WO2015102936A1

WO2015102936A1 - Poxvirus-plasmodium recombinants, compositions containing such recombinants, uses thereof, and methods of making and using same

Info

Publication number: WO2015102936A1
Application number: PCT/US2014/071386
Authority: WO
Inventors: Enzo Paoletti; Randall L. WEINBERG; Scott J. Goebel
Original assignee: V-CORE TECHNOLOGIES Inc
Current assignee: V-CORE TECHNOLOGIES Inc
Priority date: 2013-12-30
Filing date: 2014-12-19
Publication date: 2015-07-09
Anticipated expiration: 2016-06-30
Also published as: AU2014374198A1; US20200102544A1; EP3089756A4; EP3089756A1; JP2019162120A; US11168307B2; IL246539A0; CA2935341A1; PH12016501271A1; US20150191704A1; KR20160103130A; JP2017502674A

Abstract

The invention provides a recombinant or synthetic or engineered or non -naturalIy occurring poxvirus that contains and expresses DNA encoding a heterologous or exogenous antigen, epitope or immunogen and Flagellars or an operable binding portion thereof. The poxvirus can contain or be engineered to contain and express vaccinia host range gene K 1 L. The poxvirus can be attenuated as to mammals, e.g., NYVAC, NYVAC.1, NYVAC. 2, avipox, canarypox, fowlpox, ALVAC, TROVAC, MVA, or MVA-BN. The invention also provides methods for inducing an immunological response involving the poxvirus, and compositions containing the poxvirus. The antigen, epitope or immunogen that the poxvirus expresses can be at least one Plasmodium antigen. The Plasmodium antigen(s), epitope(s) or immunogen(s) can be SERA, ABRA, Pfhsp70, AMA-1, Pfs25, Pfs16, CSP, PfSSP2, LSA-1 repeatless, MSA-1, AMA-1 or combination(s) thereof. Advantageously the poxvirus contains DNA coding for and expresses Plasmodium antigen(s) CSP, PfSSP2, LSA-1 -repeatless, MSA-1, SERA, AMA-1 and Pfs25. Also, advantageously, the poxvirus is a NYVAC poxvirus. The invention thus also provides an anti-malarial immunogenic or immunological compositions comprising the poxvirus, and methods for inducing an immunogenic or immunological response against malaria or Plasmodium in a mammal comprising administering to the mammal the poxvirus or an immunological or immunogenic composition containing the poxvirus. The mammal can be a human.

Description

POXVIRUS-PLASMODIUM RECOMBINANTS, COMPOSITIONS CONTAINING SUCH RECOMBINANTS, USES THEREOF, AND METHODS OF MAKING AND USING SAME

RELATED APPLICATIONS AND/OR INCORPORATION BY REFERENCE

[0001 ] This application claims priority from US provisional application Serial No. 61/921,748, filed December 30, 2013.

[0002] The foregoing application^), and all documents cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

FIELD OF INVENTION

10003] The present invention relates to modified poxvirus and to the methods of making and using the same. In certain embodiments, the invention relates to recombinant poxvirus, which virus expresses exogenous or heterologous gene product(s), e.g., from Plasmodium, a specific poxvims replication regulator and an adjuvant for immune-response enhancement, and immunogenic compositions or vaccines containing such poxvims, and methods for providing immunity, e.g., protective immunity, against Plasmodium infections.

BACKGROUND OF INVENTION

[0004] Information concerning poxviruses, such as Chordopoxvirinae subfamily poxviruses (poxviruses of vertebrates), for instance, orthopoxviruses and avipox viruses, e.g., vaccinia virus (e.g., Wyeth Strain, WR Strain (e.g., ATCC® VR-1354), Copenhagen Strain, NY VAC, NYVAC.l, NYVAC.2, MVA. MVA-BN), canarypox virus (e.g., Wheatley C93 Strain, ALVAC), fowipox vims (e.g., FP9 Strain, Webster Strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoon pox, inter alia, synthetic or non-naturally occurring recombinants thereof, uses thereof, and methods for making and using such recombinants may be found in scientific and patent literature, such as:

I US Patents Nos. 4,603,112, 4,769,330, 5,110,587, 5,174,993, 5,364,773, 5,762,938, 5,494,807, 5,766,597, 7,767,449, 6,780,407, 6,537,594, 6,265,189, 6,214,353, 6,130,066, 6,004,777, 5,990,091, 5,942,235, 5,833,975, 5,766,597, 5,756,101, 7,045,313, 6,780,417, 8,470,598, 8,372,622, 8,268,329, 8,268,325, 8,236,560, 8,163,293, 7,964,398, 7,964,396, 7,964,395, 7,939,086, 7,923,017, 7,897,156, 7,892,533, 7,628,980, 7,459,270, 7,445,924, 7,384,644, 7,335,364, 7,189,536, 7,097,842, 6,913,752, 6,761,893, 6,682,743, 5,770,212, 5,766,882, and 5,989,562, and

Panicaii, D. Proc. Natl. Acad. Sci. 1982; 79; 4927-493, Panicaii D. Proc. Natl. Acad. Sci. 1983; 80(17): 5364-8, Mackett, M. Proc. Natl Acad. Sci. 1982; 79: 7415-7419, Smith GL. Proc. Natl. Acad. Sci. 1983; 80(23): 7155-9, Smith GL. Nature 1983; 302: 490-5, Sullivan VJ. Gen. Vir. 1987; 68: 2587-98, Perkus M Journal of Leukocyte Biology 1995; 58: 1-13, Yilma TD. Vaccine 1989; 7: 484-485, Brochier B. Nature 1991 ; 354: 520-22, Wiktor, TJ. Proc. Natl Acd. Sci. 1984; 81 : 7194-8, Rupprecht, CE. Proc. Natl Acd. Sci. 1986; 83: 7947-50, Poulet, H Vaccine 2007; 25(Jul): 5606- 12, Weyer J. Vaccine 2009; 27(Nov): 7198-201 , Buller, RY! Nature 1985; 317(6040): 813-5, Boiler RM. J. Virol. 1988; 62(3):866-74, Flexner, C. Nature 1987; 330(6145): 259-62, Shida, H. J. Virol 1988; 62(12): 4474-80, Kotwal, GJ. J. Virol. 1989: 63(2): 600-6, Child, SJ. Virology 1990; 174(2): 625-9, Mayr A. Zentraibf Bakterioi 1978; 167(5,6): 375-9, Antoine G. Virology. 1998; 244(2): 365-96, Wyatt, LS. Virology 1998: 251(2): 334-42, Saneho, MC. J. Virol. 2002; 76(16); 8313-34, Gal lego-Gomez, JC. J. Virol. 2003; 77(19); 10606-22), Goebel SJ. Virology 1990; (a,b) 179: 247-66, Tartaglia, J. Virol. 1992: 188(1 ): 217-32, Najera JL. J. Virol. 2006: 80(12): 6033-47, Najera, JL. J. Virol. 2006; 80: 6033-6047, Gomez, CE. J. Gen. Virol. 2007; 88: 2473-78, Mooij, P. Jour. Of Virol 2008: 82: 2975- 2988, Gomez, CE. Curr. Gene Ther. 2011 ; 11 : 189-217, Cox,W. Virology 1993; 195: 845-50, Perkus, M. Jour. Of Leukocyte Biology 1995; 58: 1 -13, Blanchard TJ. J Gen Virology 1998; 79(5): 1159-67, Amara R. Science 2001; 292: 69-74, Hel, Z., J. Immunol. 2001 ; 167: 7180-9, Gherardi MM. J. Virol. 2003; 77: 7048-57, Didieriaurent, A. Vaccine 2004; 22: 3395-3403, Bissht H. Proc. Nat. Aca. Sci. 2004; 101 : 6641-46, McCurdy LH. Clin. Inf. Dis 2004; 38: 1749-53, Earl PL. Nature 2004: 428: 182-85, Chen Z. J. Virol. 2005: 79: 2678-2688, Najera JL. J. Virol. 2006; 80(12): 6033-47, Nam JH. Acta. Virol. 2007; 51 : 125-30, Antonis AF. Vaccine 2007; 25: 4818~4827,B Weyer J. Vaccine 2007; 25: 4213-22, Ferrier-Rembert A. Vaccine 2008; 26(14): 1794-804, Corbett M. Proc. Natl. Acad. Sci. 2008; 105(6): 2046-51, Kaufman HI,., J. Clin. Oncol. 2004; 22: 2122-32, Amato, RJ. Clin. Cancer Res. 2008; 14(22): 7504-10, Dreicer R. Invest New Drugs 2009; 27(4): 379-86, Kantoff PW.J. Clin. Oncol . 2010, 28, 1099-1 105, Amato RJ. J. Clin. Can. Res. 2010; 16(22): 5539-47, Kim, DW. Hum, Vaccine. 2010; 6: 784-791, Oudard, S. Cancer Immunol. Immimother. 201 1 ; 60: 261-71, Wyatt, LS. Aids Res. Hum. Retroviruses. 2004; 20: 645-53, Gomez, CE. Virus Research 2004; 105: 1 1-22, Webster, DP. Proc. Natl. Acad. Sci. 2005; 102; 4836-4, Huang, X. Vaccine 2007; 25: 8874-84, Gomez, CE. Vaccine 2007a; 25: 2863-85, Esteban M. Hum. Vaccine 2009; 5: 867-871, Gomez, CE. Curr, Gene therapy 2008; 8(2): 97-120, Whelan, KT. Plos one 2009; 4(6): 5934, Scriba, TJ. Eur, Jour. Immuno. 2010; 40(1 ): 279-90, Corbett, M. Proc. Natl. Acad. Sci. 2008; 105: 2046-2051, Midgley, CM. J. Gen. Virol. 2008; 89: 2992-97, Von Krempelhuber, A. Vaccme 2010; 28: 1209-16, Perreau, Vf . J. Of Virol. 2011; Oct: 9854- 62, Pantaieo, G. Curr Opin HIV-AIDS. 2010; 5: 391-396,

each of which is incorporated herein by reference.

10005] Information on a particular YVAC-Piasmodium recombinant known as VP 1209 or NYVAC-Pf7 is discussed in Tine et al, "NYVAC-Pf7: a poxvirus-vectored, multiantigen, falciparum malaria multistage vaccine candidate for Plasmodium;' Infect. Immun, 1996, 64(9):3833, and Ockenliouse et al, "Phase I/IIa Safety, Immuiiogenicity, and Efficacy Trial of NYVAC~P†7, a Pox- Vectored, Multiantigen, Multistage Vaccine Candidate for Plasmodium falciparum Malaria," 1998; 1 77: 1664-73, each of which is incorporated herein by reference.

[0006] Despite such information, to date, there are no licensed recombinant poxvirus vaccines for use in humans; see Rollier CS. Curr. Opin. Imraun. 201 1; 23(Jun): 377-82.

[0007] In addition, malaria is considered the most important parasitic disease in the world. It is estimated that malaria caused over 200 million clinical episodes worldwide resulting in 655,000 deaths, mostlv African children; see WHO Global Malaria Program 2011. Furthermore the economic losses are magnified as most of the endemic countries are impoverished, costing some 3 billion dollars in Africa alone; see Teklehaimanot A. J. Trop. Med, Hyg. 2007; 77(6): 138-44. There have been substantial efforts and resources directed to methods and approaches for control-intervention such as indoor spraying, insecticidal nets, rapid diagnostics for testing, especially pregnant woman and children; see Aponte JJ. Lancet 2009; 374(9700): 1533-44., Menendez C, Lancet Infect. Dis, 2007; 7(2): 126-35, However, as these control interventions programs had a measured degree of success, it is with the realization that to substantially reduce disease costs and burden to society vaccines against malaria are cmcial to reduce the morbidity and mortality of this disease; see Malaria Eradication: Vaccines PloS Med. 201 1; 8(1 ): el000398. A focused effort and strategic goal was put forth by the international organization PATH, Malaria Vaccine Initiative (MVI), that by 2020 malaria vaccines provide 80% protective efficacy against P. falciparum-.

[0008] Citation or identification of any document in this application is not an admission that such document is avai lable as prior art to the present invention.

OBJECTS AND/OR SUMMARY OF THE INVENTION 0ΘΘ9] The present invention recognizes and endeavors to address poxvirus (e.g., recombi ant poxvirus) immimological or immunogenic composition or vaccine i duction of only weak or suboptimal immune correlatives; see, e.g., Smith, JM. AIDS Res. Hum. Retroviruses 2004; 20: 1335-1347, Hanke, T. J. Gen. Virol. 2007; 88: 1 -12, Sandstrom, E. J. Inf. Dis. 2008; 198: 1482-90, Walker, BD. Science 2008; 320: 760-4, Sekaly, RP. J. Exp. Med. 2008; 205: 7-12. Rerks-Ngarm S. 2009; N Engl J Med 361 : 2209-2220.

[0010] The term "poxvims" includes members of the Chordopoxvirinae subfamily, such as orthopoxviruses and avipo viruses, e.g., vaccinia virus (e.g., Wyeth Strain, WR Strain (e.g., ATCC® VR-1354), Copenhagen Strain, NYVAC, MVA, MVA-BN), canarypox virus (e.g., Wheatley C93 Strain, ALVAC), fowlpox virus (e.g., FP9 Strain, Webster Strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoon pox, inter alia; and it especially includes poxviruses of documents cited herein, including poxviruses that also express transcription and/or translation factor(s) of US Patents Nos. 5,990,091 , 6, 130,066 and 6,004,777.

[0011] In this regard, in one aspect the invention provides a poxvims that is a synthetic or non-natural iy occurring, i.e., an engineered, synthetic or a non-naturally-occurring poxvirus, e.g., through recombination, advantageously an attenuated poxvirus as to a mammal, such as NYVAC, NYVAC.1, NYVAC.2, avipox, canarypox, fowlpox, ALVAC, TROVAC, MVA, MVA-BN, that through such engineering contains DNA encoding Flagellin (or an operable binding portion thereof) and/or vaccinia host range gene K1L, and expresses such DNA. Advantageously, the poxvims contains and expresses DNA encoding Flagellin (or an operable binding portion thereof) and vaccinia host range gene K1L. [0012] Thus, as to atte uated poxviruses as to a mammals, such as NYVAC, NYVAC. l, Y VAC .2, avipox, canarypox, fowlpox, ALVAC, TROVAC, MVA, MVA-BN, the invention comprehends such a poxvirus that is synthetic or non-iiaturaily occurri g, i.e., that has been engineered or manipulated, e.g., through recombination, to contain, advantageous Sy in a nonessential region, DNA encoding Flagellin (or an operable binding portion thereof} and/or vaccinia host range gene KIT,, and express such DNA. The synthetic or non-naturally occurring or engineered or recombinant poxvirus that contains and expresses DNA encoding Flagellin (or an operable binding portion thereof) and/or vaccinia host range gene KI L can also be manipulated, engineered to contain and express DNA coding for one or more antigen(s), immunogen(s) or protein(s) that is/are foreign or exoge ous or heterologous to the poxvirus.

[0013] The invention also comprehends compositions containing such an engineered or sy thetic or noii-naturaily-occurriiig or recombmant poxvirus, e.g., immunogenic or immunological or vaccine compositions, uses of such a poxvirus or composition, e.g., to stimulate an immune response, such as a protective immune response, for example for generation of antibodies for use either in vivo, in vitro or ex vivo, and methods of making such poxviruses and compositions, and methods of using such poxviruses and compositions. Such compositions can contain an amount of poxvims akin to the amount of recombinant poxvirus found in prior art recombinant poxvirus immunogenic or immunological or vaccine compositions. Similarly, in methods for inducing an immune or protective immune response, the amount of composition and/or poxvirus to be administered can be akin to the amount administered in prior art. methods for inducing an immune or protective immune response by recombinant poxvims compositions or recombinant poxviruses. NYVAC expressing Flagelli (FliC) can be a novel vaccine directed to poxvirus infections, including smallpox.

[0014] In another aspect the invention provides a poxvirus thai is a synthetic or non-naturally occurring, i.e., an engineered, synthetic or a non-naturally-occurring poxvirus, e.g., through recombination, advantageously an attenuated poxvims as to a mammal, such as NYVAC, N YVAC.l , NYVAC.2, avipox, canarypox, fowlpox, ALVAC, TROVAC, MVA, MVA-BN, that through such engineering contains, advantageously in a non-essential region, DNA encoding Flagellin (or an operable binding portion thereof) and/or vaccinia host range gene KIL, and expresses such DNA, and DNA encoding gene product(s) of Plasmodium and expresses such DNA encoding gene product(s) of Plasmodium. Thus, as to attenuated poxviruses as to mammals, such as NYVAC, NYVAC.1, NYVAC.2, avipox, canarypox, fowlpox, ALVAC, TROVAC, MVA, MVA-BN, the invention comprehends such a poxvirus that is synthetic or non-naturally occurring, i.e., that has been engineered or manipulated, e.g., through recombination, to contain DNA encoding Flagellin (or an operable binding portion thereof) and/or vaccinia host range gene Kl L and express such DNA, and DNA encoding gene product(s) of Plasmodium and express such DNA encoding gene product(s) of Plasmodium, The engineered, synthetic, non-naturally occurring and/or recombinant poxvirus of the invention thus co-expresses gene produces) of Plasmodium, and Flagellin (or an operable binding portion thereof) (and optionally also K IL). The invention also comprehends compositions containing such an engineered or synthetic or non-naturally-occurring or recombinant poxvirus, e.g., immunogenic or immunological or vaccine compositions, uses of such a poxvirus or composition, e.g., to stimulate an immune response, such as a protective immune response, for example for generation of antibodies for use either in vivo, in vitro or ex vivo, and methods of making such poxviruses and compositions, and methods of using such poxviruses and compositions. Such compositions can contain an amount of poxvirus akin to the amount of recombinant poxvims found in prior art recombinant poxvirus immunogenic or immunological or vaccine compositions. Similarly, in methods for inducing an immune or protective immune response, the amount of composition and/or poxvirus to be administered can be akin the amount administered in prior art methods for inducing an immune or protective immune response by recombinant poxvirus compositions or recombinant poxviruses.

[0015] Immunogenic or immunological compositions stimulate an immune response that may, but need not be, protective. A vaccine stimulates a protective immune response. Advantageously, a vaccine against Plasmodium or malaria provides at least 80% protective efficacy against P, falciparum (protection in at least 80% of subjects receiving the vaccine). When other than a non-essential region is used as the locus or loci for DNA encoding Flagellin and/or DNA coding for an antigen or immunogen such as Plasmodium antigen(s) or immunogen(s), the skil led person may employ a complementing host cell or helper virus, see, e.g., US Patent No. 5,766,882.

[0016] The DNA encoding gene product(s) of Plasmodium advantageously codes for Plasmodium antigen(s) or immunogen(s), e.g., SERA, ABRA, Pflisp70, AMA-1 , Pfs25, Pfsl6, CSP, PFSSP2, I SA- ! repeatless, MSA-i, AMA-1 or combination^) thereof. The DNA encoding gene product(s) of Plasniodium advantageously codes for sequences for CSP, PfSSP2, LSA-l-repeatless, MSA-1, SERA, AMA-1 and Pfs25, akin to NYVAC-Pf7. The vector is advantageously NYVAC, The vector can also express a translation and/or transcription factor, such as in US Patents Nos. 5,990,091 , 6,130,066 and 6,004,777. Without wishing to he hound by any one particular theory, the Flageliin (or an operable binding portion thereof) when expressed in an attenuated vector, such as a NYVAC vector, may have an adjuvant or imrnunostimulatory effect. When the vector expressing Flageliin (or an operable binding portion thereof) is advantageously a NYVAC vector, this is advantageously an ''enhanced" NYVAC vector (i.e., it also contains and expresses vaccinia 1 L). Advantageously, an "enhanced" replication competent NYVAC vector that contains and expresses Flageliin (or an operable binding portion thereof) also contains and expresses Plasmodium falciparum CSP, PfSSP2, LS A- 1 -repeatless, MSA-1, SERA, AMA-1 and Pfs25. Such a vector has the capacity for a level of limited replication in humans while retaining the established vector safety profile of NYVAC with open reading frames for virulence factors deleted or disrupted, and can obtain an immunological or immunogenic response that is desired for a malaria vaccine.

[0017] ( Najera, JL. 2010; Plos one (5): el 1406, Kibler, V. Plos one 201 1; 6: e25674)

[0018] Compositions of the invention can contain an amount of engineered, synthetic, non- naturally occurring or recombinant Flagellin-Plasmodium-poxviras (that advantageously also contains and expresses vaccinia 1L) as in NYVAC -Pf7 compositions; and, in methods for inducing an immune or protective immune response of the invention, the amount of composition and/or poxvims to be administered can be akin the amount administered in prior art methods involving NYVAC~P†7.

[0019] Without wishing to be bound by any one particular theory, the invention provides self-adjuv anting immunogenic, immunological or vaccine compositions (by expression of Flageliin or an operable binding portion thereof by the poxvirus, especially with expression of vaccinia 1L). These vectors (poxviruses that express Flageliin or an operable binding portion thereof) are capable of triggering innate immunity and important pro-inflammatory cascade(s) critical for the development of robust adaptive immune responses that can provide protective immunity, e.g. against Plasmodium infection. The invention thus provides a replication competent, engineered, synthetic, non-naturally occurring or recombinant poxvirus useful for the production of Plasmodium immunogen(s) or antigen(s), in vivo or in vitro; and, the resulting immunogen(s) or a tigen(s).

[0020] Accordingly, in an aspect, the invention relates to a recombinant poxvirus containi g therein DNA encoding at least one Plasmodium antigen or irnmunogen and at least one DNA sequence encoding Flageilin or an operable binding portion thereof and/or the vaccinia host range gene K1L— and advantageously both the D A sequence encoding Flageilin or an operable binding portion thereof and the vaccinia host range gene l L advantageously in a nonessential region of the poxvirus genome. The poxvirus is advantageously NY VAC. In an advantageous aspect, the recombinant poxvirus expresses Plasmodium SERA, ABRA, Pfhsp70, AMA-1, Pfs25, Pfsl6, PfSSP2, LSA-1, LSA-l-repeatless, MSA-1, CSP, MSA-1 N-terminal p83 or MSA- 1 C-tenninai gp42 gene. Advantageously, a plurality of Plasmodium genes are co-expressed in the host by the recombinant inventive poxvirus, NYVAC e.g., CSP, PfSSP2, LSA-l-repeatless, MSA-1 , SERA, AMA-1 and Pfs25; in combination with at least one or both of the vaccinia host range gene KIL and DNA encoding Flageilin, or at least an operable binding portion of Flageilin. Advantageously, the recombinant poxvirus NYVAC contains the KIL gene providing the capacity for limited replication in humans, yet retaining attenuated virulence; and, this NYVAC contains DNA coding for and expresses the CSP, PfSSP2, LSAl-repeatless, MSA-1, SERA, AMA-1, Pfs25, ABRA, Pfhsp70, or Pfsl6, P. falciparum antigens, and advantageously this NYVAC that contains KIL and the foregoing DNA encoding P. falciparum antigens also contains DN A encoding Flagei lin, or an operable binding portion of Flageilin. While such is an advantageous embodiment, the invention comprehends recombinant poxviruses, e.g., NYVAC, expressing one or more or only some of these P, falciparum antigens, as well as Flageilin or an operable binding portion thereof and/or Kl L. The foregoing P. falciparum antigens individually or in combinations can be expressed by single poxvirus vectors (e.g., NYVACs) that also contain and express Flageilin, or an operable binding portion of Flagei lin and also advantageously KI L, and these single poxvirus vectors can be used in combination with each other in an immunogenic, immunological or vaccine composition.

[0021] The invention also comprehends poxvirus, e.g., NYVAC single recombinants expressing the CSP, PfSSP2, LSAl-repeatless, SERA, or MSA-1 N-terminal p83 and C-terminal gp42 processing fragments in combination with at least one of the genes KI L and flageilin or an operable binding portion of Fl ageilin. [0022] The invention is also directed to the methods of making and using the replication competent poxvirus expressing malaria or Plasmodium genes for the production of Plasmodium gene products, either in vivo or in vitro as well as to the recombinant gene products.

[CI023] in a further aspect, the invention relates to a composition for inducing an immunological response in a host animal inoculated with the composition. The composition can include an adjuvant for the induction of innate immunity. The composition can contain a synthetic or engineered or non-naturally occurring or recombinant poxvirus, e.g. NYVAC, that contains, advantageously in a nonessential region thereof, DNA encoding one or more antigens or imrmmogens, e.g., one or more Plasmodium antigens or immunogens, and Flagellin or an operable binding portion thereof, and optionally also 1L, as well as to methods for inducing such an immunological response in an animal by inoculating or administering to the animal the composition or a poxvirus of the composition. The immunological response can be a protective immunological response and hence the composition can be a vaccine; but, it need not elicit a protective immune response and can be an immunogenic or immunological composition. Advantageously, DNA in the poxvirus codes for and the poxvirus expresses one or more and advantageously all of SERA, ABRA, Pfhsp70, AMA-1, Pfs25, Pfsl 6, PfSSP2, LSA-1, LSA-1- repeatless, MSA-1, CSP, MSA-1 N -terminal p83 and MSA-1 C-temiinai gp42 of Plasmodium, in combination with the Flagel lin or at least an operable binding portion of Flagellin, and K1L. A portion of Flagellin that is essential to trigger the TLR5 PAMP is an operable binding portion of Flagellin. With such a poxvirus, a plurality of Plasmodium genes is advantageously co- expressed in the host or animal, e.g., CSP, PFSSP2, LSA-1 -repeatless, MSA-1 , SERA, AMA-1 , and Pfs25; and preferably the poxvirus contains the host range gene K i L and also expresses Flagellin or an operable binding portion thereof; and, preferably the poxvirus is a NYVAC poxvirus. Such a poxvirus has the capacity for limited replication in mammals, e.g., humans while retaining the attenuated virulence profi le. Accordingly, animals or hosts in this descriptio are advantageously mammals, such as humans.

[CI024] Furthermore, it is an object of the invention to not encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or meihod of using the product, which does not meet the writte description and enablement requirements of the USPTO (35 U.S.C. § 1 12, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.

[0025] It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", '"comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0026] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following Detailed Description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, incorporated herein by reference, wherein:

[CI028] Fig 1 shows primer locations with regard to Example 1 .

[0029] Fig 2 A shows the 1L expression cassette nucleotide sequence. Fig 2B is a diagram of the KIL expression cassette for insertion between the Xhol and Spel sites in Fig 1 for generation of Pf7.2

[0030] Figs 3 A, 3 B, 3C, 3D contain the results of expression by inventive recombinants.

DETAILED DESCRIPTION

[0031] Poxvirus Vectors

[0032] The success of the smallpox eradication campaign is an unprecedented medical achievement. (Henderson DA. Sci. Am 1976:235; 25-33) However, there were serious adverse effects that posed substantias risks to subpopuiations of vaccine recipients. These risks were associated with the use of virulent replication competent vaccine strains of poxvirus for immunization. These vaccine strains posed especially significant risks for those recipients, and close contacts, with abnormalities in cutaneous immunity and often caused life-threatening post vaccination adverse events. The nature and frequency of these events have been well documented. (Bray, M Antiviral research 2003; 58: 101-14, Engier, RJM J. Allergy Clinical Immunology 2002; 1 1 0: 357-65, Halsell JS JAMA 2003; 289: 3283-9, Kretzschmar, M. Plos Medicine 2006; 3(8): 1341-51 , Casey, CG. JAMA 2005; 294: 2734-43). Although significant risks were associated with vaccination against smallpox, these risks were acceptable in the Sight of the horrific pandemic smallpox posed to public health.

[0033] Concomitant with the announcement from the World Health Organization that smallpox had been eradicated, and the advent of recombinant molecular technologies, there was renewed interest in vaccinia as a recombinant eukaryotic expression vector, with the capacity to cam' and deliver heterologies target genes of interest. (Panicali, D. Proc. Natl. Acad. Sci. 1982; 79; 4927-493, Panicali D. Proc. Natl. Acad. Sci. 1983; 80(17): 5364-8, Mackett, M. Proc. Natl. Acad. Sci. 1982; 79: 7415-7419, Smith GL, Proc. Natl. Acad. Sci. 1983; 80(23): 7155-9, Smith GL. Nature 1983; 302: 490-5, Sullivan VJ. Gen. Vir. 1987; 68: 2587-98). Importantly, these pivotal studies provided the foundation highlighting the potential of recombinant vaccinia as a novel vaccine vector having the attributes of genomic stability, ease of genomic manipulation and amplification and importantly, robust storage stability, critical to address the significant unmet needs for improved tropical disease vaccines such as malaria in underdeveloped, third world countries.

[0034] In addition to its long-standing history of use in humans, the abi lity to generate synthetic recombinants expressing of any number of antigens or combinations thereof, vaccinia provides an exciting new avenue for the generation of recombinant vaccines, perhaps with the potential to be the "universal immunization vehicle". (Perkus M Journal of Leukocyte Biology 1995; 58: 1-13). Recombinant vaccinia vectors were rapidly embraced by the veterinary industry for the development of new vaccine technologies. (Yilma TD. Vaccme 1989; 7: 484-485, Brochier B. Nature 1991 ; 354: 520-22, Wiktor, TJ. Proc. Natl Acd. Sci. 1984; 81 : 7194-8, upprecht, CE. Proc, Natl Acd. Sci. 1986; 83 : 7947-50, Pouiet, H Vaccine 2007; 25(Jul): 5606- 12, Weyer J. Vaccine 2009; 27(Nov): 7198-201). However, the well documented safety issues as a vaccme in humans would remain a major hurdle that had to be addressed if recombinant vaccinia vectors were to gain regulatory approval for use in the general human population. It is with these safety concerns that rigorous ongoing clinical safety testing continues today. Further compounding safety concerns for live viral vaccines, is the fact that a significant proportion of our population is highly immuno -compromised though a variety of medical conditions such as cancer and HIV infection. (Parrino J. J. Allergy Clin. Immunol 2006:118(6): 1320-26, Jacobs BL. Antiviral Therapy 2009; 84(1): 1 -13.) To date, there are no licensed recombinant poxvirus vaccines for use in humans. (Rollier CS. Curr. Opin. Immun. 2011; 23(Jun): 377-82.)

Poxvirus Attenuation for improved viral vaccine vectors

[0035] A great deal of work has focused on the development of attenuated vaccinia virus strains. Laboratory studies have demonstrated that the deletion of certain vaccinia genes reduces the virulence of resulting recombinants in animal models ( Bul!cr, RM Nature 1985; 3 17(6040): 813-5, Bailer RM. J. Virol. 1988; 62(3):866-74, Flexner, C. Nature 1987; 330(6145): 259-62, Shida, H. J. Virol. 1988; 62(12): 4474-80, Kotwal, GJ. J. Virol. 1989; 63(2): 600-6, Child, Si. Virology 1990; 174(2): 625-9.). Two highly attenuated strains of vaccinia, Modified Vaccinia Ankara (MVA ) and NYVAC have emerged as two of the most predominately studied, non- replicating vectors in human tissues. Recombinants of both MVA and NYVAC have been extensively studied pre-clinically and many have made their way through late phase II/III clinical trials. Both viruses have been extensively studied and characterized at the genomic level.

[0036] MVA was developed during the 1970s, by high serial passage of vaccinia Ankara on primary chicken embryo fibroblasts (CEF). The high serial passage resulted in many large genomic deletions totaling some 30kb and importantly, the loss of the ability of the virus to replicate in humans and other mammals. (Mayr A. Zentralbl Bakteriol 1978; 167(5,6): 375-9, Antoine G. Virology. 1998; 244(2): 365-96, Wyatt, LS. Virology 1998; 251(2): 334-42, Sancho, MC. J. Virol. 2002; 76(16); 8313-34, Gallego-Gomez, JC. J. Virol. 2003; 77(19); 10606-22). The NYVAC strain was derived from a plaque isolate of the Copenhagen strain of vaccinia by the precise deletion of 18 open reading frames (ORFs) that were implicated in pathogenesis, virulence and host range regulatory functions. (Goebel SJ. Virology 1990; (a,b) 179: 247-66, Tartaglia, J. Virol. 1992; 188(1): 217-32, Patent 5,762,938, Najera JL. J. Virol. 2006; 80(12): 6033-47).

[0037] MVA and NYVAC strains have been directly compared in preclinical studies as to the capacity to replicate in animal models and in the clinical settings assessing the safety profile in extensive human trials. (Najera, JL. J. Virol. 2006; 80: 6033-6047, Gomez, CE. J. Gen. Virol. 2007; 88: 2473-78, Mooij, P. Jour. Of Virol. 2008; 82: 2975-2988, Gomez, CE. Curr. Gene Ther. 201 1; 1 1 : 189-217.) While avirulent and non-replicating, these vaccine vectors have repeatedly demonstrated their safety attributes but importantly, they are still competent in stimulating both cellular and humoral immune responses against a variety of expressed target antigens, (Cox,W. Virology 1993; 195: 845-50, Perkus, M. Jour. Of Leukocyte Biology 1995; 58: 1 -13, Blanchard TJ. J Gen Virology 1998; 79(5): 1159-67, Ockenhouse CF. J. Infec. Dis. 1998: 177: 1664-73, Amara R. Science 2001 ; 292: 69-74, He!, Z., J. Immunol. 2001 ; 167: 7180-9, Gherardi MM. .1. Virol. 2003; 77: 7048-57, Didierlaurent, A. Vaccine 2004; 22: 3395-3403, Bissht H. Proc. Nat. Aca. Sci. 2004; 101 : 6641-46, McCurdy LH. Clin. Inf. Dis 2004; 38: 1749-53, Earl PL. Nature 2004; 428: 182-85, Chen Z. J. Virol. 2005; 79: 2678-2688, Najera JL. J. Virol. 2006; 80( 12); 6033-47, Nam JH. Acta. Virol. 2007; 51 : 125-30, Antonis AF. Vaccine 2007; 25: 4818-4827,B Weyer J. Vaccine 2007; 25: 4213-22, Ferrier-Rembert A. Vaccine 2008; 26(14): 1794-804, Corbett M. Proc. Natl. Acad. Sci. 2008; 105(6): 2046-51.)

[0038 j The body of clinical data from late stage human trials for recombinant MVA, NYVAC and other non-replicating poxvirus vectors is growing significantly. Many of these studies have focused on two of the most challenging areas for vaccine development, cancer immtinotherapeutics (Kaufman HL., J. Clin. Oncol. 2004; 22: 2122-32, Amato, RJ. Clin. Cancer Res. 2008; 14(22): 7504-10, Dreicer R. Invest New Drugs 2009; 27(4): 379-86, Kantoff PW.J. Clin. Oncol. 2010, 28, 1099-1105, Amato RJ. J. Clin. Can. Res. 2010; 16(22): 5539-47, Kim, DW. Hum. Vaccine. 2010; 6: 784-791, Oudard, S. Cancer Immunol. Immuiiother. 2011; 60: 261- 71.) and HIV. (Wyatt, LS. Aids Res. Hum. Retroviruses. 2004; 20: 645-53, Gomez, CE, Virus Research 2004; 105: 11-22, Webster, DP. Proc. Natl Acad. Sci. 2005; 102: 4836-4, Huang, X. Vaccine 2007; 25: 8874-84, Gomez, CE. Vaccine 2007a; 25: 2863-85, Esteban M. Hum. Vaccine 2009; 5: 867-871, Gomez, CE. Curr. Gene therapy 2008; 8(2): 97- 120, Whelan, KT. Plos one 2009; 4(6): 5934, Scriba, TJ. Eur. Jour. Immimo. 2010; 40(1): 279-90, Corbett, M. Proc. Natl. Acad. Sci. 2008; 105: 2046-2051, Midgley, CM. J. Gen. Virol. 2008; 89: 2992-97, Von Krempelhuber, A. Vaccine 2010; 28: 1209-16, Perreau, M. J. Of Virol. 201 1; Oct: 9854-62, Pantaleo, G. Curr Opin H1V-AIDS. 2010; 5: 391-396).

[0039] Overall the safety data is excellent with minimal vector associated side effects, this being of great significance considering the inherent risk associated with a large portion of the target population for cancer and HIV vaccines that are potentially immunocompromised. Importantly, when scoring vaccine efficacy, the overwhelming body of clinical data suggests NYVAC and MVA and other attenuated poxvims vectors are capable of eliciting important correlative immune responses, to a degree that is both encouraging and supportive for continued development and testing of these vectors. However, studies surrounding both cancer and HIV vaccine initiatives indicate that while immune responses from vaccine recipients are indeed encouragi g, many late phase trials have failed to achieve the primary objectives necessary to go forward with further clinical development. To this point, the data surrounding vaccine induction of only weak or suboptimal immune correlatives remain the focus in assessing these failures. (Smith, JM. AIDS Res. Hum. Retroviruses 2004; 20: 1335-1347, Hanke, T. J. Gen. Virol. 2007; 88: 1-12, Sandstrom, E. J. Inf. Dis. 2008; 198: 1482-90, Walker, BD. Science 2008: 320: 760-4, Sekaly, RP. J. Exp. Med. 2008; 205: 7-32. Rerks-Ngarm S. 2009; N Engl J Med 363 : 2209- 2220.).

[0040] If recombinant vaccines targeting weakly immunogenic antigens eg., cancer Tumor Associated Antigens (TAAs) and HIV are to be effective there has to be a renewed effort focused on improving and developing second generation MVA and NYVAC viral vectors. Clearly improvements need to be focused at enhancing viral expression, possibly through more robust vaccine amplification profiles in humans, while retaining an attenuated phenotype essential for the safety of vaccine recipients. Developmental work has focused on a variety strategies including: further genomic modifications to non-replicating viral vectors such as N YVAC and MVA, routes of vaccine administration, immunization priming protocols, co-expression of immuno-stimuiatory signaling molecules and novel adjuvant strategies for enhanced immunogenicity.

Further modifications to NYVAC to elicit stronger immunogenic responses

[0041] One method of enhancing expression of target antigens from NYVAC is to re- engineer NYVAC to allow the virus to proceed later into the infectious cycle, potentially providing some limited level of avirulent replication. Importantly, replication competence does not have to exclude attenuation. (Parker SD. 2007: Vaccine 25; 6764-73) Ideally, this level of replication would be enhanced compared to N YVAC but less than that obtained from the parent Copenhagen strain. More robust replication and expression may provide more antigen load for processing and importantly, better mimicking of the naturally occurring viral infectious cycle, potentially triggering stronger innate immune responses. There are several examples of attenuated recombinant vaccinia vectors that have been engineered as aviruSent but importantly, replication competent, while exhibiting nearly the same margin of vaccine safety of the replication deficient NYVAC strain. (Verardi, PH. J. Virol. 2001; 75(1 ): 11-8, Langland, JO. Virology 2002; 299(1): 133-41, Langland, JO. Virology 2004; 324(2): 419-29, Langland, JO. J. Virology 2006; 80(20): 10083-95, Legrand, FA. Proc. Natl. Acd. Sci. 2005; 102(8): 2940-5, Denes, B. J. Gene Med. 2006; 8 (7): 814-23, Day, SL. J. Immunol. 2008; 180(11): 7158-66, Jacobs, BL, Antiviral Res. 2009; 84: 1-13, Vijaysri, S. Vaccine 2008; 26: 664-676, Dai, K. Vaccine 2008; 26: 5062-71, Huang, X. Plos One 2009; 4: e4180.)

[0042] Virally encoded genes that were specifically deleted from Copenhagen to generate NYVAC or lost upon serial passage in primary chick cells in generating MVA, were predominately viral gene functions that had evolved particularly for the modulation and or inhibition of antiviral host immune responses. Such factors are referred to as pathogenicity factors. These factors can determine viral host range, pathology and virulence in a given host. (McFadden G. Nat. Rev. 2005; 3: 201-13.) The focus of a large body of research has been devoted to study these virulence factors and their importance in determining host range. The understanding of how these host range genes interact with specific host targets has elucidated functionality with respect to viral pathogenesis and the abrogation of specific host immune responses. There are approximately 12 different host range gene families that have been identified in poxviruses. (Wcrdcn, SJ. Adv. Vir. Res. 2008; 71 : 135-171, Bratke, KA. Inf. Gen. and Evol. 2013; 14: 406-25). Many attempts to enhance immunogenicity profiles of NYVAC or MVA based vaccines have looked to restore different host range gene iterations that were originally deleted from these highly attenuated vectors. In light of the comparative studies using traditional replication competent and replication deficient NYVAC and MVA vectors, it was evident that long lasting immune responses were more robust upon immunization using the tradition first generation replication competent vaccinia vector. (Femer-Rembert, A. Vaccine 2008; 26: 1794- 1804} Furthermore, coupled with suboptimal clinical trials in humans (Rerks- Ngarm, 2009) with non-replicating vaccinia based vectors, it has been proposed by several in the field that replication deficient vectors while providing an excellent safety profile, may not provide enough antigen load to stimulate robust, long lasting, adaptive immune responses in some cases and that some level of viral replication would provide a more potent vaccine immunogen.

[0043] Host range genes C7L and K1L previously identified have been the obvious targets of choice to reinsert back into the attenuated genome of NYVAC to enable the vims to proceed further into its replicative cycle. (Perkus, ME. Virology 1990; 170: 276-86, Tartaglia, J. Virology 1992:188: 217-232, Sliisler, JL. J. Virol. 2004; 78: 3553-60, Bradley, RR. Vims Res. 2005; 114: 104-12). C7L is known to inhibit host antiviral action induced by type 1 interferons. ( VI eng. X J. Virol. 2009; 83: 10627-636, Backes, S. J. Gen. Virol. 2010; 91 : 470- 482). Additionally, C7L and KI L inhibits the phosphorylation of eIF2 alpha and the induction of apoptosis, through the inhibition of PKR activity in infected ceils. (Najera, JL. j. Virol 2006; 80: 6033-47, Willis, KL. Virology 2009; 394: 73-81). Specifically, when C7L was engineered back into NYVAC the resulting modified NYVAC-C7L vims was found to be replication-competent in both human and murine cells. In vivo, .mouse models have been used to demonstrate enhanced viral expression, while maintaining an attenuated profile, with clearly superior immune responses against expressed HIV antigens in compariso with the host restricted NYVAC vector (Najera, JL. J. Virol. 2006; 80: 6033-47, Najera, JL. 2010;). In another example, genomic modification of NYVAC was take one step further with the re-insertion of both C7L and KIL, furthermore with an additional .modification of removing B19R, a type I INF inhibitor (Kibler, KV. 2011;, Gomez, CE. Jour. Of Virol. 2012; 5026-38). The NYVAC vector containing both C7L and KIL (NYVAC +C7L, K I L), as expected, was found to be replication competent in a variety of different cultured human cells. Importantly, (NYVAC +C7L, KIL) was found to still retain the highly attenuated phenotype in comparison to wild type replicatio competent strains, such as Copenhagen and NYCBH. Bio-distribution analysis indicated that other genomic modifications such as the deletion of B19R allowed for further attenuation compared to (NYVAC +C7L, KIL), potentially through the activation of PKR through INF ί activation, resulting in the induction of the pathogen-associated molecular pattern (PAMP) sensors. (Kibler, KV. 2011)

[0044] A specific inventive embodiment of the invention is NYVAC or another attenuated (as to mammals) poxvirus containing the Host Range gene K I L, e.g., NYVAC vectors modified to contain the host range gene KI L (NYVAC+K1L) so that these vectors are further developed to specifically replicate in human tissues to a level intermediate of that of the more virulent parental replication competent strai Copenhagen and the replication deficient stain NYVAC or MVA or MVA-BN or eanarypox or fowl pox or ALVAC or TR.GVAC, and to co-express at least one vaccine target antigen(s). Advantageously such a vector also contai s DNA coding for and expresses Flagellin. or an operable binding portion thereof.

Methods to eishaisce Immune responses to vaccines

Routes of vaccine delivery [0045] Considering the suboptimai results obtained with the attenuated vectors NYVAC and MVA in the HIV trials, alternatives are sought to address these limitations with hopes of enhancing efficacy. Extensive studies have been done directly comparing MVA and NYVAC in tissue culture, (Najera et al., 2006) genome profiling studies, (Guerra et a!,, 2004, 2006,) and immunogenic! ty in human clinical trials (Gomez et al., 2007a, b). Importantly, studies comparing inoculation route and ability of the virus to disseminate in vivo have been done. (Gomez, CE. 2007; 88: 2473-2478, Gomez, CE. Vaccine 2007b: 25: 1969-92). Although NYVAC and M VA are highly attenuated they both exhibit differences in vitro and in vivo, potentially with immunological relevance (Mooij, P. J. Virol. 2008; 82(6): 2975-88.)

[0046] The normal mode of viral transmission for many viruses, including HIV and Sars CoV, is through mucosal surfaces. It is believed that cell-mediated responses at mucosal sites are critical for protection. Clinical data from some of the initial studies was unclear as to whether or not immunization with cutanous or intramuscular routes can result in important cellular responses at distal surface mucosal sites. (Benson, J. 1998; J. Virol. 72: 4170-82, Belyakov, IM. J. Clin. Invest 1998; 102: 2072-2081, Cromwell, MA. J. Virol 2000; 74: 8762-66, Stevceva, L. Genes Immun. 2000; 1 : 308-15, He!, Z. J. Immunol. 2001; 167: 7180-91, Stitielaar, KJ. Vaccine 2001; 19: 3700-09, Stevceva, L. Jour. Of Virol. 2002; 76(22): 11659-76.). Several recent studies indicate mucosal routes of vaccination may be advantageous. (Huang, X. Vaccine 2007; 25: 8874-84, Gherardi, MM. J. Gen. Virol. 2005; 86: 2925-36, Neutra, MR. Nat. Rev. Immunol. 2006; 6: 148- 158, Karkhanis, Curr. Pharm. Des 2007; 13: 2015-23, Corbett, M. Proc. Natl. Acad. Sci. 2008; 105(6): 2046-51, Belyakov, IM. J. Immunol 2009; 183: 6883-6892.) Furthermore, mucosal routes of inoculation seem to be effective at overcoming preexisting immunity. Strong anti-vector responses can result in diminished antigen load of expressed antigenic targets, therefore potentially limiting adaptive immune responses. (Naito, T. J. Gen. Virol 2007; 88: 61- 70.)

Imm unization Prim, ing

[CI047] Prime boost is routinely used in vaccination protocols to increase the immune response. Classical immune studies have shown that the immune system once activated and allowed to rest then reactivated will result in a significant boost to both B-cell and T-cell responses. (Murphy, K. Janesway C. Immunology 7^th ed. 2008; New York, NY) When using the same live recombinant virus vector repeatedly to direct antigen expression by host cells, strong anti-vector immune response (induced by the priming vaccine) can block efficacy of the following boost. Essentially, the boost vaccine is quelled before viral expression from host ceils can express foreign protein, along with immune signals and therefore provides little advantage to the original vaccination. To overcome the vector-specific immunity, it is critical to use a different vector for the boost. Prime boost protocols are well known in the art. (Hu, SL. Science 1992; 255: 456-459, Richmond, JFL. Virology 1997; 230: 265-274, Brown, S. Viruses 2010; 2: 435-467) Prime boost regimens expressing the antigens of interest from another vector system, such as DNA, then boosting with the recombinant virus vector have enhanced vaccine efficacy (Ramsay, AJ. Immunol. Cell Biol. 1997;75: 382-388, Ramshaw, IA. Immunol. Today 2000; 21 : 163-165, Estcourt, MJ. Int. Immunol. 2002; 14: 31-37, Hodge, JW. Can. Res. 2003; 63(22):7942-9, Woodland, DL, Trends Immunol. 2004; 25(2): 98-104, Webster, DP. Proc. Nat Acad. Sci. USA 2005; 102.(13): 4836-41, Harari, AJ. Exp. Med. 2008; 205: 63-77, Meiief, CJM. 2008; Immunity 29: 372-83, Brave, A. Mol. Ther. 2007; 15: 1724-1733, Robinson, HI, Hum. Vaccine 2009; 5: 436-438, Gudmundsdotter, L, Vaccine 2009; 27: 4468-4474. Ishizaki, HJ. nmunother. 2010; 33(6): 609-17, Krupa, M. Vaccine 201 1; 29(7): 1504-13).

[0048] Significantly, for recombinant NYVAC and MVA vectors, much of the late stage clinical data focuses on HIV vaccines. Clearly, for an ideal HIV⁷ vaccine it is important to stimulate both arms of the adaptive immune system eliciting strong cellular immunity, memory cells and antibodies at mucosal surfaces and throughout the body. (Demberb, T, Int. Rev. Immunol. 2009; 28(1): 20-48, Neutra, MR. Nat. Rev. Immunol. 2006; 6: 148-1 8.). Additionally clinical studies have shown thatthc ability to contain HIV virus load correlates strongly with robust cellular CD8+ T-ceil responses. (Dorrell, L. Vaccines 2005; 4(4): 513-20, Kuroda, MJ. 1999; 162: 5127-33, Shen, X. J. Immunol. 2002; 169: 4222-29, Carrington, M. Ann. Rev. Med. 2003; 54: 535-51, Frahm, N. J. Virol. 2005; 79: 10218-25, Dorrel!, L. J. Virol. 2006; 80(10): 4705-16, Dorrell, L. Vaccine 2007; 25: 3277-83, Frahm N. Nat. Immunol. 2006; 7: 173-8, Wilson NA. J. Virol. 2006; 80: 5875-85, Harari, AJ. Exp. Med. 2008; 205: 63-77, McCormack, S. Vaccine 2008; 26: 3162-74, Mooij, PJ. Virol. 2008; 82: 2975-88, Van Montfoort, N. Proc. Natl. Acad. Sci. 2009; 106; 6730-35, Quakkelaar ED. Plosone 6; 2011 : el6819) Clearly immunization regimens and modes of antigen delivery that stimulate both arms of the adaptive immune system, would provide a clear advantage in recombinant vaccine design. (Dorrell, L. 2005 and 2007, Sekaly, RP. J. Exp. Med. 2008; 205: 7-12) Immuno-stimulatory gene expression as an adjuv&sst

Co-stim ulatkm molecules

[0049] Another approach to optimizing immunization is through T-celi co-stimulation. T- cel l activation depends on the interaction of MHC peptide complexes with T-cell receptors, along with the interaction of co-stimulatory molecules with antigen presenting cells (APC) and the corresponding receptors on T-ce!ls. Co-stimulation is particularly important when expressed antigens are only poorly immunogenic such as TAAs. The use of co-stimulatory molecules such as B7.1 , the ligand for T-cell surface antigens CD28 and CTLA-4 and a triad of human co- stimulatory molecules (TRICOM) have been studied extensively. (Damle, NK. J. Immunol, 1992: 148: 1985-92, Hodge, JW. Can. Res. 1999; 59: 5800-7, Von Mehren, M. Clin. Cancer Res. 2000; 6: 2219-28, Lu, M. Proc. Natl . Acad. Sci. 2004; 101(suppl2): 14567-71 , Gulley, JL. Clin. Can. Res. 2005; 11 : 3353-62, Arlen, PM. J. Urol. 2005; 174: 539-46, Nam, JH. Acta. Viri, 2007 51 : 125-30, Madan, RA. Exp. Opin. Invest. Drugs 2009; 18: 1001 -11). The data suggests enhanced immune responses through co-stimulatory molecules leads to sustained activation and signaling in T-celis. Furthermore, it has been suggested that co-stimulation increases CTL avidity resulting in more effective targeted cell lysis (Oh, S. J. Immunol. 2003; 170: 2523-30, Hodge, JW. J. Immunol. 2005; 174: 5994-6004,). Innate immune activation can drive co- stimulatory molecule expression.

Cytokines

OOSOj The rational for using an immune adjuvant is to enhance the immune response to a vaccine by interaction with Antigen Presenting Cells (APCs) and T-celis. The co-expression of a variety of cytokines such as GM-CSF, IL-2, and FLT-3 ligand, has been studied extensively. The co-expression of cytokines in the vicinity with targeted expressed antigens was found to enhance the recruitment of dendritic cells (DCs) to the site of immunization resulting in enhanced presentation to APCs. The co-expression of cytokines has been highly utilized in oncology based vaccines, to boost responses, again to poorly immunogenic TAAs. ( ass, E. Cancer Res. 2001; 61 : 206-14, Davis, ID. J. Immunothere. 2006; 29: 499-511, Arlen, PM. .1. Urol. 2007; 178: 1515-20, Lechleider, RJ. Clin. Can. Res. 2008; 14: 5284-91, Gulley, JL. Can. Immunol. Immunother. 2010; 59: 663-74. KantofF, PW. N. Eng. Jour. Med. 2010; 363-411-22, Lutz, E. Ann. Sur. 2011; 253: 328-35). Innate immune activation can drive cytokine expression.

Innate Immunity Activation Role of Toll-Like Receptors in Innate Immunity

OOSlj Immune responses have been classically categorized into innate and adaptive immunity. Adaptive responses are further subdivided into cellular and humoral. In comparative analysis of innate and adaptive responses, adaptive immunity is driven by the specificity of the T-cell and B-celi antigen specific receptors resulting in further induction of immune cell, cytokine and antibody trafficking to converge on the invading pathogen. Additionally, memory T and B-cell responses are generated so that any subsequent adaptive response to the same pathogen can be more rapidly regenerated (Janeway 2002). Innate immunity is found in all vertebrates. Originally, innate responses were viewed as a vestige of ancient host defenses and were simply used as an immediate host defense, a temporary and highly non-specific reaction until more important adaptive responses could take over. However, recent studies have shown that the innate immune system has a high degree of specificity with the ability to identify important signatures of foreign pathogens. The ability to identify signatures of foreign pathogens is associated with a highly conserved family of receptors designated, Toll-Like Receptors (TLRs) for their homology to the Toll protein identified in Drosophila (Lemaitre, B. Cell 86; 973-83).

[0052] TLRs are type one integral membrane glycoproteins, with an exceiiular domain having a leucine rich repeat region (LRR) and a cytoplasmic signaling domain. The LRR domain is important for ligand binding. (Akira, S. Phil, Trans R. Soc. B 2011; 366: 2748-55). Initial studies indicated that specific TLRs (TLR-4) were involved with the recognition of lipopolysaeeharidc (LPS), the cell wall component of gram-negative bacteria. The connection of mammalian TLRs with LPS recognition provided the important link necessary between TLRs and Pathogen-Associated Molecular Pattern (PAMP) recognition. (Poltorak, A. Science 1998; 282(5396): 2085-88, Qureshi, ST. J. Exp. Med. 1999; 189(4): 615-25. Hoshino, . J. Immunol. 1999; 162(7): 3749-52).

[0053] To date, 12 members of the TLR family have been identified in mammals. (Akira, S. Ceil 2006; 124: 783-01 , Beutler, B. Nature 2004; 430: 257-63, Medzhitov, R. Nature 2007; 449: 819-826). TLRs can recognize a variety of components derived from bacteria and viral pathogens. In addition to LPS a ceil wail component, bacterial and viral DNA are recognized through (CpG) by TLR-9 (Hemmi, H. Nature 2000; 408: 740-5.), ssRNA by TLRs 7 and 8 (Hemmi, H. Nat. Immunol. 2002; 3: 196-200, Diebold, S. Science 2004; 505: 1529-31.) dsRNA by TLR-3 (Alexopoulou, L. Nature 2001; 413: 732-38.) and bacterial proteins such as Flagellin, a component of bacterial Flagella. FlageSla are responsible for bacterial motility, and are detected by TLR-5 (Hayashi, F. Nature 2001; 410: 1099-1103, Uematsu, S. Nat. Immunol 2006; 7: 868- 874.) TLRs can be divided as to cellular localization, TLR 1 ,2,4-6 are on the cell surface, TLR 3,7-9 are within endosomcs. (Kumar H. Biochem. Biophy. Res. Commun. 2009; 388: 621-5).

[0054] Once triggered by the TLR specific ligand, the signaling process occurs through adapter molecules called TIR-Domaincontaining Inducing interferon-B (TRIF) or Myeloid Differentiation Primary Response Gene (MyD88). This results in cytosolic signaling complexes through TRIF and MyD88 activating F- B and IRF transcription factors resulting in the productio of inflammatory cytoki es and type I interferon (IFN). (Yamamoto M. Scie ce 2003; 301 (5633): 640-3, Kawai T. Semin. Immunol 2007; 19(1): 24-32. O'Neill LA. Nat. Rev. Immunol. 2007; 7: 353-64.) Furthermore, activation of these transcription factors results in the activation of the complement and coagulation cascades and induction of phagocytosis and apoptosis. (Adams, S. Immunotherapy 2009; (6): 949-64) All these processes play a critical role in initiating innate and adaptive arms of immune protection. ( oebe K. Nat. Immunol 2004; 5( 10): 971-4. Akira S. Nat. Immunol. 2001; 2(8): 675-80, Medzhitov, R. Nature 1997; 388(6640): 394-7.)

Toll-Like Receptors ami Viral Infection

[0055] Initial evidence that TLRs were involved in controlling viral infection came from the ^■finding that some viruses expressed genes specifically targeting and blocking TLR signaling responses. TLRs have been shown to be involved in antiviral responses to a wide variety of virus families, in context with many different viral macromolecules; the list is long and reviewed extensively (Carry, M. Clinical and Exp. Immunol. 2010; 161 : 397-406). Plasmacytoid dendritic cells ( DC) are specialized immune cells that produce type I IFN and are critical for antiviral responses. (Gilliet M. Nat. Rev. Immunolo. 2008; 8: 594-606, Theofilopoulos AN. Ann. Rev. Immunol. 2005; 23: 307-36), It has been shown that TLRs 7 a d 9 signaling by viral nucleic acids in the endosome promotes activation of pDCs. TLR9 detects CpG in DNA, while TLRs 7 and 8 detect G/U rich ssRNA. (Diebold SS. Scie ce 2004; 303: 1529-3 L Krieg AM. Ann. Rev. Immunol. 2002; 20: 709-60, Heil F. Science 2004; 303: 1526-29). TLR 7-9 signaling is mediated through adaptor MyD88. (Akira S. Nat. Rev. Immunol. 2004; 4: 499-511.)

[0056] Vaccinia has been shown to activate pDCs upon infection. In human cells such as monocytes, macrophages and keratinocytes, activation of NF-KB is mediated through TLR 2, 3 and 4 . (Bauernfeind, F. Nat. Immunol. 2009; 10: 1 139-41 , Howel l, MD. Immunity 2006; 24: 3 1-8, Carty, M. Clinical and Exp, Immunol. 2010: 161 : 397-406). In comparison, in mice, A/T rich viral DNA was detected by TLR 8, resulting in !NF responses from activated pDCs. (Martinez, J. Proc. Nat. Acad. Sci. 2010: 107: 6442-7.) Importantly, this response in mice was shown to be independent of TLR-9. Interestingly, human pDCs do not express TLR8, only TLR- 7 and 9. However, human conventional DCs do express TLR8 and these may play a role in IFN responses. (Iwasaki A. Nat. Immunol 2004; 5: 987-95.) It is important to note that vaccinia encodes several genes targeting modes of TLR signaling, A46R has been shown to inhibit the activity of MyD88, while A52R and C4L mhibits TLR mediated NF-KB activation. (Stack, J. J. Exp. Med. 2005; 201 : 1007-18, Maloney, G. J. Biol, ( hem 2005; 280: 30838-44, Stuart W. Jour. Gen. Virol. 2012; 93: 2098-108.) Other viruses have developed methods to block TLR activity, HCV has been shown to inhibit TLR. signaling though the activity of its protease NS3/4a that cleaves the TRIP complex while NS5a directly inhibits MyD88. (Li, K. Proc. Nat. Acad. Sci. 2005; 102: 2992-7, Abe T. J. Virol. 2007; 81 : 8953-66.)

TLRs Expression profile

[0057] TLRs lie at the forefront of the host defense system, and provide a system wide network for the detection of pathogens, in humans, the network of 10 different expressed TLRs have been determined for a variety of different cell types. Importantly, TLRs are found not only on cel ls of the immune system but are also expressed on epithelial cells of the intestine, urogenital and respiratory tracts, areas potentially important to the site of invading pathogens. (Guillot, L. J. Bio. Chem. 2004; 280: 5571 -80, Vora, P. J. Immunol. 2004; 173(9): 5398-405.) The TLR expression profile by cell type has been well established: mDCs express TLRs (1-6, 8), pDCs express TLR (7, 9), neutrophils express TLR (1, 2, 4-10), NK cells express TLR1, monocytes express all except TLR 3, B-lymphocytes express TLR (9,10), activated T-cells express TLR 2, regulatory T-eells express TLR (8, 10) . ( adowaki, N. J. Exp. Med 2001; 194(6): 863-869, Bernasconi, NL. Blood 2003; 101(1 1 ): 4500-04, Hayashi, F. Blood 2003; 102(7): 2660-69, Muzio M. J. Immunol. 2000; 164(11): 5998-6004, Hasan, U. J. Immunol. 2005; 174(5): 2942-50, Peng, G. Science 2005; 309(5739): 1380-84.)

TLR ago ists important for designer vaccine adjuvants

[0058] Having the information as to the TLR specific activating ligand and the complement

?? of TL s expressed on different cell types, it is now possible to specifically target TLRs by using activating ligands in vaccine formulations as adjuvants to enhance vaccine immunogemcity. In such a designer vaccine, it is possible toincorporate TLR activating components for vaccine optimization; as to site or route of vaccine inoculation (dermal, mucosol intranasal), type of desired immune response (cellular, humoral or both, TH1 or 2 ), type of vaccine (subunit, viral or bacterial, live, killed). In the present example, using live recombinant vaccinia vectors that naturally activate virus specific TLRs it would be of significant advantage to co-express one or more additional TLR iigand(s) (or at least the operable binding portion of that ligand) to recruit additional TLR activation providing an adjuvanting effect to further stimulate immune responses to the vaccine. It is critically important to recognize the tight regulation dictated by activation of the innate immune system to control a specific class of infection and limit immune response induced damage to the host. For example, viral innate immune activation can induce interferon and programmed cell death while bacterial innate immune activation can induce reactive oxygen species and promote cell survival; adaptive immune responses follow this pattern. Innate immune activation is optimized for each class of infection thai directs appropriate acquired immune responses to similar infections. Most immune responses to antigens expressed by a viral vector, such as NYVAC, would be expected to be anti-viral. However, novel inclusion of a bacterial PAMP, such as flagellin, to a viral vector would induce the expected antiviral responses plus an additional array of anti bacterial responses - this vaccine induction of two classes of innate responses should enhance the vigor and breadth of immune responses when encountering Plasmodium infection with induction of both antiviral and antibacterial responses (an unnatural response dictated by the nature of the novel vaccine). In the future, innate Plasmodium immune activators may be used to further enhance vaccine efficacy.

Flagellin, TLRS ligand as a vaceisie adjuvant

|0059| Flagel lin is the integral component of Flagel Sa, structures that certain bacteria have that are responsible for motility. In isolates of Salmonella there are two genes that encode the flagellar antigens. FliC encodes phase I flagellin and FljB encodes phase II flagellin. (Zieg J. Science 1977; 196: Γ70-2.) Both the FliC and FljB encode N and C domains that form part of the flagellar structure. (McQuiston JR. J. Clin. Microbio. 2004; 42: 1923-32.) Importantly, both contain motifs that are recognized by TLRS.

[0060] In addition to flagelli detection by TLR5, there is a second flagellin detection system based on the NLRC4 iiiflammasome complex. (Zhao Y. Nature 2011; 477: 596-600.) The mechanism of NLRC4 inflammasome complex activation has been extensively studied. (Franchi, L. Nat. Immunol. 2006; 7: 576-682., Franchi, L. Eur. J. Immunol. 2007: 37: 3030-39, Miao, EA. Nat. Immunol. 2006; 7: 569-75., Miao EA., Proc. Nat, Acad. Sci. 2008; 105: 2562-67, Miao EA, Proc. Nat. Acad. Sci. 2010; 107: 3076-80). Activation of the inflammasome leads to release of mature IL-IB, IL-18, and pro-inflammatory cytokines. (Jordan, JA. J. Immunol. 2001 ; 167: 7060-68, Birreil MA. Pharmacol. Ther. 2011; 130: 364-70, Stittwaia, FS. J. Exp. Med. 2007; 204: 3235-45). The components of the inflammasome are found in the cytosol, thus the signaling flagel lin is detected in the cytosol. (Franchi L. Nat. Immunol 2012; 13: 325-32.)

[0061] Interestingly, Flagellin the iigand for TLR5, has shown utility in vaccine formulations as an adjuvant. (Wang, BZ. J. Virol. 2008; 82: 11813-23, Huleatt, JW. Vaccin 2008; 26: 201- 14, Le Moigne, V. Mol. Immunoio, 2008; 45: 2499-2507, Wang, BZ. Clin, and Vaccine Immunol. 2012; 19(8): 3 119-25). Furthermore, Flagellin has been shown to be an effective adjuvant in physical association (formulation mixtures) within vaccine antigen preparations, or expressed as a fusion with targeted antigens or lastly, co-incorporated into viral particles with target antigens such as in vims-like particles, (VLPs). (Huleatt, JW. Vaccine 2008; 26: 201-14, Mizel, SB. Clin. Vaccine. Immunol. 2009; 16: 21-28, Wang, BZ. J. Virol. 2008; 82: 11813-23). The recognition of flagel lin and TL 5 is not associated with the central variable region (Anderson -Nissen E. Proc. Nat. Acd. Sci. 2005; 102: 9247-52.). However, there are conflicting reports as to the importance of removing the hyperimmune central variabl e region. (Ben-Yedidia T. Immuno Lett. 1998; 64: 9-15, Ncmpont C. J. Immunoio. 2008; 181 : 2036-43.) Interestingly, there are reports that suggest the adjuvant effects of Flagellin may drive specific mucosal immune responses, and furthermore suggestions are that these would be more effective via specific routes of immunization, e.g., mucosal surfaces such as intranasal. (De FiletteM. Virology 2008; 337: 149-61 , Liang B, J. Virol, 2001 ; 75: 5416-20).

[0062] Specific Inventive embodiments of the invention accordingly include: Coexpression by a recombinant or synthetic or engineered or no s -naturall occurring poxvirus of one or more exogenous or heterologous antigens or Immuiiogeiis and one or more PAMP modulators as an adjuvant. Accordingly, the invention comprehends a poxvirus vector developed to specifically express the Flagellin PAMP responsible for activation of TLR5 for enhanced adaptive immune responses to co-expressed antigen(s) or immunogen(s). In certain embodiments the poxvirus is an attenuated (as to mammals) poxvirus, such as NYVAC, MVA, MVA-BN, canarypox, fowfpox, ALVAC, TROVAC. In such embodiments, to specifically target and trigger the Flagellin PAMP responsible for activation of TLR5 for enhanced adaptive immune responses to co-expressed antigen(s) or immunogen(s), the poxvirus contains DNA coding for and expresses the entire or operable binding portion of the bacterial protein Flagellin. The operable binding portion of the Flagellin, is the portion responsible for binding to and activating the TLR5 receptor, resulting in a cascade of immune stimulatory pro-inflammatory responses to the targeted vaccine antigen. The Flagellin or operable binding portion is expressed either as peptide or fusion with antigen(s) or immunigen(s) provides for a multiplicity of options; the key to Flagellin or operable binding portion thereof expression is that the Flagellin operably and specifically agonize TLR5 to further stimulate "adjuvant" adaptive immune responses to expressed antigeii(s) or immunogen(s).

[0063] The invention thus comprehends a synthetic, engineered, recombinant or non- naturally occurring poxvirus, e.g., vaccinia, vector developed to specifically replicate in human tissues to a level intermediate of that of the parental replication competent strain, e.g., Copenhagen, and the replication deficient stain e.g., NY VAC, MVA, MVA-BN (e.g., via K1 L being present in the vector) and further developed to co-express Flagellin or an operable bindi g portion thereof (e.g., to deliver the Flagellin PAMP responsible for activation of TLR5) and at least one a tigen or immunogen for which an adaptive immune response is desired whereby the poxvirus provides agonist(s) for one or several TLRs, e.g., TLR5 and a resulting cascade of immune stimulatory pro -inflammatory responses to the antigen(s) or immunogen(s). In such embodiments NYVAC vectors are preferred.

Malaria

[0064] Malaria is considered one of the most important parasitic diseases in the world. It is estimated that malaria caused over 200 million clinical episodes worldwide resulting in 655,000 deaths, mostly African children (WHO Global Malaria Program 2011). Furthermore the economic losses are magnified as most of the endemic countries are impoverished, costing some 3 billion dollars in Africa alo e. (Teklehaimanot A. J. Trop. Med. Hyg. 2007; 77(6): 138-44. There have been substantial efforts and resources directed to methods and approaches for control-intervention such as indoor spraying, insecticidal nets, rapid diagnostics for testing, especially pregnant woman and children (Aponte JJ. Lancet 2009; 374(9700): 1533-44., Meiiendez C. La cet Infect. Dis. 2007; 7(2): 126-35), However, as these control interventions programs have had a measured degree of success, it is with the realization that to substantially reduce disease costs and burden to society vaccines agai st malaria are crucial to reduce the morbidity and mortality of this disease. (Malaria Eradication: Vaccines PloS Med. 2011 ; 8(1 ): el000398.) A focused effort and strategic goal was put forth by the international organization PATH, Malaria Vaccine Initiative (MVI), that by 2020 malaria vaccines provide 80% protective efficacy against P. falciparum. Importantly, if vaccines are to contribute to malaria eradication, they need to have an impact on preventing malaria transmission, these are known as transmission blocking vaccines.

[0065] Intensive malaria vaccine research has encompassed several decades and has yet to overcome substantial hurdles associated with complexities of the parasite life cycle, specifically, antigen expression during different parasite life stages and variability of a tigens or important epitopes from different parasite isolates. Although many develop anti-parasitic immunity by repeated natural exposure, reproducing this by vaccination has been difficult. (Langhome, i. Nat. Immunity 2008; 9: 725-32., Goodman AL. Ann. imp Med. Parasitoi. 2010; 104: 189-211). Vaccine candidates have targeted the pre-erythrocytic liver stage, blood stage or transmission blocking stage, (Dubovsky F. In: Plotkin SA Vaccines 2004; pl283-9„ Plos Med 2011; 8(1): el 000400., Aide P. Arch Dis. Child. 2007; 92(6): 476-9.)

[0066] In pre-erythrocytic vaccines, the immune response would direct antibodies to invading Plasmodium sporozoites delivered by mosquitoes and target infected liver cells with humoral and cellular immunity with the hope to prevent or limit parasites from entering red blood cells, thus avoiding clinical symptomology and any risk of further infection and transmission. Pre-erythrocytic vaccines were the first attempted modern vaccines against malaria, and currently the basis of the GlaxoSmithKline (GSK) RTS,S vaccine, the furthest along the clinical pipeline currently in phase III trials. (Nussenzweig RS. Nature 1967; 216(5111): 160- 2,, Rieckmann H. Bull. WHO 1979; 57(Suppl. 1 : 261-5.) The GSK vaccine uses the central repeat region of the circumsporozoite protein (CSP) and hepatitis B surface antigen (H BsAg) as an immunogenic carrier. Efficacy results of the RTS,S in adults and children are reviewed. ( Bojang KA. Vaccine 2005; 23(32): 4148-57., Macete E. Trap. Int. Med. Health 2007; 12(1): 37- 46., Alonso PL. Lancet 2004; 364(9443): 1411-20., Alonso PL. Lancet 2005; 366 (9502): 2012- 8.) Several vaccines have focused on delivering attenuated sporozoites, or sporozoite antigens, through a variety of methods including viral vectors such as MVA. (Hoffman SL. J. Infectious Dis. 2002; 185(8): 1 155-64., Bejon P. N. Jour. Med. 2008; 359:2521-32., I oestenberg M. Lancet 2011; 377(9779): 1770-6., Hoffman SL. Hum. Vacc. 2010; 6(1): 97-106., Hill AV. Philos. Trans. Soc. Lond. B. Biol Sci. 201 1; 366(1579): 2806-14., Liu MA. Immunity 2010; 33(4): 504-15., Draper SJ. Cell Host Microb 2009; 5(1): 95-105). NYVAC-Pf7 directs immune responses to sporozoites, and all other life cycle stages, including induction of antibodies that have been shown to block Plasmodium transmission by mosquitoes - NYVAC-Pf7.1 and NYVAC-Pf7.2 are expected to enhance immunogenicity.

[0067] The asexual blood stage vaccines attempt to block parasite infection of red blood cells. It is this stage of rapid parasite replication that leads to the onset of clinical symptoms of the disease. Vaccines directed to this stage would only hope to limit or reduce the level of infection and therefore the severity of the symptoms, therefore blood stage vaccines should only be considered as part of a multi-component malaria vaccine. (Thera MA N. Jour. Med. 201 1; 365(1 1): 1004-13). Targeted blood stage antigens that have been evaluated are the apical membrane protein (AMA1), merozoite surface protein (MSP)l, 2 and 3, (SERA5) erythrocyte binding antigen 175(EBA 175), glutamme-rieh protein long synthetic peptide (GRURP) and ring-infected erythrocyte surface antigen (RESA), (WHO, "The Rainbow Tables" Initiative for Vaccine Research 2010). The results of 40 phase 1/11 trials directed to blood-stage candidate vaccines have been very disappointing, showing at best "reduced parasite density". (Goodman, AL. Ann. Trap. Med. Parasitol. 2010; 104(3): 189-21 1 , Genton B. J. Inf. Dis. 2002; 185(6):820- 7., Ogutu, BR. Plos ONE 2009; 4(3) e4708, Thera MA. N. Eng. J. Med. 2011; 365(11 ): 1004-13. Sheehy, SH. Mol. Ther. 2012; 20(12): 2355-68.) Antigenic variation of the blood stage antigens represents one of the biggest hurdles for vaccines directed to these antigens. (Ellis RD. Human Vaccines 2010; 6(8): 627-34). However, naturally acquired immunity (or the bites of one thousand irradiated mosquitoes) induces resistance to Plasmodium infection - this encourages development of novel vaccines such as YVAC~Pf7.1..

A great deal of malaria vaccine research (pre-erythrocytic) has been devoted to studies using rodent malaria species for the development of chimeric rodent/human models with hopes of better assessing a variety of vaccine candidates and vaccine delivery platforms applicable to human P. falciparu before entering clinical trials. (Miambo, G. Eukaryot. Cell 2008;7(11); 1875-1879, Langhome J. Chem. Immunol. 2002; 80: 204-228) P. yoelii and P. chabaudi rodent malaria species have been utilized to demonstrate protection against blood stage parasitemia by vaccines expressing MSP1 and AMA1. (Draper Si. Nat. Med. 2008; 14: 819-821, Biswas, S. J. Immunol. 2012; 188(10): 5041-53.) A third rodent model, P. herghei has been widely used to stud)' pre-erythrocytic and transmission blocking vaccines. (Kaba SA. J, Immunol. 2009; 183(1 1): 7268-77, Sridhar, S. J. Virol 2008; 82(8): 3822-33, Blagborough AM. Vaccine 2009; 27(38): 5187-94) P. herghei has proven to be much more difficult to generate protective responses against than either P. yoe!ii or P. chabaudi. (Yoshida S. Pios ONE 2010; 5( 10) el 3727, Weiss R. Vaccine 2010; 28(28): 4515-22) It is this difficulty that makes the P. herghei system of great interest as a model, possibly leading to better preclinical analysis for potential pre- erythocytic vaccines for P. falciparum. (Goodman, AL. Sci Rep. 2013; 3: 1706.) The complexity of immune responses induced by different poxvirus vaccine vector strains is not ful ly understood. In the case of immunization with different poxvirus vectors expressing CSP, NYVAC stands out by inducing high levels of protection of mice. A full understanding of poor results from vaccinia virus strains WR and Wyeth expressing CSP has not been achieved. High levels of protection induced by NYVAC-K!L expressing P. berghei CSP has furthered the notion that NYVAC based vectors have potential as human malaria vaccine candidates (Lanar DE, Infect Immun. 1996; May;64(5): 1666-71).

[0068] Sexual Stage vaccines, or transmission blocking vaccines are vaccines that target the sexual stage of Plasmodia by blocking the fertilization of gametes in the mosquito midgut, thus preventing further development in the vector and subsequent rounds of new infections. Although not fully understood, it is believed that ingested sexual stage antibodies, complement and cytokines inhibit oocyst development in the vector. There are four main sexual stage antigens that have been targeted in early preclinical studies, antigens from the gametocyte P230, P48/45 and antigens from zygote P28 and P25. (Arevalo-Herrera M. Mem. Inst. Oswaldo Cruz. 2011 ; 106 suppl. 1 :202-1 1). P25 is the only sexual stage antigen to reach later stage vaccine clinical trials. Additional transmission blocking vaccine targets would include antigens of the ookinete, (DinglasanRR. Trends Parasitol. 2008;24(8) :364-70). Interestingly, it has been shown that antibodies generated against the mosquito mid-gut antigen aminopeptidase-N (AgAPNl) are effective in blocking ookinete invsion. (Dinglasan RR. Proc. Nat. Acd. Sci. 2007; 104(33): 13461-6.)

[0069] Great hopes have been placed in the GSK RTS,S malaria vaccine, currently in late phase III trials. Data from the most recent RTS.S trial (2011) have included a target population of children from 5 -17 months old. Using a 14-month follow up, the vaccine was found to have an efficacy of 50.4% as scored by the first clinical episode. (N. Eng. Jour. Med 2011 First Results of Phase 3 trial of RTS,S/AS01). Currently an additional large Phase III RTS,S trial is underway looking at establishing efficacy in a target population of children just 6-12 weeks old. However, as the results are encouraging from the RTS,S trials, it is understood that this vaccine will not be fully efficacious. It is already apparent even before licensure, that second generation vaccines are desperately needed to provide greater protection. As discussed above, there is a focused effort and strategic goal put forth by the international organization PATH, and the Malaria Vaccine Initiative (MVI), that by 2020 malaria vaccines provide efficacy approaching 80%. it. is clear that non-vaccine approaches and measures such as vector control and drug treatments have failed in controlling malaria and several other infectious diseases (Henderson DA. Vaccine 1999; 17(sup3): 53-55). Many investigators believe a successful malaria vaccine will only be achieved with multistage, multi-component vaccines targeting several stages of this complex parasitic organism. (Richie TL. Nature 2002; (415):694-70L, Heppner DG. Vaccine 2005; 23: 2243-50., Malaria Eradication: Vaccines PloS Med. 201 1; 8(1): el 000398.) Interestingly, natural protection in endemic areas seems to be achieved by the slow acquisition of immune responses acquired over years of uncomplicated exposure to a variety of diverse malaria antigens. Semi-immune adults remain susceptible to asymptomatic parasitemia, but importantly, are protected against clinical disease. However, this protective immunity is short-lived and lost after only a few years without repeated malaria exposures. (Thera MA. A nu. Rev. Med. 2012; 63: 345-357)

[0070] Specific embodiments of the invention include: Coexpression of Flageilm or an operable binding portion thereof and Plasmodium antigen(s) or immunogen(s), advantageously by a poxvirus vector, and more advantageously by a poxvirus vector that has reproductive capability via 1L. The invention comprehends poxvirus, e.g., vaccinia, vectors developed to specifically deliver the Flageilm PAMP responsible for activation of TLR5 for enhanced adaptive immune responses to co-expressed Malaria aiitigen(s). The invention thus comprehends a non-naturally occurring or synthetic or engineered or recombinant poxvirus, e.g., vaccinia vector that contains DNA for and expression of multiple P. falciparum antigens for which adaptive immune responses are desired and the entire or a binding portion of the bacterial protein Flagelliii, and advantageously the poxvirus vector has reproductive capability via K1L. The binding portion of Fiagel Sin, is the portion responsible for binding to and activating the TLR5 receptor, resulting i a cascade of immune stimulating pro-inflammatory responses to the co- expressed P. falciparum antigen(s). The mode in which Flagellin or the operable binding portion thereof is expressed (either as peptide or fusion) with P. falciparum antigen(s) provides for a multiplicity of options; the key is that the expressed Flagellin or portion thereof is operable to specifically agonize TLR5 to further stimulate adjuvant adaptive immune responses to co- expressed Malaria antigens. A particularly preferred embodiment is a non-naturaily occurring or recombinant or synthetic or engineered poxvirus, e.g., vaccinia, that co-expresses 1 L, Flagel lin or an operable binding portion thereof and o e or more Malaria antigen(s) or immunoge (s). An enhanced NYVAC or MVA or MVA-BN vector (e.g., one that expresses 1 L) replicates in human tissues to a level intermediate of that of the more virulent parental replication competent strain Copenhagen and the replication deficient stain NYVAC or MVA or MVA-BN. When such an enhanced vector further co-express at least one P. falciparum antigen(s) or immunogen(s) for which adaptive immune responses are desired and the entire or a binding portion of the bacterial protein Flageilin (wherein the binding portion of the Flagellin is the portion responsible for binding to and activating the TLR5 receptor), a cascade of immune stimulating pro-inflammatory responses to the co-expressed P. falciparum antigen(s) or immunogen(s) results. The Flageilin sequence and species and mode in which Flagellin is expressed (either as peptide or fusion) is selected to specifically agonize TLR5 to further stimulate adaptive immune responses to P. falciparum.

[0071] The invention also comprehends P. falciparum antigen(s) or immunogen(s) co- expressed with Flagellin or an operable binding portion thereof in vitro. After infecting cells in vitro with an inventive recombinant, the expression products are collected and the collected malarial expression products can then be employed in a vaccine, antigenic or immunological composition which also contains a suitable carrier.

[0072] Alternatively, the ^"viral, vector system, especially the preferred poxvirus vector system, of the invention can itself be employed in a vaccine, immunological or immunoge ic composition which also contains a suitable earner. The recombinant poxvirus in the composition expresses the malarial products and Flageilin or a binding operable portion thereof in vivo after administration or inoculation. Advantageously, the poxvirus has some reproductive capacity, e.g., from KIL being present in an attenuated (as to mammals) poxvirus such as a NYVAC, ALVAC, TROVAC, VI VA. MVA-BN, avipox, eanarypox, or fowlpox,

[0073] The antigenic, immunological or vaccine composition of the invention either containing products expressed or con taming a recombinant poxvirus is administered in the same fashion as typical malarial antigenic immunological or vaccine compositions (e.g., NYVAC- Pf7). One skilled in the medical arts can determine dosage from this disclosure without undue experimentation, taking into consideration such factors as the age, weight, and general health of the particular individual.

[CI074] Additionally, the inventive recombinant poxvirus and the expression products therefrom stimulate an immune or antibody response in animals. From those antibodies, by techniques well-known in the art, monoclonal antibodies can be prepared and, those monoclonal antibodies, can be employed in well known antibody binding assays, diagnostic kits or tests to determine the presence or absence of particular malarial an tigen(s) and theref om the presence or absence of malaria or, to determine whether an immune response to malaria or malarial antigen(s) has simply been stimulated. Monoclonal antibodies are immunoglobulins produced by hybridoma ceils. A monoclonal antibody reacts with a single antigenic determinant and provides greater specificity than a conventional, serum-derived antibody. Furthermore, screening a large number of monoclonal antibodies makes it possible to select an individual antibody with desired specificity, avidity and isotype. Hybridoma ceil lines provide a constant, inexpensive source of chemically identical antibodies and preparations of such antibodies can be easily standardized. Methods for producing monoclonal antibodies are well known to those of ordinary skill in the art, e.g., Koprowski, PL et al., U.S. Pat. No. 4,196,265, issued Apr. 1, 1989, incorporated herein by reference. Uses of monoclonal antibodies are known. One such use is in diagnostic methods, e.g., David, G. and Greene, H., U.S. Pat. No. 4,376,110, issued Mar. 8, 1983, incorporated herein by reference. Monoclonal antibodies have also been used to recover materials by immunoadsorption chromatography, e.g. Milstein, C, 1980, Scientific American 243:66, 70, incorporated herein by reference.

[0075] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

j l Examples

[0076] Embodiments of this invention include: NYVAC-PF7.1 (AMA1 repair + FliC) and NYVAC-PF7.2 (AMA1 repair + FliC + K1L).

[0077] EXAMPLE 1: The development of the Improved YVAC vaccine vectors for Malaria NYVAC-PF7J (AMA1 repair + FHC) and NYVAC-PF7.2 (AM 1 repair + FliC + K1L) are based on NYVAC-PF7 that is described in US Patent No. 5766597, incorporated herein by reference. Modification to these genetic sequences, description of the donor plasmids and methods used for the construction of recombinant vims, are detailed and set forth as follows.

[0078] Doraor Plasmid constructions ami primer sequences for YVAC-PF7.1

[0079] FliC

[0080] Dry pellets of Salmonella enterica are readily available and were obtained from the University of New Hampshire (e.g., Robert ooney). The S. enterica coding sequence and flanking sequences were amplified using primers RW3 and RW4 then digested with BamHI and EcoRI generating a 1.5kb fragment.

[0081] RW3: TATTCAAGCTTGAATTCGTGTCGGTGAATCAATCG

[0082] R.W4 : AACTCT AG AGG ATCC AAT A AC ATCAAGTTGTAATTG

[0083] The 1 ,5kb BamHI-EcoRl fragment containing the FliC coding sequence was inserted into the 2.7kbp BamHI-EcoRI fragment of plasmid pSV~ Gal (Promega, Madison, WI), yielding plasmid pRW2.

[0084] The Pi promoter, previously described in US Patent No. 5766597, incorporated herein by reference, was used to drive the expression of FliC.

[0085] The Pi promoter sequence:

ACTGTAAAAATAGAAACTATAATCATA.TAA.TAGTGTAGGTTGGTAGTAGGGTACTCG

1 GAI TAATTT 1 ΛΤΊ GTi ΛΛΛΓΠ Π (Ί ΤΛΛ< Ύ( ^"ΓΊ^'ΛΛ(Π (Ί ΤΛ 1 ΤΛΛΤΛ 1 G

[0086] A Pi promoted fragme t was synthesized by IDT (Coraiville, IA). The Pi promoted synthetic fragment contained the 5 ' and 3' FliC coding sequences. This fragment was inserted between the Hindlll-Xbal of pZErO-2 (Invitrogen, Carlsbad, CA) yielding plasmid pRW8.

[0087] The sequence of the pRW8 insertion:

AGATCTACTGTAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTAGG

GTACTCGTGATTAATTTTATTGTTAAACnTGTCrrTAACn TTAAGTCri ATTAATATG

GCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAACAA

XL ATCCCAGTCCGCTCTGGGCACCGCTATCGAGCGTCTGTCTTCCGGTCTGGTACCTCC (ίΊ^'Ί I^'i iC C !(^'Α( )(ί( ^'(}ΛΛί C ^'Ai iC i l (^'(^'( ·( ^'ΛΛΛΛ(^'( !^'!^'( ( ^'' IX ΊΧ ΊΊΎΛΓΪ(Χ ΟΪΤΛΑΤΊΊΎ TATCTCGAGGCCAATTAGGCCTATTATATTTTTTATCTAAAAAACTAAAAATAAACA ΤΤ⁽ ;ΛΊΊΛΛΛΤ⁾Ύί\\ΛΤΛΤΛΛΤΛ(Ύ1 ΛΛΛΛΤ^^^{^}

TATGTATTTTGAGGAAATTGATAATGAGTTAGATTACGAACCAGAAAGTGCAAATG

AGGTCGCAAAAAAACTGCCGTATCAAGGACAGTTAAAAGAATTC

[0088] AMA1 Repairs

[0089] ΑΜΑ coding sequences from in the original NYVAC-PF7 had several regions that needed to be modified for complete authentic AM A! expression. Firstly, the constructed repairs removed a 5-amino acid (RRI S also called IKSRR, both the same insert with reading from different ends) accidental insertion between amino acids 377 and 378 of AMA1 , secondly, it was necessary to modify sequences encodi g a early tra scription termi ation signal (T5NT) fou d between nucleotide positions (1436-1442) in the AMAl coding sequences and lastly to remove unnecessary DNA sequences 3' of the original NYVAC-Pf7, AMAl coding sequences. Preliminary experiments repairing IKSRR demonstrated a change of small Pf7 plaques on CEF ceils to an increase of plaque size approaching the size of NYVAC plaques.

[0090] pRW55 construction

[00 J ] Plasmid pRW55, containing AMAl repairs and Pi promoted FliC, was constructed in the following manner. Full length Pi promoted FliC was constructed by insertion of a L3kb pRW2 Bbsl-Kpnl fragment, containing the central coding portion of FliC, between the Bbsl and Kpnl sites of pRW8 followed by PGR with the primers VC106/VC107. The product of PGR from NYVAC with the primers VC68/VC105 was combined with the VC 106/107 fragment for PGR with the primers VC98/VC106. Three PGR fragments derived from Pf7 with the primer pairs VC1 10/VC91 , VC103/VC109 and VC 108/104 were combined for PGR with the primers VC110/VC104. Fragments derived with the primers VC98/VC1 Q6 and VC ! H) VC I 04 were combined for PCR with the primers VC97/VC98, followed by digestion with Sail for insertion into the Sail site of pUC19 (Yanisch-Perron, C. Gene 1985; 33(1 ): 103-19.), yielding plasmid pRW55.

[0092] Primer Sequences

[0093] VC68 : AATAGACCTGCTTCGTTGGCCTC

[0094] VC 1 ; AGCACTTTTG ATC AT ACTAGCGTTCTTATTTTTG [0095] VC97 : CCTAC AGGTCGACC ATTACACCAGGAACATACATACC

[00961 VC98: ( C ^"i^'ACAGC JTC XiACCATA!XXX ΠΎΤΠΧΧ X ^'ΛΛΤΛΤί ^'Λί^'

[0097] VC 103 : GAACGCTAGTATGATCAAAAGTGCTTTTCTTCCCACTGGTGCT

[0098] VC I04:

ΤΛΟΥί ^"Ι^'ί X ^'Ί^'ί G AG( Ί (;Λ( 7\0ΛΤ( ^"Ι^'Λ I ΛΛΛ ΑΑΤΤΛ ΑΊ AG I ΛΤί id I 1 ^'ΓΊ^'ί X ^'ΛΊ ( AG

[0099] VC105:

GATCTGTCAGCTCGAGGAGACTAGTCGTAGGGCCCGGCCGTGGCAATATTCTGTA

[00100] VC106:

(ΪΑΊΧΪΟΛΛΛΛΛίΧ ΛΊ^\( Ί^'ΛΊΊ ΛΛΊΊ ΓΊΊΑ ΓΛίίΛΊΧ Ί^'Λ(ΊΧΓ! ΛΛΑΛΑΊΑΟΛΛΛ(Ί Λ^'Ι^' [00101] VC107:

ΑΊΑ ΓΊΧ Λ(Χ ^'( (;θ(;(Χ^'ίΊ^·Λ(Χ_ίΛ(Ί Λ(ΠΧΊ(ΧΊ(Χ_!Λί_!ΛΊ ΛΛΑΛΑ·Γ!ΛΛ( Ci(-A<;

[00102] VC 108: AGCTCC AAGAATATTC ATTTC AGATGATAAAGACAGTTTAAAATG [00103] VC109:

CATCTGAAATGAATATTCTTGGAGCTATAATTTTTTTATTCCCTTCATCATC

[00104] VC 110: TGACTAAATATTTAACATTCCCAAGATGATTC

[OOlOSj Sequence of 3.9kb pRW55 insertion:

CATCCACTATATTGTTTTGCACATCTCTACCATTAACTAGAAACAAATCAAAGAAAA

TCAAAAA(LACAATGAC rAAATATTTAA(LATTCXXi;AAGATGATTCATTTTATATTGTA.

ATTATATATTTTCAATTTTGAGGATCAGCTTACATCATGCAGTGGTTAAACAAAAAC

ATTTTTATTCriX^AAATGAGATAAAGTGAAAATATATATCATTATATTACAAAGTACA

ΛΊΊΑΊΊΊ ΛθίΠΊΊ ΛΛΊ ΛΊΧίΛ(ίΛΛΛΛΓΓΛ ! Λ(ΊΧ( ίΠ^\ΓΓΛ ! lAl ! CiACiCXK (ΊΊΊΟΛ

GTTTACATATATGATAAACTTTGGAAGAGGACAGAATTATTGGGAACATCCATATCA

AAATAGTGATGTGTATCGTCCAATCAACGAACATAGGGAACATCCAAAAGAATACG

AATATCCATTACACCAGGAACATACATACCAACAAGAAGATTCAGGAGAAGACGAA

ΑΛΊ AC ΛΊ ^'ΓΛί A..\( Λ( ί JCATATC ΧΆΛΊ A AC X ^'A( GAAGC ΠΧΧ X G AA( X X G( ^'ACX^'AC ^'Λ

AGAACAAAATTTATTTTCAAGCATTGAAATAGTAGAAAGAAGTAATTATATGGGTA

ATCX^ATGGACGGAATATATGGC^AAAATATGATATTGAAGAAGTTCATGGTTCAGGT

ATAAGAGTAGATTTAGGAGAAGATGCTGAAGTAGCTGGAACTCAATATAGACTTCC

ATCAGGGAAATGTCCAGTA.TTTGGTAAAGGTATAATTATTGAGAATTCAAATACTAC

TTTTTTAACACCGGTAGCTACGGGAAATCAATATTTAAAAGATGGAGGTTTTGCTTT

TCCTCCAACAGAACCTCTTATGTCACCAATGACATTAGATGAAATGAGACATTTCTA

^4 TAAAGATAATAAATATGTAAAAAATTTAGATGAATTGACTTTATGTTCAAGACATGC

AGGAAATATGATTCXLAGATAATGATAAAAATTCAAATTATAAATATCCAGCTGTTTA

TGATGACAAAGATAAAAAGTGTCATATATTATATATTGCAGCTCAAGAAAATAATG

GT(XTrAGATATTGTAATAAAGA(XiAAAGTAAAAGAAACAGCATGTTTTGTTTTAGAC

CAGCAAAAGATATATCATTTCAAAACTATACATATTTAAGTAAGAATGTAGTTGATA

ACTGGGAAAAAGTTTGCCCTAGAAAGAATTTACAGAATGCAAAATTCGGATTATGG

GTCGATGGAAATTGTGAAGATATACCACATGTAAATGAATTTCCAGCAATTGATCTT

TTTGAATGTAATAAATTAGTTTTTGAATTGAGTGCTTCGGATCAACCTAAACAATAT

GAACAACATTTAACAGATTATGAAAAAATTAAAGAAGGTTTCAAAAATAAGAACGC

TAGTATGATCAAAAGTGCTTTTCTTCCCACTGGTGCTTTTAAAGCAGATAGATATAA

AAGTCATGGTAAGGGTTATAATTGGGGAAATTATAACACAGAAACACAAAAATGTG

AAATTTTTAATGTCAAACCAACATGTTTAATTAACAATTCATCATACATTGCTACTAC

TGCTTTGTCCCATCCCATCGAAGTTGAAAACAATTTTCCATGTTCATTATATAAAGAT

GAAATAATGAAAGAAATCGAAAGAGAATCAAAACGAATTAAATTAAATGATAATG

ATGATGAAGGGAATAAAAAAATTATAGCTCCAAGAATATTCATTTCAGATGATAAA

GACAGTTTAAAATGCCCATGTGACCCTGAAATGGTAAGTAATAGTACATGTCGTTTC

TTTGTATGTAAATGTGTAGAAAGAAGGGCAGAAGTAACATCAAATAATGAAGTTGT

AGTTAAAGAAGAATATAAAGATGAATATGCAGATATTCCTGAACATAAACCAACTT

ATGATAAAATGAAAATTATAATTGCATCATCAGCTGCTGTCGCTGTATTAGCAACTA

TTTTAATGGTTTATCrrrTATAAAAGAAAAGGAAATGCTGAAAAATATGATAAAATGG

ATGAACCACAAGATTATGGGAAATCAAATTCAAGAAATGATGAAATGTTAGATCCT

GAGGCATCTTTTTGGGGGGAAGAAAAAAGAGCATCACATACAACACCAGTTCTGAT

GGAAAAACCATACTATTAAT TTTATAGATCTACTGTAAAAATAGAAACTATAATCA

TATAATAGTGTAGGTTGGTAGTAGGGTACTCGTGATTAATTTTATTGTTAAACTTGTC

Ί^{^}Ί^{^}ΑΑ :ΊΧ:Ί ^,ΑΑΟΊ ΓΓΊ^ΓΊ ΑΑΊ^,ΑΤΟΟ( Α :ΑΑΟΊ ::ΑΊ^{^}Ί^,ΑΑΊΑ€ΑΑΑ :Α(;ΧΧ Ί^'αΊ Χ Τ

GTTGACCCAGAATAACCTGAACAAATCCCAGTCCGCTCTGGGCACCGCTATCGAGC

( ) ] (^' ] ( TTCX ί JGT( T( ·( ^'CiTATC Λ AC ^'Λί XX sC ^'G AAGACG/YFGi < sC ^'Λί -G^'l ( AGi ·( ^'G

ATTGCTAACCGTTTTACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCT

AACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAA

CAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACCAACTCCCA

GTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACC GTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACC

(I CiAC A rcXAC nii JI C ^'AAC iAC^'i rriiAAAi Ί^'ΛΊΪ (ΪΛΊΑ Γί ιΛΊΧΊαΛΛί -Λ

GATCAACTCTCAGACCCTGGGTCTGGATACGCTGAATGTGCAACAAAAATATAAGG

^'!^'( A(sC ί ΑΤΛ( X ·(}{ Ί^'ί ^'ΛΛΓΊ GTTAC ^'Λί iGATATGi X ΧλΛΤΛ( TAC ΧίΛ^'ΠΧ ΧΎΤΓΛί · Α( Λ

ATAGTACTTTTAAAGCCTCGGCTACTGGTCTTGGTGGTACTGACCAGAAAATTGATG

GCGATTTAAAATTTGATGATACGACTGGAAAATATTACGCCAAAGTTACCGTTACGG

GGGGAACTGGTAAAGATGGCTATTATGAAGTTTCCGTTGATAAGACGAACGGTGAG

GTGACTCTTGCTGGCGGTGCGACTTCCCCGCTTACAGGTGGACTACCTGCGACAGCA

ACrrGAGGATGTGAAAAATGTACAAGTTGCAAATGCTGATTTGACAGAGGCTAAAGC

CGCATTGACAGCAGCAGGTGTTACCGGCACAGCATCTGTTGTTAAGATGTCTTATAC

TGATAATAACGGTAAAACTATTGATGGTGGTTTAGCAGTTAAGGTAGGCGATGATTA

CTATTCTGCAACTCAAAATAAAGATGGTTCCATAAGTATTAATACTACGAAATACAC

TGCAGATGACGGTACATCCAAAACTGCACTAAACAAACTGGGTGGCGCAGACGGCA

AAACCGAAGT GTTTCTATTGGTGGTAAAACTTACGCTGCAAGTAAAGCCGAAGGTC

ACAACTTTAAAGCACAGCCTGATCTGGCGGAAGCGGCTGCTACAACCACCGAAAAC

CCGCTGCAGAAAATTGATGCTGCTTTGGCACAGGTTGACACGTTACGTTCTGACCTG

GGTGCGGTACAGAACCGTTTCAACTCCGCTATTACCAACCTGGGCAACACCGTAAAC

ΛΑ(ΧΊΧ}Λ<ΊΊΧ ^'ΊΧ ( (Π Λί ^'ίΠ^'ΛΊ ( ίΛΛ()ΛΊ^'Ί^'( ( (ΪΛ( ^'Ί^'Λ(^'ί :ΧίΛ(Χ ί ;ΛΛ(}^'ΓΊ ^'!^'(^'( ^'

AACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACCTCCGTTCTGGCGCAGGCG

ΛΛ( ί Λ(ί( ΠΊΧ ΧΧίί^{' '}ΛΛΛΛί Χ.Π ( ί Ί^'ί Ί^'ί ^'ΠΊ AC ΊΧ ΧΠΎΛΛΊ ΊΤΓ ΛΊΧ ^"f^'C XJAGGAGAC ^'Τ

AGTCGTAGGGCCCGGCCGTGGCAATATTCTGTATTACGTATTATATATGTAATAAAC

GTTCACGTAAATACAAAACAGAGAACAAAGTCTAGATTTTTGACTTACATAAATGTC

TGGGATAGTAAAATCTATCATATTGAGCGGACCATCTGGTTCAGGAAAGACAGCCA

TAGCCAAAAGACTATGGGAATATA.TTTGGA.TTTGTGGTGTCCCA.TACCACTAGATTT

( X Ί ( C ΠΧ ΧΊ ΛΊ ( ;G Λ( GAGAAGC sTGTC X JATTAC X ^'Λ^'Π Λ( GTTAACAC Ϊ AC Ϊ AC ΧΚ X^' ΆΤ(

TGGAAGGGAATAGCCGCCGGAAACTTTCTAGAACATACTGAGTTTTTAGGAAATATT

ΤΛ(Χί<;ΑΛ(^'ΊΊ <Ί ΛΛΛΛ(Ί Gi ΊΧΊΎΟΑΛ

ATGG ΑΤΤΤ AAAC ATC G AC GGTGTT AG AAGTTTT A ΑΑΑΑΤ ACTT AC CT AATGC CTT AC TCGGTGTATATAAGACCTACCTCTCTTAAAATGGTTGAGACCAAGCTTCGTTGTAGA AACACTGAAGCTAACGATGAGATTCATCGTCGCGTGATATTGGCAAAAACGGATAT GGATGAGGCCAACGAAGCAGGTCTATTCGACACTATTATTATTGAAGATGATGTGA ATTTAGCATATAGTAAGTTAATTCAGATACTACAGGACCGTATTAGAATGTATTTTA

ACACTAATTAAAGACTTAAGACTTAAAACJrTGATAATTAATAATATAACTCGTTTTT

ATATGTGGCTATTTCAACGTCTAATGTATTAGTTAAATATTAAAACTTACCACGTAA

AACTTAAAATTTAAAATGATATTTCATTGACAGATAGATCACACATTATGAACTTTC

AAGGACTTGTGTTAACTGACAATTGCAAAAATCAATGGGTCGTTGGACCATTAATAG

GAAAAGG

[00106] Figure 1 illustrates primer locations.

[00107] Additions! Donor Plasmid construction asid primer sequences specific for NYVAC-PF7.2 (AMAl repair + FliC + K1L)

[00108] The vaccinia virus Copenhagen strain 1L promoted 1L coding sequence (Giliard et al., 1986) was synthesized at TOP Gene Technologies (Montreal, Canada) as a fragment similar to the Bgill (partial)-Hpal fragment described in Perkus et al., 1989: Xhol was added to the 5' end and Spel was added 3' of HpaL The synthetic DNA was inserted between the Ascl and Pad sites of an intermediate cloning shuttle pAPGlO, yielding plasmid p ! L.

[00109] Plasmid pR.W56 was constructed by insertion of the 1 Kb Xho!-Spel fragment from p IL, containing the K1 L expression cassette, between the Xhol and Spel sites of pRW55. The synthetic DNA sequence and its position are illustrated in Figs 2 A, 2B.

Generation of recombinant virus

[00110] in vivo recombination (IVR) was performed by transfection of donor plasmid (8ug) with Lipofectamine 2000 as per manufacturer specification (invitrogen, Carlsbad, California) into 1 E6 poxvirus infected Vero cells using a multiplicity of infection (MO I) of 0.1. Donor plasmid pR.W55 was used in an IVR. with NYVAC-PF7 to generate the recombinant NYVAC- PF7. 1 containing AMAl repairs plus FliC. Donor plasmid pRW56 was used in an IVR with NYVAC-PF7.1 to generate NYVAC-PF7.2 containing AMAl repairs, FliC plus K1L.

[00111] Recombinants were identified by polymerase chain reaction (PGR). Briefly, one PGR primer was located within newly inserted sequences not present in NYVAC-Pf7. The second primer, directed toward the first, was located in sequences outside of the donor plasmid. Location of the primer outside of the donor plasmid ensured no amplification of the donor plasmid. [00112] After the IVR, virus was serially diluted in 96 well plates. Between 1-10% of each single well was used in PGR analysis. Wells identified as positive by PGR were repeatedly serially diluted for several rounds of infection and further tested by PGR.

[00113] After several rounds of PGR analysis, a second set of PGR primers were used to assess purity. The second primer set contained sequences present in the original NYVAC-Pf7 that flanked the insertion site; input NYVAC-P17 control virus would yield a PGR fragment smaller than a NYVAC-Pf7.1 recombinant containing an insertion. Following detection of a high level of purity by PGR using the 96 well, format, virus was further purified by plaquing under agarose (Perkus M. et a!,, .1993). Well isolated plaques were picked from agarose, amplified and screened by PGR with both sets of PGR primers. Purificatio sometimes requires more than one round of plaque purification under agarose.

[00114] Once a pure recombi ant was identified, the vims stock was amplified. All insertions were assessed for correct size by PGR. fragment analysis on agarose gels, and finally nucleotide sequence of all insertions and flanking sequences were confirmed. Expression analysis was confirmed by Western blotting. Figs 3A, 3B, 3C, 3D demonstrate expression by inventive recombinants. Compared with NYVAC-Pf7, construction of YVAC-Pf7.1 did not involve modifications of Pfs25 or CSP; it is important to note increased expression levels demonstrated in the Figs that may be due to FliC survival signals expressed by the novel vaccine candidate YVAC-Pf7.1 or by removal of potentially deleterious effects of RRIKS from AMAl .

[00115] Fig 3A shows expression of P. falciparum CSP from cell lysates two days post, infection: Lysates were separated by 10% SDS-PAGE for western blotting with colorimetric detection. Rabbit antibody (Alpha Diagnostics, San Antonio, TX) directed to P, falciparum CSP repeat sequence (NANP)₅. Lanes: 1 & 4, NYVAC; 2 & 5, NYVAC Pf?.l; 3 & 6, NYVAC Pf7. Compared with lanes 1-3, 20% of lysat.es were loaded on lanes 4-6.

[00116] Fig 3B shows expression of secreted P. falciparum CSP from infected cel l media two days post infection: Ceil media was separated by 10% SDS-PAGE for western blotting with colorimetric detection. Rabbit, antibody (Alpha Diagnostics, San Antonio, TX) directed to P. falciparum CSP repeat sequence (NA P)₅. Lanes: 1, NYVAC Pf7; 2, NYVAC; 3, NYVAC Pi-7.1 .

[00117] Fig 3C show^rs expression of P. falciparum Pfs25 two days post infection: Lysates were separated by 10% SDS-PAGE for western blotting and colorimetric detection: Rabbit anti- Pfs25 antiserum (ATCC, Manassas, VA). Lanes: 1, NYVAC Pf7 supernatant; 2, NY VAC Pf7 cell pellet; 3, N YVAC supernatant; 4, NYVAC Pf7.I supernatant; 5, NYVAC PfZ. l ceil pellet; 6, NYVAC cell pellet; 7, uninfected ceil pellet; 8, molecular weight marker.

[001 J 8] Fig 3D shows FliC expression two days post infection : 10% SDS-PAGE analyzed by western blotting and colorimetric detection. Mouse anti-FliC (BioLegend, San Diego, CA). Lane 1 , NYVAC Pf7.1 cells; 2, NYVAC Pf7.1 supernatant; 3, NYVAC supernatant; 4, NYVAC Pf? supernatant; 5 NYVAC Pf7 ceils; 6 NYVAC cells.

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[00120] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A recombinant or synthetic or engineered or non-naturaily occurring poxvirus that contains and expresses DNA encoding a heterologous or exogenous antigen, epitope or immunogen and Flageilin or an operable binding portion thereof.

2. The poxvirus of claim 1 that contains and expresses vaccinia host range gene K1L.

3. The poxvirus of claim 2 that is engineered to contain and expresses vaccinia host range gene K1L.

4. The poxvirus of claim 3 wherein the poxvirus is attenuated as to mammals.

5. The poxvirus of claim 4 wherein the poxvirus is a NYVAC, YVAC.l, NYVAC.2, avipox, canarypox, fowl pox, ALVAC, TRQVAC, MVA, or MVA-BN.

6. A method of inducing an immunological response against the antigen, epitope or immunogen in a mammal comprising administering to the mammal the poxvirus of any one of claims 1-5 or an immunological or immunogenic composition containing the poxvirus.

7. An immunological or immunogenic composition containing the poxvirus of any one of claims 1-5.

8. The poxvirus of any one of claims 1-5 wherein the antigen, epitope or immunogen is at least one Plasmodium antigen.

9. The poxvirus of claim 8 wherein the Plasmodium antigen(s), epitope(s) or immunogen(s) comprise SERA, ABRA, Pfhsp70, AMA-1 , Pfs25, Pfs36, CSP, PfSSP2, LSA-1 repeatiess, MSA-1, AMA-1 or combination' ) thereof.

10. The poxvirus of claim 9 wherein poxvirus contains DNA coding for and expresses Plasmodium antigen(s) CSP, PFSSP2, LSA-1 -repeatiess, MSA-1 , SERA, AMA-1 and Pfs25.

11. The poxvirus of claim 10 wherein the poxvirus is a NYVAC poxvirus.

12. An anti-malarial immunogenic or immunological composition comprising a poxvirus of claim 8.

13. An anti-malarial immunogenic or immunological composition comprising a poxvirus of claim 9.

14. An anti-malarial immunogenic or immunological composition comprising a poxvirus of claim 10.

15. An anti-malarial immunogenic or immunological composition comprising a poxvirus of claim 11.

16. A method for inducing an immunogenic or immunological response against malaria or Plasmodium in a mammal comprising administering to the mammal the poxvirus of claim 8 or an immunological or immunogenic composition containing the poxvirus.

17. A method for inducing an immunogenic or immunological response against malaria or Plasmodium in a mammal comprising administering to the mammal the poxvirus of claim 9 or an immunological or immunogenic composition containing the poxvirus.

18. A method for inducing an immunogenic or immunological response against malaria or Plasmodium in a mammal comprising administering to the mammal the poxvims of claim 10 or an immunological or immunogenic composition containing the poxvirus.

19. A method for inducing an immunogenic or immunological response against malaria or Plasmodium in a mammal comprising administering to the mammal the poxvirus of claim 11 or an immunological or immunogenic composition containing the poxvirus.