EP4598943A2 - Nouveaux antigènes vaccinaux recombinants à base de protéines multiples chimériques pour la prévention de la maladie de lyme chez des animaux et des êtres humains - Google Patents

Nouveaux antigènes vaccinaux recombinants à base de protéines multiples chimériques pour la prévention de la maladie de lyme chez des animaux et des êtres humains

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Publication number
EP4598943A2
EP4598943A2 EP23875683.7A EP23875683A EP4598943A2 EP 4598943 A2 EP4598943 A2 EP 4598943A2 EP 23875683 A EP23875683 A EP 23875683A EP 4598943 A2 EP4598943 A2 EP 4598943A2
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EP
European Patent Office
Prior art keywords
protein
seq
amino acid
sequence
proteins
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Pending
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EP23875683.7A
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German (de)
English (en)
Inventor
Richard T. Marconi
Nathaniel O'BIER
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Virginia Commonwealth University
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Virginia Commonwealth University
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Publication of EP4598943A2 publication Critical patent/EP4598943A2/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0225—Spirochetes, e.g. Treponema, Leptospira, Borrelia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • LD BACKGROUND OF THE INVENTION 02941631TA Lyme disease
  • LymeRix® The human LD vaccine, LymeRix® was available between 1998 and 2002. LymeRix® consisted of a single Borreliella protein called outer surface protein A (OspA). Sales of LymeRix® plummeted shortly after its introduction, leading to its voluntary withdrawal from the market by the manufacturer in 2002. The scientific limitations of LymeRix® were many. Most notably, it failed to induce long-term protection, mandating the need for frequent booster vaccinations. The requirement for multiple boosters can be traced to the mechanism of action of the vaccine and the environment-specific production of Borreliella outer surface proteins.
  • OspA outer surface protein A
  • the antibody titer to OspA is sufficiently high, preformed antibodies in the blood can bind to OspA on the cell surface and kill the bacteria in the tick through an antibody-dependent complement-mediated mechanism.
  • protective OspA antibody levels wane within 6 months of vaccination.
  • the antibody levels to OspA in the blood fall below a critical titer, some Borreliella cells are able to transmit into the vaccinated 02941631TA individual and establish an infection. Since the Borreliella are not producing OspA in mammals, they cannot be targeted by OspA antibodies in that environment.
  • a vaccine that can elicit memory immune responses would be highly beneficial since it would reduce the number and frequency of required boosters.
  • the vaccine formulation comprises two unique custom-designed recombinant proteins (chimeritopes) that are comprised of antigenic regions derived from four different B. burgdorferi proteins: OspA, OspB, OspC, and FtlA.
  • the vaccine antigens are designated herein as BAF and CE-BBB19 chimeritopes.
  • Chimeritopes are novel recombinant proteins created in the laboratory 02941631TA that comprise epitopes and/or specific protein segments derived from multiple different proteins or protein variants, and usually some unrelated but useful sequences.
  • An advantage of chimeritopes in general, which is well-documented in the literature, is that they can be designed to elicit antibodies that target the numerous species of Borreliella that cause Lyme disease in humans, companion animals, wildlife, and other mammals.
  • a vaccine that targets spirochetes in both ticks and mammals, kills through both complement-dependent and complement-independent immune mechanisms, and elicits memory immune responses, such as the vaccine disclosed herein, is even more beneficial because it increases vaccine efficacy and reduces the number and frequency of required boosters.
  • BRIEF DESCRIPTION OF THE FIGURES Figure 1. Demonstration that FtlA and FtlB are widely distributed among Lyme disease isolates and that antibodies to each protein cross-react. Borreliella isolates (indicated by labeling across the top of the figure) were grown to the mid-log phase, and then the cells were harvested and prepped for SDS-PAGE in AnyKda SDS-PAGE gels.
  • the fractionated cell lysates were transferred to membranes and screened with the antisera indicated to the left. MW markers are shown on the left (in kDa).
  • Figure 2. ELISA analyses of the Ftl proteins. The immunoreactivity of recombinant FtlA (r-FtlA), r-FtlB, r-FtlC, and r-FtlD with antiserum raised against each protein was assessed by ELISA (top panel) and immunoblotting. (Bottom panel). BSA served as the negative-control immobilized protein in the ELISA analyses. The antisera used in the assays are indicated along the x-axis, and the y-axis indicates the absorbance measured at 405 nm.
  • ELISAs were run in triplicate.
  • the recombinant Ftl proteins were also screened using an immunoblot format with each antiserum (from left to right: anti-FtlA, anti-FtlB, anti-FtlC, and anti-FtlD).
  • an immunoblot was screened with preimmune serum (not shown).
  • Figure 3A and B Bactericidal Activity of FtlA and FtlB antibodies. Hyperimmune sera raised against FtlA, FtlB, FtlC, and FtlD were tested for bactericidal activity against B.
  • the loop domain into which the A1 and A15 epitopes of OspA were inserted is indicated by the box on the left.
  • the small box on the top indicates the C-terminal domain of OspB.
  • the N-terminal domain of FtlA was joined to the construct at this site. Note that the location of introduced W residues is not indicated but can be found in the BAF amino acid sequence in the text. 02941631TA Figure 8.
  • Antibodies to CE-BBB19 kill through both complement-dependent and complement-independent mechanisms. Antiserum was generated against CE-BBB19 in rats and tested for bactericidal antibodies. Percent killing is indicated on the y-axis.
  • PI preimmune
  • GPS guinea pig serum
  • HI-GPS heat inactivated GPS.
  • Figure 9 Coadministration of BAF and CE-BBB19 induces strong IgG antibody responses. Mice were immunized with BAF and CE-BBB19 in alum (two doses, two weeks apart), and then serum was collected and screened by ELISA for IgG antibodies to each protein. The numbers below each bar are arbitrary numbers assigned to track each mouse. Dark and light gray bars indicate the absorbance reading obtained with a 1:1000 dilution of serum with the BAF and CE-BBB19 chimeritopes, respectively.
  • Figure 10 IgG isotyping of mice immunized with BAF and CE-BBB19.
  • the immobilized proteins were screened using standard ELISA approaches with preimmune serum (left bar in each pair) and pooled serum from mice immunized with BAF/CE-BBB19.
  • the results obtained by screening OspC types derived from North American and European Borreliella isolates are shown.
  • the strong reactivity of the hyperimmune serum with diverse OspC types is indicative of a broad protective range.
  • Figure 12. Immunization of rhesus macaques induces bactericidal antibodies that provide protection through synergistic antibody-mediated complement and complement- independent mechanisms. Serum from Rhesus macaques vaccinated with BAF/CE- BBB19 (adjuvanted with alum) was tested for bactericidal activity using the identical protocol described above.
  • EM lesions are not always present or evident, and the early stages of infection are generally non-descript. As a result, a diagnosis based on clinical presentation is often difficult. If not diagnosed and treated early, the LD spirochetes disseminate and establish a persistent and debilitating infection that is characterized by neurologic, cardiac, and/or rheumatologic manifestations. In light of the challenges associated with the diagnosis and treatment of LD and the potentially serious consequences of long-term infection, prevention through vaccination is an attractive path forward. The present disclosure describes vaccine development efforts using an approach referred to as chimeritope technology.
  • Chimeritopes are novel recombinant proteins created in the laboratory that comprise epitopes and/or specific protein segments derived from multiple different proteins or protein variants.
  • the advantage to chimeritopes is that they can be designed to elicit antibodies that target the numerous species of Borreliella that can cause Lyme disease in humans, companion animals, wildlife, and other mammals, all in a single or a few recombinant proteins.
  • the chimeritopes described here also possess the unique and highly desirable feature of being able to kill the LD spirochetes through both antibody-mediated complement dependent and complement-independent mechanisms.
  • the present disclosure provides two unique custom-designed recombinant proteins (also referred to herein as chimeritopes, vaccine antigens, and vaccinogens) that include antigenic regions derived from five different B. burgdorferi proteins: OspA, OspB, OspC, FtlA, and FtlB.
  • a novel two-protein vaccine formulation 02941631TA comprising both of the two recombinant proteins is provided, as are methods of its use to vaccinate mammals against LD.
  • Antigen a term used historically to designate an entity that is bound by an antibody and also to designate the entity that induces the production of the antibody. More current usage limits the meaning of antigen to that entity bound by an antibody, while the word “immunogen” is used for the entity that induces antibody production.
  • an antigen, immunogen or epitope is generally a portion of a protein (e.g., a peptide or polypeptide).
  • Antibody-dependent complement-mediated killing the lysis or killing of a bacterial cell by antibody through a mechanism that requires, and is dependent on, active proteins of the complement system.
  • Different subtypes of IgG are more efficient at complement fixation than others.
  • IgG1 and IgG3 are best at complement fixation. Note - we show immunization with CE-BBB19+BAF induces a strong IgG1 response.
  • linker sequences 02941631TA include but are not limited to an amino acid spacer, an amino acid linker, a signal sequence, a stop transfer sequence, a transmembrane domain, and a protein purification ligand.
  • Tags Recombinant protein sequences that can be added to the N- or C-terminus of a recombinant protein for the purpose of identification or for purifying the recombinant protein for subsequent uses.
  • GST glutathione-S-transferease
  • MBP maltose binding protein
  • FLAG FLAG
  • V5 halo, myc
  • HA hemaglutinin
  • SBP streptavidin binding protein
  • Softag1TM Softag3TM
  • Xpress tag isopeptag
  • Spy Tag biotin carboxyl carrier protein (BCCP)
  • BCCP biotin carboxyl carrier protein
  • tags are well-known to those of ordinary skill in the art of recombinant protein production and may or may not be removed before using tag-specific cleavage protocols.
  • a hexa-histidine tag is typically removed by incubation of the protein with the enzyme enterokinase, which recognizes a specific amino acid motif. Cleavage of this motif releases the His tag from the recombinant protein.
  • Epitope a specific chemical domain on an antigen that is recognized by a B-cell receptor and which can be bound by a secreted antibody.
  • Residues in conformational epitopes may be located far from other resides in the epitope with respect to primary sequence but may be spatially located near other residues in the conformational epitope due to protein folding.
  • Chimeric or fusion peptide or polypeptide a recombinant or synthetic peptide or polypeptide whose primary sequence comprises two or more amino acid sequences that do not occur together in a single molecule in nature.
  • the two or more sequences may be, 02941631TA for example, a peptide (e.g., an epitope or antigenic region) and a linker sequence, or two or more peptides (which may be the same or different) which are either contiguous or separated by a linker sequence, etc.
  • Original or native or wild-type sequence The sequence of a peptide, polypeptide, protein or nucleic acid as found in nature.
  • Recombinant peptide, polypeptide, protein, or nucleic acid A man-made, non-natural peptide, polypeptide, protein, or nucleic acid that has been produced and/or manipulated using molecular biology techniques such as cloning, polymerase chain reaction (PCR), etc.
  • Synthetic peptide, polypeptide, protein or nucleic acid A peptide, polypeptide, protein or nucleic acid that has been produced using chemical synthesis procedures.
  • a sequence of a peptide, polypeptide, or protein is “similar” to a reference sequence if the amino acid sequence possesses a specified amount of identity compared to the reference sequence.
  • the similarity of two sequences can be compared along their entire lengths by aligning the residues to optimize the number of identical amino acids; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
  • a pair-wise comparison analysis of amino acid sequences can be carried out using the BESTFIT algorithm in the GCG package (version 10.2, Madison Wis.).
  • polypeptides may be compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website.
  • similarity may be referred to by e.g.
  • Nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine.
  • Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • Positively charged (basic) amino acids include arginine, lysine and histidine.
  • Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • the first vaccine antigen includes OspB as the backbone.
  • OspB is an outer surface protein that is specifically produced by Lyme disease spirochete during residence in the midgut of an unfed tick.
  • this sequence was inserted at the C-terminal end of the construct to yield a chimeric protein comprising OspB, OspA epitopes (A#1 and A#15) and the N-terminal domain of FtlA.
  • the final product (OspB backbone, OspA epitopes (A#1 and A#15), and the N-terminal domain of FtlA) is informally designated herein as the “BAF” construct, and the chimeric gene and protein were designated as the baf gene and the BAF protein, respectively.
  • W tryptophan residues
  • BAF construct amino acid sequence AQKGAESIGSQKENDLNLEDSSKKSHQNAKQDLPAVTEDSVSLFNGNKIFVSKEKNSSG KYDLRATIDQVELKGTSDKNNGSGTLEGSKPDKSKVKLTVSADLNTVTLEAFDASNQKI SSKVTKKQGSITEETLKANKLDSKKLTRSNGTTLEYSQITDADNATKAVETLKNSIKLE GSLVGGKTTVEIKEGTVTLKREIEKDGKVKVFLNDTAGSNKKTGKWEDSTSTLTISADS KKTKDLVFLTDSTLTITVNSKKTKDLVFTKEKTLTVSADSKKIKDFVFLTDGTITVQQY NTAGTSLEGSASEIKNLSELKNALKWWNLDSKLSSNKEQKNNNNVKEVSDSVQEDGLND LYNNQEKQKSFTKNFGERKYEDLINPIEPI
  • the second protein is a modified variant of a chimeric epitope-based protein (chimeritope) comprised of different variants of two OspC epitopes, L5 and H5, that were identified through epitope mapping, as described in the Examples section.
  • the amino acid sequence of CE-BBB19 is presented below.
  • CE-BBB19 amino acid sequence SETFTNKLKEKHTDLGKEGVTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSSEEFS TKLKDNHAQLGIQGVTKGVEELEKLSGSLESLSSEDFTKKLEGEHAQLGIENVTAAELE KLFKAVENLAKAAKEMAKLKGEHTDLGKEGVTKGADELEKLFESVKNLSKAAKEMLTNS 02941631TA KESEKFAGKLKNEHASLGKKDATKGAKELKDLSDSVESLVKASDDFTKKLQSSHAQLGV AGGATTADELEKLFKSVESLAKAAQDALANSVNELTSKKLKEKHTDLGKKDATAAELEK LFESVENLAKAAKEMLSNSNKAFTDKLKSSHAELGIANGAATKGAQELEKLFESVKNLS KAAQETLNNSVKESESFTSEKFTKKLSESHADIGIQALKTNPTKGAEELDKLFKAVE NLSKAAKEMLANSSEDFTNKLKNGNAQ
  • amino acid sequences may be altered somewhat and still be suitable for use as described herein.
  • certain conservative amino acid substitutions may be made without having a deleterious effect on the ability of the recombinant proteins to elicit an immune response.
  • substitution of a positively charged amino acid for another positively charged amino acid e.g., K for R or vice versa
  • substitution of a negatively charged amino acid for another negatively charged amino acid e.g. D for E or vice versa
  • substitution of a hydrophobic amino acid for another hydrophobic amino acid e.g. substitution of A, V, L, I, W, etc. for one another
  • substitution of a positively charged amino acid for another positively charged amino acid e.g., K for R or vice versa
  • substitution of a negatively charged amino acid for another negatively charged amino acid e.g. D for E or vice versa
  • substitution of a hydrophobic amino acid for another hydrophobic amino acid e.g. substitution of A, V, L, I, W, etc. for one another
  • Recombinant proteins resulting from all such substitutions or alterations of the sequences of the recombinant proteins that are disclosed herein are encompassed by the present invention, as long as the resulting recombinant proteins still function to elicit a suitable immune response an antibody response, preferably a protective antibody response, in a subject to whom the vaccine formulation is administered.
  • the amino acid sequences of the recombinant proteins of the invention need not encompass a full-length sequence as disclosed herein. Certain small N- and/or C-terminal and/or internal deletions of amino acids (e.g., from about 1-10 amino acids, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) may be tolerated without decreasing the ability of the proteins to elicit the desired immune response.
  • CE-BBB19 (SEQ ID#2), which is a linear and unstructured protein as determined using nuclear magnetic 02941631TA resonance techniques, harbors several imperfect repeats of the L5 and H5 epitope domains from 10 different OspC types. Hence, it was designed with some degree of inherent sequence redundancy. While the full L5 and H5 repeats in the sequence are imperfect, some share segments with perfect identity. Some instances of this are the 8 amino acid sequence, NSVKELTSF (bold), which is repeated twice; the 6 amino acid sequence, KAAKEM (italicized), which is repeated 5 times; the 4 amino acid sequence, HDTL (underlined), which is repeated 3 times; and the 3 amino acid sequence, KLF (underlined and bolded), which is repeated 7 times.
  • NSVKELTSF bold
  • KAAKEM italicized
  • HDTL underlined
  • KLF underlined and bolded
  • Deletion of one or more of a given repeat sequence or pair of different repeat sequences may not attenuate the recombinant proteins' function to elicit a suitable immune response, preferably a protective antibody response, in a subject to whom the vaccine formulation is administered.
  • a suitable immune response preferably a protective antibody response
  • This also applies in a similar manner to the recombinant chimeritope of SEQ ID NO: 1. It is noted that, in some aspects, when such deletions are made, the remaining contiguous amino acids of the primary sequence remain the same. All such forms of the recombinant sequences disclosed herein are encompassed herein. SEQ ID#2 with selected repeated sequences indicated.
  • Proteins resulting from all such changes are encompassed by the present invention as long as the resulting recombinant protein functions to elicit an antibody response, preferably a protective antibody response, in a subject to whom the vaccine formulation is administered.
  • substituted or modified protein sequences will be at least about 50% identical or similar to the corresponding sequence in the recombinant protein disclosed herein, or about 60 to 70, or 70 to 80, or 80 to 90% identical to the disclosed sequences (including all integers within these ranges to 0.1 decimal places), or even about 95, 96, 97, 98 or 99% identical (including all decimal fractions between these values).
  • sequences may elicit antibody production.
  • Such sequences may or may not be present between the segments in a chimera. If present, they may, for example, serve to separate the segments and contribute to the steric isolation of the segments from each other.
  • sequences may be simply artifacts of recombinant processing procedures, e.g., cloning procedures.
  • Such sequences are typically known as linker or spacer peptides, many examples of which are known to those of skill in the art. See, for example, Crasto, C. J. and J. A. Feng. 2000.
  • LINKER a program to generate linker sequences for fusion proteins. Protein Engineering 13(5): 309-312, which is a reference that describes unstructured linkers.
  • Structured (e.g., helical) sequence linkers may also be designed using, for example, existing sequences that are known to have that secondary structure or using basic known biochemical principles to design the linkers.
  • 02941631TA other elements may be present in the chimeric proteins, for example, leader sequences or sequences that “tag” the protein to facilitate purification or detection of the protein, examples of which include but are not limited to histidine (e.g., hexahistidine) tags, detection tags (e.g., S-tag, or Flag-tag), other antigenic amino acid sequences such as known T-cell epitope containing sequences and protein stabilizing motifs, etc.
  • the chimeric proteins may be chemically modified, e.g., by amidation, sulfonylation, lipidation, or other techniques that are known to those of skill in the art, as long as the activity of the proteins to elicit a suitable antibody response is not vitiated.
  • the naturally occurring leader sequences of the native proteins on which the chimeritopes are based are not included in the chimera.
  • chimeras in which one or more leader sequences are included are also encompassed.
  • compositions for use in eliciting an immune response, preferably a protective immune response.
  • the compositions may be utilized as vaccines to prevent or treat Borreliella infection, particularly when manifested as Lyme disease (Lyme borreliosis).
  • non-vaccine compositions e.g., for laboratory purposes such as storage, purification protocols, etc.
  • the vaccine compositions generally include both of the recombinant proteins disclosed herein (SEQ ID NO: 1 and SEQ ID NO: 2, or a variant of one or both of SEQ ID NO: 1 and/or SEQ ID NO: 2 as described herein), which have been isolated and substantially purified, together with a pharmacologically suitable carrier.
  • compositions comprising only one of the proteins SEQ ID NO: 1 or SEQ ID NO: 2 or a 02941631TA variant of one or both of SEQ ID NO: 1 and/or SEQ ID NO: 2 as described herein
  • Such compositions may elicit an immune response.
  • compositions for use as vaccines are well known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions; however, solid forms such as tablets, pills, powders, and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared.
  • the preparation may also be emulsified.
  • the active ingredients may be mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, or combinations thereof.
  • the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like.
  • compositions of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration.
  • the final amount of one or both recombinant proteins in the formulations may vary. However, in general, the amount in the formulations will be from about 0.01-99%, weight/volume.
  • the vaccine preparations disclosed herein may further comprise an adjuvant, suitable examples of which include but are not limited to Seppic, Quil A, Alhydrogel, etc.
  • lipid nanoparticles include but are not limited to: lipid nanoparticles, azalides, ‘MF59’ (a submicron oil-in-water emulsion of squalene, polysorbate 80, and sorbitan trioleate), 1H-imidazo[4,5-c]quinolin-4-amine, various saponins, substituted pyrimidines (e.g., see issued US patent 11/266,738, the complete contents of which are hereby incorporated by reference herein), CpG based adjuvants delivered in combination with other adjuvants that may include various formulations of alum and the like as known in the art.
  • MF59 a submicron oil-in-water emulsion of squalene, polysorbate 80, and sorbitan trioleate
  • 1H-imidazo[4,5-c]quinolin-4-amine include various saponins, substituted pyrimidines (e.g., see issued US patent 11/266,738, the complete contents
  • the present invention provides methods of eliciting an immune response to Borreliella and/or to vaccinate against Borreliella infection in mammals.
  • the methods involve administering, to a mammal, a therapeutically effective amount of a composition 02941631TA comprising at least one, and usually both of the recombinant proteins disclosed herein.
  • Administration elicits or causes an immune response, preferably a protective immune response.
  • compositions comprising the recombinant proteins disclosed herein causes the synthesis of specific antibodies at high titer and/or immune cell proliferation, as measured, e.g., by 3 H thymidine incorporation or by other known techniques.
  • protection immune response we mean that the vaccine elicits an immune response that results in the protection of a vaccinated organism against challenge with Borreliella.
  • the protective response either wholly or partially prevents or arrests the development of symptoms related to Borreliella infection in an individual that has been vaccinated with the preparation disclosed herein and is then later exposed to the bacteria, in comparison to a non-vaccinated (e.g., adjunct alone) control organism, in which or in whom disease progression is not prevented.
  • a non-vaccinated (e.g., adjunct alone) control organism in which or in whom disease progression is not prevented.
  • one or more symptoms of LD are prevented in vaccinated individuals who are later exposed to the bacteria and who could otherwise develop LD but for having been vaccinated.
  • administration of the vaccine wholly or partially prevents and/or arrests symptoms already present in an individual who already has LD.
  • the present vaccine formulations advantageously provide protection by killing Borreliella bacteria through antibody-mediated complement-dependent and complement-independent mechanisms.
  • Examples include but are not limited to companion “pets” such as 02941631TA dogs, cats, etc.; food source, work and recreational animals such as cattle, horses, oxen, sheep, pigs, goats, and the like; wild animals that are protected in zoos or preserves; or wild animals that serve as a reservoir of Borreliella (e.g., mice, deer, etc.).
  • the vaccine preparations may be administered by any of the many suitable means that are well known to those of skill in the art, including but not limited to injection, inhalation, orally, intranasally, ingestion of a food product, etc. In most aspects, the mode of administration is subcutaneous, intraperitoneal, or intramuscular.
  • compositions may be administered in conjunction with other treatment modalities, such as substances that boost the immune system, various anti-bacterial chemotherapeutic agents, antibiotics, and the like.
  • the amount of the two proteins in a dose of the vaccine is a therapeutically effective amount.
  • a “therapeutically effective amount” refers to an amount that is sufficient to elicit an immune response to both of the recombinant proteins, preferably an antibody response, more preferably a protective immune response.
  • the response is an antibody-mediated complement-dependent response.
  • the therapeutic and preventative value can be further enhanced by synergism with complement-independent responses that the vaccines elicit.
  • the amount of each protein in a single dose of vaccine generally ranges from about 1 to about 90 ⁇ g, for example, from about 0.1 to 300 ⁇ g, such as from about 0.5 to about 200 ⁇ g, or from about 1 to about 100 ⁇ g, such as about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 ⁇ g per dose. In some aspects, the dose ranges from about 1 to about 90 ⁇ g.
  • the vaccine compositions disclosed herein may be administered according to any suitable (effective) schedule. Because of the excellent immune response elicited by the compositions, the administration of “boosters” (e.g., repeat administration) is less stringent than with previous LD vaccines.
  • the vaccine may be administered once every 6 months, or once a year, or once every 2-5 years, or even once every ten years.
  • a “prime-boost” strategy is used at least during the first year of use, in which the initial vaccine dose is followed by a second dose that serves to generate immune memory cells and boost the response to future exposure to the disease-causing agent, in this case, Borreliella species.
  • a prime-boost strategy may include, for example, 02941631TA a first dose followed by a second dose, generally between about 3 to about 8 weeks after the first, and thereafter as needed to provide immune protection, for example, yearly, every 5 years, or every 10 years.
  • the present invention also encompasses antibodies to the epitopes and/or to the chimeric polypeptides disclosed herein.
  • Such antibodies may be polyclonal, monoclonal, or chimeric and may be generated in any manner known to those of skill in the art.
  • the antibodies are bactericidal (borreliacidal), i.e., exposure of Borreliella spirochetes to the antibodies causes the death of the spirochetes, both within a tick or within the body of a vaccinated mammal.
  • Such antibodies may be used in a variety of ways, e.g., as detection reagents to diagnose prior exposure to Borreliella, as a reagent in a kit for the investigation of Borreliella, to treat Borreliella infections, etc.
  • the antibody response is protective, i.e., prevents or lessens the development of symptoms of disease in a vaccinated subject that is later exposed to Borreliella, compared to an unvaccinated subject. Infections caused by several species and/or strains of Borreliella are prevented by the administration of the compositions. For example, infection by B. burgdorferi, B, garinii, B. mayonii, B. afzelii, B. carolinesis, B. americanum, B.
  • the invention further provides nucleic acid sequences that encode the recombinant proteins disclosed herein. Such nucleic acids include DNA, RNA and hybrids thereof, and the like. Further, the invention comprehends vectors that contain or house such coding sequences. Examples of suitable vectors include but are not limited to plasmids, cosmids, viral-based vectors, expression vectors, etc. In a preferred embodiment, the vector is a plasmid expression vector.
  • nucleic acid sequences capable of 02941631TA encoding SEQ ID NO: 1 and SEQ ID NO: 2, and/or variants thereof as described herein, are encompassed herein.
  • polynucleotide or “nucleic acid” refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids.
  • Polynucleotides may be single-stranded or double-stranded, are recombinant and may be synthetic, or isolated from an organism that produces them, for example, a genetically engineered bacterial, yeast, insect or mammalian.
  • Polynucleotides include, but are not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), plus-strand RNA (RNA(+)), minus-strand RNA (RNA(-)), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.
  • pre-mRNA pre-messenger RNA
  • mRNA messenger RNA
  • RNA(+) plus-strand RNA
  • RNA(-) minus-strand RNA
  • cDNA complementary DNA
  • synthetic DNA or recombinant DNA.
  • polynucleotides are codon-optimized for gene expression in E. coli or any other suitable expression cell (insect, mammalian, other bacteria).
  • cognidized refers to substituting codons in a polynucleotide encoding a polypeptide or protein in order to increase the expression, stability and/or activity of the polypeptide based on, e.g.
  • codon biases between two or more organisms or genes or synthetically constructed bias tables variation in the degree of codon bias within an organism, gene, or set of genes, systematic variation of codons including context, variation of codons according to their decoding tRNAs, variation of codons according to GC %, either overall or in one position of the triplet, variation in degree of similarity to a reference sequence, for example, a naturally occurring sequence, variation in the codon frequency cutoff, structural properties of mRNAs transcribed from the DNA sequence, systematic variation of codon sets for each amino acid, and/or isolated removal of spurious translation initiation sites.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide over a window of comparison, similar to that of amino acid “identity” discussed herein.
  • a "percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • Sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not 02941631TA comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (including but not limited to GAP, BESTFIT, FASTA, and TFASTA) etc. or the BLAST family of programs as for example, disclosed in the GCG (Genetics Computer Group) product, the Wisconsin Package 11.0 (or later when available), a recognized industry standard for sequence analysis of nucleic acids and protein sequences; and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 2012.
  • algorithms including but not limited to GAP, BESTFIT, FASTA, and TFASTA
  • nucleic acid cassette which may be (but is not necessarily) an “expression cassette.”
  • expression cassette refers to genetic sequences which can express an RNA and, subsequently, a polypeptide.
  • the nucleic acid cassette contains at least one gene(s)-of interest, e.g., a polynucleotide(s) of interest that encodes at least one chimera as disclosed herein.
  • the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter (e.g.
  • a cassette encoding the polypeptide can be inserted into an appropriate vector, such as an expression vector.
  • the nucleic acid cassette is positionally and sequentially oriented within a vector such that the nucleic acid in the cassette can be transcribed into RNA.
  • the cassette can be removed and inserted into a plasmid or viral vector as a single unit.
  • vectors include, but are not limited to plasmids, autonomously replicating sequences, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), various episomal vectors, bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses.
  • viruses useful as vectors include, without limitation, retrovirus 02941631TA (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).
  • retrovirus 02941631TA including lentivirus
  • adenovirus including lentivirus
  • adeno-associated virus e.g., adeno-associated virus
  • herpesvirus e.g., herpes simplex virus
  • poxvirus baculovirus
  • papillomavirus papillomavirus
  • papovavirus e.g., SV40
  • Exemplary expression vectors include but are not limited to pClneo vectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST.TM., pLenti6/V5-DEST.TM., and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.
  • pClneo vectors Promega
  • pLenti4/V5-DEST.TM. pLenti6/V5-DEST.TM.
  • pLenti6.2/V5-GW/lacZ Invitrogen
  • a wide range of vectors can be used, for example pET vectors which are widely used for expression in bacteria and which were used in the Examples described herein.
  • a polynucleotide sequence of interest is generally “operably linked” to other elements of the cassette or vector so as the intended function of all elements is possible.
  • the phrase refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, and/or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide-of-interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • a nucleic acid expression control sequence such as a promoter, and/or enhancer
  • a second polynucleotide sequence e.g., a polynucleotide-of-interest
  • Exemplary expression control sequences suitable for use according to the present disclosure include but are not limited to: cytomegalovirus (CMV) immediate early promoter, viral simian virus 40 (SV40) (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, short elongation factor 1-alpha (EF1a-short) promoter, long elongation factor 1-alpha (EF1a-long) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5)
  • the vaccine antigens may be produced by any suitable method, many of which are known to those of skill in the art. For example, they may be chemically synthesized or produced using recombinant DNA technology (e.g., via a vector encoding a vaccine antigen) in bacterial cells, in cell culture (mammalian, yeast, or insect cells), in plants or plant cells, or by cell-free prokaryotic or eukaryotic-based expression systems, by other in vitro systems, etc. In some embodiments, the vaccine antigens are produced using an E. coli recombinant expression system.
  • the disclosure also encompasses bacterial, mammalian, yeast, and/or insect host cells, which comprise at least one copy of a nucleotide sequence encoding a recombinant vaccine antigen disclosed herein.
  • the host cell is a bacterial cell.
  • the bacterial cell is an E. coli host that has been genetically engineered to contain and express at least one nucleic acid sequence disclosed herein in a manner that results in the production of the encoded recombinant vaccine antigen, such as via a vector located therein. Methods of making the recombinant vaccine antigens are also encompassed herein.
  • the methods include genetically engineering a host cell to contain and express a nucleic acid encoding at least one recombinant vaccine antigen disclosed herein, e.g., by introducing a suitable vector into the host cell.
  • the host cell is genetically engineered so that at least one vaccine antigen is produced by the host cell, either within the host cell or excreted into the medium in which the host cell is grown.
  • Methods of making recombinant proteins using various types of host cells are known in the art, e.g., by growing/cultivating the host cell in a medium compatible with growth and then i) harvesting and lysing the host cells to release the proteins or, for excreted proteins ii) harvesting the growth medium.
  • EXAMPLE 1 Identification of candidate tick phase and mammalian phase proteins for use in the construction of multi-protein, multi-valent chimeric vaccine antigens. To design chimeric proteins that could target the LD spirochetes in a) the tick environment and or b) during infection of mammals, an extensive literature and database search was conducted. These analyses did not provide clear guidance for selecting the optimal vaccine candidate.
  • the gene-carrying plasmids were propagated in Escherichia coli NovaBlue DE3 cells, purified, and transformed into E. coli BL21/DE3 cells. Protein expression was induced by overnight autoinduction or with IPTG (1 mM). When IPTG was used, a 4-6 hour induction timeframe was used.
  • IPTG IPTG was used, a 4-6 hour induction timeframe was used.
  • the 02941631TA induced cells were lysed using a high-pressure homogenizer, and the cell lysate was fractionated into soluble or insoluble phases using standard methods. Recombinant proteins that fractionated with the soluble phase were purified using non-denaturing conditions via an N-terminal hexahistidine tag (the sequence of which is presented below) using nickel affinity chromatography ( ⁇ KTA purifier).
  • the elution buffer which contained imidazole, was removed by dialysis into phosphate-buffered saline (PBS; pH 7.4) across a 6 to 8 kDa molecular weight cut-off membrane.
  • PBS phosphate-buffered saline
  • Cellular debris was removed by centrifugation (15,500 x g; 30 m; 4 o C), and the supernatants were loaded onto a Poly-Prep Chromatography column pre-equilibrated in 50 mM Tris-HCl, 300 mM NaCl, pH 8.0, with 8 M urea.
  • the protein-bound resin was washed, and the proteins were eluted using 250 mM imidazole. Urea and imidazole were removed by stepwise dialysis into phosphate-buffered saline (PBS; pH 7.4) with decreasing concentrations of urea. Protein concentrations were determined using a BCA assay. To identify proteins that are expressed during infection in mammals, the recombinant proteins were immobilized in the wells of ELISA plates and tested for reactivity with several hundred sera from infected humans, dogs, wild canids (Eastern coyotes), mice, raccoons, foxes, eastern black bears, and other wildlife.
  • PBS phosphate-buffered saline
  • SEQ ID NO: 3 Protein name: His tag sequence with enterokinase cleavage site.
  • SEQ ID NO: 4 Protein name: FtlA (BBK01) B. burgdorferi B31 Protein ID: AAC66147.1
  • the protein was readily expressed in E. coli as a soluble protein and was purified to homogeneity. As detailed below, the underlined sequence spanning residues 19-143 is the domain we identified that induces high titer, immunodominant, bactericidal antibodies.
  • FtlC with leader SEQ ID NO: 25
  • FtlC with leader SEQ ID NO: 26
  • 02941631TA Based on the high identity between FtlA and FtlB, particularly in their N-terminal domains, we speculated that antibodies that recognize one would recognize the other. This was verified by immunoblot and ELISA analyses. Immunoblots of Borreliella cell lysates were screened with anti-FtlA, anti-FtlB, and anti-FtlC antiserum ( Figure 1). The immunoreactivity patterns were the same for FtlA and FtlB but differed for FtlC. The results were further verified using ELISA assays.
  • the Ftl proteins were immobilized in the ELISA plate wells and on immunoblots and screened with antisera generated against each Ftl protein (Figure 2). Strong cross-reactivity of the FtlA and FtlB antisera with both FtlA and FtlB but not with FtlC and FtlD proteins was observed. Antibodies to FtlC were highly specific for FtlC, with only weak reactivity with FtlA and FtlB. The significance of this finding is that antibodies elicited by recombinant FtlA bind to both FtlA and FtlB and will therefore act synergistically to kill LD spirochete strains by binding to two protein targets on the cell surface.
  • anti-FtlC and anti- FtlD antisera had low bactericidal activity consistent with the low levels of expression of FtlC and FtlD during cultivation.
  • full-length FtlA minus the leader peptide
  • overlapping fragments spanning the length of the protein F1, F2, and F3 were produced and screened with B. burgdorferi peptide C6 Ab-positive serum samples from client-owned dogs. Of the 50 dogs screened, 74% (37/50) were Ab positive for fragment F1, whereas only 44% and 28% were Ab positive for the F2 and F3 fragments, respectively (Figure 4A).
  • Serum was collected from the mice, and sub-fragments or peptides derived from each protein were screened with the sera by ELISA and immunoblotting.
  • Peptides corresponding to these domains induced bactericidal antibodies but only when conjugated to the carrier protein, Keyhole limpet hemocyanin (KLH). This demonstrated that the context in which the immunogenic domains are presented is a critical factor.
  • Figure 5 demonstrated that antibodies directed at OspB kill in a complement-dependent manner.
  • the two epitope variants were derived from different Borreliella isolates. This epitope chimeric was not produced as a stand-alone protein but was introduced into different chimeric proteins, as detailed below, in a location that would present these epitopes on the surface of the proteins.
  • STLTITVNSKKTKDLVFTKEKTLIVSADSKKIKDFKTVFLTD SEQ ID NO: 11
  • SEQ ID NO: 12 Protein name: OspB (BBA16) B. burgdorferi B31 Protein ID: AAC66243.2
  • the leader peptide is omitted. The protein was readily expressed in E.coli as a soluble protein and was purified to homogeneity.
  • This epitope chimeric was not produced as a stand-alone protein but was introduced into different chimeric proteins, as detailed below, in a location that would present these epitopes on the surface of the proteins.
  • STLTISADSKKTKDLVFLTDNTLTVSADSKKIKDFVFLTD SEQ ID NO: 27.
  • Development of chimeric vaccine antigens Based on the data presented above, several different chimerics were generated that carried domains from two or more of the following proteins: OspC, OspA, OspB, and FtlA. The chimeric proteins, their sequences, and their properties are listed below.
  • SEQ ID NO: 15 Protein designation: OspB A1/A15 MW: 34.5 kDa Amino acids: 320 Gene length: 960bp Tag: His Tag Vector: pET45 Codon optimized for expression in E. coli. Soluble or insoluble upon expression: Soluble NOTES: This chimeric consists of a His tag (not shown) followed by OspB amino acids 17-243, followed by the A1/A15 OspA chimeric epitope (underlined), followed by amino acids 264-296 of OspB. The protein was produced at high levels in E. coli and was 02941631TA purified to homogeneity using FPLC. It was advanced to immunogenicity and protective efficacy analyses.
  • the nucleotide sequence was codon optimized for expression in E. coli. Soluble or insoluble upon expression: Not applicable (see notes below). Description: Chimeric consisting of his tag (not shown) followed by FtlA residues 19- 143, followed by CE-BBB19 (underlined), followed by OspA amino acid residues 211- 260 (italicized). The W residues that were added to increase detection sensitivity are bolded. The inclusion of two pairs of W residues increased the extinction coefficient. Extinction coefficient values were calculated using Protparam. The protein was predicted to be stable. However, using several different methods, expression vectors, and E. coli strains, the protein was not expressed.
  • the nucleotide sequence was codon optimized for expression in E. coli. Description: Chimeric consisting of his tag (not shown) followed by FtlA residues 19- 143 followed by CE-BBB19 (underlined). As described above, W residues (bolded) were added to allow for increased detection sensitivity. Important note: In spite of predictive analyses that suggested this protein could be readily expressed, stable, and present the desired immunogenic domains on its surface, we were unable to achieve sufficient expression in E. coli. It was clear from the research done on this chimeric that predictive algorithms are of limited value. This chimeric was abandoned, but the information obtained highlighted the importance of chimeric domain organization and informed the future design of other chimerics.
  • Soluble Description: The chimeric consists of a His tag (not shown) followed by CE-BBB19, followed by A1/A15 chimeric epitope (underlined), followed by the OspB chimeric epitope, B1/B3 (italicized).
  • B1/B3 italicized
  • the nucleotide sequence was codon optimized for expression in E. coli optimized.
  • BAF was soluble upon expression in E. coli.
  • the structural organization of BAF is depicted in Figure 7. The two pairs of W residues were added to increase detection sensitivity.
  • BAF Bacillus coli and purified to homogeneity by FPLC with high yield.
  • BAF was advanced for further evaluation, including immunogenicity analyses, and the ability to induce protective immunity in mice, rats and Rhesus macaques.
  • BAF is one of the two chimerics in the vaccine formulation. Data demonstrating the protective efficacy of the BAF/CE-BBB19 formulation are detailed below.
  • the BAK construct is designed to elicit antibodies that target FtlA, FtlB, OspA, and OspB. This is a defining aspect of this novel vaccine antigen and represents a significant advancement in the art. The linkage of several epitopes derived from several different proteins is a significant advancement.
  • SEQ ID NO: 21 Gene name: OspC (type A) Protein ID: AAC66329 02941631TA NOTES: We have identified two immunodominant epitopes in the Borreliella OspC protein designated as L5 (loop 5) and H5 (helix 5).
  • CE-BBB19 was designed to include L5H5 chimeric epitopes from 10 distinctly different OspC types derived from North American Borreliella OspC proteins. Although the total number of amino acids varies among OspC types, here we use the amino acid numbering assigned to B. burgdorferi B31 OspC type A sequence. Note that the leader peptide was omitted.
  • the L5 epitope spans amino acids 136 to 150, and the H5 epitope spans 168 to 203.
  • the region of the OspC that contains the L5 and H5 epitopes is indicated by underlining and italicizing, respectively.
  • the coding sequence was codon optimized for expression in E. coli. Description: Consists of L5 and H5 epitope chimeras from OspC types that are associated with human infection. Below, the sequence is presented in contiguous form.
  • CE-BBB19 amino acid sequence SETFTNKLKEKHTDLGKEGVTKGAEELGKLFESVEVLSKAAKEMLANSVKELTSSEEFS TKLKDNHAQLGIQGVTKGVEELEKLSGSLESLSSEDFTKKLEGEHAQLGIENVTAAELE KLFKAVENLAKAAKEMAKLKGEHTDLGKEGVTKGADELEKLFESVKNLSKAAKEMLTNS 02941631TA KESEKFAGKLKNEHASLGKKDATKGAKELKDLSDSVESLVKASDDFTKKLQSSHAQLGV AGGATTADELEKLFKSVESLAKAAQDALANSVNELTSKKLKEKHTDLGKKDATAAELEK LFESVENLAKAAKEMLSNSNKAFTDKLKSSHAELGIANGAATKGAQELEKLFESVKNLS KAAQETLNNSVKESESFTSEKFTKKLSESHADIGIQALKTNPTKGAEELDKLFKAVE NLSKAAKEMLANSSEDFTNKLKNGNAQ
  • CE-BBB19 amino acid sequence SEQ ID NO: 2
  • CE-BBB19 DNA sequence Note that the CE-BBB19 sequence was codon optimized for expression in E. coli and thus does not match the wild-type natural sequence of each epitope.
  • any nucleotide sequence that encodes the protein sequence above is suitable for protein production.

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Abstract

L'invention concerne une formulation de vaccin pour des humains ou d'autres mammifères (notamments, les chiens, les chevaux et les chats). La formulation de vaccin comprend deux protéines chimériques conçues pour éliciter des anticorps qui se lient à plusieurs cibles sur la surface de spirochètes de la maladie de Lyme pendant leur résidence chez des tiques et chez des mammifères, et agissent de manière synergique pour tuer les bactéries au travers des deux mécanismes dépendant du complément et indépendant du complément à médiation par anticorps.
EP23875683.7A 2022-10-03 2023-10-03 Nouveaux antigènes vaccinaux recombinants à base de protéines multiples chimériques pour la prévention de la maladie de lyme chez des animaux et des êtres humains Pending EP4598943A2 (fr)

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