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  Pharmaceutical Patents  

 

Title:  Recombinant constructs of Borrelia burgdorferi
United States Patent: 
7,605,248
Issued: 
October 20, 2009

Inventors:
 Dattwyler; Raymond J. (Setauket, NY), Gomes-Solecki; Maria J. C. (New York, NY), Luft; Benjamin J. (East Setauket, NY), Dunn; John J. (Bellport, NY)
Assignee: 
Research Foundation of the State University of New York (Stony Brook, NY), Brookhaven Sciences Associates, LLC (Upton, NY)
Appl. No.: 
11/527,161
Filed:
 September 26, 2006


 

Patheon


Abstract

Novel chimeric nucleic acids, encoding chimeric Borrelia proteins comprising OspC or an antigenic fragment thereof and OspA or an antigenic fragment thereof, are disclosed. Chimeric proteins encoded by the nucleic acid sequences are also disclosed. The chimeric proteins are useful as vaccine immunogens against Lyme borreliosis, as well as for immunodiagnostic reagents.

Description of the Invention

SUMMARY OF THE INVENTION

The current invention pertains to chimeric Borrelia proteins which include two or more antigenic Borrelia polypeptides which do not occur naturally (in nature) in the same protein in Borrelia, as well as the nucleic acids encoding such chimeric proteins. The proteins from which the antigenic polypeptides are derived can be from the same strain or genospecies of Borrelia, from different strains or genospecies, or from combinations of proteins from the same and from different strains or genospecies. Particular chimeric proteins, and the nucleotide sequences encoding them, are set forth in FIGS. 30-37 and 55-72 (see Original Patent).

The chimeric proteins of the current invention provide antigenic polypeptides of a variety of Borrelia strains and/or proteins within a single protein. Such proteins are particularly useful in immunodiagnostic assays to detect the presence of antibodies to native Borrelia in potentially infected individuals as well as to measure T-cell reactivity, and can therefore be used as immunodiagnostic reagents. These chimeric proteins are also useful in the generation of immune responses (such as antibody production) against proteins expressed by Borrelia burgdorferi. The chimeric proteins of the current invention are additionally useful as vaccine immunogens against Borrelia infection.

In one embodiment of the present invention, the chimeric proteins are made up of polypeptide fragments from Lyme Disease-causing strains of Borrelia. In another embodiment, the polypeptide fragments that make up the chimeric protein are from outer surface protein A (OspA) and outer surface protein C (OspC), which have the general structure of OspC linked via a peptide bond to the N-terminus of OspA. The present invention encompasses both lipidated and unlipidated chimeric proteins. In one embodiment, the OspA and OspC portions of the chimeric protein possess a lipidation signal. In other embodiments, either the OspA polypeptide portion, the OspC polypeptide portion, or both, do not include a lipidation signal.

The OspA portion of the chimeric polypeptide can itself comprise OspA portions from two or more strains of Lyme Disease-causing Borrelia as described herein and provided, for example, in FIGS. 23-29 and 43-46 (see Original Patent). In another embodiment, the OspA polypeptide comprises OspA portions from two or more genospecies of Lyme Disease-causing Borrelia, for example, wherein the genospecies are defined as Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. In this manner, the OspC and OspA polypeptide fragments that make up the chimeric protein can be from the same strain or genospecies of Borrelia, from different strains or genospecies of Borrelia, or from combinations of proteins from the same and from different strains or genospecies of Borrelia.

The present invention is also drawn to nucleic acids which encode a Borrelia chimeric protein. In a particular embodiment, the composition comprises a nucleic acid that encodes a chimeric protein of at least two polypeptides, wherein the first polypeptide comprises Borrelia burgdorferi OspC, and the second polypeptide comprises Borrelia burgdorferi OspA, such that OspC is upstream of OspA. The OspC and OspA nucleic acid fragments that make up the chimeric protein can be from the same strain or genospecies of Borrelia, from different strains or genospecies of Borrelia, or from combinations of proteins that are from the same and/or different strains or genospecies of Borrelia.

The present invention is also drawn to expression vectors which comprise an isolated DNA encoding a Borrelia chimeric protein. In one embodiment, the composition includes an expression vector comprising an isolated DNA which encodes an OspC/OspA chimeric protein as described herein. The present invention also encompasses host cells which comprise a recombinant nucleic acid encoding an OspC/OspA chimeric protein as described herein.

The present invention is also drawn to methods of making the Borrelia chimeric polypeptides described herein. In one embodiment, the method of making a chimeric Borrelia protein comprises selecting a polynucleotide sequence encoding OspC, or an antigenic portion thereof, selecting a polynucleotide sequence encoding OspA, or an antigenic portion thereof, and ligating these polynucleotide sequences together.

The present invention is also drawn to methods of delivering the Borrelia chimeric polypeptides described herein. In one embodiment, the method comprises administering the chimeric protein in a physiologically-acceptable carrier to an individual. As a result of the administration of the chimeric protein, the individual develops at least some immune response to the chimeric protein, e.g., the individual generates a humoral immune response, wherein antibodies are produced by the individual that recognize at least a portion of said chimeric polypeptide.

The present invention is also drawn to methods of delivering nucleic acids which encode the chimeric polypeptides described herein. In one embodiment, the method comprises administering the nucleic acid in a physiologically-acceptable carrier to an individual. As a result of the administration of the nucleic acid, the individual expresses the chimeric protein at least transiently and develops at least some immune response to the chimeric protein encoded by the nucleic acid, e.g., the individual generates a humoral immune response, wherein antibodies that recognize at least a portion of the chimeric polypeptide produced from the nucleic acid, are produced by the individual.

The invention also encompasses methods of using the chimeric proteins described herein in a diagnostic assay. As described herein, the method can be used to detect the presence of OspA- and/or OspC-specific antibodies in a sample, e.g., a host sample of interest. The method comprises contacting a sample, e.g., a host sample of interest, with the chimeric protein, under conditions, wherein antibodies, if present in the host sample, bind to the chimeric protein thereby forming antigen-antibody complexes. The antigen-antibody complexes are then detected. In this manner, the chimeric proteins of the present invention can be used to detect an immune response to Lyme Disease causing Borrelia.

The present invention is also drawn to diagnostic kits which comprise the chimeric polypeptides described herein. In one embodiment, the kit comprises a Borrelia burgdorferi OspC/OspA chimeric protein. The kit also includes reagents for detecting antibody-antigen complexes that are formed between the OspC/OspA chimeric protein and antibodies that are present in a sample, e.g., a user-supplied host sample.

The present invention is also drawn to pharmaceutical compositions which can be used to vaccinate and/or treat Borrelia infection in an animal or human. The pharmaceutical composition can be administered together with a physiologically-acceptable carrier and/or with suitable excipients and/or adjuvants.

The present invention is also drawn to methods of immunizing an animal or human against Lyme disease. In a particular embodiment, the method comprises administering a Borrelia chimeric OspC/OspA protein. The chimeric protein can be administered together with a physiologically-acceptable carrier, a suitable excipient and/or a suitable adjuvant, to an animal or human such that the animal or human develops an immune response to at least one of the OspC and/or OspA polypeptides of the composition.

By incorporating polypeptide fragments from multiple Borrelia burgdorferi proteins, the present invention provides a composition that has great utility for vaccines and diagnostic kits. As a result of the present invention, there exist diagnostic tools and vaccines that comprise both OspA and OspC antigens from various Borrelia burgdorferi strains and/or genospecies in a single protein. Since OspA is primarily expressed in the tick vector, and OspC is upregulated in response to the feeding of an infected tick on a mammal, this allows for a diagnostic tool or vaccine that can recognize antigens that are expressed at different stages of the life cycle of Borrelia burgdorferi. Thus, the chimeric proteins of the present invention can act at the level of the tick as well as the level of the host, in preventing infection and/or disease caused by Borrelia. Moreover, by incorporating unique polypeptide fragments from pathogenic families of Borrelia, such as Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, an improved diagnostic tool or vaccine is produced which can detect clinically important exposure to a wider variety of pathogenic Borrelia, while overlooking the remainder of non-pathogenic families of Borrelia. Furthermore, OspC polypeptides can be selected from strains of Borrelia that are associated with disseminated disease, as described in WO 00/78966, the teachings of which are incorporated herein in their entirety.

The present invention also provides a combination of Borrelia antigens in a single polypeptide that, when used as a vaccine, are expected to prevent Lyme disease from becoming systemic. The chimeric proteins of the present invention can be effective in preventing Lyme disease, as well as having a therapeutic effect on established infection, for example after the tick bite is noticed by the patient.

The present invention is drawn to both lipidated and unlipidated chimeric proteins. Unlipidated chimeric proteins, such as the OspC/OspA chimeric proteins described herein, have certain advantages over their lipidated counterparts. These advantages include simpler production methods, improved yields of protein and simpler purification methods. While the lack of a lipidation signal provides several advantages, it had been thought that the lipidation signal was required for immunogenicity. However, as described herein, the non-lipidated OspC/OspA chimeric proteins of the present invention elicit an immune response that is at least as broadly reactive as that of lipidated OspA and lipidated OspC control proteins. Moreover, the unlipidated OspC/OspA chimeric proteins of the present invention unexpectedly elicit an immune response to more than one genospecies and/or strain of Lyme disease-causing Borrelia, including genospecies and/or strains that were not used to generate the particular chimeric OspC/OspA immunogen.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to chimeric proteins comprising various antigenic Borrelia polypeptides. In a preferred embodiment, the chimeric protein comprises Borrelia outer surface protein C (OspC) and outer surface protein A (OspA). These chimeric proteins have the general structure of OspC linked to OspA via a peptide bond. Each of the OspA and OspC portions of the chimeric OspC/OspA protein can be lipidated or unlipidated. In a preferred embodiment, the OspC/OspA chimer comprises OspC and OspA polypeptide fragments that do not possess their lipidation signals.

The chimeric forms of the OspA and OspC proteins described herein were bioengineered such that the resultant chimeric protein maintained at least some antigenicity of one or both of the parent molecules. As described herein, antigenic refers to the ability of a compound to bind products of an immune response, such as antibodies, T-cell receptors or both. Such responses can be measured using standard antibody detection assays, such as ELISA or standard T-cell activation assays. In a particular embodiment, the chimeric OspC/OspA proteins comprise OspA polypeptides which lack the putative autoreactive domain that has similarity to a region of human leukocyte function-associated antigen-1 (hLFA-1) (Gross, D. M. et al., Science, 281: 703-706 (1998)).

The current invention pertains to chimeric proteins comprising antigenic Borrelia polypeptides which do not occur in nature in the same Borrelia protein. The chimeric proteins are a combination of two or more antigenic polypeptides derived from Borrelia proteins. The antigenic polypeptides can be derived from different proteins from the same species of Borrelia, or different proteins from different Borrelia species, as well as from corresponding proteins from different species. As used herein, the term "chimeric protein" describes a protein comprising two or more polypeptides which are derived from corresponding and/or non-corresponding native Borrelia protein. A polypeptide "derived from" a native Borrelia protein is a polypeptide which has an amino acid sequence the same as an amino acid sequence present in a Borrelia protein, an amino acid sequence equivalent to the amino acid sequence of a naturally occurring Borrelia protein, or an amino acid sequence substantially similar to the amino acid sequence of a naturally occurring Borrelia protein (e.g., differing by a few amino acids), such as when a nucleic acid encoding a protein is subjected to site-directed mutagenesis. "Corresponding" proteins are equivalent proteins from different species or strains of Borrelia, such as outer surface protein A (OspA) from strain B31 and OspA from strain K48. The invention additionally pertains to nucleic acids encoding these chimeric proteins.

In one embodiment, the present invention is drawn to chimeric proteins comprising antigenic polypeptides from Lyme Disease-causing strains of Borrelia. In another embodiment, the chimeric proteins described herein comprise antigenic polypeptides from different pathogenic genospecies of Borrelia, such as Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. In a preferred embodiment, the chimeric proteins comprise antigenic polypeptides from each of the pathogenic genospecies of Borrelia, including Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii.

The OspA portion of the chimeric molecules of the present invention can themselves be chimeric combinations of more than one OspA polypeptide. Similarly, the OspC portion of the chimeric molecules of the present invention can themselves be chimeric combinations of more than one OspC polypeptide. As described below, Applicants have identified two separate antigenic domains of OspA and OspB which flank the sole conserved tryptophan present in OspA and in OspB. These domains share cross-reactivity with different genospecies of Borrelia. The precise amino acids responsible for antigenic variability were determined through site-directed mutagenesis, so that proteins with specific amino acid substitutions are available for the development of chimeric versions of OspA which can be included in the OspC/OspA chimeric proteins of the present invention. Furthermore, Applicants have identified immunologically important hypervariable domains in OspA proteins, as described below in Example 2. The first hypervariable domain of interest for chimeric proteins, Domain A, includes amino acid residues 120-140 of OspA, the second hypervariable domain, Domain B, includes residues 150-180 and the third hypervariable domain, Domain C, includes residues 200-216 or 217 (depending on the position of the sole conserved tryptophan residue in the OspA of that particular species of Borrelia) (see FIG. 3 (see Original Patent)). In addition, Applicants have sequenced the genes for several Borrelia proteins.

These discoveries have aided in the development of novel recombinant Borrelia proteins which include two or more amino acid regions or sequences which do not occur in the same Borrelia protein in nature. The recombinant proteins comprise polypeptides from a variety of Borrelia proteins, including, but not limited to, OspA, OspB, OspC, OspD, p12, p39, p41, p66, and p93. Preferred combinations include all or a portion of OspC linked to all or a portion of OspA. Antigenically relevant polypeptides from each of a number of proteins are combined into a single chimeric protein.

In one embodiment of the current invention, chimeras are now available which include antigenic OspA polypeptides flanking a tryptophan residue. OspB has a similar primary structure as OspA and is included in the following discussion. The antigenic polypeptides are derived from either the proximal portion from the tryptophan (the portion of the OspA protein present between the amino terminus and the conserved tryptophan of the protein), or the distal portion from the tryptophan (the portion of the OspA protein present between the conserved tryptophan of the protein and the carboxy terminus) in OspA. The resultant chimeras can be OspA-OspA chimeras (e.g., chimeras incorporating polypeptides derived from OspA from different strains of Borrelia), OspA-OspB chimeras, or OspB-OspB chimeras, and are constructed such that amino acid residues amino-proximal to an invariant tryptophan are from one protein and residues carboxy-proximal to the invariant tryptophan are from the other protein. For example, one available chimer consists of a polypeptide derived from the amino-proximal region of OspA from strain B31, followed by the tryptophan residue, followed by a polypeptide derived from the carboxy-proximal region of OspA from strain K48 (SEQ ID NO. 92). Another available chimer includes a polypeptide derived from the amino-proximal region of OspA from strain B31, and a polypeptide derived from the carboxy-proximal region of OspB from strain B31 (SEQ ID NO. 104). If the polypeptide proximal to the tryptophan of these chimeric proteins is derived from OspA, the proximal polypeptide can be further subdivided into the three hypervariable domains (Domains A, B, and C), each of which can be derived from OspA from a different strain of Borrelia. These chimeric proteins can further comprise antigenic polypeptides from another protein, e.g., OspC, in addition to the antigenic polypeptides flanking the tryptophan residue.

In one embodiment, the chimeric OspC/OspA proteins of the present invention comprise at least a first and a second polypeptide, wherein the first polypeptide comprises Borrelia burgdorferi OspC and wherein the second polypeptide comprises Borrelia burgdorferi OspA, such that OspC comprises the N-terminus of the protein.

In a particular embodiment, the first polypeptide comprises a Borrelia burgdorferi OspC polypeptide from about amino acid residue 19 to about amino acid residue 213, and the second polypeptide comprises a Borrelia burgdorferi OspA polypeptide. In another embodiment, the first polypeptide comprises a Borrelia burgdorferi OspC polypeptide from about amino acid residue 19 to about amino acid residue 211. In another embodiment, the first polypeptide comprises a Borrelia burgdorferi OspC polypeptide from about amino acid residue 19 to about amino acid residue 208. In another embodiment, the first polypeptide comprises a Borrelia burgdorferi OspC polypeptide from about amino acid residue 19 to about amino acid residue 204. The numbering of the OspC residues is according to the numbering of SEQ ID NO: 30 (FIGS. 12A and 12B (see Original Patent)). It is evident that the person of skill in the art recognizes that OspC genes from different strains and/or genospecies may differ in their primary sequence and that based on homology, similar regions of such OspC proteins could be identified and used in the present invention with no or only routine experimentation.

In one embodiment, the invention is drawn to chimeric OspC/OspA proteins wherein the first polypeptide comprises a Borrelia burgdorferi OspC polypeptide and the second polypeptide comprises a Borrelia burgdorferi OspA polypeptide from about amino acid residue 18 to about amino acid residue 273. In other embodiments, the chimeric OspC/OspA protein comprises a first polypeptide which is a Borrelia burgdorferi OspC polypeptide and a second polypeptide which is a Borrelia burgdorferi OspA polypeptide selected from the group consisting of an OspA polypeptide from about amino acid residue 132 to about amino acid residue 216, an OspA polypeptide from about amino acid residue 218 to about amino acid residue 273, an OspA polypeptide from about amino acid residue 18 to about amino acid residue 216 and an OspA polypeptide from about 132 to about amino acid residue 273. The numbering of the OspA residues is according to the numbering of SEQ ID NO: 7 (FIGS. 7A and 7B (see Original Patent)). It is evident that the person of skill in the art recognizes that OspA genes from different strains and/or genospecies may differ in their primary sequence and that based on homology, similar regions of such OspA proteins could be identified and used in the present invention with no or only routine experimentation.

The present invention is also drawn to OspC/OspA chimeric proteins wherein the first polypeptide comprises a Borrelia burgdorferi OspC polypeptide and the second polypeptide comprises a Borrelia burgdorferi OspA polypeptide, wherein the OspA polypeptide comprises two or more OspA polypeptide fragments as described above. In a preferred embodiment, the OspA polypeptide comprises portions of OspA from two or more strains of Borrelia. In another preferred embodiment, the OspA polypeptide comprises portions of OspA from two or more Lyme Disease-causing genospecies of Borrelia, e.g., wherein the genospecies are Borrelia burgdorferi sensu stricto, Borrelia afzelii and/or Borrelia garinii. In still another preferred embodiment, the OspC/OspA chimeric protein comprises one or more polypeptides from each of the pathogenic genospecies, Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii.

The chimeras described herein can be produced so that they are highly soluble, hyper-produced in E. coli, and non-lipidated. Lipidated chimeric proteins can also be produced. In addition, the chimeric proteins can be designed to end in an affinity tag (His-tag) to facilitate purification. The recombinant proteins described herein have been constructed to maintain antigenicity of at least one of the parent polypeptides. In addition, recombinant proteins specific for the various genospecies of Borrelia that cause Lyme disease are now available, because the genes from each of the major genospecies have been sequenced. These recombinant proteins with their novel biophysical and antigenic properties will be important diagnostic reagent and vaccine candidates.

The chimeric proteins of the current invention are advantageous in that they retain at least some specific reactivity to monoclonal or polyclonal antibodies that recognize wild-type Borrelia proteins. The proteins are immunogenic, and elicit antibodies that inhibit growth and/or induce lysis of Borrelia in vitro. Furthermore, in some embodiments, the proteins provide antigenic domains of two or more Borrelia strains and/or proteins within a single protein. Such proteins are particularly useful in immunodiagostic assays. For example, proteins of the present invention can be used as reagents in assays to detect the presence of antibodies to native Borrelia in potentially infected individuals. These proteins can also be used as immunodiagnostic reagents, such as in dot blots, Western blots, enzyme-linked immunosorbent assays (ELISA), or agglutination assays. The chimeric proteins of the present invention can be produced by known techniques, such as by recombinant methodology, polymerase chain reaction, or mutagenesis.

Furthermore, the proteins of the current invention are useful as vaccine immunogens against Borrelia infection. Because Borrelia has been shown to be clonal, a protein comprising antigenic polypeptides from a variety of Borrelia proteins and/or species, will provide immunoprotection for a considerable time when used in a vaccine. The lack of significant intragenic recombination, a process which might rapidly generate novel epitopes with changed antigenic properties, ensures that Borrelia can only change antigenic type by accumulating mutational change, which is slow when compared with recombination in generating different antigenic types. The chimeric protein can be combined with a physiologically-acceptable carrier and administered to a vertebrate animal through standard methods (e.g., intravenously or intramuscularly, for example).

In addition to the chimeric proteins described herein, the present invention is also drawn to nucleic acids which encode the Borrelia chimeric protein described herein. In one embodiment of the present invention, the composition comprises a nucleic acid that encodes a chimeric protein of at least two polypeptides, wherein the first polypeptide comprises Borrelia burgdorferi OspC, and the second polypeptide comprises Borrelia burgdorferi OspA, such that OspC is upstream of OspA. The OspC and OspA nucleic acid fragments that make up the chimeric protein can be from the same strain or genospecies of Borrelia, from different strains or genospecies of Borrelia, or from combinations of nucleic acids that are from the same and/or different strains or genospecies of Borrelia.

It is understood that the nucleic acids that encode the polypeptides that comprise the chimeric protein can include extra nucleotides or fewer nucleotides in order to simplify the construction of the gene encoding the chimeric polypeptide, e.g., to allow for the use of convenient restriction endonuclease sites or to allow the ligation of the gene fragments such that a contiguous coding region is created. Based on the guidance provided herein, one of ordinary skill in the art would readily be able to add or remove nucleotides from the termini of the gene fragments encoding the polypeptides of the chimeric OspC/OspA protein in order to generate the chimeric proteins of the present invention with no or only routine experimentation. Furthermore, there can be an extra about 1 to about 10 amino acids on the N- and/or C-terminus of the polypeptides and chimeric proteins of the present invention and still retain the properties of the present invention. It is also understood that those of skill in the art, using art-known methods and/or the methods described herein, could generate additional OspC-OspA chimeric proteins, and that these chimeric proteins are encompassed by the invention.

The present invention is also drawn to expression vectors which comprise an isolated DNA encoding the Borrelia chimeric protein described herein. In one embodiment, the composition includes an expression vector comprising an isolated DNA which encodes an OspC/OspA chimeric protein, wherein the OspC portion of the protein is upstream of the OspA portion. The present invention also encompasses host cells which comprise a recombinant nucleic acid that encodes an OspC/OspA chimeric protein, as described herein.

The present invention is also drawn to methods of making the Borrelia chimeric polypeptides described herein. In one embodiment, the method of making a chimeric Borrelia protein comprises selecting a polynucleotide sequence encoding OspC, or an antigenic portion thereof, selecting a polynucleotide sequence encoding OspA, or an antigenic portion thereof, and ligating these polynucleotide sequences together, such that OspC comprises the N-terminus of the protein. The polypeptides of the present invention can also be recombinantly expressed in suitable microbial hosts, wherein said hosts include, but are not limited to, bacterial hosts, such as E. coli, fungal hosts, such as S. cerevisiae or cell culture hosts, such as those of mammalian cell culture or insect cell culture.

The present invention is also drawn to methods of delivering the Borrelia chimeric polypeptides described herein. In one embodiment, the method comprises administering the chimeric protein in a physiologically-acceptable carrier to an individual. The individual develops at least some immune response to the chimeric protein. As an example, the individual could generate a humoral immune response, wherein antibodies that recognize at least a portion of said chimeric polypeptide are produced by the individual. The antibodies that recognize the chimeric polypeptide can be of any class of immunoglobulin, such as IgM, IgD, IgA and IgG or combinations thereof.

The present invention is also drawn to methods of delivering a nucleic acid which encodes a chimeric polypeptide described herein. In one embodiment, the method comprises administering the nucleic acid in a physiologically-acceptable carrier to an individual using art-accepted methods of DNA delivery, including but not limited to, biolistic delivery and lipid encapsulation. The chimeric polypeptide is at least transiently expressed and the individual develops at least some immune response to the chimeric protein encoded by the nucleic acid.

The invention also encompasses methods of using the chimeric proteins described herein in diagnostic assays. In one embodiment, the method can be used to detect the presence of OspA- and/or OspC-specific antibodies in a sample, e.g., a host sample of interest. In one embodiment, the method comprises contacting a host sample of interest with the chimeric OspC/OspA protein, under conditions, wherein antibodies, if present in the host sample, bind to the chimeric protein thereby forming antigen-antibody complexes. The antigen-antibody complexes are then detected. In this manner, an immune response to Lyme-Disease causing Borrelia can be detected.

As described herein, the chimeric proteins of the present invention incorporate antigenic domains from different Borrelia proteins, as well as from different Borrelia strains and/or genospecies. As such, they are useful in the detection or diagnosis of the presence of Lyme disease-causing Borrelia, especially Borrelia from groups capable of causing disseminated symptoms of Lyme disease. Disseminated symptoms refer to infection outside of the erythema migrans skin lesion, e.g., infection in blood, CNS or synovia.

The chimeric polypeptides of the present invention elicit specific immune responses to OspC and OspA. In one embodiment, the chimeric polypeptides elicit immune responses to strains of Lyme disease-causing Borrelia of the same genospecies as that represented by the OspC/OspA chimeric protein. In another embodiment, the chimeric polypeptides elicit immune responses to strains of Lyme disease-causing Borrelia of different genospecies than that represented by the OspC/OspA chimeric protein, as well as to Lyme disease-causing Borrelia of the same genospecies as that represented by the OspC/OspA chimeric protein. The immune response includes, but is not limited to, a humoral response, a secretory response, a cell-mediated response, or any combination thereof.

The immunogenic compositions of the present invention can also be used to immunize animals, e.g., mammals, including humans. Immunization is understood to elicit specific immunogenic responses as described herein. In one embodiment, administration of an immunogenic composition, e.g., an OspC/OspA chimeric protein, an OspC/OspA chimeric nucleic acid, to an animal results in the animal developing immunity to infection by Lyme disease-causing Borrelia, e.g., Borrelia burgdorferi, Borrelia afzelii and/or Borrelia garinii.

Immunity, as described herein, is understood to mean the ability of the treated animal to resist infection (e.g., systemic infection), to overcome infection (e.g., systemic infection) or to overcome infection (e.g., systemic infection) more easily and/or more quickly when compared to non-immunized and/or non-treated individuals. Immunity can also include an improved ability of the treated individual to sustain an infection with reduced or no clinical symptoms of systemic infection. The individual may be treated with the chimeric proteins of the present invention either proactively, e.g., once a year or alternatively after sustaining a tick bite.

In one embodiment, the OspC/OspA chimeric protein of the present invention, together with suitable excipients and/or adjuvants, is administered to an animal such that the animal develops an immune response to at least one of the OspC and/or OspA polypeptides of the composition. The pharmaceutical composition can also be administered with other components suitable for in vitro and/or in vivo use. These additional components include buffers, carrier proteins, adjuvants, excipients, preservatives and combinations thereof.

The present invention is also drawn to pharmaceutical compositions which can be used to vaccinate and/or treat Borrelia infection in an animal or human. In a particular embodiment, the pharmaceutical composition comprises a Borrelia burgdorferi OspC/OspA chimeric protein. The pharmaceutical composition can also be administered together with a physiologically-acceptable carrier, an excipient and/or an adjuvant. Suitable adjuvants are well known in the art (see for example PCT Publication WO 96/40290, the entire teachings of which are incorporated herein by reference), and can be used, for example, to enhance immunogenicity, potency or half-life of the chimeric proteins in the treated animal.

The pharmaceutical compositions used to vaccinate and/or treat Borrelia infection can be prepared using methods for preparing vaccines which are well known in the art. For example, the OspC/OspA chimeric proteins described herein can be isolated and/or purified by known techniques, such as by size exclusion chromatography, affinity chromatography, ion exchange chromatography, preparative electrophoresis, selective precipitation or combinations thereof. The prepared chimeric proteins can be mixed with suitable other reagents as described herein, such that the chimeric protein is at a suitable concentration. The dosage of the chimeric protein will vary and depends upon the age, weight and/or physical condition of the animal, e.g., mammal, human, to be treated. The optimal dosage can be determined by routine optimization techniques, using suitable animal models.

Administration of the pharmaceutical composition to be used as a vaccine can be by any suitable technique. Suitable techniques for administration of the pharmaceutical composition include, but are not limited to, injection, e.g., subcutaneous injection, intramuscular injection, intravenous injection, intra peritoneal injection; mucosal administration, e.g., exposing nasal mucosa to nose drops containing the proteins or chimeric proteins of the present invention; oral administration; and DNA immunization.

The incorporation of polypeptide fragments from different strains and/or genospecies of Borrelia burgdorferi allows for a greater detection range and a more effective vaccination tool. The present invention provides a chimeric combination of proteins that, when used as a vaccine, can prevent Lyme disease from becoming systemic. The chimeric proteins of the present invention can be effective in preventing Lyme disease, as well as having a therapeutic effect on established infection, for example, after the tick bite is noticed by the patient. Since the chimeric proteins of the present invention comprise both OspC and OspA polypeptides, they are expected to act at the level of the tick as well as the level of the host in preventing both infection and disease due to Borrelia burgdorferi, Borrelia afzelii and/or Borrelia garinii.

The present invention is also drawn to diagnostic kits which comprise the chimeric polypeptides described herein. In one embodiment, the kit comprises a chimeric protein comprising at least a first and a second polypeptide, wherein the first polypeptide comprises Borrelia burgdorferi OspC and wherein the second polypeptide comprises Borrelia burgdorferi OspA, such that OspC comprises the N-terminus of the protein. The kit also includes reagents for detecting antibody-antigen complexes that are formed between the OspC/OspA chimeric protein and antibodies that are present in a sample, e.g., a user-supplied host sample.

As a result of the present invention, it is now possible to prepare improved diagnostic tools comprising both OspA and OspC antigens from various Borrelia burgdorferi strains and/or genospecies. Since OspA is primarily expressed in the tick vector, and OspC is upregulated in response to the feeding of an infected tick on a mammal, the diagnostic compositions of the invention can recognize antigens that are expressed at different stages of the life cycle of Borrelia burgdorferi. Moreover, by incorporating unique polypeptide fragments from pathogenic families of Borrelia, the present invention allows for improved diagnostic compositions which can detect clinically important exposure to pathogenic Borrelia while overlooking the remainder of non-pathogenic families of Borrelia.

As described herein, the OspC/OspA chimeric proteins were bioengineered such that the protective domains of each protein were maintained. In experiments described herein, mice were either immunized with OspA, OspC or OspC/OspA chimeric proteins in aluminum hydroxide. Mice were then bled and tested for antibody responses against OspC and OspA derived from various strains of Borrelia. In additional experiments, these immunized mice were challenged with ticks infected with Borrelia burgdorferi and transmission of infection was assessed.

Mice immunized with the OspC/OspA chimeric protein gave a remarkable and equivalent antibody response to both OspA and OspC, as compared to mice immunized with OspA and OspC control proteins (FIGS. 47 and 48 (see Original Patent)). In addition, antibodies in the sera of mice immunized with the OspC/OspA chimeric protein were also reactive against antigens derived from different strains of Borrelia burgdorferi (FIGS. 49-50 and 52-54 (see Original Patent)). Chimer-immunized mice were fully protected against challenge with ticks infected with Borrelia burgdorferi, as compared to sham-vaccinated controls (infection rates of 100%) (Table VI (see Original Patent)).

In other experiments described herein, mice were either immunized with a lipidated OspA chimeric protein, a lipidated OspC chimeric protein, or a non-lipidated OspC/OspA chimeric protein, once again in the presence of aluminum hydroxide. Mice were then bled and tested for antibody responses against OspA and OspC derived from various strains of Borrelia. Surprisingly, the results of these studies indicate that mice immunized with the non-lipidated OspC/OspA chimeric protein have antibody responses to OspA and OspC that are equivalent or greater than those generated by mice immunized with the corresponding lipidated OspA or lipidated OspC chimeric proteins (FIGS. 49-51 (see Original Patent)).

The results of the studies presented herein indicate that mice immunized with OspC-OspA chimeric proteins generate a potent antibody response against two immunoprotective targets that are expressed at different stages of the life cycle of Borrelia burgdorferi.
 

Claim 1 of 12 Claims

1. A nucleic acid encoding a protein comprising at least a first and a second polypeptide, wherein the first polypeptide comprises OspC from Borrelia burgdorferi sensu lato and wherein the second polypeptide comprises OspA from Borrelia burgdorferi sensu lato, such that the first polypeptide is linked to the N-terminus of the second polypeptide.
 

 

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