CANINE LYME DISEASE VACCINE

Abstract

The present invention provides a vaccine for canine Lyme disease and methods of making and using the vaccine alone, or in combinations with other protective agents.

Claims

1. An immunogenic composition comprising two or more alphavirus RNA replicon particles each individually encoding one or more Borrelia burgdorferi antigens or antigenic fragments thereof; wherein one alphavirus RNA replicon particle encodes a one or more Borrelia burgdorferi outer surface protein A (OspA) or an antigenic fragment thereof, and wherein another alphavirus RNA replicon particle encodes one or more Borrelia burgdorferi outer surface protein C (OspC) or an antigenic fragment thereof.

2. An immunogenic composition comprising an alphavirus RNA replicon particle that encodes two or more Borrelia burgdorferi antigens; wherein at least one Borrelia burgdorferi antigen is an outer surface protein A (OspA) or an antigenic fragment thereof; and wherein at least one other Borrelia burgdorferi antigen is an outer surface protein C (OspC) or an antigenic fragment thereof.

3. The immunogenic composition of claim 2 comprising a first and a second alphavirus RNA replicon particle each individually encoding an OspA or an antigenic fragment thereof and an OspC or an antigenic fragment thereof wherein the first RNA replicon particle comprises a nucleic acid sequence encoding OspA or an antigenic fragment thereof that is located upstream of a nucleic acid sequence encoding OspC or an antigenic fragment thereof, and the second RNA replicon particle comprises a nucleic acid sequence encoding OspC or an antigenic fragment thereof that is located upstream of a nucleic acid sequence encoding OspA or an antigenic fragment thereof.

4. The immunogenic composition of claim 2, wherein at least one of the alphavirus RNA replicon particles is a Venezuelan Equine Encephalitis Virus (VEEV) RNA replicon particle.

5. The immunogenic composition of claim 4, that comprises one or more additional alphavirus RNA replicon particles which encode a Borrelia burgdorferi antigen selected from the group consisting of a second OspA or an antigenic fragment thereof that originates from a different strain of Borrelia burgdorferi than the OspA, a second OspC or an antigenic fragment thereof that originates from a different strain of Borrelia burgdorferi than the OspC, and any combination thereof.

6. The immunogenic composition of claim 5, wherein the one or more additional alphavirus RNA replicon particles are VEEV RNA replicon particles.

7. The immunogenic composition of claim 4, wherein at least one OspA originates from a B. burgdorferi strain 297 and at least one OspC originates from a B. burgdorferi strain 50772.

8. The immunogenic composition of claim 7, wherein the OspA comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO: 2, and the OspC comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO: 4.

9. A vaccine to aid in the prevention of disease due to Borrelia burgdorferi infection comprising the immunogenic composition of claim 8 and a pharmaceutically acceptable carrier.

10. The vaccine of claim 9, wherein one or more antibodies are induced in a canine when said canine is immunized with the vaccine, and wherein said one or more antibodies is selected from the group consisting of an OspA-borreliacidal antibody, an OspC-borreliacidal antibody, or both an OspA-borreliacidal antibody, an OspC-borreliacidal antibody.

11. The vaccine composition of claim 9, further comprising at least one non-Borrelia immunogen for eliciting protective immunity to a non-Borrelia pathogen.

12. The vaccine composition of claim 9, further comprising an alphavirus RNA replicon particle comprising a nucleotide sequence encoding at least one protein antigen or an antigenic fragment thereof from a non-Borrelia immunogen.

13. The vaccine of claim 12, wherein the non-Borrelia immunogen comes from a non-Borrelia pathogen selected from the group consisting of canine distemper virus, canine adenovirus, canine parvovirus, canine parainfluenza virus, canine coronavirus, canine influenza virus, Leptospira serovars, Leishmania organisms, Bordetella bronchiseptica, Mycoplasma species, rabies virus, Ehrlichia canis, an Anaplasma species, and any combination thereof.

14. The vaccine of claim 11, wherein the non-Borrelia immunogen is a killed or attenuated non-Borrelia pathogen selected from the group of killed or attenuated non-Borrelia pathogens consisting of canine distemper virus, canine adenovirus, canine parvovirus, canine parainfluenza virus, canine coronavirus, canine influenza virus, Leptospira serovars, Leishmania organisms, Bordetella bronchiseptica, Mycoplasma species, rabies virus, Ehrlichia canis, an Anaplasma species, and any combination thereof.

15. The vaccine of claim 14, wherein the Mycoplasma species comprises Mycoplasma cynos.

16. The vaccine of claim 15, wherein the Leptospira serovars are selected from the group consisting of Leptospira kirschneri serovar grippotyphosa, Leptospira interrogans serovar canicola, Leptospira interrogans serovar icterohaemorrhagiae, Leptospira interrogans serovar pomona, and any combination thereof.

17. The vaccine composition of claim 9, that is a nonadjuvanted vaccine.

18. A method of immunizing a mammal against a pathogenic Borrelia genospecies comprising administering to the mammal an immunologically effective amount of the vaccine of claim 9.

19. The method of claim 18, wherein the mammal is a canine.

20. The method of claim 18, wherein the mammal is selected from the group consisting of an equine and a feline.

21. (canceled)

Description

DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention provides immunogenic compositions and/or vaccines that include an immunologically effective amount of an alphavirus RNA replicon particle encoding a Borrelia burgdorferi outer surface protein A (OspA) or an antigenic fragment thereof and a Borrelia burgdorferi outer surface protein C (OspC) or an antigenic fragment thereof, an immunologically effective amount of two or more vectors, with at least one alphavirus RNA replicon particle encoding a Borrelia burgdorferi outer surface protein A (OspA) or an antigenic fragment thereof and at least another alphavirus RNA replicon particle encoding a Borrelia burgdorferi outer surface protein C (OspC) or an antigenic fragment thereof, or a combination of the alphavirus RNA replicon particles that encode both Osp A or an antigenic fragment thereof and OspB or an antigenic fragment thereof, with alphavirus RNA replicon particles that encode Osp A or an antigenic fragment thereof and/or encode Osp C or an antigenic fragment thereof. All of such immunogenic compositions may be used in mammalian vaccines. In one aspect of the present invention, the vaccine aids in the protection of the vaccinated subject (e.g., mammal) against Lyme disease. In a particular embodiment of this type, the vaccinated subject is a canine. Accordingly, the present invention provides new immunologic compositions that improve the reliability of vaccination to prevent canine Lyme disease by (i) significantly reducing the potential for untoward side effects by eliminating vaccination with unrelated antigens from bacterins and (ii) still provide comprehensive protection. The Lyme Disease vaccine formulations of the present invention should also significantly lengthen the window of effectiveness by inducing an effective anamnestic memory response.

[0038] In order to more fully appreciate the invention, the following definitions are provided.

[0039] The use of singular terms for convenience in description is in no way intended to be so limiting. Thus, for example, reference to a composition comprising a polypeptide includes reference to one or more of such polypeptides. In addition, reference to an organism includes reference to a plurality of such organisms, unless otherwise indicated.

[0040] As used herein the term approximately is used interchangeably with the term about and signifies that a value is within fifty percent of the indicated value i.e., a composition containing approximately 110.sup.8 alphavirus RNA replicon particles per milliliter contains from 510.sup.7 to 1.510.sup.8 alphavirus RNA replicon particles per milliliter.

[0041] As used herein the term, canine includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise indicated.

[0042] The term genospecies, was first used and defined by G. Baranton et al., 1992, International J. of Systematic Bacteriology 42: 378-383, and is used herein in the same way that the term, species is employed in describing the taxonomy of non-Borrelia organisms.

[0043] The term non-Borrelia, is used to modify terms such as organism, pathogen, and/or antigen (or immunogen) to signify that the respective organism, pathogen, and/or antigen (or immunogen) is not a Borrelia organism, not a Borrelia pathogen, and/or not a Borrelia antigen (or immunogen) respectively, and that a non-Borrelia protein antigen (or immunogen) does not originate from a Borrelia organism.

[0044] The terms originate from, originates from and originating from are used interchangeably with respect to a given protein antigen and the pathogen or strain of that pathogen that naturally encodes it, and as used herein signify that the unmodified and/or truncated amino acid sequence of that given protein antigen is encoded by that pathogen or strain of that pathogen. The coding sequence, within a nucleic acid construct of the present invention for a protein antigen originating from a pathogen may have been genetically manipulated so as to result in a modification and/or truncation of the amino acid sequence of the expressed protein antigen relative to the corresponding sequence of that protein antigen in the pathogen or strain of pathogen (including naturally attenuated strains) it originates from.

[0045] Standard growth conditions for culturing Borrelia genospecies require growth at a temperature ranging from about 33 C. to about 35 C., in BSK (Barbour Stoenner Kelly) medium. BSK medium as described herein was prepared according to Callister et al. [Detection of Borreliacidal Antibodies by Flow Cytometry, Sections 11.5.1-11.5.12, Current Protocols in Cytometry, John Wiley and Sons, Inc. Supplement 26, (2003) hereby incorporated by reference herein in its entirety]. (BSK medium is also commercially available, e.g., from Sigma, St. Louis, Mo.).

[0046] As used herein OspC7 is an immunodominant OspC borreliacidal antibody epitope located in a 7 amino acid region [Lovrich et al., Clin. Diagn. Lab. Immunol., 12:746-751, (2005)] within the C-terminal 50 amino acids of OspC, as disclosed by Callister et al. [U.S. Pat. No. 6,210,676 B1 and U.S. Pat. No. 6,464,985 B1 that is conserved among the known pathogenic Borrelia spp. This conservation is readily confirmed by a BLAST search of the codon segment encoding the 7 amino acid segment described by Lovrich et al. [Clin. Diagn. Lab. Immunol., 12:746-751, (2005)]. Such a search, when conducted on Oct. 9, 2006 generated a results list of 100 Borrelia species containing the above noted OspC 7-mer epitope coding segment. In particular embodiments, an alphavirus RNA replicon particle encodes an antigenic fragment of Osp C that comprises OspC7.

[0047] As used herein, the terms protecting or providing protection to or eliciting protective immunity to and aids in the protection do not require complete protection from any indication of infection. For example, aids in the protection can mean that the protection is sufficient such that, after challenge, symptoms of the underlying infection are at least reduced, and/or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced and/or eliminated. It is understood that reduced, as used in this context, means relative to the state of the infection, including the molecular state of the infection, not just the physiological state of the infection.

[0048] As used herein, a vaccine is a composition that is suitable for application to an animal, e.g., a canine, feline, or equine (including, in certain embodiments, humans, while in other embodiments being specifically not for humans) comprising one or more antigens typically combined with a pharmaceutically acceptable carrier such as a liquid containing water, which upon administration to the animal induces an immune response strong enough to minimally aid in the protection from a disease arising from an infection with a wild-type micro-organism, i.e., strong enough for aiding in the prevention of the disease, and/or preventing, ameliorating or curing the disease.

[0049] As used herein, a multivalent vaccine is a vaccine that comprises two or more different antigens. In a particular embodiment of this type, the multivalent vaccine stimulates the immune system of the recipient against two or more different pathogens.

[0050] As used herein, the term replicon refers to a modified RNA viral genome that lacks one or more elements (e.g., coding sequences for structural proteins) that if they were present, would enable the successful propagation of the parental virus in cell cultures or animal hosts. In suitable cellular contexts, the replicon will amplify itself and may produce one or more sub-genomic RNA species.

[0051] As used herein, the term alphavirus RNA replicon particle, abbreviated RP, is an alphavirus-derived RNA replicon packaged in structural proteins, e.g., the capsid and glycoproteins, which also are derived from an alphavirus, e.g., as described by Pushko et al., [Virology 239(2):389-401 (1997)]. An RP cannot propagate in cell cultures or animal hosts (without a helper plasmid or analogous component), because the replicon does not encode the alphavirus structural components (e.g., capsid and glycoproteins). The heterologous nucleic acid sequences in the RNA RPs encoding OspA and/or OspC, or antigenic fragments thereof, are under the transcriptional control of an alphavirus subgenomic (sg) promoter, in particular the 26S sg promoter, preferably the VEEV 26S sg promoter.

[0052] In case of dual RP constructs of OspA and OspC coding sequences, each of the coding sequences in a construct can be under the transcriptional control of separate subgenomic promoters. In such a dual construct the upstream coding sequence corresponds to the 5 promoter position and the downstream coding sequence corresponds to the 3 promoter position (positive sense RNA; FIGS. 1 and 2). Preferably the upstream- and downstream coding sequences are adjacent.

[0053] As used herein, the term pharmaceutically acceptable is used adjectivally to mean that the modified noun is appropriate for use in a pharmaceutical product. When it is used, for example, to describe an excipient in a pharmaceutical vaccine, it characterizes the excipient as being compatible with the other ingredients of the composition and not disadvantageously deleterious to the intended recipient animal, e.g., canine.

[0054] Parenteral administration includes subcutaneous injections, submucosal injections, intravenous injections, intramuscular injections, intradermal injections, and infusion.

[0055] As used herein the term antigenic fragment in regard to a particular protein (e.g., a protein antigen) is a fragment of that protein (including large fragments that are missing as little as a single amino acid from the full-length protein) that is antigenic, i.e., capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. For example, an antigenic fragment of an outer surface protein A (OspA) is a fragment of the OspA protein that is antigenic. Preferably, an antigenic fragment of the present invention is immunodominant for antibody and/or T cell receptor recognition. In particular embodiments, an antigenic fragment with respect to a given protein antigen is a fragment of that protein that retains at least 25% of the antigenicity of the full length protein. In preferred embodiments, an antigenic fragment retains at least 50% of the antigenicity of the full length protein. In more preferred embodiments, it retains at least 75% of the antigenicity of the full length protein. Antigenic fragments can be as small as 7-20 amino acids (see above) or at the other extreme, be large fragments that are missing as little as a single amino acid from the full-length protein. In particular embodiments, the antigenic fragment comprises 25 to 150 amino acid residues. In other embodiments, the antigenic fragment comprises 50 to 250 amino acid residues.

[0056] An OspC-specific borreliacidal antibody is one that is found, e.g., in the serum of an animal vaccinated with B. burgdorferi ss 50772 (ATCC No. PTA-439), and is one that selectively binds to any epitope of the OspC antigen and kills the spirochetes dependent or independent of complement. An OspC7-specific borreliacidal antibody is one that is found, e.g., in the serum of an animal vaccinated with B. burgdorferi ss 50772 (ATCC No. PTA-439), and is one that selectively binds to the 7 C-terminal amino acids of OspC as described by Lovrich et al. [Clin. Diagn. Lab. Immunol., 12:746-751, (2005)] and kills the spirochetes (generally by inducing a complement-mediated membrane attack complex). The specificity of OspC borreliacidal antibodies has been well-established. For example, OspC borreliacidal antibodies are detected commonly in Lyme disease sera by measuring the susceptibility of B. burgdorferi ss 50772 in a borreliacidal antibody test. Sera from human patients with closely-related illnesses only rarely (2%) contain cross-reactive antibodies that also kill strain 50772 [described in detail by Callister, et al., Clinical and Diagnostic Laboratory Immunology 3(4): 399-4021(1996)]. Moreover, a peptide ELISA that uses the OspC7 borreliacidal epitope accurately captures borreliacidal antibodies in Lyme disease sera, and sera from patients with other closely related illnesses only rarely (<2%) contain cross-reactive antibodies that also bind the OspC7 peptide.

[0057] When a significant proportion of the OspC-specific borreliacidal antibodies in sera induced by a vaccine are specific for the conserved epitope OspC7, it means that there is a measurable reduction in the OspC-specific borreliacidal antibodies in the sera following the absorption of that sera with OspC7. It is preferably defined as at least a 2-fold reduction in the borreliacidal antibody titer of the sera detected by using B. burgdorferi ss 50772, and more preferably as a 2- to 4-fold, or greater reduction in the borreliacidal antibody titer of the sera following the absorption of that sera with OspC7.

[0058] A complement specific reaction is an antibody reaction that requires serum complement to be present in order for Borrelia spp. organism(s) to be killed by a borreliacidal antibody.

[0059] As used herein, the term inactivated microorganism is used interchangeably with the term killed microorganism. For the purposes of this invention, an inactivated Borrelia burgdorferi ss organism is an organism which is capable of eliciting an immune response in an animal, but is not capable of infecting the animal. The Borrelia burgdorferi ss isolates may be inactivated by an agent selected from the group consisting of binary ethyleneimine, formalin, beta-propiolactone, thimerosal, or heat. In a particular embodiment, the Borrelia burgdorferi ss isolates are inactivated by binary ethyleneimine.

[0060] As used herein, a nonadjuvanted vaccine is a vaccine or a multivalent vaccine that does not contain an adjuvant.

[0061] B. burgdorferi ss 50772 (ATCC No. PTA-439) as stated in U.S. Pat. No. 6,210,676, and B. burgdorferi ss S-1-10 (ATCC No. PTA-1680) as stated in U.S. Pat. No. 6,316,005, were deposited with the American Type Culture Collection, 10801 University Boulevard Manassas (Va.) 20110 on Jul. 30, 1999, and Apr. 11, 2000, respectively.

[0062] As used herein one amino acid sequence is 100% identical or has 100% identity to a second amino acid sequence when the amino acid residues of both sequences are identical. Accordingly, an amino acid sequence is 50% identical to a second amino acid sequence when 50% of the amino acid residues of the two amino acid sequences are identical. The sequence comparison is performed over a contiguous block of amino acid residues comprised by a given protein, e.g., a protein, or a portion of the polypeptide being compared. In a particular embodiment, selected deletions or insertions that could otherwise alter the correspondence between the two amino acid sequences are taken into account.

[0063] As used herein, nucleotide and amino acid sequence percent identity can be determined using C, MacVector (MacVector, Inc. Cary, N.C. 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters. Alternatively, an Advanced Blast search under the default filter conditions can be used, e.g., using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program using the default parameters.

[0064] It is also to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

Alternative OspA Strains

[0065] Strains providing the OspA antigen, can be a conventional pathogenic laboratory B. burgdorferi ss isolate [Barbour et al., J. Clin. Microbiol. 52:478-484 (1985)] such as B. burgdorferi ss B-31 (ATCC No. 35210). A particular second organism is the exemplified B. burgdorferi ss S-1-10 strain (ATCC No. PTA-1680). Additional strains suitable for use as the second organism for vaccine compositions optimized for regions outside of North America include, e.g., the strains: B. burgdorferi ss B-31 (ATCC No. 35210), B. afzelii (e.g., available as ATCC No. 51567) and B. garinii (e.g., available as ATCC Nos. 51383 and 51991), as well as those listed in Table 1 below.

TABLE-US-00001 TABLE 1 Strain Country Cultured from B. burgdorferi ss DK7 .sup.1 Denmark skin B. burgdorferi ss 61BV3 .sup.1 Germany skin B. burgdorferi ss ZS7 .sup.1 Switzerland tick B. burgdorferi ss Pka .sup.1 Germany tick B. burgdorferi ss IP1, IP2, IP3 .sup.1 France CSF B. burgdorferi ss HII .sup.1 Italy blood B. burgdorferi ss P1F .sup.1 Switzerland synovia B. burgdorferi ss Mil .sup.1 Slovakia tick B. burgdorferi ss 20006 .sup.1 France tick B. burgdorferi ss 212 .sup.1 France tick B. burgdorferi ss ESP1 .sup.1 Spain tick B. burgdorferi ss Ne-56 .sup.1 Switzerland tick B. burgdorferi ss Z136 .sup.1 Germany tick B. burgdorferi ss ia .sup.2 Finland CSF .sup.1 Lagal et al., J. Clin. Microbiol. 41: 5059-5065 (2003) .sup.2 Heikkila et al., J. Clin. Microbiol. 40: 1174-1180 (2002)

[0066] The vaccine composition is readily administered by any standard route including intravenous, intramuscular, subcutaneous, oral, intranasal, intradermal, and/or intraperitoneal vaccination. The artisan will appreciate that the vaccine composition is preferably formulated appropriately for each type of recipient animal and route of administration.

[0067] Thus, the present invention also provides methods of immunizing a canine against B. burgdorferi ss and other Borrelia spp. One such method comprises injecting a canine with an immunologically effective amount of a vaccine of the present invention, so that the canine produces appropriate OspA and/or OspC. In particular embodiments the antibodies are borreliacidal antibodies.

EXAMPLES

[0068] The following examples serve to provide further appreciation of the invention, but are not meant in any way to restrict the effective scope of the invention.

Example 1

Construction of OspA and OspC Vaccines Delivered by Alphavirus RNA Replicon Particles

[0069] RNA viruses have been used as vector-vehicles for introducing vaccine antigens, which have been genetically engineered into their genomes. However, their use to date has been limited primarily to incorporating viral antigens into the RNA virus and then introducing the virus into a recipient host. The result is the induction of protective antibodies against the incorporated viral antigens. For example, the alphavirus replicon vector has been used to protect mice against botulinum neurotoxin and anthrax via expression of C. botulinum neurotoxin Hc or the B. anthracis protective antigen, respectively [Lee et al., Vaccine 24(47-48) 6886-6892 (2006)]. Alphavirus RNA replicon particles have been used to encode pathogenic antigens. Such alphavirus replicon platforms have been developed from several different alphaviruses, including Venezuelan equine encephalitis virus (VEE) [Pushko et al., Virology 239:389-401 (1997)], Sindbis (SIN) [Bredenbeek et al., Journal of Virology 67:6439-6446 (1993) the contents of which are hereby incorporated herein in their entireties], and Semliki Forest virus (SFV) [Liljestrom and Garoff, Biotechnology (NY) 9:1356-1361 (1991), the contents of which are hereby incorporated herein in their entireties]. Moreover, alphavirus RNA replicon particles are the basis for several USDA-licensed vaccines for swine and poultry. These include: Porcine Epidemic Diarrhea Vaccine, RNA Particle (Product Code 19U5.P1), Swine Influenza Vaccine, RNA (Product Code 19A5.D0), Avian Influenza Vaccine, RNA (Product Code 1905.D0), and Prescription Product, RNA Particle (Product Code 9PP0.00). As disclosed below, the ability of an alphavirus RNA replicon vector system to induce canines to produce borreliacidal antibodies specific for OspA, OspC, and DbpA has been examined.

Incorporation of the Coding Sequences for OspA or OspC, into the Alphavirus Replicon:

[0070] Amino acid sequences for OspA (strain 297), and OspC (strain 50772) were used to generate codon-optimized (Canis lupus codon usage) nucleotide sequences in silico. Optimized sequences were prepared as synthetic DNA by a commercial vendor (ATUM, Newark, Calif.).

[0071] The VEE replicon vectors designed to express OspA or OspC were constructed as previously described [see, U.S. Pat. No. 9,441,247 B2; the contents of which are hereby incorporated herein by reference], with the following modifications. The TC-83-derived replicon vector pVEK [disclosed and described in U.S. Pat. No. 9,441,247 B2] was digested with restriction enzymes Ascl and Pacl. A DNA plasmid containing the codon-optimized open reading frame sequence of the OspA, or OspC, [0072] with 5 flanking sequence (5-GGCGCGCCGCACC-3) [SEQ ID NO: 5] and 3 flanking sequence (5-TTAATTAA-3),
was similarly digested with restriction enzymes Ascl and Pacl. The synthetic gene cassette was then ligated into the digested pVEK vector, and the resulting clones were re-named pVHV-OspA and pVHV-OspC.

[0073] Production of TC-83 RNA replicon particles (RP) was conducted according to methods previously described [U.S. Pat. No. 9,441,247 B2 and U.S. Pat. No. 8,460,913 B2; the contents of which are hereby incorporated herein by reference]. Briefly, pVHV replicon vector DNA and helper DNA plasmids were linearized with Notl restriction enzyme prior to in vitro transcription using MegaScript T7 RNA polymerase and cap analog (Promega, Madison, Wis.). Importantly, the helper RNAs used in the production lack the VEE subgenomic promoter sequence, as previously described [Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727 (2010)]. Purified RNA for the replicon and helper components were combined and mixed with a suspension of Vero cells, electroporated in 4 mm cuvettes, and returned to serum-free cell culture media obtained from Thermo Fisher, Waltham Mass. sold under the name OptiPro SFM. Following overnight incubation, alphavirus RNA replicon particles were purified from the cells and media by passing the suspension through a ZetaPlus BioCap depth filter (3M, Maplewood, Minn.), washing with phosphate buffered saline containing 5% sucrose (w/v), and finally eluting the retained RP with 400 mM NaCl buffer. Eluted RP were formulated to a final 5% sucrose (w/v), passed through a 0.22 micron membrane filter, and dispensed into aliquots for storage. Titer of functional RP was determined by immunofluorescence assay on infected Vero cell monolayers. Batches of RP were identified according to the gene encoded by the packaged replicon: RP-OspA or RP-OspC.

Example 2

Vaccine with RP-OspA Construct

Materials and Methods

Construct:

[0074] The RP-OspA construct was produced as described above using a nucleotide sequence encoding an antigen comprising the immunogenic epitopes of outer surface protein A.

Animals:

[0075] Five month old beagles (Marshall Bioresources) were housed communally in raised dog runs, and food and water was available ad libitum.

Preparation of the RP-OspA Vaccine:

[0076] The OspA RNA was electroporated in conjunction with helper RNAs into Vero cells. The OspA was packaged into RPs following the co-electroporation process generating the RP-OspA. The RP-OspA was then blended with stabilizer (sucrose, N-Z Amine, gelatin), 0.9% saline, amphotericin B, and gentamicin so that a 1.0 mL dose contained a target of 1.010.sup.8 replicon particles/mL. The vaccine was then freeze dried.

Vaccination and Collection of Serum:

[0077] Dogs were vaccinated subcutaneously in the neck with a 1 mL dose of the RP-OspA vaccine and boosted with an additional 1 mL dose after 21 days. Whole blood was collected on study days 7, 14, 20, 29, 35, and 42 by venipuncture of the jugular vein. The serum was separated by centrifugation and stored at 10 C. or colder until tested.

Detection of OspA Borreliacidal Antibodies:

[0078] OspA borreliacidal antibodies were detected using a flow cytometric procedure and B. burgdorferi ss S-1-10 [Callister et al., Arch. Intern. Med. 154:1625-1632 (1994)].

Detection of OspA IgG Antibodies:

[0079] OspA IgG opsonizing antibodies were detected by ELISA.

Results

[0080] Vaccination with the RP-OspA vaccine reliably induced high levels of IgG antibodies, and the antibody response included a significant amount of borreliacidal OspA antibodies at 2 weeks post-booster vaccination.

TABLE-US-00002 TABLE 2 Mean Antibody Titers (n = 5) after Vaccination with RP-OspA Antibody Type Day 1 Day 35 IgG ND.sup.a 7610 Borreliacidal ND.sup.a 3044 .sup.aND = none detected

[0081] The results in Table 2 above, demonstrate the ability of a vaccine comprising RP-OspA to induce significant levels of OspA borreliacidal antibodies.

Example 3

Vaccine with RP-OspC Construct

Materials and Methods

Construct:

[0082] The RP-OspC construct was produced as described above using a nucleotide sequence encoding an antigen comprising the immunogenic epitopes of outer surface protein C.

Animals:

[0083] Five month old beagles (Marshall Bioresources) were housed communally in raised dog runs, and food and water was available ad libitum.

Preparation of the RP-OspC Vaccine:

[0084] The OspC RNA was electroporated in conjunction with helper RNAs into Vero cells. The OspC was packaged into RPs following the co-electroporation process generating the RP-OspC. The RP-OspC was then blended with stabilizer (sucrose, N-Z Amine, gelatin), 0.9% saline, amphotericin B, and gentamicin so that a 1.0 mL dose contained a target of 1.010.sup.8 replicon particles/mL. The vaccine was then freeze dried.

Vaccination and Collection of Serum:

[0085] Dogs were vaccinated subcutaneously in the neck with a 1 mL dose of the RP-OspC vaccine and boosted with an additional 1 mL dose after 21 days. Whole blood was collected on study days 7, 14, 20, 29, 35, and 42 by venipuncture of the jugular vein. The serum was separated by centrifugation and stored at 10 C. or colder until tested.

Detection of OspC Borreliacidal Antibodies:

[0086] OspC borreliacidal antibodies were detected using a flow cytometric procedure and B. burgdorferi ss 50772 [Callister et al., Arch. Intern. Med. 154:1625-1632 (1994)].

Detection of OspC IgG Antibodies:

[0087] OspC IgG antibodies were detected by ELISA.

Results

[0088] Vaccination with the RP-OspC vaccine reliably induced high levels of IgG antibodies, and the antibody response included a significant amount of borreliacidal OspC antibodies at 2 weeks post-booster vaccination.

TABLE-US-00003 TABLE 3 Mean Antibody Titers (n = 5) after Vaccination with RP-OspC Antibody Type Day 1 Day 35 IgG ND.sup.a 696 Borreliacidal ND.sup.a 1940 .sup.aND = none detected

[0089] The results demonstrate the ability of a vaccine comprising RP-OspC to induce significant levels of OspC borreliacidal antibodies.

Example 4

Combination Vaccine with RP-OspA and RP-OspC

Materials and Methods

Construct:

[0090] The RP-OspA and RP-OspC constructs were produced as described above.

Animals:

[0091] Five month old beagles (Marshall Bioresources) were housed communally in raised dog runs, and food and water was available ad libitum.

Preparation of the RP-OspA, and RP-OspC Combination Vaccine:

[0092] The RP-OspA and RP-OspC antigens were blended with stabilizer (sucrose, N-Z Amine, gelatin), 0.9% saline, amphotericin B, and gentamicin so that a 1.0 mL dose contained a target of 1.010.sup.8 replicon particles/mL of each construct. The vaccine was then freeze dried.

Vaccination and Collection of Serum:

[0093] Dogs were vaccinated subcutaneously in the neck with a 1 mL dose of the combination vaccine and boosted with an additional 1 mL dose after 21 days. Whole blood was collected on study days 7, 14, 20, 29, 35, and 42 by venipuncture of the jugular vein. The serum was separated by centrifugation and stored at 10 C. or colder until tested.

Detection of Borreliacidal Antibodies:

[0094] OspA and OspC borreliacidal antibodies were detected using a flow cytometric procedure and B. burgdorferi ss S-1-10 or B. burgdorferi ss 50772, respectively [Canister et al., Arch. Intern. Med. 154:1625-1632 (1994)]. Detection of IgG antibodies: OspA and OspC antibodies were detected by ELISA.

Results

[0095] Vaccination with the combination vaccine reliably induced high levels of IgG and borreliacidal OspA and OspC antibodies at 2 weeks post-booster vaccination. The results demonstrate the ability of a combination vaccine comprising RP-OspA and RP-OspC to induce high levels of OspA and OspC borreliacidal antibodies and to induce high levels of RP-OspC opsonizing IgG antibodies.

Example 5

Combination Vaccine with RP-OspA and RP-OspC

Constructs:

[0096] The RP-OspA and RP-OspC constructs were produced as described above.

Animals:

[0097] Three month old beagles (Ridglan Farms) were housed communally in dog runs, and food and water was available ad libitum.

Preparation of the RP-OspA, and RP-OspC Combination Vaccine:

[0098] Treatment Group A received a combination of BEI inactivated bacterin of strains S-1-10 and 50772 (blended with 5% Emulsigen adjuvant solution-MVP Laboratories Inc., Omaha, US). The RP-OspA and RP-OspC antigens were blended with stabilizer (sucrose, N-Z Amine, gelatin), 0.9% saline, amphotericin B, and gentamicin so that a 1.0 mL dose contained a target of either 5.0107 (Treatment Group B), 5.0106 (Treatment Group C), or 5.0105 (Treatment Group D) replicon particles/mL of each construct. The vaccines were freeze dried.

Vaccination and Injection Site Reactions:

[0099] Dogs were vaccinated subcutaneously in the neck with a 1 mL dose of the combination vaccine and boosted with an additional 1 mL dose after 21 days. Dogs were monitored for injection site reactions on study days 3 and 4 after the first vaccination and study days 24 and 25 after the second vaccination until no reaction could be felt (Table 4). The injection site reactions were evaluated based on type and size. Reactions were scored as visible, thickening, soft, hard, or tender, and reaction size was scored as S1=<1.0 cm, S2=1.0-2.0 cm, or S3=>2.0 cm.

Results

[0100]

TABLE-US-00004 TABLE 4 Injection site reactions Treatment Group Day 3 Day 4 Day 5 Day 24 Day 25 Day 26 Day 27 Day 28 A S2 (T) S1 (T) S1 (S) Whole Cell S1 (T) S1 (S) S1 (S) S1 (S) S1 (H) Bacterin S1 (H) S1 (H) S1 (H) Min. S1 (S) S1 (T) Protective S1 (S) S1 (S) Dose B OspA, OspC7, S1 (S) DbpA-tpA 5.0 10.sup.7 C OspA, OspC7, S2 (T) DbpA-tPA 5.0 10.sup.6 D OspA, OspC7, Dbpa-tpA 5.0 10.sup.5 S1 = <1.0 cm; S2 = 1.0-2.0 cm; S3 = >2.0 cm; T = thickening; S = Soft; H = Hard; V = Visible = no reaction

Example 6

Combination Vaccine with Dual Insert RP-OspA/C Constructs

Materials and Methods

[0101] Incorporation of the Coding Sequences for OspA and OspC, into the Alphavirus Replicon:

[0102] Ascl and Pacl digested TC-83-derived replicon vector pVEK was prepare as described in Example 1. Two DNA plasmids containing the codon-optimized open reading frame sequences of both the OspA and OspC, with 5 flanking sequence (5-GGCGCGCCGCACC-3) [SEQ ID NO: 5] and 3 flanking sequence (5-TTAATTAA-3), were similarly digested with restriction enzymes Ascl and Pacl. The design of the synthetic gene cassettes incorporates one of the open reading frame sequences (OspA or OspC), a non-coding sequence containing the alphavirus subgenomic promoter and flanking sequences, and then the other desired open reading frame (OspA or OspC). The alphavirus subgenomic promoter and flanking sequences are 5-GTTTAAACTGTAAAACGACGGCCAGTAGTCGTCATAGCTGTTTCCTGGCTACCTGAGA GGGGCCCCTATAACTCTCTACGGCTAACCTGAATGGACTACGACATAGTCTAGTCCGC CAAGATATCTTCAGCACCGGTGGCACC-3 [SEQ ID NO: 6]. This design duplicates a short portion of the 3 nsP4 open reading frame, the native alphavirus subgenomic promoter, and the 5 untranslated portion of the native subgenomic sequence. Also incorporated are restriction enzyme recognition sites, noncoding random sequences, primer binding sites, and a Kozak consensus sequence immediately 5 proximal to the second open reading frame. The synthetic gene cassettes were then ligated into the digested pVEK vector, and the resulting clones were re-named pVDG-OspA-OspC or pVDG-OspC-OspA, with the order of the names denoting the relative 5 and 3 position within the cassette.

[0103] Production of RPs was conducted as described in Example 1.

Animals:

[0104] 7-8 week old beagles (Ridglan Farms) were housed communally in dog runs, and food and water were available ad libitum.

Preparation of the RP-OspA/OspC or RP-OspC/OspA Vaccines:

[0105] The replicon RNA for each construct was electroporated in conjunction with helper RNAs (derived from VEE capsid helper and glycoprotein sequences) into Vero cells. Each replicon was packaged into RPs following the co-electroporation process generating the RP antigens. The resulting RPs were then collected in 0.4M NaCl phosphate buffer, formulated with 5% (w/v) sucrose, and quantified by immunofluorescence assay.

[0106] Three separate vaccines were blended with stabilizer (sucrose, N-Z Amine, gelatin) and 0.9% saline in a 1 mL dose. The vaccine in Treatment Group A contained a target dose of 5.0107 for each separate RP-OspA and RP-OspC antigen. The vaccine in Treatment Group B contained a target dose of 5.0107 for the RP-OspA/OspC dual construct antigen. The vaccine in Treatment Group C contained a target dose of 5.0107 for the RP-OspC/OspA dual construct antigen. The vaccines were freeze-dried.

Vaccination and Collection of Serum:

[0107] Dogs were vaccinated subcutaneously in the neck with a 1 mL dose of the vaccine and boosted with an additional 1 mL dose after 21 days. Whole blood was collected on study days 1, 28, 35, 70, 92, and 119 by venipuncture of the jugular vein. The serum was separated by centrifugation and stored at 10 C. or colder until tested.

Detection of OspA and OspC Borreliacidal Antibodies:

[0108] OspA borreliacidal antibodies were detected using a flow cytometric procedure and B. burgdorferi ss

[0109] S-1-10 [Callister et al., Arch. Intern. Med. 154:1625-1632 (1994)]. OspC borreliacidal antibodies were detected using a flow cytometric procedure and B. burgdorferi ss 50772 [Callister et al., Arch. Intern. Med. 154:1625-1632 (1994)].

Results

[0110] A vaccine containing separate RP-OspA and RP-OspC antigens induced moderate levels of borreliacidal antibodies at 1 week post-booster vaccination. At 1 week post-booster vaccination, a vaccine containing the RP-OspA/OspC dual construct antigen induced high levels of borreliacidal antibodies to OspC but relatively low levels of borreliacidal antibodies to OspA. In contrast, a vaccine containing the RP-OspC/OspA dual construct antigen induced high levels of borreliacidal antibodies to OspA, but relatively low levels of borreliacidal antibodies to OspC. The data suggest that a more robust borreliacidal antibody response to OspA or OspC was induced when that gene was in the downstream position of the construct (Table 5).

TABLE-US-00005 TABLE 5 Borreliacidal Data OspA Borreliacidal OspC Borreliacidal Treatment Group Titers (Day 28) Titers (Day 28) Treatment Group A 5120 10240 OspA + OspC 2560 1280 (Separate Constructs) 10240 10240 2560 1280 320 5120 5120 5120 1280 <80* Geomean 2560 2100 Treatment Group B 5120 20480 OspA/OspC 5120 20480 (Dual Construct) 80 20480 40 2560 320 20480 640 1280 5120 20480 Geomean 707 10240 Treatment Group C 5120 <80* OspC/OspA 1280 80 (Dual Construct) 20480 2560 20480 10240 10240 80 2560 80 2560 640 Geomean 5653 320 Treatment Group D <80 <80 Placebo <80 <80 <80 <80 <80 <80 <80 <80 <80 <80 <80 <80 Geomean <80 <80 *A value of 40 was used to determine the Geomean
Challenge with B. burgdorferi Infected Ixodes scapularis Ticks:

[0111] The experimental challenge with B. burgdorferi-infected ticks was conducted approximately 2 weeks after the second vaccination. Briefly, 9 female and 8 male adult ticks were placed onto the shaved side of each dog in a rubber cup that was held in place with tape and bandage wrap. The ticks were allowed to feed on the dogs for 7 days and removed. At 1, 2, and 3 months post-challenge, a skin biopsy was taken using a 4 mm puncture device from each dog, at a site adjacent to tick attachment site, for isolation of B. burgdorferi. The skin biospies were incubated in BSA rich media and observed for 4 weeks for the growth of B. burgdorferi. Tissue samples from the left side of the dog or from a limb that demonstrated limping and/or lameness were collected from the elbow, carpus, stifle, and tarsus and processed for isolation of B. burgdorferi by PCR (Table 6).

TABLE-US-00006 TABLE 6 Number of Dogs Positive for B. burgdorferi from Either the Skin or Joints No. of Dogs No. of Dogs Treatment Group Skin Biopsy Positive Joint Positive Treatment Group A: 0/7 0/7 OspA + OspC (Separate Constructs) Treatment Group B: 0/7 0/7 OspA/OspC (Dual Construct) Treatment Group C: 0/7 0/7 OspC/OspA (Dual Construct) Treatment Group D: 6/7 5/7 Placebo

TABLE-US-00007 SEQUENCETABLE SEQID NO: Description Type 1 OuterSurfaceProteinA nucleicacid 2 OuterSurfaceProteinA aminoacid 3 OuterSurfaceProteinC nucleicacid 4 OuterSurfaceProteinC aminoacid 5 ggcgcgccgcacc nucleicacid 6 GTTTAAACTGTAAAACGACGGCCAGTAGTCGTCATAGCTGT nucleicacid TTCCTGGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGC TAACCTGAATGGACTACGACATAGTCTAGTCCGCCAAGATA TCTTCAGCACCGGTGGCACC

TABLE-US-00008 SEQUENCES OuterSurfaceProteinA(SEQIDNO:1) atgaaaaagtaccttttgggaatcggactcattctcgccctgatcgcctgcaagcaaaacgtgtcct ccctcgacgaaaagaactcagtgtcggtggatctgcccggcgaaatgaaggtgctcgtgtccaaaga gaagaacaaggatggaaaatacgacctgattgccaccgtggacaagctggagttgaagggcacctca gacaagaacaacgggtctggagtgctggaaggagtcaaagcggacaagtccaaggtcaagctgacta tttcggacgacctgggccagactaccctggaagtgttcaaggaggacggaaagaccctggtgtccaa gaaggtcacctccaaggataagtcgagcaccgaagagaagttcaatgagaagggagaagtgtcggag aagatcatcacccgcgccgatggaacccggctggagtacaccgagatcaagtccgatggttcgggga aggctaaggaagtcctgaagggctacgtgcttgagggtactctgactgcggaaaagaccactctggt ggtcaaggaaggcaccgtgactctgtcaaagaacatctccaagagcggagaagtcagcgtggaactg aacgacacagattcctccgctgccacgaaaaagaccgccgcctggaacagcgggaccagcactctca ccattaccgtgaacagcaaaaagactaaggacctggtgttcaccaaggagaacacgatcaccgtgca gcagtatgactccaacggtaccaagctcgaagggtccgccgtggagatcactaagctggacgagatt aagaatgcactgaagtga OuterSurfaceProteinA(SEQIDNO:2) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDG KYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLE VFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTEI KSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVE LNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDS NGTKLEGSAVEITKLDEIKNALK* OuterSurfaceProteinCSEQIDNO:3 atgaagaagaatactctctccgccattctgatgaccctgttcctgtttatctcctgcaacaactccg ggaaggatggcaacacctcggccaactccgccgatgaaagcgtcaagggtcccaacctgactgagat ctcgaagaaaatcaccgagtccaacgcggtggtgttggcagtgaaggaggtcgaaactctgctgact agcatcgacgagcttgccaaggccattggaaagaagattaagaacgacgtgtcactggacaacgaag ctgaccataacggatctcttatctcgggcgcttacctgatttcgaccctcatcaccaagaagatctc cgcgatcaaggacagcggggagctcaaggccgaaattgagaaagcaaagaagtgctccgaagagttc accgcgaagctcaagggagaacacaccgacctgggaaaggaaggcgtcaccgatgataacgcgaaga aggccatcctcaaaaccaacaacgacaagacaaagggcgccgacgaactggagaagctgttcgagag cgtgaagaatctgtccaaggccgccaaggaaatgttgacgaacagcgtgaaggaactgacctcccct gtggtggccgagtcaccgaaaaagccatga OuterSurfaceProteinC(SEQIDNO:4) MKKNTLSAILMTLFLFISCNNSGKDGNTSANSADESVKGPNLTEISKKIT ESNAVVLAVKEVETLLTSIDELAKAIGKKIKNDVSLDNEADHNGSLISGA YLISTLITKKISAIKDSGELKAEIEKAKKCSEEFTAKLKGEHTDLGKEGV TDDNAKKAILKTNNDKTKGADELEKLFESVKNLSKAAKEMLTNSVKELTS PVVAESPKKP*

[0112] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0113] It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.