcccatatagccagatttatggttggtaccgcgtaaattttggtgtgattgatgaacgtttgcatcgtaaccgtgaata ccgcgatcgctactaccgtaacttgaacattgcacctgccgaggacggctatcgttagcgggattcccacccgatcatcaggcgtgg cgtgaggaaccgtggatccatcacgcccctcaggggtgcgggaacagtagtcgcgggtccgcggcatccatgcttaatattcaaaatt attccgcttctcctcatccggggatcgttgccgaacggccgcagactccctcggcgagcgagcacgtcgagactgccgtggtaccgt ctaccacagaacatcgcggtacagatatcatttcattatcgcaggcggctactaaaatccaccaggcacagcagacgctgcagtcaac gccaccgatctctgaagagaataatgacgagcgcacgctggcgcgccagcagttgaccagcagcctgaatgcgctggcgaagtcc ggcgtgtcattatccgcagaacaaaatgagaacctgcggagcgcgttttctgcgccgacgtcggccttatttagcgcttcgcctatggc gcagccgagaacaaccatttctgatgctgagatttgggatatggtttcccaaaatatatcggcgataggtgacagctatctgggcgtttat gaaaacgttgtcgcagtctataccgatttttatcaggccttcagtgatattctttccaaaatgggaggctggttattaccaggtaaggacgg taataccgttaagctagatgttacctcactcaaaaatgatttaaacagtttagtcaataaatataatcaaataaacagtaataccgtttatttc cagcgcagtcaggcagcggcgttaaagtagccactgaagcggaagcgagacagtggctcagtgaattgaattaccgaatagctgc ctgaaatcttatggatccggttatgtcgtcaccgttgatctgacgccattacaaaaaatggttcaggatatgatggtttaggcgcgccgg gaaaagactcaaaactcgaaatggataacgccaaatatcaagcctggcagtcgggttttaaagcgcaggaagaaaatatgaaaacca cattacagacgctgacgcaaaaatatagcaatgccaattcattgtacgacaacctggtaaaagtgctgagcagtacgataagtagcagc ctggaaaccgccaaaagcttcctgcaaggagtcgacatggtaaatgacgcaagtagcattagccgtagcggatatacccaaaatccg cgcctcgctgaggcggcttttgaaggcgttcgtaagaacacggactttttaaaagcggcggataaagctttaaagatgtggtggcaac gaaagcgggcgaccttaaagccggaacaaagtccggcgagagcgctattaatacggtgggtciaaagccgcctacggacgccgcc cgggaaaaactctccagcgaagggcaattgacattactgctggcaagttaatgaccctactgggcgatgtttcgctgtctcaactgga gtctcgtctggcggtatggcaggcgatgattgagtcacaaaaagagatggggattcaggtatcgaaagaattccagacggctctggg aactgacgcaggcgcaaaataaattgcaatcgctggacccggctgaccccggctatgcacaagctgaagccgcggtagaacaggc cggaaaagaagcgacagaggcgaaagaggccttagataaggccacggatgcgacggttaaagcaggcacagacgccaaagcga aagccgagaaagcggataacattctgaccaaattccagggaacggctaatgccgcctctcagaatcaggtttcccagggtgagcagg ataatctgtcaaatgtcgcccgcctcactatgctcatggccatgtttattgagattgtgggcaaaaatacggaagaaagcctgcaaaacg atcttgcgcttttcaacgccttgcaggaagggcgtcaggcggagatggaaaagaaatcggctgaattccaggaagagacgcgcaaa gccgaggaaacgaaccgcattatgggatgtatcgggaaagtcctcggcgcgctgctaaccattgtcagcgtgtggccgctgtttttac cggtggggcgagtctggcgctggctgcggtgggacttgcggtaatggtggccgatgaaattgtgaaggcggcgacgggagtgtcgt ttattcagcaggcgctaaacccgattatggagcatgtgctgaagccgttaatggagctgattggcaaggcgattaccaaagcgctgga aggattaggcgtcgataagaaaacggcagagatggccggcagcattgttggtgcgattgtcgccgctattgccatggtggcggtcatt gtggtggtcgcagttgtcgggaaaggcgcggcggcgaaactgggtaacgcgctgagcaaaatgatgggcgaaacgattaagaagt tggtgcctaacgtgctgaaacagttggcgcaaaacggcagcaaactcttacccaggggatgcaacgtattactagcggtctgggtaa tgtgggtagcaagatgggcctgcaaacgaatgccttaagtaaagagctggtaggtaataccctaaataaagtggcgttgggcatggaa gtcacgaataccgcagcccagtcagccggtggtgttgccgagggcgtatttattaaaaatgccagcgaggcgcttgctgattttatgct cgcccgttttgccatggatcagattcagcagtggctaaacaatccgtagaaatatttggtgaaaaccagaaggtaacggcggaactgc aaaaagccatgtcttctgcggtacagcaaaatgcggatgcttcgcgttttattctgcgccagagtcgcgcataaCTCGAG

SEQ ID NO: 58 LTA1-GSAAS-S1 Amino acid sequence

MDN GDRL YRAD SRPPDEIKRS GGLMPRGHNE YFDRGT QMNINL YDHARGT Q

TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSP

HPYEQEVSALGGIPYSQIYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYR

LAGFPPDHQAWREEPWIHHAPQGCGNSSRGSAASMLNIQNYSASPHPGIVAERPQTP SASEHVETAVVPSTTEHRGTDIISLSQAATKIHQAQGTLQSTPPISEENNDERTLARQQ LTS SLNALAKSGV SLS AEQNENLRS AF S APTSALF S ASPMAQPRTTISD AEIWDM V SQ NISAIGDSYLGVYENVVAVYTDFYQAFSDILSKMGGWLLPGKDGNTVKLDVTSLKN

VATEAEARQWLSELNLPNSCLKSYGSGY

WTVDLTPLQKMVQDIDGLGAPGKDSKLEMDNAKYQAWQSGFKAQEENMKTTLQ TLTQK Y SNANSLYDNL VKVLS STIS S SLET AKSFLQGVDMVND AS SISRSGYTQNPRL

AEAAFEGVRKNTDFLKAADKAFKDVYATKAGDLKAGTKSGESAINTVGLKPPTDA

AREKLSSEGQLTLLLGKLMTLLGDVSLSQLESRLAVWQAMffiSQKEMGIQVSKEFQT

ALGEAQEATDLYEASIKKTDTAKSVYDAATKKLTQAQNKLQSLDPADPGYAQAEA

AVEQAGKEATEAKEALDKATDATVKAGTDAKAKAEKADNILTKFQGTANAASQNQ

VSQGEQDNLSNVARLTMLMAMFIEIVGKNTEESLQNDLALFNALQEGRQAEMEKKS

AEFQEETRKAEETNRIMGCIGKVLGALLTIVSVVAAVFTGGASLALAAVGLAVMVA

DEIVKAATGVSFIQQALNPIMEHVLKPLMELIGKAITKALEGLGVDKKTAEMAGSIV

GAIVAALAMVAVIVVVAVVGKGAAAKLGNALS MMGETIKKLVPNVLKQLAQNGS

KLFTQGMQRITSGLGNVGSKMGLQTNALSKELVGNTLNKVALGMEVTNTAAQSAG

GVAEG IKNASEALADFMLARFAMDQIQQWLKQSVEIFGENQKVTAELQKAMSSA VQQNAD A SRF I LRQ SRA *

SEQ ID NO: 59 His-SscA chaperone for S2 nucleic acz7/_sequence

ATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGatgaaaaaagac ccgaccctacaacaggcacatgacacgatgcggtttttccggcgtggcggctcgctgcgtatgttgtggatgacgatgttacacagcc gcttaatactctgtatcgctatgccacgcagcttatggaggtaaaagaattcgccggcgcagcgcgactttttcaattgctgacgatatat gatgcctggtcatttgactactggtttcggttaggggaatgctgccaggctcaaaaacattggggggaagcgatatacgcttatggacg cgcggcacaaattaagattgatgcgccgcaggcgccatgggccgcagcggaatgctatctcgcgtgtgataacgtctgttatgcaatc aaagcgttaaaggccgtggtgcgtatttgcggcgaggtcagtgaacatcaaattctccgacagcgtgcagaaaagatgtacagcaac tttctgacaggagctaaAAGCTT SEQ ID NO: 60 His-SscA chaperone for S2 Amino add sequence

MGS SHHHHHHSQDPMKKDPTLQQ AHDTMRFFRRGGSLRMLLDDD VTQPLN TLYRYATQLMEVKEFAGAARLFQLLTIYDAWSFDYWFRLGECCQAQKHWGEAIYA YGRAAQIKIDAPQAPWAAAECYLACDNVCYAIKALKAVVR1CGEVSEHQILRQRAE

KMLQQLSDRS*

SEQ ID NO: 61 SseB nucleic acid sequence

Atgtcttcaggaaacatcttatggggaagtcaaaaccctattgtgtttaaaaatagcttcggcgtcagcaacgctgataccgg gagccaggatgacttatcccagcaaaatccgtttgccgaagggtatggtgttttgcttattctccttatggttattcaggctatcgcaaataa taaatttattgaagtccagaagaacgctgaacgtgccagaaatacccaggaaaagtcaaatgagatggatgaggtgattgctaaagca gccaaaggggatgctaaaaccaaagaggaggtgcctgaggatgtaattaaatacatgcgtgataatggtattctcatcgatggtatgac cattgatgattatatggctaaatatggcgatcatgggaagctggataaaggtggcctacaggcgatcaaagcggctttggataatgacg ccaaccggaataccgatcttatgagtcaggggcagataacaattcaaaaaatgtctcaggagcttaacgctgtccttacccaactgaca gggcttatcagtaagtggggggaaatttccagtatgatagcgcagaaaacgtactca

SEQ ID NO: 62 SseB amino acid sequence

MSSGNILWGSQNPIVFKNSFGVSNADTGSQDDLSQQNPFAEGYGVLLILLMVI

QAIANNKFffiVQKNAERARNTQEKSNEMDEVIAKAAKGDAKTKEEVPEDVIKYMRD

NGILIDGMTIDDYMAKYGDHGKLDKGGLQAIKAALDNDANRNTDLMSQGQITIQK MSQELNAVLTQLTGLISKW GEIS SMIAQKTY S

SEQ ID NO: 63 SseC nucleic acid sequence

atgaatcgaattcacagtaatagcgacagcgccgcaggagtaaccgccttaacacatcatcacttaagcaatgtcagttgcg tttcctcgggttcgctgggaaagcgccagcatcgtgtgaattctacttttggcgatggcaacgccgcgtgtctgctatccgggaaaatta gtcttcaggaggcaagcaatgcgttgaagcaactgcttgatgccgtacccggaaatcataagcgtccatcattgcctgactttttgcaga ccaatcccgcggttttatcaatgatgatgacgtcataatactcaacgtctttggtaataacgctcaatcgttatgccaacagcttgagcgg gcaactgaggtgcaaaatgcatacgtaataagcaggtaaaggagtatcaggagcagatccagaaagcgatagagcaggaggataa agcgcgtaaagcgggtatttttggcgctatttttgactggattaccggcatatttgaaaccgtgattggcgccttaaaagttgtggaaggtt ttctgtccggaaatcccgcagaaatggctagcggcgtagcttatatggccgcaggttgtgcaggaatggttaaagccggagccgaaa cggcaatgatgtgcggtgctgaccacgatacctgtcaggcaattattgacgtgacaagtaagattcaatttggttgtgaagccgtcgcg ctggcactggatgttttccagattggccgtgcttttatggcgacgagaggtttatctggcgcagctgcaaaagtgcttgactccggttttg gcgaggaagtggttgagcgtatggtaggtgcaggggaagcagaaatagaggagttggctgaaaagtttggcgaagaagtgagcga aagtttttccaaacaatttgagccgcttgaacgtgaaatggctatggcgaatgagatggcagaggaggctgccgagttttctcgtaacgt cctggaaaaatgtgtgcaagaaggtggaaagttcctgttaaaaaaattccgtaataaagttctcttcaatatgttcaaaaaaatcctgtatg ccttactgagggattgttcattaaaggcttacaggctatcagatgtgcaaccgagggcgccagtcagatgaatactggcatggttaaca cagaaaaagcgaagatcgaaaagaaaatagagcaattaataactcagcaacggtttctggatttcataatgcaacaaacagaaaacca gaaaaagatagaacaaaaacgcttagaggagctttataaggggagcggtgccgcgcttagagatgtattagataccattgatcactata gtagcgttcaggcgagaatagctggctatcgcgcttaa

SEQ ID NO: 64 SseC amino acid sequence

MNRIHSNSDSAAGVTALTHHHLSNVSCVSSGSLGKRQHRVNSTFGDGNAAC LLSGKISLQEASNALKQLLDAVPGNHKRPSLPDFLQTNPAVLSMMMTSLILNVFGNN AQ SLCQQLERAIE VQNALR KQ VKE Y QEQIQK AIEQEDKARK AGIF GAIFDWITGIFE TVIGALKVVEGFLSGNPAEMASGVAYMAAGCAGMVKAGAETAMMCGADHDTCQ AIIDVTSKIQFGCEAVALALDVFQIGRAFMATRGLSGAAAKVLDSGFGEEVVERMVG AGEAEIEELAEKF GEE V SESF SKQFEPLEREMAMANEMAEEAAEF SRNVENNMTRS A GKSFTKEGVKAMAKEAAKEALEKCVQEGGKFLLKKFRNKVLFNMFKKILYALLRD CSFKGLQAIRCATEGASQMNTGMVNTEKAKIEKKIEQLITQQRFLDFIMQQTENQKKI EQKRLEELYKGSGAALRDVLDTIDHYSSVQARIAGYRA

SEQ ID NO: 65 S2 nucleic acid sequence

atgtcttcaggaaacatcttatggggaagtcaaaaccctattgtgtttaaaaatagcttcggcgtcagcaacgctgataccgg gagccaggatgacttatcccagcaaaatccgtttgccgaagggtatggtgttttgcttattctccttatggttattcaggctatcgcaaataa taaatttattgaagtccagaagaacgctgaacgtgccagaaatacccaggaaaagtcaaatgagatggatgaggtgattgctaaagca gccaaaggggatgctaaaaccaaagaggaggtgcctgaggatgtaattaaatacatgcgtgataatggtattctcatcgatggtatgac cattgatgattatatggctaaatatggcgatcatgggaagctggataaaggtggcctacaggcgatcaaagcggctttggataatgacg ccaaccggaataccgatcttatgagtcaggggcagataacaattcaaaaaatgtctcaggagcttaacgctgtccttacccaactgaca gggcttatcagtaagtggggggaaatttccagtatgatagcgcagaaaacgtactcaGAGCTCatgaatcgaattcacagtaata gcgacagcgccgcaggagtaaccgccttaacacatcatcacttaagcaatgtcagttgcgtttcctcgggttcgctgggaaagcgcca gcatcgtgtgaattctactttggcgatggcaacgccgcgtgtctgctatccgggaaaattagtcttcaggaggcaagcaatgcgttgaa gcaactgcttgatgccgtacccggaaatcataagcgtccatcattgcctgactttttgcagaccaatcccgcggttttatcaatgatgatg acgtcattaatactcaacgtctttggtaataacgctcaatcgttatgccaacagcttgagcgggcaactgaggtgcaaaatgcattacgta ataagcaggtaaaggagtatcaggagcagatccagaaagcgatagagcaggaggataaagcgcgtaaagcgggtatttttggcgct atttttgactggattaccggcatatttgaaaccgtgattggcgcctaaaagttgtggaaggttttctgtccggaaatcccgcagaaatggc tagcggcgtagctatatggccgcaggtgtgcaggaatggtaaagccggagccgaaacggcaatgatgtgcggtgctgaccacga tacctgtcaggcaattattgacgtgacaagtaagattcaatttggttgtgaagccgtcgcgctggcactggatgttttccagattggccgt gcttttatggcgacgagaggtttatctggcgcagctgcaaaagtgcttgactccggttttggcgaggaagtggttgagcgtatggtaggt gcaggggaagcagaaatagaggagttggctgaaaagtttggcgaagaagtgagcgaaagtttttccaaacaatttgagccgcttgaa cgtgaaatggctatggcgaatgagatggcagaggaggctgccgagttttctcgtaacgtagaaaataatatgacgcgaagcgcggga aaaagctttacgaaagagggggtgaaagcaatggcaaaagaagcggcaaaagaagccctggaaaaatgtgtgcaagaaggtgga aagttcctgttaaaaaaattccgtaataaagttctcttcaatatgttcaaaaaaatcctgtatgccttactgagggattgttcatttaaaggctt acaggctatcagatgtgcaaccgagggcgccagtcagatgaatactggcatggttaacacagaaaaagcgaagatcgaaaagaaaa tagagcaattaataactcagcaacggtttctggatttcataatgcaacaaacagaaaaccagaaaaagatagaacaaaaacgcttagag gagctttataaggggagcggtgccgcgcttagagatgtattagataccattgatcactatagtagcgttcaggcgagaatagctggctat cgcgcttaa

SEQ ID NO: 66 S2 amino acid sequence

MSSGNILWGSQ PIVFKNSFGVSNADTGSQDDLSQQNPFAEGYGVLLILLMVI

QAIA NKFIEVQKNAERARNTQEKSNEMDEVIAKAAKGDAKTKEEVPEDVIKYMRD

NGILIDGMTIDDYMAKYGDHGKLDKGGLQAIKAALDNDANRNTDLMSQGQITIQK

MSQELNAVLTQLTGLISKWGEISSMIAQKTYSELMNRIHSNSDSAAGVTALTHHHLS

NVSCVSSGSLGKRQHRVNSTFGDGNAACLLSGKISLQEASNALKQLLDAVPGNHKR

PSLPDFLQTNPAVLSMMMTSLILN GNNAQSLCQQLERATEVQNALRNKQVKEYQ

EQIQKAIEQEDKARKAGIFGAIFDWITGIFETVIGALKVVEGFLSGNPAEMASGVAYM

MATRGLSGAAAKVLDSGFGEEVVERMVGAGEAEIEELAEKFGEEVSESFSKQFEPLE

REMAMANEMAEEAAEFSRNVENNMTRSAGKSFTKEGVKAMAKEAAKEALEKCVQ

KAKffiKKIEQLITQQRFLDFIMQQTENQKKIEQKRLEELYKGSGAALRDVLDTIDHYS S V Q ARIAGYRA

SEQ ID NO: 67 LTA1-GSAAS-S2 nucleic acid sequence

CATatggacaatggcgatcgtttataccgtgccgactcgcgtcccccagatgagattaaacgtagcggtgggttaatgcc acgtgggcacaatgagtattttgaccgtggaacacagatgaacattaacctttacgatcatgcccgtgggacccagaccgggtttgtcc gttatgatgacgggtatgttagtacgagtttgtcctacgctccgcacacctgcgggacaaagtattttatcaggctacagcacatattac atttatgtgatcgccactgccccaaacatgttcaatgtgaacgatgtgttgggggttacagcccccatccatatgaacaagaagtctcg gcccttggggggatcccatatagccagatttatggttggtaccgcgtaaattttggtgtgatgatgaacgtttgcatcgtaaccgtgaata ccgcgatcgctactaccgtaacttgaacatgcacctgccgaggacggctatcgtttagcgggattcccacccgatcatcaggcgtgg cgtgaggaaccgtggatccatcacgcccctcaggggtgcgggaacagtagtcgcgggtccgcggcatccatgtcttcaggaaacat cttatggggaagtcaaaaccctattgtgtttaaaaatagcttcggcgtcagcaacgctgataccgggagccaggatgacttatcccagc aaaatccgtttgccgaagggtatggtgttttgcttattctccttatggttattcaggctatcgcaaataataaatttattgaagtccagaagaa cgctgaacgtgccagaaatacccaggaaaagtcaaatgagatggatgaggtgattgctaaagcagccaaaggggatgctaaaacca aagaggaggtgcctgaggatgtaattaaatacatgcgtgataatggtattctcatcgatggtatgaccattgatgattatatggctaaatat ggcgatcatgggaagctggataaaggtggcctacaggcgatcaaagcggcttggataatgacgccaaccggaataccgatctatg agtcaggggcagataacaattcaaaaaatgtctcaggagctaacgctgtccttacccaactgacagggcttatcagtaagtgggggg aaatttccagtatgatagcgcagaaaacgtactcaGAGCTCatgaatcgaattcacagtaatagcgacagcgccgcaggagtaa ccgccttaacacatcatcactaagcaatgtcagttgcgttcctcgggtcgctgggaaagcgccagcatcgtgtgaattctactttggc gatggcaacgccgcgtgtctgctatccgggaaaattagtcttcaggaggcaagcaatgcgttgaagcaactgcttgatgccgtacccg gaaatcataagcgtccatcattgcctgactttttgcagaccaatcccgcggttttatcaatgatgatgacgtcattaatactcaacgtctttg gtaataacgctcaatcgttatgccaacagcttgagcgggcaactgaggtgcaaaatgcattacgtaataagcaggtaaaggagtatca ttgaaaccgtgattggcgccttaaaagttgtggaaggttttctgtccggaaatcccgcagaaatggctagcggcgtagcttatatggcc gcaggttgtgcaggaatggttaaagccggagccgaaacggcaatgatgtgcggtgctgaccacgatacctgtcaggcaattatgac gtgacaagtaagattcaatttggttgtgaagccgtcgcgctggcactggatgttttccagattggccgtgcttttatggcgacgagaggtt tatctggcgcagctgcaaaagtgcttgactccggttttggcgaggaagtggttgagcgtatggtaggtgcaggggaagcagaaatag aggagttggctgaaaagtttggcgaagaagtgagcgaaagtttttccaaacaatttgagccgcttgaacgtgaaatggctatggcgaat gagatggcagaggaggctgccgagttttctcgtaacgtagaaaataatatgacgcgaagcgcgggaaaaagctttacgaaagaggg ggtgaaagcaatggcaaaagaagcggcaaaagaagccctggaaaaatgtgtgcaagaaggtggaaagttcctgttaaaaaaattcc gtaataaagttctcttcaatatgttcaaaaaaatcctgtatgcctactgagggattgttcatttaaaggcttacaggctatcagatgtgcaac cgagggcgccagtcagatgaaiactggcatggttaacacagaaaaagcgaagatcgaaaagaaaatagagcaataataactcagc aacggtttctggatttcataatgcaacaaacagaaaaccagaaaaagatagaacaaaaacgcttagaggagcttataaggggagcgg tgccgcgcttagagatgtattagataccatgatcactatagtagcgtcaggcgagaatagctggctatcgcgcttaaCTCGAG

SEQ ID NO: 68 LTA1-GSAAS-S2 Amino acid sequence

MDNGDRLYRADSRPPDEIKRSGGLMPRGHNEYFDRGTQMNINLYDHARGTQ TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDYLGVYSP HPYEQEVSALGGIP YSQIY GWYRVNF GVIDERLHRNREYRDRYYRJSILNIAPAEDGYR LAGFPPDHQ AWREEPWIHH APQGCGNS SRGS AASMS SGNILWGSQNPIVFKN SFGV S NADTGSQDDLSQQNPFAEGYGVLLILLMVIQAIANNKFIEVQKNAERARNTQEKSNE

MDEVIAKAAKGDAKTKEEVPEDVIKYMRDNGILIDGMTIDDYMAKYGDHGKLDKG

GLQAIKAALDNDANRNTDLMSQGQITIQKMSQELNAVLTQLTGLISKWGEISSMIAQ

KTYSELMNRIHSNSDSAAGVTALTHHHLSNVSCVSSGSLGKRQHRVNSTFGDGNAA

CLLSGKISLQEASNALKQLLDAVPGNHKRPSLPDFLQTNPAVLSMMMTSLILNVFGNNAQSLCQQLERATEVQNALRNKQVKEYQEQIQKAIEQEDKARKAGIFGAIFDWITGI

FET VIGALK VVEGFL SGNP AEMASGVAYM AAGC AGMVK AGAET AMMC GADHDTC

QAIIDVTSKIQFGCEAVALALDVFQIGRAFMATRGLSGAAAKVLDSGFGEEVVERMV

GAGEAEIEELAEKFGEEVSESFSKQFEPLEREMAMANEMAEEAAEFSRNVENNMTRS

AGKSFTKEGVKAMAKEAAKEALEKCVQEGGKFLLKKFKNKVLFNMFKKILYALLR

DCSFKGLQAIRCATEGASQMNTGMVNTEKAKIEKKIEQLITQQRFLDFEMQQTENQK

KIEQKRLEELYKGSGAALRDVLDTIDHYSSVQARIAGYRA*

SEQ ID NO: 69 LTAl-SseB Nucleic add sequence

AT GGGC AGC AGCC ATC AT CATC AT C ATC AC AGC AGCGGCCT GGT GCC GCG CGGCAGCCATatggacaatggcgatcgtttataccgtgccgactcgcgtcccccagatgagattaaacgtagcggtgggtta atgccacgtgggcacaatgagtattttgaccgtggaacacagatgaacattaaccttacgatcatgcccgtgggacccagaccgggt tgtccgttatgatgacgggtatgttagtacgagtttgtccttacgctccgcacaccttgcgggacaaagtattttatcaggctacagcacat attacatttatgtgatcgccactgccccaaacatgttcaatgtgaacgatgtgttgggggttacagcccccatccatatgaacaagaagt ctcggcccttggggggatcccatatagccagatttatggttggtaccgcgtaaattttggtgtgattgatgaacgtttgcatcgtaaccgtg

..... 86— aataccgcgatcgctactaccgtaacttgaacattgcacctgccgaggacggctatcgtttagcgggattcccacccgatcatcaggcg tggcgtgaggaaccgtggatccatcacgcccctcaggggtgcgggaacagtagtcgcgggtccgcggcatccatgtcttcaggaaa catcttatggggaagtcaaaaccctattgtgtttaaaaatagcttcggcgtcagcaacgctgataccgggagccaggatgacttatccca gcaaaatccgtttgccgaagggtatggtgttttgcttattctccttatggttattcaggctatcgcaaataataaatttatgaagtccagaag aacgctgaacgtgccagaaatacccaggaaaagtcaaatgagatggatgaggtgattgctaaagcagccaaaggggatgctaaaac caaagaggaggtgcctgaggatgtaattaaatacatgcgtgataatggtattctcatcgatggtatgaccatgatgatatatggctaaat atggcgatcatgggaagctggataaaggtggcctacaggcgatcaaagcggctttggataatgacgccaaccggaataccgatcttat gagtcaggggcagataacaattcaaaaaatgtctcaggagcttaacgctgtccttacccaactgacagggcttatcagtaagtggggg gaaatttccagtatgatagcgcagaaaacgtactcataaGGATCC

SEQ ID NO: 70 LTAl-SseB Amino add sequence

MG S SHHHHHH S S GL VPRG SHMDN GDRL YRAD S RPPDEIKRS GGLMPRGHNE YFDRGTQMNINLYDHARGTQTGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIY VIATAPNMFNVNDVLGVYSPHPYEQEVSALGGIPYSQIYGWYRVNFGVIDERLHRNR

EYRDRYYR .NIAPAEDG YRL AGFPPDHQ AWREEPWfflHAPQGCGNS SRG S AASMS

SGMLWGSQNPIVFKNSFGVSNADTGSQDDLSQQNPFAEGYGVLLILLMVIQAIANN

KFIEVQKNAERARNTQEKSNEMDEVIAKAAKGDAKTKEEVPEDVIKYMRDNGILED

GMTIDDYMAKYGDHGKLDKGGLQAIKAALDNDANRNTDLMSQGQITIQKMSQELN A VLTQLT GLISKW GEI S SMI AQKT Y S *

SEQ ID NO: 71 HisScc2 chaperone for LTAl-CT053-CopB nucleic acid sequence

ATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGatgagcactccat cttctaataattctaaaaaaccttcggcctcttttaataaaaaatcacgtagccgcttggccgagattgctgcacaaaaaaaagcaaaagc tgaggatttggaacaaaaatatcctgttcctacggaagaggagacaaaacaagttctcatggacatcctacaggggttaagcaacgga ttaactcttcagcaaattttaggtctctccgacgtcctccttgaagagatctacaccgtagcatataccttctactcccaagggaaatatcg ggaagctatcggtctttccaaatcttaacagcctccaaacctcaatgctacaaatacatcttaggtcttagctcttgctatcaccagctaaa aatgtatgatgaagccgcttttggtttcttcctagctttcgatgctcaacccgaaaaccccatccctccttactacatcgccgatagcttgat gaagctaaaccaacccgaagaatctcaagacttcctcgatattacgatcgatatgtgtaagaacaagccggaatataaagttcttaaaga tcgctgcagcattatgaagcaatctttagatgccgtgctgaaaaaagagaaatctgcaaaaggctctgaaacacaagcctcctctcctaa aaacacaaaagctaaaaaagctgcttctaacaagaaaaaagcaaagtaaGCGGCCGC

SEQ ID NO: 72 His See 2 chaperone for LTAl-CT053-CopB Amino add sequence

MGSSHHHHHHSQDPMSTPSSNNSKKPSASFNKKSRSRLAEIAAQKKAKAEDL EQKYPWTEEETKQVLMDILQGLSNGLTLQQILGLSDYLLEEIYTVAYTFYSQGKYRE AIGLFQILTASKPQCYKYILGLSSCYHQLKMYDEAAFGFFLAFDAQPENPIPPYYiADS LMKLNQPEESQDFLDITIDMCKNKPEYKVLKDRCSIMKQSLDAVLKKEKSAKGSETQ AS SPKNTKAKKAASNKKKAK*

SEQ ID NO: 73 CT053 nucleic acid sequence

Aaaagtgagcgtttaaaaaaattagaatcagagcttcatgatcttacccagtggatgcaacttggccttgttcctaaaaaaga aatcgagagacaccaggaagaaatccgtctgctagaaagcaaaatcctgaagagaaagaacgtctacaacttctcaaagaaagcgg tgagatcaaagagtacgtaacccctcgaagaactccagctaaaaccatttacccagatggccccagcgtttcagacgttgagtttgtag aatcctcggatacagaagtggatctcgatgccggtgacacaattgagattgacctaggtgatgaggcaagagaagaaagcggaaac gaactcgactactctagtgaagacgatgaggatcctttcagcgatcgcaatcgttggcgccgaggaggcatcatagatcctgacgcga atgaatgg

SEQ ID NO: 74 CT053 amino acid sequence

MKSERLKKLESELHDLTQWMQLGLVPKKEIERHQEEIRLLESKILEEKERLQL LK ESGEIKE Y VT PRRTP AK TI YPDG PS VSD VEF VES SDTE VDLDAGDnEIDLGDEARE E S GNELD Y S SEDDE DPF S DR_NR WRRGG IIDPD ANE W SEQ ID NO: 75 CopB nucleic acid sequence

atgagcttgtcatccagcagcagctcggatagttcgaatctgaaaaatgtgttatctcaggtcatcgcgtctacaccAcagg gggttcctaatgctgacaaattaaccgacaatcaggtaaaacaagtccagcagacccgtcaaaaccgtgatgatctgtccatggagag cgacgtcgcggtggcgggaacagccggaaaagatcgtgctgcgtcggcgtcccagatcgagggacaagagctgattgagcaaca gggactgcggctgggaaagagacggcttctgctgatgctacatcattgacccagtcggcatccaaaggcgcttccagtcagcagtgt attgaggataccagtaagtccctggagctttcttcgctttcgagcctgtcaagcgtagatgcgacacatttgcaggaaatccaatcgatc gtgtcttcagcaatgggcgccaccaacgaattgtcatgacgaacttagagacaccgggattaccaaagccgagtaccactccAcgc caggaagtatggagatcagccttgccttagcgaaggccatcactgcattgggtgagagcactcaggctgccttggaaaattttcagtc cactcagagtcagtccgcgaacatgaataagatgagtttggaatcccaaggcttgaaaatcgacaaggagcgtgaagaatttaagaaa atgcaggagattcagcaaaagagcggcacaaattcaaccatggatactgtgaataaagttatgattggcgtgacagtggcaattacagt aatctctgttgtttcagcattgtttacctgcggtttgggcttgattggcacagccgctgcgggtgccacagccgccaccgctggggcaac ggccgccgccacgaccgctacctctgtgacgaccacagtcgctacccaggtgacgatgcaagcggtggtccaagtcgttaagcagg ctattatccaagcagtaaaacgcgccatcgtccaagcgattaaacaggggattaagcaaggcattaaacaagcgatcaaacaggcag tcaaggcaagcgtgaagacacttgccaaaaatgtaggcaagattttcagcgcaggcaagaacgctgtgagtaagtccttcccAaaatt gtctaaggtgattaatacacttggttccaaatgggttactcttggcgtgggggcccttacagcggtgccgcagttagtcagtggcattac ctcccttcaattgtctgatatgcaaaaagaacttgcacaaatccaaaaggaagtgggtgcacttacggcgcagagtgagatgatgaaa gcgtttacactgttctggcagcaagcttcgaaaatcgcggccaaacaaacggaatcaccttcagagacgcaacaacaggcagctaag accggcgcccagatcgctaaagcgttgtccgccatttcgggtgctttagctgctgctgctTAG

SEQ ID NO: 76 CopB amino acid sequence

MSLSSSSSSDSSNLKNVLSQVIASTPQGVPNADKLTDNQVKQVQQTRQNRDD LSMESDVAVAGTAGKDRAASASQIEGQELIEQQGLAAGKETASADATSLTQSASKG A SSQQCIEDTSKSLEL S SL S SL S S VD ATHLQEIQ SI VS S AMGATNEL SLTNLETPGLPKP STTPRQEVMEISLALAKAITALGESTQAALENFQSTQSQSANMNKMSLESQGLKIDK EREEFKKMQEIQQKSGTNSTMDTVNKVMIGVTVArrVISWSALFTCGLGLIGTAAA GATAATAGATAAATTATSVTTTVATQVTMQAWQVVKQAIIQAVKRAIVQAIKQGI KQGIKQAIKQAVKASVKTLAKNVGKIFSAGKNAVSKSFPKLSKVINTLGSKWVTLGV GALTAVPQLVSGITSLQLSDMGKELAQIQKEVGALTAQSEMMKAFTLFWQQASKIA AKQTESPSETQGQAAKTGAQIAKALSAISGALAAAA

SEQ ID NO: 77 CT053-CopB nucleic acid sequence

aaaagtgagcgtttaaaaaaattagaatcagagcttcatgatcttacccagtggatgcaacttggccttgttcctaaaaaagaa atcgagagacaccaggaagaaatccgtctgctagaaagcaaaatcctgaagagaaagaacgtctacaacttctcaaagaaagcggt gagatcaaagagtacgtaacccctcgaagaactccagctaaaaccatttacccagatggccccagcgtttcagacgttgagtttgtaga atcctcggatacagaagtggatctcgatgccggtgacacaattgagattgacctaggtgatgaggcaagagaagaaagcggaaacg aactcgactactctagtgaagacgatgaggatcctttcagcgatcgcaatcgttggcgccgaggaggcatcatagatcctgacgcgaa tgaatggGGTTCAGCTGCTTCAatgagcttgtcatccagcagcagctcggatagttcgaatctgaaaaatgtgttatctca ggtcatcgcgtctacaccAcagggggttcctaatgctgacaaattaaccgacaatcaggtaaaacaagtccagcagacccgtcaaaa ccgtgatgatctgtccatggagagcgacgtcgcggtggcgggaacagccggaaaagatcgtgctgcgtcggcgtcccagatcgag ggacaagagctgattgagcaacagggacttgcggctgggaaagagacggcttctgctgatgctacatcattgacccagtcggcatcc aaaggcgcttccagtcagcagtgtattgaggataccagtaagtccctggagctttcttcgctttcgagcctgtcaagcgtagatgcgaca catttgcaggaaatccaatcgatcgtgtcttcagcaatgggcgccaccaacgaattgtcattgacgaacttagagacaccgggattacc aaagccgagtaccactccAcgccaggaagttatggagatcagccttgccttagcgaaggccaicacigcattgggtgagagcactca ggctgccttggaaaattttcagtccactcagagtcagtccgcgaacatgaataagatgagtttggaatcccaaggcttgaaaatcgaca aggagcgtgaagaatttaagaaaatgcaggagattcagcaaaagagcggcacaaattcaaccatggatactgtgaataaagttatgat tggcgtgacagtggcaattacagtaatctctgttgtttcagcattgtttacctgcggtttgggcttgattggcacagccgctgcgggtgcc acagccgccaccgctggggcaacggccgccgccacgaccgctacctctgtgacgaccacagtcgctacccaggtgacgatgcaag cggtggtccaagtcgttaagcaggetattatceaagGagtaaaaegcgccatcgtGcaagcgattaaacaggggattaageaaggcat taaacaagcgatcaaacaggcagtcaaggcaagcgtgaagacacttgccaaaaatgtaggcaagattttcagcgcaggcaagaacg ctgtgagtaagtccttcccAaaattgtctaaggtgataatacacttggttccaaatgggttactcttggcgtgggggcccttacagcggt gccgcagttagtcagtggcattacctcccttcaattgtctgatatgcaaaaagaacttgcacaaatccaaaaggaagtgggtgcacttac ggcgcagagtgagatgatgaaagcgtttacactgttctggcagcaagcttcgaaaatcgcggccaaacaaacggaatcaccttcaga gacgcaacaacaggcagctaagaccggcgcccagatcgctaaagcgttgtccgccatttcgggtgctttagctgctgctgctTAG

SEO ID NO: 78 CT053-CopB amino acid sequence

MKSERLKKLESELHDLTQWMQLGLVPKKEIERHQEEIRLLESKILEEKERLQL

LKESGEIKEYVTPRRTPAKTIYPDGPSVSDVEFVESSDTEVDLDAGDTffilDLGDEARE

E S GNELD Y S S EDDED PF SD RNR WRRG GIIDPD ANEW GS A A S M S L S S S S S SD S S NLKN

VLSQVIASTPQGVPNADKLTDNQVKQVQQTRQNRDDLSMESDVAVAGTAGKDRAA

SASQIEGQELIEQQGLAAGKETASADATSLTQSASKGASSQQCIEDTSKSLELSSLSSL

SSVDATHLQEIQSIVSSAMGATNELSLTNLETPGLPKPSTTPRQEVMEISLALAKAITA

LGESTQAALENFQSTQSQSANMNKMSLESQGLKIDKEREEFKKMQEIQQKSGTNST

MDTVNKVMIGVTVAITVISVVSALFTCGLGLIGTAAAGATAATAGATAAATTATSVT

TTVATQVTMQAVVQVVKQAIIQAVKRAIVQAIKQGIKQGIKQAIKQAVKASVKTLA

KNVGKIFSAGKNAVSKSFPKLSKVINTLGSKWVTLGVGALTAVPQLVSGITSLQLSD

MQKELAQIQKEVGALTAQSEMMKAFTLFWQQASKIAAKQTESPSETQQQAAKTGA

QIAKALSAISGALAAAA

SEO ID NO: 79 LTAl-CT053-CopB nucleic acid sequence

CATatggacaatggcgatcgttataccgtgccgactcgcgtcccccagatgagattaaacgtagcggtgggttaatgcc acgtgggcacaatgagtatttgaccgtggaacacagatgaacataaccttacgatcatgcccgtgggacccagaccgggtttgtcc gttatgatgacgggtatgttagtacgagtttgtccttacgctccgcacaccttgcgggacaaagtattttatcaggctacagcacatattac atttatgtgatcgccactgccccaaacatgttcaatgtgaacgatgtgttgggggtttacagcccccatccatatgaacaagaagtctcg gccctggggggatcccatatagccagatttatggttggtaccgcgtaaattttggtgtgattgatgaacgtttgcatcgtaaccgtgaata ccgcgatcgctactaccgtaacttgaacattgcacctgccgaggacggctatcgtttagcgggattcccacccgatcatcaggcgtgg cgtgaggaaccgtggatccatcacgcccctcaggggtgcgggaacagtagtcgcCATatgaaaagtgagcgtttaaaaaaattag aatcagagcttcatgatcttacccagtggatgcaactggccttgttcctaaaaaagaaatcgagagacaccaggaagaaatccgtctg ctagaaagcaaaatccttgaagagaaagaacgtctacaacttctcaaagaaagcggtgagatcaaagagtacgtaacccctcgaaga actccagctaaaaccatttacccagatggccccagcgtttcagacgttgagtttgtagaatcctcggatacagaagtggatctcgatgcc ggtgacacaattgagattgacctaggtgatgaggcaagagaagaaagcggaaacgaactcgactactctagtgaagacgatgagga tcctttcagcgatcgcaatcgttggcgccgaggaggcatcatagatcctgacgcgaatgaatggGGTTCAGCTGCTTCA atgagcttgtcatccagcagcagctcggatagttcgaatctgaaaaatgtgttatctcaggtcatcgcgtctacaccAcagggggttcct aatgctgacaaataaccgacaatcaggtaaaacaagtccagcagacccgtcaaaaccgtgatgatctgtccatggagagcgacgtc gcggtggcgggaacagccggaaaagatcgtgctgcgtcggcgtcccagatcgagggacaagagctgattgagcaacagggactt gcggctgggaaagagacggcttctgctgatgctacatcatgacccagtcggcatccaaaggcgcttccagtcagcagtgtattgagg ataccagtaagtccctggagctttcttcgctttcgagcctgtcaagcgtagatgcgacacatttgcaggaaatccaatcgatcgtgtcttc agcaatgggcgccaccaacgaattgtcattgacgaacttagagacaccgggattaccaaagccgagtaccactccAcgccaggaa gttatggagatcagcctgccttagcgaaggccatcactgcattgggtgagagcactcaggctgccttggaaaattttcagtccactcag agtcagtccgcgaacatgaataagatgagtttggaatcccaaggcttgaaaatcgacaaggagcgtgaagaatttaagaaaatgcag gagattcagcaaaagagcggcacaaattcaaccatggatactgtgaataaagttatgattggcgtgacagtggcaattacagtaatctct gttgtttcagcattgtttacctgcggtttgggcttgattggcacagccgctgcgggtgccacagccgccaccgctggggcaacggccg ccgccacgaccgctacctctgtgacgaccacagtcgctacccaggtgacgatgcaagcggtggtccaagtcgttaagcaggctattat ccaagcagtaaaacgcgccatcgtccaagcgattaaacaggggattaagcaaggcattaaacaagcgatcaaacaggcagtcaagg caagcgtgaagacacttgccaaaaatgtaggcaagattttcagcgcaggcaagaacgctgtgagtaagtccttcccAaaattgtctaa ggtgattaatacactggttccaaatgggttactcttggcgtgggggcccttacagcggtgccgcagttagtcagtggcattacctccctt caattgtctgatatgcaaaaagaacttgcacaaatccaaaaggaagtgggtgcacttacggcgcagagtgagatgatgaaagcgttta cactgttctggcagcaagcttcgaaaatcgcggccaaacaaacggaatcaccttcagagacgcaacaacaggcagctaagaccggc gcccagatcgctaaagcgttgtccgccatttcgggtgctttagctgctgctgctTAGCTCGAG SEQ ID NO: 80 LTA l-CT053-CopB Amino add sequence

MDN GDRL YBAD SRPPDEIKRS GGLIviPRGHNE YFDRGT QMNINL YDHARGT Q TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSP HPYEQEVSALGGIPYSQIYGWYRVNFGYIDERLURNREYRDRYYRNL IAPAEDGYR LAGFPPDHQAWREEPWIHHAPQGCGNSSRMKSERLKKLESELHDLTQWMQLGLVP KKEIERHQEEIRLLESKILEEKERLQLLKESGEIKEYVTPRRTPAKTIYPDGPSVSDVEF VES SDTFi VDLD AGDTIEIDLGDEAREE SGNELD YS SEDDEDPF SDRNRWRRGGIIDPD ANEWGSAASMSLSSSSSSDSSNLKNVLSQVIASTPQGVPNADKLTDNQVKQVQQTR QNRDDLSMESDVAVAGTAGKDRAASASQIEGQELIEQQGLAAGKETASADATSLTQ SASKGASSQQCIEDTSKSLELSSLSSLSSVDATHLQEIQSIVSSAMGATNELSLTNLETP GLPKPSTTPRQEVMEISLALAKAITALGESTQAALENFQSTQSQSANMNKMSLESQG LKIDKEREEFKKMQEIQQKSGTNSTMDTVNKVMIGVTVAITVISWSALFTCGLGLIG TAAAGATAATAGATAAATTATSVTTTVATQVrMQAWQWKQAnQAVKRAIVQAI KQGIKQGIKQAEKQAVKASVKTLAKNVGKIFSAGKNAVSKSFPKLSKVINTLGSKWV TLGVGALTAVPQLVSGITSLQLSDMQKELAQIQKEVGALTAQSEMMKAFTLFWQQA SKIAAKQTESPSETQQQAAKTGAQIAKALSAISGALAAAA*

SEQ ID NO: 81 HisSccl chaperone for LTAl-CT668-CopB nucleic acid sequence

ATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGatgagcactecat ctctaataattctaaaaaaccttcggcctcttttaataaaaaatcacgtagccgcttggccgagattgctgcaeaaaaaaaagcaaaagc tgaggatttggaacaaaaatatcctgttcctacggaagaggagacaaaacaagttctcatggacatcctacaggggttaagcaacgga ttaactcttcagcaaatttaggtetctccgacgtcctcctgaagagatctaeaccgiagcatataccttciactcecaagggaaatateg ggaagctatcggtctttccaaatcttaaeagcctecaaacctcaatgctacaaatacatcitaggtcttagctcttgctatcaccagctaaa aatgtatgatgaagccgcttttggtttcttcctagctttcgatgctcaacccgaaaaccccatccctccttactacatcgccgatagcttgat gaagctaaaecaaeccgaagaatcteaagactcctcgatatacgatcgatatgtgtaagaacaagccggaatataaagttcttaaaga tcgctgcagcattatgaageaatetttagatgccgtgctgaaaaaagagaaatctgcaaaaggctctgaaacacaagcctcctctcctaa aaacacaaaagctaaaaaagctgcttctaacaagaaaaaagcaaagtaaGCGGCCGC

SEQ ID NO: 82 HisSccl chaperone for LTAl-CT668-CopB Amino add sequence

MGSSHHHHHHSQDPMSTPSSNNSKKPSASFNKKSRSRLAEIAAQKKAKAEDL

EQKYPVPTEEETKQVLMDILQGLSNGLTLQQE GLSDVLLEEIYTVAYTFYSQGKYRE

AIGLFQILTASKPQCYKYILGLSSCYHQLKMYDEAAFGFFLAFDAQPENPIPPYYIADS

LMKLNQPEESQDFLDITIDMCKNKPEYKVLKDRCSIMKQSLDAVLKKEKSAKGSETQ

ASSPKNTKAKKAASNKKKAK*

SEQ ID NO: 83 CT668 nucleic acid sequence

Atagatcctcttaagctttttccaaattttgatggggataaggagagtgctgcggtgaataaaccttcagcatctcctatgccc agcgaattaagtaaaaatgttgcctcattctetttagggggtggaggtgctgcgttggattcgacagtgtccaeagaaaagctatcgttga tggctatgatgcaggataaaaattcgcagttgatcgatcctgagttggaggaagctctgaactctgaagagttacaagagcagatccatt tgttaaaaagtcgtttgtgggatgcacaaacgcagatgcaaatgcaagatcecgacaagttggcctctgagcatgtagatgctttagga gtcatgttgatttaatcaatggggattttcaagcgatagctgaacatacacaacagacggtcaagcagggtaatggtgacgaagaaaa atctgttacacgcaagatagtcgattgggtctcttcaggagaagaaattttgaatcgtgctttgttgtatttctccgatcgtaatggagaaag agaaacattagccgatttcttaaaagttcagtatgccgttcaaagagctacacaacgcgccgagttatttgccagtattctaggtgccacg gtgagtagtgtaaaaacgattatgacaacccagttaggt

SEQ ID NO: 84 CT668 amino acid sequence

MIDPLKLFPNFDGDKESAAVNKPSASPMPSELSKNVASFSLGGGGAALDSTVS TEKLSLMAMMQDKNSQLIDPELEEALN SEELQEQIHLLKSRLWD AQT QMQMQDPD KLASEHVDALGVIVDLINGDFQAIAEHTQQTVKQGNGDEEKSVTRKIVDWVSSGEEI LNRALL YF SDRN GERETL ADFLK V Q Y AV QRATQRAELF ASILGAT V S S VKTIMTTQL SEQ ID NO: 85 CT668-CopB nucleic acid sequence

atagatcctcttaagctttttccaaattttgatggggataaggagagtgctgcggtgaataaaccttcagcatctcctatgccca gcgaattaagtaaaaatgttgcctcattctctttagggggtggaggtgctgcgttggattcgacagtgtccacagaaaagctatcgttgat ggctatgatgcaggataaaaattcgcagttgatcgatcctgagttggaggaagctctgaactctgaagagttacaagagcagatccattt gttaaaaagtcgttgtgggatgcacaaacgcagatgcaaatgcaagatcccgacaagttggcctctgagcatgtagatgctttaggag tcattgttgatttaatcaatggggattttcaagcgatagctgaacatacacaacagacggtcaagcagggtaatggtgacgaagaaaaat ctgttacacgcaagatagtcgattgggtctcttcaggagaagaaattttgaatcgtgctttgttgtatttctccgatcgtaatggagaaaga gaaacattagccgatttcttaaaagttcagtatgccgttcaaagagctacacaacgcgccgagttatttgccagtattctaggtgccacgg tgagtagtgtaaaaacgattatgacaacccagttaggtGGTTCAGCTGCTTCAatgagcttgtcatccagcagcagctcg gatagttcgaatctgaaaaatgtgttatctcaggtcatcgcgtctacaccAcagggggttcctaatgctgacaaattaaccgacaatcag gtaaaacaagtccagcagacccgtcaaaaccgtgatgatctgtccatggagagcgacgtcgcggtggcgggaacagccggaaaag atcgtgctgcgtcggcgtcccagatcgagggacaagagctgattgagcaacagggacttgcggctgggaaagagacggcttctgct gatgctacatcattgacccagtcggcatccaaaggcgcttccagtcagcagtgtatgaggataccagtaagtccctggagctttcttcg ctttcgagcctgtcaagcgtagatgcgacacatttgcaggaaatccaatcgatcgtgtcttcagcaatgggcgccaccaacgaatgtc attgacgaacttagagacaccgggattaccaaagccgagtaccactccAcgccaggaagttatggagatcagccttgccttagcgaa ggccatcactgcattgggtgagagcactcaggctgccttggaaaattttcagtccactcagagtcagtccgcgaacatgaataagatga gtttggaatcccaaggcttgaaaatcgacaaggagcgtgaagaatttaagaaaatgcaggagattcagcaaaagagcggcacaaatt caaccatggatactgtgaataaagttatgattggcgtgacagtggcaattacagtaatctctgttgtttcagcattgttacctgcggtttgg gcttgattggcacagccgctgcgggtgccacagccgccaccgctggggcaacggccgccgccacgaccgctacctctgtgacgac cacagtcgctacccaggtgacgatgcaagcggtggtccaagtcgttaagcaggctattatccaagcagtaaaacgcgccatcgtcca agcgattaaacaggggataagcaaggcattaaacaagcgatcaaacaggcagtcaaggcaagcgtgaagacactgccaaaaatg taggcaagattttcagcgcaggcaagaacgctgtgagtaagtccttcccAaaattgtctaaggtgattaatacacttggttccaaatggg ttactcttggcgtgggggcccttacagcggtgccgcagttagtcagtggcattacctcccttcaattgtctgatatgcaaaaagaacttgc acaaatccaaaaggaagtgggtgcactacggcgcagagtgagatgatgaaagcgtttacactgttctggcagcaagcttcgaaaatc gcggccaaacaaacggaatcaccttcagagacgcaacaacaggcagctaagaccggcgcccagatcgctaaagcgttgtccgcca tttcgggtgctttagctgctgctgctTAG

SEQ ID NO: 86 CT668-CopB amino acid sequence

MIDPLKLFPNFDGDKESAAVNKPSASPMPSELSKNVASFSLGGGGAALDSTVS

TEKLSLMAMMQDKNSQLIDPELEEALNSEELQEQIHLLKSRLWDAQTQMQMQDPD

KLASEH\^/DALG\^I T)LINGDFQAIAEHTQQT\^/KQGNGDEEKSVTRKI T)WVSSGEEI

LNRALLYFSDRNGERETLADFLKVQYAVQRATQRAELFASILGATVSSVKTIMTTQL

GGSAASMSLSSSSSSDSSNLKNVLSQVIASTPQGVPNADKLTDNQVKQVQQTRQNRD

DLSMESDVAVAGTAGKDRAASASQIEGQELIEQQGLAAGKETASADATSLTQSASK

GASSQQCIEDTSKSLELSSLSSLSSVDATHLQEIQSIVSSAMGATNELSLTNLETPGLPK

PSTTPRQEVMEISLALAKAITALGESTQAALENFQSTQSQSA MNKMSLESQGLKIDK

EREEFKKMQEIQQKSGTNSTMDTVNKVMIGVTVAITVISWSALFTCGLGLIGTAAA

GATAATAGATAAATTATSVTTTVATQVTMQAVVQVVKQAIIQAVKRAIVQAIKQGI

KQGIKQAIKQAVTiASVKTLAKNVGKIFSAGKNAVSKSFPKLSKVINTLGSKWVTLGV

GALTAVPQLVSGITSLQLSDMQKELAQIQKEVGALTAQSEMMKAFTLFWQQASKIA

AKQTESPSETQQQAAKTGAQIAKALSAISGALAAAA

SEQ II) NO: 87 LTA l-CT668-CopB nucleic acid sequence

CATatggacaatggcgatcgtttataccgtgccgactcgcgtcccccagatgagattaaacgtagcggtgggttaatgcc acgtgggcacaatgagtattttgaccgtggaacacagatgaacattaacctttacgatcatgcccgtgggacccagaccgggtttgtcc gttatgatgacgggtatgttagtacgagtttgtcctacgctccgcacacctgcgggacaaagtattttatcaggctacagcacatattac atttatgtgatcgccactgccccaaacatgttcaatgtgaacgatgtgttgggggtttacagcccccatccatatgaacaagaagtctcg gcccttggggggatcccatatagccagatttatggttggtaccgcgtaaatttggtgtgatgatgaacgtttgcatcgtaaccgtgaata ccgcgatcgctactaccgtaacttgaacatgcacctgccgaggacggctatcgtttagcgggattcccacccgatcatcaggcgtgg cgtgaggaaccgtggatccatcacgcccctcaggggtgcgggaacagtagtcgcCATatgatagatcctcttaagcttttccaaatt ttgatggggataaggagagtgctgcggtgaataaaccttcagcatctcctatgcccagcgaattaagtaaaaatgttgcctcattctcttta gggggtggaggtgctgcgttggattcgacagtgtccacagaaaagctatcgttgatggctatgatgcaggataaaaattcgcagttgat cgatcctgagttggaggaagctctgaactctgaagagttacaagagcagatccatttgttaaaaagtcgtttgtgggatgcacaaacgc agatgcaaatgcaagatcccgacaagttggcctctgagcatgtagatgcttaggagtcattgttgatttaatcaatggggattttcaagc gatagctgaacatacacaacagacggtcaagcagggtaatggtgacgaagaaaaatctgtacacgcaagatagtcgatgggtctct tcaggagaagaaattttgaatcgtgctttgttgtatttctccgatcgtaatggagaaagagaaacatagccgatttcttaaaagttcagtat gccgttcaaagagctacacaacgcgccgagttatttgccagtattctaggtgccacggtgagtagtgtaaaaacgattatgacaaccca gttaggtGGTTCAGCTGCTTCAatgagcttgtcatccagcagcagctcggatagttcgaatctgaaaaatgtgttatctcag gtcatcgcgtctacaccAcagggggttcctaatgctgacaaattaaccgacaatcaggtaaaacaagtccagcagacccgtcaaaac cgtgatgatctgtccatggagagcgacgtcgcggtggcgggaacagccggaaaagatcgtgctgcgtcggcgtcccagatcgagg gacaagagctgattgagcaacagggactgcggctgggaaagagacggcttctgctgatgctacatcattgacccagtcggcatcca aaggcgcttccagtcagcagtgtattgaggataccagtaagtccctggagctttcttcgctttcgagcctgtcaagcgtagatgcgacac atttgcaggaaatccaatcgatcgtgtcttcagcaatgggcgccaccaacgaattgtcatgacgaacttagagacaccgggattacca aagccgagtaccactccAcgccaggaagttatggagatcagccttgccttagcgaaggccatcactgcattgggtgagagcactca ggctgccttggaaaattttcagtccactcagagtcagtccgcgaacatgaataagatgagtttggaatcccaaggcttgaaaatcgaca aggagcgtgaagaatttaagaaaatgcaggagattcagcaaaagagcggcacaaattcaaccatggatactgtgaataaagttatgat tggcgtgacagtggcaattacagtaatctctgttgtttcagcattgtttacctgcggttgggcttgattggcacagccgctgcgggtgcc acagccgccaccgctggggcaacggccgccgccacgaccgctacctctgtgacgaccacagtcgctacccaggtgacgatgcaag cggtggtccaagtcgttaagcaggctattatccaagcagtaaaacgcgccatcgtccaagcgattaaacaggggattaagcaaggcat taaacaagcgatcaaacaggcagtcaaggcaagcgtgaagacacttgccaaaaatgtaggcaagattttcagcgcaggcaagaacg ctgtgagtaagtccttcccAaaattgtctaaggtgataatacacttggttccaaatgggttactcttggcgtgggggcccttacagcggt gccgcagttagtcagtggcattacctcccttcaattgtctgatatgcaaaaagaacttgcacaaatccaaaaggaagtgggtgcacttac ggcgcagagtgagatgatgaaagcgtttacactgttctggcagcaagcttcgaaaatcgcggccaaacaaacggaatcaccttcaga gacgcaacaacaggcagctaagaccggcgcccagatcgctaaagcgttgtccgccatttcgggtgctttagctgctgctgctTAG

CTCGAG

SEQ ID NO: 88 LTA l-CT668-CopB Amino add sequence

MDN GDRL YRAD SRPPDEIKRS GGLMPRGHNE YFDRGT QMNINL YDHARGT Q TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSP HPYEQEVSALGGIPYSQIYGWYRVNFGYIDERLURNREYRDRYYRNLNIAPAEDGYR L AGFPPDHQ AWREEPWIHHAPQGCGN S SRA1IDPLKLFPNFDGDKES AAVNKPS ASP MPSELSKNVASFSLGGGGAALDSTVSTEKLSLMAMMQDKNSQLIDPELEEALNSEEL QEQIHLLKSRLWDAQTQMQMQDPDKLASEHVDALGVIVDLINGDFQAIAEHTQQTV KQGNGDEEKS VTRKIVDWV S SGEEILNRALLYF SDRNGERETL ADFLK V Q YAV QRA TQRAELFASILGATVSSVKTIMTTQLGGSAASMSLSSSSSSDSSNLKNVLSQVIASTPQ G NADKLTDNQVKQVQQTRQNRDDLSMESDVAVAGTAGKDRAASASQIEGQELI EQQGL AAGKET AS AD ATSLTQS ASKGAS SQQCIEDTSK SLEL S SLS SLS S VD ATHLQEI QSIVSSAMGATNELSLTNLETPGLPKPSTTPRQEVMEISLALAKAITALGESTQAALEN FQSTQSQSANMNKMSLESQGLKIDKEREEFKKMQEIQQKSGTNSTMDTVNKVMIGV TVAITVISVVSALFTCGLGLIGTAAAGATAATAGATAAATTATSVTTTVATQVTMQA V V Q VVKQ AIIQ AVKRAI VQ AIKQGIKQGIKQ AIKQ AVK AS VKTLAKNV GKIF S AGKN AVSKSFPKLSKVINTLGSKWVTLGVGALTAVPQLVSGITSLQLSDMQKELAQIQKEV GALTAQSEMMKAFTLFWQQASKIAAKQTESPSETQQQAAKTGAQIAKALSAISGAL AAAA*

SEQ ID NO: 89 HisScc3 chaperone for CT053-CopB2 Nucleic acid sequence AT GGGC AGC AGCC AT C ACC ATC ATC ACC AC AGC C AGGATCCGatgccaccaagc aagatccaatgtcttgaaacttttgaaagaactatggacacctttatctacaacatgcgtccctaatgcgtcatttagcctatctactcgata aaattgctcgctcttaccctcatatgtgtccgcttcccgataatatggaagcgtactttgagaatatatccccaataaagatatccctctgg

92 -- acacctatcaaaaaattttcaaactgtcctcagaagatctgaacaagtctacaaggaaggatacaacgcctattacaaggagactatg aggaaagttctaccgctttttactggttgattttcttaacccatttgtgtctaaattttggttttcattaggagcttcgctccatatgcgccaaaa atatcaacaagctcttcatgcttatggtgtagctgctttgctaagagaaaaagacccttatcctcattactatgcctacatctgctacaccct gctcaataatcctgaagaagctgaaaaagctcttgatcttgcttggcaaaaagtaaaaacaagctctgcctatagctctttaaaagaaga aattttagcgatcaaatcgtacgcctaaGCGGCCGC

SEQ ID NO: 90 HisSccJ chaperone for CT053-CopB2 amino add sequence

MGSSHHHHHHSQDPMPPSKIQCLETFERTYGHLYLQHASLMRHLAYLLDKIA RSYPHMCPLPDNMEAYFENYIPNKDIPLDTYQKIFKLSSEDLEQVYKEGYNAYLQGD YEES ST AF YWLIFFNPF VSKFWF SLGASLHMRQKY QQALHAY GVAALLREKDP YPH Y Y AYIC YTLLNNPEEAEK ALDL AW QK VKT S S A YS SLKEEIL AIK S YA*

SEQ ID NO: 91 CopB2 nucleic acid sequence

atgagctcttggtttgcacaggcgacggacgtcgctttgagccagacccttgatctgcctgacgcttcattggcggttcaaac cgaaaaatttccAtacagctgttcaatctctaaggaatccgccccAtcatgtattcgtaaaatcttcgcccatttagcatctcagaaggaa agtgctccgctgtctttttctcgtttacaaccgactactccgaaagaacgcatcctgtttttcgggtcatcgccttcctcccaattgtcctcga ctgtccgcaccacaacctcttctccatggaatctttttagcaactcccaggcacgcaactcgacccgtaaattgtcggagaagcttcattt gagctcagagttatccgcccgtgactccactaagccttcgtcgagcgaaccggttaaaccatcggaaaatcttttgcacacccctgagc atcataaggaatccttctcaagtttgaaaaaggataacttatctcctatcatggaggagatcgactcattctctgcagagacagagtccctt gaagagcgtttggtcacccagaaaaaggaggagacggtggcccaggagcaaaagcacccAttgctgcgtacatctactccgccat caaaggccagcggggaatcacaagattctagcgaacacagctcaaaggaagatccttatagtcaacaaccgagccataaaatccaac gccgtaaagagcgtgctaagcgcgtcgtcccAattattactccgccaacggtgggtatctttagtttgagctaccttctacaaaacagg ggatcttagcggatttcagcgcctattcggcatacaaggataatttagaaacaactcagcaagagctgaccatgttgcatcaagaacgta tcgagcaagtccaaaaGatcgtggataaaagtaagacaatgcgcttttgggattcattagcatccattgtggccacaatcattccatgga tcgaaatgggtgttgcagtaaccatcatcgcactgggaggtggaatcctttcctggtgctctctttttgctgcgcttatcatgatgtaatttc attattggaagcattcgacgggtggcgtgcaatcgctaagcatttaccaggtaacgatcttgaaaagaagatgcgttatttaggttacgta aagttggccttaactgtgttctcgtgcttactgagtttaagcgccttgtatgtagcaaaataggaatgagtccgcttttggagggggtgtg aagagtatcgcaccAgcattaagtggtatgctgggtttgactcaaggcgtagcactgtatttacaatcttcatcgcaaaagattcgtgcc cgctgcactcagatcgacgcacgcattgaattgattaactgggaacgcgatgagtatttcttgcgtgctgaacaacttcttgattcaatgc aaacgtccttcgaacaacttactgaaacattacagttacaacgtgaaattgatcagacattacagacgctttgcgcTAG

SEQ ID NO: 92 CopB2 amino acid sequence

MSSWFAQATDVALSQTLDLPDASLAVQTEKFPYSCSISKESAPSCIRKIFAHLA SQKESAPLSFSRLQPTTPKERILFFGSSPSSQLSSTVRTTTSSPWNLFSNSQARNSTRKL SEKLHL S SEE SARD STKP S S SEP VKP SENLLHTPEHHKESF S SLKKDNL SPIMEEID SF S

AETESLEERLVTQKKEETVAQEQKHPLLRTSTPPSKASGESQDSSEHSSKEDPYSQQP SHKIQRRKERAKRVVTIITPPT VGIF SLS YLLTKQGIL ADF S AY S AYKDNLETT QQELT MLHQERIEGVGKIVDKSKTMRFWDSLASIVATIIPWIEMGVAVTIIALGGGILSWCSLF AALIMIVISLLEAFDGWRAIAKHLPGNDLEKKMRYLGYVKLALTVFSCLLSLSALYV

AKLGM SPLLEG VVK SI AP AL S GMLGLTQGVAL YLQ S S S QKIR ARC T QID ARIELINWE

RDEYFLRAEQLLDSMQTSFEQLTETLQLQREIDQTFTDALR

SEQ ID NO: 93 CT053-CopB2 nucleic acid sequence

aaaagtgagcgtttaaaaaaattagaatcagagcttcatgatcttacccagtggatgcaacttggccttgttcctaaaaaagaa atcgagagacaccaggaagaaatccgtctgctagaaagcaaaatccttgaagagaaagaacgtctacaacttctcaaagaaagcggt gagatcaaagagtacgtaacccctcgaagaactccagctaaaaccatttacccagatggccccagcgtttcagacgttgagtttgtaga atcctcggatacagaagtggatctcgatgccggtgacacaattgagattgacctaggtgatgaggcaagagaagaaagcggaaacg aactcgactactctagtgaagacgatgaggatcctttcagcgatcgcaatcgttggcgccgaggaggcatcatagatcctgacgcgaa tgaatggGGTTCAGCTGCTTCAatgagctcttggtttgcacaggcgacggacgtcgctttgagccagacccttgatctgc ctgacgcttcattggcggttcaaaccgaaaaatttccAtacagctgttcaatctctaaggaatccgccccAtcatgtattcgtaaaatctt

..... 93 ...... cgcccatttagcatctcagaaggaaagtgctccgctgtctttttctcgtttacaaccgactactccgaaagaacgcatcctgtttttcgggt catcgccttcctcccaattgtcctcgactgtccgcaccacaacctcttctccatggaatctttttagcaactcccaggcacgcaactcgac ccgtaaattgtcggagaagcttcatttgagctcagagttatccgcccgtgactccactaagccttcgtcgagcgaaccggttaaaccatc ggaaaatcttttgcacacccctgagcatcataaggaatccttctcaagtttgaaaaaggataacttatctcctatcatggaggagatcgac tcattctctgcagagacagagtcccttgaagagcgttggtcacccagaaaaaggaggagacggtggcccaggagcaaaagcaccc

Attgctgcgtacatctactccgccatcaaaggccagcggggaatcacaagattctagcgaacacagctcaaaggaagatcctatagt caacaaccgagccataaaatccaacgccgtaaagagcgtgctaagcgcgtcgtcccAattattactccgccaacggtgggtatcttta gtttgagctaccttcttacaaaacaggggatcttagcggatttcagcgcctattcggcatacaaggataatttagaaacaactcagcaag agctgaccatgtgcatcaagaacgtatcgagcaagtccaaaaGatcgtggataaaagtaagacaatgcgcttttgggattcattagca tccattgtggccacaatcattccatggatcgaaatgggtgttgcagtaaccatcatcgcactgggaggtggaatcctttcctggtgctctc tttttgctgcgcttatcatgattgtaatttcattattggaagcattcgacgggtggcgtgcaatcgctaagcatttaccaggtaacgatcttga aaagaagatgcgttatttaggttacgtaaagttggccttaactgtgttctcgtgcttactgagtttaagcgcctgtatgtagcaaaattagg aatgagtccgcttttggagggggttgtgaagagtatcgcaccAgcattaagtggtatgctgggttgactcaaggcgtagcactgtattt acaatcttcatcgcaaaagattcgtgcccgctgcactcagatcgacgcacgcattgaattgataactgggaacgcgatgagtatttcttg cgtgctgaacaacttcttgattcaatgcaaacgtccttcgaacaacttactgaaacattacagttacaacgtgaaattgatcagacatttac agacgctttgcgcTAG

SEQ ID NO: 94 CT053-CopB2 amino acid sequence

MKSEKLKKLESELHDLTQWMQLGLVPKKEIERHQEEIRLLESKILEEKERLQL LKESGEIKEYVTPRRTPAKTIYPDGPSVSDVEFVESSDTEVDLDAGDTIEIDLGDEARE ESGNELDYS SEDDEDPF SDRNRWRRGGHDPDANEWGSAASMSSWFAQATDVALSQ TLDLPDASLAVQTEKFPYSCSISKESAPSCIRKIFAHLASQKESAPLSFSRLQPTTPKERI LFFGSSPSSQLSSTVRTTTSSPWNLFSNSQARNSTRKLSEKLHLSSELSARDSTKPSSSE P VKP SENLLHTPEHHKESF S SLKKDNL SPIMEEID SF S AETESLEERL VTQKKEET V AQ EQKHPLLRTSTPPSKASGESQDSSEFISSKEDPYSQQPSHKIQRRKERAKRVWIITPPT VGIFSLSYLLTKQGILADFSAYSAYKDNLETTQQELTMLHQERIEQVQKIVDKSKTM

RFWDSLASIVATIIPWIEMGVAVTIIALGGGILSWCSLFAALIMIVISLLEAFDGWRAIA

KHLPGNDLEKKMRYLGYVKLALTVFSCLLSLSALYVAKLGMSPLLEGVVKSIAPALS

GMLGLTQGVALYLQSSSQKIRARCTQIDARIELINWERDEYFLRAEQLLDSMQTSFE

QLTETLQLQREIDQTFTDALR

SEQ ID NO: 95 LTAl-CT053-CopB2 nucleic acid sequence

CATatggacaatggcgatcgtttataccgtgccgactcgcgtcccccagatgagattaaacgtagcggtgggttaatgcc acgtgggcacaatgagtatttgaccgtggaacacagatgaacataacctttacgatcatgcccgtgggacccagaccgggtttgtcc gttatgatgacgggtatgttagtacgagtttgtccttacgctccgcacaccttgcgggacaaagtattttatcaggctacagcacatattac atttatgtgatcgccactgccccaaacatgttcaatgtgaacgatgtgttgggggtttacagcccccatccatatgaacaagaagtctcg gcccttggggggatcccatatagccagatttatggttggtaccgcgtaaatttggtgtgattgatgaacgtttgcatcgtaaccgtgaata ccgcgatcgctactaccgtaacttgaacattgcacctgccgaggacggctatcgtttagcgggattcccacccgatcatcaggcgtgg cgtgaggaaccgtggatccatcacgcccctcaggggtgcgggaacagtagtcgcCATatgaaaagtgagcgtttaaaaaaattag aatcagagcttcatgatcttacccagtggatgcaacttggccttgttcctaaaaaagaaatcgagagacaccaggaagaaatccgtctg ctagaaagcaaaatccttgaagagaaagaacgtctacaacttctcaaagaaagcggtgagatcaaagagtacgtaacccctcgaaga actccagctaaaaccatttacccagatggccccagcgtttcagacgttgagtttgtagaatcctcggatacagaagtggatctcgatgcc ggtgacacaattgagattgacctaggtgatgaggcaagagaagaaagcggaaacgaactcgactactctagtgaagacgatgagga tcctttcagcgatcgcaatcgttggcgccgaggaggcatcatagatcctgacgcgaatgaatggGGTTCAGCTGCTTCA atgagctcttggtttgcacaggcgacggacgtcgctttgagccagacccttgatctgcctgacgcttcattggcggttcaaaccgaaaa atttccAtacagctgttcaatctctaaggaatccgccccAtcatgtattcgtaaaatcttcgcccatttagcatctcagaaggaaagtgct ccgctgtctttttctcgtttacaaccgactactccgaaagaacgcatcctgtttttcgggtcatcgccttcctcccaattgtcctcgactgtcc gcaccacaacctcttctccatggaatcttttagcaactcccaggcacgcaactcgacccgtaaattgtcggagaagcttcatttgagctc agagttatccgcccgtgactccactaagccttcgtcgagcgaaccggttaaaccatcggaaaatcttttgcacacccctgagcatcataa ggaatccttctcaagtttgaaaaaggataacttatctcctatcatggaggagatcgactcattctctgcagagacagagtcccttgaagag

..... 94 ...... cgtttggtcacccagaaaaaggaggagacggtggcccaggagcaaaagcacccAttgctgcgtacatctactccgccatcaaaggc cagcggggaatcacaagattctagcgaacacagctcaaaggaagatccttatagtcaacaaccgagccataaaatccaacgccgtaa agagcgtgctaagcgcgtcgteccAattattactccgceaacggtgggtatctttagtttgagctacettcttacaaaacaggggatctta gcggatttcagcgcctattcggcatacaaggataatttagaaacaactcagcaagagctgaccatgttgcatcaagaacgtatcgagca agtccaaaaGatcgtggataaaagtaagacaatgcgcttttgggattcattagcatccattgtggccacaatcattccatggatcgaaat gggtgtgcagtaaccatcatcgcactgggaggtggaatcctttcctggtgctctcttttgctgcgcttatcatgatgtaatttcattattgg aagcattcgacgggtggcgtgcaatcgctaagcatttaccaggtaacgatcttgaaaagaagatgcgttatttaggttacgtaaagttgg ecttaactgtgttctcgtgcttactgagtttaagcgcettgtatgtagcaaaattaggaatgagtccgcttttggagggggttgtgaagagt atcgcaccAgcataagtggtatgctgggtttgactcaaggcgtagcactgtatttacaatcttcatcgcaaaagattcgtgcccgctgc actcagatcgacgcacgcattgaattgattaactgggaacgcgatgagtatttcttgcgtgctgaacaacttcttgattcaatgcaaacgt ccttcgaacaacttactgaaacattacagttacaacgtgaaattgatcagacatttacagacgctttgcgcTAGCTCGAG

SEQ ID NO: 96 LTAl-CT053-CopB2 Amino add sequence

MDNGDRLYRADSRPPDEIKRSGGLMPRGHNEYFDRGTQMNINLYDHARGTQ TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPTSIMFNVNDVLGYYSP HPYEQEVSALGGIPYSQIYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYR LAGFPPDHQAWREEPWIHHAPQGCGNSSRMKSERLKKLESELHDLTQWMQLGLVP KKEIERHQEEIRLLESKILEEKERLQLLKESGEIKEYVTPRRTPAKTIYPDGPSVSDVEF VE S SDTE VDLD AGDTIEIDLGDEAREE SGNELD YS SEDDEDPF SDRNRWRRGGIIDPD ANEWGSAASMSSWFAQATDVALSQTLDLPDASLAVQTEKFPYSCSISKESAPSCIRKI FAHLASQKESAPLSFSRLQPTTPKERILFFGSSPSSQLSSTVRTTTSSPWNLFSNSQARN STRKLSEKLHLSSELSARDSTKPSSSEPVKPSENLLHTPEHHKESFSSLKKDNLSPIMEE IDSFSAETESLEERLVTQKKEETVAQEQKHPLLRTSTPPSKASGESQDSSEHSSKEDPY SQQPSHKIQRRKERAKRVVPIITPPTVGIFSLSYLLTKQGILADFSAYSAYKD LETTQ QELTMLHQERIEQVQKIVDKSKTMRFWDSLASIVATIIPWIEMGVAVTIIALGGGILS WCSLFAALIMIVISLLEAFDGWRAIAKHLPGNDLEKKMRYLGYVKLALTVFSCLLSL SALYVAKLGMSPLLEGWKSIAPALSGMLGLTQGVALYLQSSSQKIRARCTQIDARIE LINWERDEYFLRAEQLLDSMQTSFEQLTETLQLQREIDQIFTDALR*

SEQ ID NO: 97 HisScc3 chaperone for CT668~CopB2 nucleic acid sequence

ATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGatgccaccaagc aagatccaatgtcttgaaacttttgaaagaacttatggacacctttatctacaacatgcgtccctaatgcgtcatttagcctatctactcgata aaattgctcgctcttaccctcatatgtgtccgcttcccgataatatggaagcgtacttgagaatatatccccaataaagatatccctctgg acacctatcaaaaaattttcaaactgtcctcagaagatctgaacaagtctacaaggaaggatacaacgcctatttacaaggagactatg aggaaagttctaccgctttttactggttgatttcttaacccatttgtgtctaaattttggttttcattaggagcttcgctccatatgcgccaaaa atatcaacaagctcttcatgctatggtgtagctgctttgctaagagaaaaagaccctatcctcattactatgcctacatctgctacaccct gctcaataatcctgaagaagctgaaaaagctcttgatcttgcttggcaaaaagtaaaaacaagctctgcctatagctctttaaaagaaga aattttagcgatcaaatcgtacgcctaaGCGGCCGC

SEQ ID NO: 98 His See 3 chaperone for CT668-CopB2 Amino acid sequence

MGSSHHHHHHSQDPMPPSKIQCLETFERTYGHLYLQHASLMRHLAYLLDKIA

RSYPHMCPLPDNMEAYFENYIPNKDIPLDTYQKIFKLSSEDLEQVYKEGYNAYLQGD

YEESSTAFYWLIFFNPFVSKFWFSLGASLHMRQKYQQALHAYGVAALLREKDPYPH

YYAYICYTLLNNPEEAEKALDLAWQKVKTSSAYSSLKEEILAIKSYA*

SEQ ID NO: 99 CT668~CopB2 nucleic acid sequence

aaaagtgagcgtttaaaaaaattagaatcagagcttcatgatcttacccagtggatgcaacttggccttgttcctaaaaaagaa atcgagagacaccaggaagaaatccgtctgctagaaagcaaaatccttgaagagaaagaacgtctacaacttctcaaagaaagcggt gagatcaaagagtacgtaacccctcgaagaactccagctaaaaccatttacccagatggccccagcgtttcagacgttgagtttgtaga atcctcggatacagaagtggatctcgatgccggtgacacaattgagattgacctaggtgatgaggcaagagaagaaagcggaaacg

9_{5 .....} aactcgactactctagtgaagacgatgaggatcctttcagcgatcgcaatcgttggcgccgaggaggcatcatagatcctgacgcgaa tgaatggGGTTCAGCTGCTTCAatgagctcttggtttgcacaggcgacggacgtcgctttgagccagacccttgatctgc ctgacgcttcattggcggttcaaaccgaaaaatttccAtacagctgttcaatctctaaggaatccgccccAtcatgtattcgtaaaatctt cgcccatttagcatctcagaaggaaagtgctccgctgtctttttctcgtttacaaccgactactccgaaagaacgcatcctgtttttcgggt ccgtaaattgtcggagaagcttcattgagctcagagttatccgcccgtgactccactaagccttcgtcgagcgaaccggtaaaccatc ggaaaatcttttgcacacccctgagcatcataaggaatccttctcaagtttgaaaaaggataacttatctcctatcatggaggagatcgac tcattctctgcagagacagagtcccttgaagagcgtttggtcacccagaaaaaggaggagacggtggcccaggagcaaaagcaccc

Attgctgcgtacatctactccgccatcaaaggccagcggggaatcacaagattctagcgaacacagctcaaaggaagatccttatagt caacaaccgagccataaaatccaacgccgtaaagagcgtgctaagcgcgtcgtcccAattattactccgccaacggtgggtatcttta gtttgagctaccttcttacaaaacaggggatcttagcggatttcagcgcctattcggcatacaaggataatttagaaacaactcagcaag agctgaccatgttgcatcaagaacgtatcgagcaagtccaaaaGatcgtggataaaagtaagacaatgcgcttttgggattcattagca tccattgtggccacaatcattccatggatcgaaatgggtgttgcagtaaccatcatcgcactgggaggtggaatcctttcctggtgctctc tttttgctgcgcttatcatgattgtaatttcattattggaagcattcgacgggtggcgtgcaatcgctaagcatttaccaggtaacgatcttga aaagaagatgcgttatttaggttacgtaaagttggccttaactgtgttctcgtgcttactgagtttaagcgccttgtatgtagcaaaattagg aatgagtccgcttttggagggggttgtgaagagtatcgcaccAgcattaagtggtatgctgggtttgactcaaggcgtagcactgtattt acaatcttcatcgcaaaagattcgtgcccgctgcactcagatcgacgcacgcattgaattgattaactgggaacgcgatgagtatttcttg cgtgctgaacaacttcttgattcaatgcaaacgtccttcgaacaacttactgaaacattacagttacaacgtgaaattgatcagacatttac agacgctttgcgcTAG

SEQ ID NO: 100 CT668~CopB2 amino acid sequence

MKSERLKKLESELHDLTQWMQLGLVPKKEIERHQEEIRLLESKILEEKERLQL

LKESGEIKEYVTPRRTPAKTIYPDGPSVSDVEFVESSDTEVDLDAGDTIEIDLGDEARE

ESGNELDYS SEDDEDPF SDRNRWRRGGIIDPD ANEWGS AASM S SWF AQ ATD VAL SQ TLDLPDASLAVQTEKFPYSCSISKESAPSCiRKlFAHLASQKESAPLSFSRLQPTTPKERI LFF GSSPSSQLSS T VRTTT S SP WNLF SN S Q ARN S TRKL SEKLHL S SEE S ARD S TKP S S SE

EQKHPLLRTSTPPSKASGESQDSSEHSSKEDPYSQQPSHKIQRRKERAKRWPnTPPT VGIF SLS YLLTKQGILADF SAY S AYKDNLETT QQELTMLHQERIEQ VQKIVDKSKTM

RFWDSLASIVATUPWIEMGVAVTnALGGGILSWCSLFAALIMIVISLLEAFDGWRAIA

KHLPGNDLEKKMRYLGYVKLALTVFSCLLSLSALYVAKLGMSPLLEGVVKSIAPALS GMLGLTQGVAL YLQ S S S QKIRARC TQID ARIELINWERDEYFLRAEQLLD SMQ T SFE QLTETLQLQREIDQTFTDALR

SEQ ID NO: 101 LTAl-CT668-CopB2 nucleic acid sequence

CATatggacaatggcgatcgtttataccgtgccgactcgcgtcccccagatgagattaaacgtagcggtgggttaatgcc acgtgggcacaatgagtattttgaccgtggaacacagatgaacattaacctttacgatcatgcccgtgggacccagaccgggtttgtcc gttatgatgacgggtatgttagtacgagtttgtccttacgctccgcacaccttgcgggacaaagtattttatcaggctacagcacatattac atttatgtgategccactgccccaaacatgttcaatgtgaacgatgtgttgggggtttacagcccceatccatatgaacaagaagtctcg gcccttggggggatcccatatagccagatttatggttggtaccgcgtaaattttggtgtgattgatgaacgtttgcatcgtaaccgtgaata ccgcgatcgctactaccgtaacttgaacattgcacctgccgaggacggctatcgtttagcgggattcccacccgatcatcaggcgtgg cgtgaggaaccgtggatccatcacgcccctcaggggtgcgggaacagtagtcgcCATatgaaaagtgagcgtttaaaaaaattag aatcagagcttcatgatcttacccagtggatgeaacttggccttgttcctaaaaaagaaatcgagagaeaccaggaagaaatccgtctg ctagaaagcaaaatccttgaagagaaagaacgtctacaacttctcaaagaaagcggtgagatcaaagagtacgtaacccctcgaaga actccagctaaaaccatttacccagatggccccagcgtttcagacgttgagtttgtagaatcctcggatacagaagtggatctcgatgcc ggtgacacaattgagattgacctaggtgatgaggcaagagaagaaagcggaaacgaactcgactactctagtgaagacgatgagga tcctttcagcgatcgcaatcgttggcgccgaggaggcatcatagatcctgacgcgaatgaatggGGTTCAGCTGCTTCA atgagctcttggtttgcacaggcgacggacgtcgctttgagccagacccttgatctgcctgacgcttcattggcggttcaaaccgaaaa atttccAtacagctgttcaatctctaaggaatccgccccAtcatgtattcgtaaaatcttcgcccatttagcatctcagaaggaaagtgct ccgctgtctttttctcgttacaaccgactactccgaaagaacgcatcctgtttttcgggtcatcgccttcctcccaattgtcctcgactgtcc gcaccacaacctcttctccatggaatctttttagcaactcccaggcacgcaactcgacccgtaaattgtcggagaagcttcattgagctc agagttatccgcccgtgactccactaagccttcgtcgagcgaaccggttaaaccatcggaaaatctttgcacacccctgagcatcataa ggaatccttctcaagtttgaaaaaggataacttatctcctatcatggaggagatcgactcattctctgcagagacagagtcccttgaagag cgtttggtcacccagaaaaaggaggagacggtggcccaggagcaaaagcacccAttgctgcgtacatctactccgccatcaaaggc cagcggggaatcacaagattctagcgaacacagctcaaaggaagatccttatagtcaacaaccgagccataaaatccaacgccgtaa agagcgtgctaagcgcgtcgtcccAattattactccgccaacggtgggtatcttagtttgagctaccttcttacaaaacaggggatctta gcggatttcagcgcctattcggcatacaaggataatttagaaacaactcagcaagagctgaccatgttgcatcaagaacgtatcgagca agtccaaaaGatcgtggataaaagtaagacaatgcgcttttgggattcattagcatccattgtggccacaatcattccatggatcgaaat gggtgttgcagtaaccatcatcgcactgggaggtggaatcctttcctggtgctctctttttgctgcgcttatcatgattgtaatttcattattgg aagcattcgacgggtggcgtgcaatcgctaagcatttaccaggtaacgatcttgaaaagaagatgcgttatttaggttacgtaaagttgg ccttaactgtgttctcgtgcttactgagtttaagcgccttgtatgtagcaaaattaggaatgagtccgctttggagggggttgtgaagagt atcgcaccAgcataagtggtatgctgggtttgactcaaggcgtagcactgtatttacaatcttcatcgcaaaagattcgtgcccgctgc actcagatcgacgcacgcattgaattgattaactgggaacgcgatgagtatttcttgcgtgctgaacaacttcttgattcaatgcaaacgt ccttcgaacaacttactgaaacattacagttacaacgtgaaattgatcagacatttacagacgctttgcgcTAGCTCGAG

SEQ ID NO: 102 LTAl-CT668-CopB2Ammo acid sequence

MDN GDRL YRAD SRPPDEIKRS GGLMPRGHNE YFDRGT QMNINL YDHARGT Q TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSP HPYEQEVSALGGIPYSQIYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYR

LAGFPPDHQAWREEPWIHHAPQGCGNSSRMKSERLKKLESELHDLTQWMQLGLVP

KKEIERHQEEIRLLESKILEEKERLQLLKESGEIKEYVTPRRTPAKTIYPDGPSVSDVEF VES SDTE VDLD AGDTIEIDLGDEAREE SGNELD Y S SEDDEDPF SDRNRWRRGGIIDPD

ANEWGSAASMSSWFAQATDVALSQTLDLPDASLAVQTEKFPYSCSISKESAPSCIRKI FAHLASQKESAPLSFSRLQPTTPKERILFFGSSPSSQLSSTVRTTTSSPWNLFSNSQARN S TRKL SEKLHL S SEE SARD S TKP S S SEP VKP SENLLHIPEHHKE SF S SLKKDNL SP IMEE

ID SF S AETESLEERL VT QKKE ET V AQE QKHPLLRT S TPP SKA S GE S QD S SEHS SKEDP Y SQQPSHKIQRRKERAKRVVPHTPPTVGIFSLSYLLTKQGILADFSAYSAYKD LETTQ QELTMLHQERIEQVQKIVDKSKTMRFWDSLASIVATUPWIEMGVAVTIIALGGGILS

WCSLFAALIMIVISLLEAFDGWRAIAKHLPGNDLEKKMRYLGYVKLALTVFSCLLSL

SALYVAKLGMSPLLEGWKSIAPALSGMLGLTQGVALYLQSSSQKIRARCTQIDARIE

LINWERDEYFLRAEQLLDSMQTSFEQLTETLQLQREIDQTFTDALR

SEQ ID NO: 113 dmLT eltA (LTa)nucleic acid sequence

atgattgaca tcatgttgca tataggttag ataaaacaag tggttatctt tccggattgt cttcttgtat gatatataag ttttcctcga tgaaaaatat aactttcatt ttttttattt tattagcatc gccattatat gcaaatggcg acagattata ccgtgctgac tctagacccc cagatgaaat aaaacgtttc cggagtctta tgcccagagg taatgagtac ttcgatagag gaactcaaat gaatattaat ctttatgatc acgcgagagg aacacaaacc ggctttgtca gatatgatga cggatatgtt tccacttctc ttagtttgag aagtgctcac ttagcaggac agtatatatt atcaggatat tcacttacta tatatatcgt tatagcaaat atgtttaatg ttaatgatgt aattagcgta tacagccctc acccatatga acaggaggtt tctgcgttag gtggaatacc atattctcag atatatggat ggtatcgtgt taattttggt gtgattgatg aacgattaca tcgtaacagg gaatatagag accggtatta cagaaatctg aatatagctc cggcagagga tggttacaga ttagcaggtt tcccaccgga tcaccaagct tggagagaag aaccctggat tcatcatgca ccacaaggtt gtggagattc atcaGgaaca atcacaggtg atacttgtaa tgaggagacc cagaatctga

..... 97 ...... gcacaatata tGCcagggaa tatcaatcaa aagttaagag gcagatattt tcagactatc agtcagaggt tgacatatat aacagaattc gggatgaatt atgaataaag taaaatgt

SEQ ID NO: 114 e!tB (LTb) nucleic acid sequence

gttgacatat ataacagaat tcgggatgaa ttatgaataa agtaaaatgt tatgttttat ttacggcgtt actatcctct ctatatgcac acggagctcc ccagactatt acagaactat gttcggaata tcgcaacaca caaatatata cgataaatga caagatacta tcatatacgg aatcgatggc aggcaaaaga gaaatggtta tcattacatt taagagcggc gaaacatttc aggtcgaagt cccgggcagt caacatatag actcccagaa aaaagccatt gaaaggatga aggacacatt aagaatcaca tatctgaccg agaccaaaat tgataaatta tgtgtatgga ataataaaac ccccaattca attgcggcaa tcagtatgaa aaactagttt gctttaaaag catgtctaat gctaggaacc tatataacaa ctactgtact tatactaatg agccttatgc tgcatttgaa aaggcggtag aggaggcaat accgatcctt aaactgtaac actataacag cttccactac agggagctgt tatagcacac agaaaaaact aagctaggct ggaggggcaa gctt

Claims

.· Ji-. What is claimed is:

1. A fusion polypeptide comprising a fusion of a needle tip protein or an antigenic fragment thereof and a translocator protein or an antigenic fragment thereof from a Type III secretion system (T3SS) of a Gram negative bacteria, wherein the gram negative bacteria is not a Salmonella enter ica. or Shigella spp.

2. The polypeptide of claim I, wherein the fusion polypeptide is arranged so that the needle tip protein is 5’ of the translocator protein.

3. The polypeptide of claim 1, wherein the gram negative bacteria comprises Bordetella spp., Burkholderia spp., Chlamydia spp., Pseudomonas spp., Vibrio spp., or Yersinia spp

4. The polypeptide of claim 1, wherein the needle tip protein comprises Bsp22, LcrV, BipD, PcrV, CT053, or CT668.

5. The polypeptide of claim 1, wherein the translocator protein comprises BopB, YopB, BipB, PopB, CopB, or CopB2.

6. The polypeptide of claim 1, wherein the fusion comprises the Bordetella spp. needle-tip protein (Bsp22) and translocator protein (BopB), or antigenic fragments thereof.

7. The polypeptide of claim 1, wherein the fusion comprises the Yersinia spp.

needle-tip protein (LcrV) and translocator protein (YopB), or antigenic fragments thereof.

8. The polypeptide of claim 1, wherein the fusion comprises the Burkholderia spp. needle-tip protein (BipD) and translocator protein (BipB), or antigenic fragments thereof.

9. The polypeptide of claim 1, wherein the fusion comprises the Pseudomonas spp. needle-tip protein (PcrV) and translocator protein (PopB), or antigenic fragments thereof.

10. The polypeptide of claim 1 , wherein the fusion comprises the Chlamydia spp. needle-tip protein (CT053 or CT668) and translocator protein (CopB or CopB2), or antigenic fragments thereof

1 1. The polypeptide of claim 1, wherein the fusion further comprises double mutant labile toxin (dmLT) or an antigenic fragment thereof from Enterotoxigenic Escherichia coli or cholera toxin or an antigenic fragment thereof.

12. The polypeptide of claim 1, wherein the dmLT comprises the active moiety

LTA1

13. The polypeptide of claim 1, wherein the dmLT retains its ADP ribosylation activity.

14. The polypeptide of claim 1, wherein the dmLT is 5’ of the needle tip protein and translocator protein fusion.

15. A vaccine comprising one or more of the fusion polypeptides of any of claims 1- 14.

16. The vaccine of claim 15, further comprising one or more components of an acellular pertussis vaccine.

17. The vaccine of claim 15, further comprising pertussis toxoid (PTd).

18. A method of treating, inhibiting, or preventing an infection of a Gram negative bacteria in a subject comprising administering to the subject the fusion polypeptide of any of claims 1-14 or vaccine of claims 15-17.

19. The method of claim 18, wherein the method further inhibits or prevents colony formation of the bacteria and/or transmission of the bacteria to another subject.

20. A method of eliciting an immune response in a subject to a Gram negative bacteria comprising administering to the subject the fusion polypeptide of any of claims 1-14 or vaccine of claims 15-17.

21. The method of claim 20, wherein the immune response comprises a sterilizing immune response.

22. The method of claims 18-21, wherein the bacteria compri ses Bordetella spp., Burkholderia spp., Chlamydia spp., Pseudomonas spp., Vibrio spp., or Yersinia spp.

23. A method of eliciting an immune response against at least one Gram negative bacteria serovar in a subject in need thereof, comprising administering to the subject a composition comprising at least one needle tip protein or an antigenic fragment thereof and/or at least one translocator protein or an antigenic fragment thereof, wherein said composition is administered in an amount sufficient to elicit an immune response to said at least one Gram negative bacteria serovar in said subject; and wherein the Gram negative bacteria is not a Shigella spp. or Salmonella enter ica.

24. The method of claim 23, wherein the bacteria comprises Bordetella spp., Burkholderia spp., Chlamydia spp., Pseudomonas spp., Vibrio spp., or Yersinia spp. loo