ABSTRACT
Escherichia coli is the mainstay tool for fundamental microbiology research due to its ease of cultivation and safety. Auxotrophic strains of the K-12 and B lineages of E. coli are the organisms of choice to produce recombinant proteins. Components present in the cell envelope of bacteria are also potent immune modulators and have been used to develop adjuvants. We used live E. coli, after induction of recombinant protein expression, to develop a vehicle which has a dualistic function of producing vaccine while presenting itself as the adjuvant to deliver oral vaccines against a number of infectious diseases, including Lyme disease. Here, we give an example using E. coli expressing B. burgdorferi Outer Surface Protein A, which was proven effective in reducing B. burgdorferi burden in infected ticks after a 5-year field trial of a baited formulation containing this reservoir targeted vaccine.
Subject(s)
Antigens, Surface/genetics , Bacterial Outer Membrane Proteins/genetics , Bacterial Vaccines/genetics , Borrelia burgdorferi/genetics , Escherichia coli/genetics , Lipoproteins/genetics , Lyme Disease Vaccines/genetics , Lyme Disease/prevention & control , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/genetics , Administration, Oral , Animals , Freeze Drying , Gene Expression , Humans , Immunization/methods , Lyme Disease/microbiology , Lyme Disease Vaccines/administration & dosage , Mice , Recombinant Proteins/genetics , Transformation, BacterialABSTRACT
The causative agent of Lyme borreliosis, Borrelia burgdorferi, is transmitted by Ixodes ticks. During tick feeding, B. burgdorferi migrates from the tick gut to the salivary glands from where transmission to the host occurs. B. burgdorferi-interacting tick proteins might serve as vaccine targets to thwart B. burgdorferi transmission. A previous screening for B. burgdorferi-interacting Ixodes scapularis gut proteins identified an I. scapularis putative dystroglycan protein (ISCW015049). Here, we describe the ISCW015049's protein structure and its cellular location in the tick gut in relation to B. burgdorferi migration. Secondly, in vivo B. burgdorferi-tick attachment murine models were performed to study the role of ISCW015049 during B. burgdorferi migration and transmission. In silico analysis confirmed that ISCW015049 is similar to dystroglycan and was named I. scapularis dystroglycan-like protein (ISDLP). Confocal microscopy of gut tissue showed that ISDLP is expressed on the surface of gut cells, is upregulated during tick feeding, and is expressed significantly higher in infected ticks compared to uninfected ticks. Inhibition of ISDLP by RNA interference (RNAi) resulted in lower B. burgdorferi transmission to mice. In conclusion, we have identified a dystroglycan-like protein in I. scapularis gut that can bind to B. burgdorferi and promotes B. burgdorferi migration from the tick gut. Key messages: B. burgdorferi exploits tick proteins to orchestrate its transmission to the host. B. burgdorferi is able bind to an I. scapularis dystroglycan-like protein (ISDLP). Inhibition of ISDLP in ticks results in lower B. burgdorferi transmission to mice. ISDLP is a potential target to prevent Lyme borreliosis.
Subject(s)
Arthropod Proteins/metabolism , Borrelia burgdorferi/physiology , Dystroglycans/metabolism , Ixodes/microbiology , Lyme Disease/prevention & control , Lyme Disease/transmission , Animals , Arthropod Proteins/genetics , Arthropod Proteins/therapeutic use , Dystroglycans/genetics , Dystroglycans/therapeutic use , Humans , Immunization , Ixodes/genetics , Ixodes/physiology , Lyme Disease/metabolism , Lyme Disease Vaccines/genetics , Lyme Disease Vaccines/metabolism , Lyme Disease Vaccines/therapeutic use , Mice , RNA, Small Interfering/genetics , RNA, Small Interfering/therapeutic use , RNAi Therapeutics , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/therapeutic useABSTRACT
Lyme disease is a major human health problem which continues to increase in incidence and geographic distribution. As a vector-borne zoonotic disease, Lyme disease may be amenable to reservoir targeted strategies for control. We have previously reported that a vaccinia virus (VV) based vaccine expressing outer surface protein A (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease, protects inbred strains of laboratory mice against infection by feeding ticks and clears the ticks of infection when administered by gavage. Here we extend these studies to develop an effective bait formulation for delivery of the VV based vaccine and test its characteristics under simulated environmental conditions. We show that this vaccine is efficacious in decreasing acquisition of B. burgdorferi by uninfected larval ticks as well as in decreasing transmission from infected ticks to its natural reservoir, Peromyscus leucopus, when fed to mice in oral baits. Using live, in vivo imaging techniques, we describe the distribution of vaccinia virus infection after ingestion of the baited vaccines and establish the use of in vivo imaging technology for optimization of bait delivery. In summary, a VV based OspA vaccine is stable in an oral bait preparation and provides protection against infection for both the natural reservoir and the tick vector of Lyme disease.
Subject(s)
Disease Reservoirs , Disease Transmission, Infectious/prevention & control , Lyme Disease Vaccines/immunology , Lyme Disease/prevention & control , Vaccination/methods , Zoonoses/transmission , Animals , Antigens, Surface/genetics , Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines/genetics , Bacterial Vaccines/immunology , Borrelia burgdorferi/genetics , Borrelia burgdorferi/immunology , Chemistry, Pharmaceutical , Drug Carriers , Genetic Vectors , Larva/immunology , Lipoproteins/genetics , Lipoproteins/immunology , Lyme Disease/immunology , Lyme Disease Vaccines/administration & dosage , Lyme Disease Vaccines/genetics , Mice , Mice, Inbred BALB C , Peromyscus/immunology , Ticks/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccinia virus/geneticsABSTRACT
Borrelia burgdorferi OspC is an outer membrane lipoprotein required for the establishment of infection in mammals. Due to its universal distribution among B. burgdorferi sensu lato strains and high antigenicity, it is being explored for the development of a next-generation Lyme disease vaccine. An understanding of the surface presentation of OspC will facilitate efforts to maximize its potential as a vaccine candidate. OspC forms homodimers at the cell surface, and it has been hypothesized that it may also form oligomeric arrays. Here, we employ site-directed mutagenesis to test the hypothesis that interdimeric disulfide bonds at cysteine 130 (C130) mediate oligomerization. B. burgdorferi B31 ospC was replaced with a C130A substitution mutant to yield strain B31::ospC(C130A). Recombinant protein was also generated. Disulfide-bond-dependent oligomer formation was demonstrated and determined to be dependent on C130. Oligomerization was not required for in vivo function, as B31::ospC(C130A) retained infectivity and disseminated normally. The total IgG response and the induced isotype pattern were similar between mice infected with untransformed B31 and those infected with the B31::ospC(C130A) strain. These data indicate that the immune response to OspC is not significantly altered by formation of OspC oligomers, a finding that has significant implications in Lyme disease vaccine design.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Outer Membrane Proteins/metabolism , Disulfides , Lyme Disease Vaccines/metabolism , Protein Multimerization , Virulence Factors/metabolism , Amino Acid Substitution/genetics , Animals , Antibodies, Bacterial/blood , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Borrelia burgdorferi Group/genetics , Borrelia burgdorferi Group/immunology , Cysteine/genetics , Immunoglobulin G/blood , Lyme Disease Vaccines/chemistry , Lyme Disease Vaccines/genetics , Lyme Disease Vaccines/immunology , Mice , Mice, Inbred C3H , Mutagenesis, Site-Directed , Virulence Factors/chemistry , Virulence Factors/genetics , Virulence Factors/immunologyABSTRACT
RT-PCR evaluation of the activity of eight Ixodes persulcatus salivary gland genes shows clear distinctions in their expression depending of the stage of tick feeding. Out of them, only Salp 10 and Salp 15 proteins may be regarded as candidates for protective antigens to develop anti-tick and anti-Borrelia vaccines. Firstly they play an important role in feeding a tick and modifying a host's immune response. Secondly, the increasing expression of the salp 10 and salp 10 genes begins at early tick feeding stages. Thirdly, the activity of these genes increases with the beginning of feeding by tens and hundreds times and keeps at this level until the third tick feeding stage is over.
Subject(s)
Immunodominant Epitopes/genetics , Ixodes/genetics , Lyme Disease Vaccines/genetics , Salivary Glands/metabolism , Salivary Proteins and Peptides/genetics , Animals , Female , Gene Expression Regulation , Ixodes/physiology , Lyme Disease/prevention & control , Reverse Transcriptase Polymerase Chain Reaction , Vaccines, Combined/geneticsABSTRACT
Lyme disease is the most common vector-borne disease in North America and Europe and, if untreated, has significant arthritic, cardiac, dermatological and neurological sequelae. There is no currently available human Lyme disease vaccine. Outer surface protein C, because of its antigenicity, protective ability, and expression characteristics has emerged as a promising second generation vaccine candidate; however, significant sequence heterogeneity has impeded its development. Analyses of OspC sequences have revealed the existence of stable phylogenetic clusters or types, and that the type-defining sequence variation occurs within defined domains of the protein. Recent data indicating that immunodominant, and potentially protective OspC epitopes are located in these hypervariable regions has allowed development of a tetravalent, epitope-based, chimeric vaccine. In this report, we have extended that previously described tetravalent construct to include four additional OspC types. We demonstrate that the construct is highly immunogenic, and elicits type-specific antibodies that recognize each of the eight incorporated OspC type-specific epitopes. Antibody raised to the octavalent construct readily binds to the surface of strains expressing each component OspC type, indicating that the incorporated epitopes are presented on the surface of intact cells. In addition, the construct elicits antibody isotypes associated with complement-dependent bactericidal activity. These results represent an important step forward in the design of a broadly protective polyvalent OspC-based Lyme disease vaccine.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/immunology , Borrelia burgdorferi/immunology , Lyme Disease Vaccines/chemistry , Lyme Disease Vaccines/immunology , Lyme Disease/prevention & control , Amino Acid Sequence , Animals , Antibodies, Bacterial , Antibody Specificity , Antigens, Bacterial/chemistry , Bacterial Outer Membrane Proteins/chemistry , Bacterial Vaccines/chemistry , Borrelia burgdorferi/isolation & purification , Chimera/immunology , Epitopes/immunology , Lyme Disease/immunology , Lyme Disease Vaccines/genetics , Mice , Mice, Inbred C3H , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
Lyme disease is the most common arthropod-borne disease in North America and Europe. At present, there is no commercially available vaccine for use in humans. Outer surface protein C (OspC) has antigenic and expression characteristics that make it an attractive vaccine candidate; however, sequence heterogeneity has impeded its use as a vaccinogen. Sequence analyses have identified 21 well defined OspC phyletic groups or "types" (designated A-U). In this report we have mapped the linear epitopes presented by OspC types B, K, and D during human and murine infection and exploited these epitopes (along with the previously identified type A OspC linear epitopes) in the development of a recombinant, tetravalent, chimeric vaccinogen. The construct was found to be highly immunogenic in mice and the induced antibodies surface labeled in vitro cultivated spirochetes. Importantly, vaccination induced complement-dependent bactericidal antibodies against strains expressing each of the OspC types that were incorporated into the construct. These results suggest that an effective and broadly protective polyvalent OspC-based Lyme disease vaccine can be produced as a recombinant, chimeric protein.
Subject(s)
Borrelia burgdorferi/immunology , Lyme Disease Vaccines/immunology , Lyme Disease/immunology , Lyme Disease/prevention & control , Phosphatidylcholines/immunology , Amino Acid Sequence , Animals , Base Sequence , Borrelia burgdorferi/isolation & purification , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Fluorescent Antibody Technique , Immunoblotting , Lyme Disease Vaccines/genetics , Male , Mice , Mice, Inbred C3H , Models, Molecular , Molecular Sequence Data , Phosphatidylcholines/genetics , Reverse Transcriptase Polymerase Chain Reaction , Species Specificity , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
The immunogenicity of peptides and protein fragments can be considerably enhanced by their presentation on particulate carriers such as capsid-like particles (CLP) from hepatitis B virus (HBV). Here we tested the suitability of the HBV capsid protein as a carrier for a relevant full-length pathogen-derived protein antigen. The entire 255-amino acid ectodomain of the outer surface protein A (OspA) from Borrelia burgdorferi, the causative agent of Lyme disease, was inserted into the major B cell epitope of the HBV capsid, yielding a multimerization-competent fusion protein, termed coreOspA. CoreOspA, consisting only in part of regular CLP, induced antibodies to OspA, including the Ig isotype profile and specificity for the protective epitope LA-2, with an efficiency similar to that of recombinant lipidated OspA, the first generation vaccine against Lyme disease. Moreover, coreOspA actively and passively protected mice against subsequent challenge with B. burgdorferi. The data demonstrate the capacity of the HBV capsid protein to act as a potent immunomodulator even for full-length and structurally complex polypeptide chains and thus opens new avenues for novel vaccine designs.
Subject(s)
Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/immunology , Capsid Proteins/immunology , Hepatitis B Antigens/immunology , Lipoproteins/immunology , Lyme Disease Vaccines/immunology , Recombinant Fusion Proteins/immunology , Animals , Antigens, Surface/genetics , Antigens, Surface/metabolism , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Bacterial Vaccines , Borrelia burgdorferi/immunology , Borrelia burgdorferi/metabolism , Capsid Proteins/genetics , Female , Hepatitis B Antigens/genetics , Hepatitis B virus/immunology , Lipid Metabolism , Lipoproteins/genetics , Lipoproteins/metabolism , Lyme Disease/immunology , Lyme Disease/prevention & control , Lyme Disease Vaccines/genetics , Mice , Mice, Inbred BALB C , Mice, SCID , Protein Structure, Tertiary , Recombinant Fusion Proteins/geneticsABSTRACT
An ideal vaccine delivery system would elicit persistent protection following a single administration, preferably by a noninvasive route, and be safe even in the face of immunosuppression, either inherited or acquired, of the recipient. We have exploited the unique life cycle of the autonomous parvoviruses to develop a nonproliferating vaccine platform that appears to both induce priming and continually boost a protective immune response following a single inoculation. A crippled parvovirus vector was constructed, based on a chimera between minute virus of mice (MVM) and LuIII, which expresses Borrelia burgdorferi outer surface protein A (OspA) instead of its coat protein. The vector was packaged into an MVM lymphotropic capsid and inoculated into naive C3H/HeNcr mice. Vaccination with a single vector dose, either intravenously or intranasally, elicited high-titer anti-OspA-specific antibody that provided protection from live spirochete challenge and was sustained over the lifetime of the animal. Both humoral and cell-mediated Th(1) immunity was induced, as shown by anti-OspA immunoglobulin G2a antibody and preferential gamma interferon production by OspA-specific CD4(+) T cells.
Subject(s)
Antibodies, Bacterial/blood , Borrelia burgdorferi/immunology , Genetic Vectors/immunology , Lipoproteins , Lyme Disease Vaccines/immunology , Minute Virus of Mice/genetics , Parvovirus/genetics , Administration, Intranasal , Animals , Antigens, Surface/genetics , Antigens, Surface/immunology , Antigens, Surface/metabolism , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/metabolism , Bacterial Vaccines , CD4-Positive T-Lymphocytes/immunology , Cell Line , Genetic Vectors/administration & dosage , Humans , Immunologic Memory , Injections, Intravenous , Lyme Disease/immunology , Lyme Disease/prevention & control , Lyme Disease Vaccines/administration & dosage , Lyme Disease Vaccines/genetics , Mice , Minute Virus of Mice/physiology , Parvovirus/physiology , Recombination, GeneticABSTRACT
The outer surface protein C (OspC) of Borrelia burgdorferi, the spirochete that causes Lyme disease, is a promising candidate for a vaccine against borreliosis. BALB/c and C3H/HeJ mice were immunized either with recombinant OspC protein or with plasmid DNA encoding OspC fused to the human tissue plasminogen activator leader sequence (pCMV-TPA/ZS7). The influence of the route of administering the DNA and the use of oligodeoxynucleotides containing CpG-motifs on the development of the immune response was investigated. In both mouse strains, protein as well as gene-gun immunization induced Th2 type responses, whereas needle injection of plasmid DNA resulted in Th1 type antibody production. Co-injection of CpG-motifs did not significantly modify the response type in any immunization group, as indicated by only marginal changes of antibody subclass distribution. The protection rate after challenge with 10(4) B. burgdorferi organisms per mouse was between 80% and 100% for all groups. These results demonstrate, for the first time, that a DNA vaccine encoding OspC of B. burgdorferi is suitable for inducing protection against Lyme borreliosis.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/immunology , Borrelia burgdorferi/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Vaccines, DNA/administration & dosage , Animals , Antigens, Bacterial/genetics , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Bacterial Vaccines/immunology , Borrelia burgdorferi/chemistry , Borrelia burgdorferi/genetics , Lyme Disease/immunology , Lyme Disease/prevention & control , Lyme Disease Vaccines/genetics , Lyme Disease Vaccines/immunology , Mice , Mice, Inbred BALB C , Plasmids/administration & dosage , Plasmids/genetics , Ticks/microbiology , Vaccines, DNA/immunologyABSTRACT
The overall efficacy of a recombinant vaccine for Lyme disease that is effective worldwide will depend upon the selection of one or more immunoprotective target(s) and the frequency of genetic variation, which can alter the antigenicity of the immunoprotective epitopes of the target proteins. Careful delineation of these protective epitopes on target antigens is essential for the development of vaccine candidates as well as for understanding the limitations of such vaccines. Structural models of these targets will provide critical information about conformation and specific residue surface accessibility for defining protective epitopes. Co-crystal structures with Fab fragments of protective antibodies will further delineate critical antigen surfaces. Population genetics will provide vital information on the heterogeneity of these proteins. Detailed epitope mapping will provide the information needed for the bioengineering of antigens needed to expand the specificity of a candidate vaccine.
Subject(s)
Antigens, Surface/chemistry , Bacterial Outer Membrane Proteins/chemistry , Borrelia burgdorferi/immunology , Drug Design , Lipoproteins , Lyme Disease Vaccines/chemistry , Lyme Disease/prevention & control , Antigens, Surface/genetics , Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines , Lyme Disease Vaccines/genetics , Lyme Disease Vaccines/immunology , Models, Molecular , Protein ConformationABSTRACT
A total of 152 ixodid ticks (Acari: Ixodidae) consisting of nine species was collected from 82 passerine birds (33 species) in 14 locations in Canada from 1996 to 2000. The Lyme disease spirochete Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwaldt & Brenner was cultured from the nymph of a blacklegged tick, Ixodes scapularis Say, that had been removed from a common yellowthroat, Geothlypis trichas L., from Bon Portage Island, Nova Scotia. As a result of bird movement, a nymphal I. scapularis removed from a Swainson's thrush, Catharus ustulatus incanus (Godfrey), at Slave Lake, Alberta, during spring migration becomes the new, most western and northern record of this tick species in Canada. Amblyomma longirostre Koch, Amblyomma sabanerae Stoll, and Ixodes baergi Cooley & Kohls are reported for the first time in Canada. Similarly, Amblyomma americanum L., Arnblyomma maculatum Koch, and ixodes muris Bishopp & Smith are reported for the first time on birds in Canada. After removal of an I. muris gravid female from a song sparrow, Melospiza melodia Wilson, at St. Andrews, New Brunswick, eggs were laid, which developed into larvae, and this new tick-host record demonstrates that birds have the potential to start a new tick population. We conclude that passerine birds disperse several species of ixodid ticks in Canada, and during spring migration translocate ticks from the United States, and Central and South America, some of which are infected with B. burgdorferi.
Subject(s)
Antigens, Bacterial , Bird Diseases/parasitology , Borrelia burgdorferi Group/isolation & purification , Ixodes/microbiology , Lipoproteins , Songbirds/parasitology , Tick Infestations/veterinary , Ticks/microbiology , Animals , Antigens, Surface/genetics , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Bacterial Vaccines , Borrelia burgdorferi Group/genetics , Canada , Female , Humans , Lyme Disease Vaccines/genetics , Male , Rabbits , Tick Infestations/parasitologyABSTRACT
Borrelia burgdorferi outer surface protein (Osp) A is preferentially expressed by spirochetes in the Ixodes scapularis gut and facilitates pathogen-vector adherence in vitro. Here we examined B. burgdorferi-tick interactions in vivo by using Abs directed against OspA from each of the three major B. burgdorferi sensu lato genospecies: B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. Abs directed against B. burgdorferi sensu stricto (isolate N40) destroy the spirochete and can protect mice from infection. In contrast, antisera raised against OspA from B. afzelii (isolate ACA-1) and B. garinii (isolate ZQ-1) bind to B. burgdorferi N40 but are not borreliacidal against the N40 isolate. Our present studies assess whether these selected OspA Abs interfere with B. burgdorferi-tick attachment in a murine model of Lyme disease with I. scapularis. We examined engorged ticks that had fed on B. burgdorferi N40-infected scid mice previously treated with OspA (N40, ACA-1, ZQ-1, or mAb C3.78) or control Abs. OspA-N40 antisera or mAb C3.78 destroyed B. burgdorferi N40 within the engorged ticks. In contrast, treatment of mice with OspA-ACA-1 and OspA-ZQ-1 antisera did not kill B. burgdorferi N40 within the ticks but did effectively interfere with B. burgdorferi-I. scapularis adherence, thereby preventing efficient colonization of the vector. These studies show that nonborreliacidal OspA Abs can inhibit B. burgdorferi attachment to the tick gut, highlighting the importance of OspA in spirochete-arthropod interactions in vivo.
Subject(s)
Anti-Bacterial Agents/administration & dosage , Antibodies, Bacterial/administration & dosage , Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/immunology , Borrelia burgdorferi Group/immunology , Ixodes/immunology , Ixodes/microbiology , Lipoproteins , Lyme Disease Vaccines/immunology , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Antibodies, Bacterial/chemistry , Antibodies, Bacterial/metabolism , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Antigens, Surface/genetics , Antigens, Surface/metabolism , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Bacterial Vaccines , Binding Sites, Antibody/genetics , Borrelia burgdorferi Group/genetics , Digestive System/immunology , Digestive System/metabolism , Digestive System/microbiology , Epitopes/genetics , Epitopes/metabolism , Female , Immune Sera/administration & dosage , Immune Sera/chemistry , Immune Sera/metabolism , Injections, Intraperitoneal , Injections, Subcutaneous , Ixodes/anatomy & histology , Ixodes/metabolism , Lyme Disease/immunology , Lyme Disease/prevention & control , Lyme Disease Vaccines/genetics , Lyme Disease Vaccines/metabolism , Mice , Mice, SCID , Mutation , Protein Structure, Tertiary/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolismABSTRACT
Here we describe a protocol for purifying Borrelia burgdorferi from feeding ticks by velocity centrifugation and Percoll density gradient centrifugation. The purified spirochetes were motile and 10- to 20-fold purer than the bacteria in crude tick homogenates. The purified bacteria were present in sufficient quantity for protein and gene expression studies. In comparison to culture-grown bacteria, tick-borne spirochetes had several proteins that were upregulated and a few that were downregulated. When the levels of B. burgdorferi outer surface proteins OspA and OspC were measured, OspC protein and mRNA levels were lower in cultured bacteria than in bacteria purified from ticks. Although differences in OspA mRNA levels were observed between cultured and tick-borne bacteria, no differences were observed at the protein level. These experiments demonstrate that tick-transmitted borreliae display a gene expression and antigen profile different from that of spirochetes cultured in vitro.
Subject(s)
Borrelia burgdorferi Group/isolation & purification , Ixodes/microbiology , Lipoproteins , Lyme Disease/transmission , Animals , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Antigens, Bacterial/metabolism , Antigens, Surface/genetics , Antigens, Surface/immunology , Antigens, Surface/metabolism , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/metabolism , Bacterial Vaccines , Bacteriological Techniques , Borrelia burgdorferi Group/growth & development , Centrifugation, Density Gradient/methods , Culture Media , Feeding Behavior , Female , Ixodes/physiology , Lyme Disease/microbiology , Lyme Disease Vaccines/genetics , Lyme Disease Vaccines/immunology , Lyme Disease Vaccines/metabolism , Mice , Mice, Inbred C3HABSTRACT
In about 10% of patients with Lyme arthritis in the United States, joint inflammation persists for months or even several years after the apparent eradication of the spirochete, Borrelia burgdorferi, from the joint with antibiotic treatment. We propose a model of molecular mimicry affecting genetically susceptible individuals to explain this treatment-resistant course. The majority of patients with treatment-resistant Lyme arthritis have HLA-DRB1*0401 or related alleles, and the severity and duration of their arthritis correlate with cellular and humoral immune responses to outer-surface protein A OspA) of the spirochete. Using an algorithm, the immunodominant epitope of OspA presented by the DRB1*0401 molecule was predicted to be located at aa 165-173. In a search of the Genetics Computer Group gene bank, only one human protein was identified, lymphocyte function associated antigen-1 (hLFA-1), that had sequence homology with OspA(165-173)and predicted binding in the DRB1*0401 molecule. Synovial fluid T cells from most patients with treatment-resistant arthritis responded to both OspA and hLFA-1, whereas those from patients with other forms of chronic inflammatory arthritis did not. Molecular mimicry between a dominant T cell epitope of OspA and hLFA-1 may be an important factor in the persistence of joint inflammation in genetically susceptible patients with treatment-resistant Lyme arthritis.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Arthritis, Infectious/immunology , Autoimmunity/immunology , Lipoproteins , Lyme Disease/immunology , Antigens, Surface/genetics , Antigens, Surface/immunology , Arthritis, Infectious/drug therapy , Arthritis, Infectious/microbiology , Arthritis, Infectious/physiopathology , Autoantigens/immunology , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines , Borrelia burgdorferi Group/immunology , Borrelia burgdorferi Group/pathogenicity , Drug Resistance, Microbial , Genetic Variation , HLA-DR Antigens/immunology , HLA-DR4 Antigen/immunology , HLA-DRB1 Chains , Humans , Lyme Disease/drug therapy , Lyme Disease/microbiology , Lyme Disease/physiopathology , Lyme Disease Vaccines/genetics , Lyme Disease Vaccines/immunology , Lymphocyte Function-Associated Antigen-1/immunology , United StatesABSTRACT
Genetic transformation of Borrelia spp. is limited in development and has found application in only one species. For a non-genetic approach for manipulating the phenotype of these spirochaetes, we determined whether exogenous recombinant lipoproteins would incorporate in the cell's outer membrane. Using unlabelled or 125I-labelled Osp proteins, Osp-specific monoclonal antibodies, proteinase K and formaldehyde as reagents, we found that decoration of spirochaetes had the following characteristics. (i) Purified recombinant OspA or OspD lipoproteins associated with Borrelia burgdorferi and B. hermsii cells that lacked abundant lipoproteins of their own. (ii) This decoration of the cells with exogenous OspA did not affect cell's viability. (iii) The decoration was concentration and temperature dependent and stable for at least 24 h. (iv) Like native OspA, the recombinant OspA decorating the cells was accessible to antibodies and proteases and could be cross-linked to the integral outer membrane protein, P66. (v) Decoration of viable B. burgdorferi and B. hermsii with OspA rendered the cells susceptible to killing by OspA-specific antiserum. Such non-genetic alteration of the surface of a bacterium may be used to study functions and properties of lipoproteins in situ.
Subject(s)
Antigens, Surface/metabolism , Bacterial Outer Membrane Proteins/metabolism , Borrelia/classification , Borrelia/metabolism , Cell Membrane/immunology , Cell Membrane/metabolism , Lipoproteins , Lyme Disease Vaccines/metabolism , Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Antigens, Surface/genetics , Bacterial Outer Membrane Proteins/genetics , Bacterial Vaccines , Borrelia/growth & development , Borrelia/immunology , Borrelia burgdorferi Group/classification , Borrelia burgdorferi Group/growth & development , Borrelia burgdorferi Group/immunology , Borrelia burgdorferi Group/metabolism , Cross-Linking Reagents , Endopeptidase K/metabolism , Formaldehyde , Lyme Disease Vaccines/genetics , Phenotype , Surface PropertiesABSTRACT
Different species of pathogenic Borrelia show different symptoms and tick vector specificity. Even within regions where only one species is found, Lyme disease progresses very differently from one patient to another. Since Borrelia shows very little recombination either within or between species, alleles of a gene can be used to mark clones. The ospC gene is highly variable within each species and can be used to define groups of related clones. It has been previously shown that only four out of seventeen ospC groups of Borrelia burgdorferi sensu stricto cause invasive forms of the disease. Other groups cause erythema migrans, a skin rash at the site of the tick bite, but not invasive disease, while still other groups seem to be nonpathogenic to humans. In this study we extend the analysis of the ospC gene to the other pathogenic species, Borrelia garinii and Borrelia afzelii. Only two groups in B. afzelii and four groups in B. garinii cause invasive disease. Thus, only ten out of the 58 defined ospC groups cause invasive and presumably chronic Lyme disease.
Subject(s)
Antigens, Bacterial , Bacterial Outer Membrane Proteins/genetics , Borrelia burgdorferi Group/genetics , Borrelia burgdorferi Group/pathogenicity , Borrelia/genetics , Genetic Variation , Lipoproteins , Animals , Antigens, Surface/genetics , Antigens, Surface/immunology , Antigens, Surface/metabolism , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/metabolism , Bacterial Vaccines , Borrelia/pathogenicity , Erythema Chronicum Migrans/microbiology , Genes, Bacterial , Humans , Lyme Disease/microbiology , Lyme Disease Vaccines/genetics , Lyme Disease Vaccines/immunology , Lyme Disease Vaccines/metabolism , Phylogeny , Ticks/microbiology , VirulenceABSTRACT
DNA vaccines encoding the outer surface protein A (OspA) of Borrelia burgdorferi have been shown to induce protective humoral responses capable of preventing but not curing infection in mice. Subsequent studies showed that an established infection or disease could be resolved by passive transfer of antibodies to OspC. In the present study, DNA vaccines encoding either the OspC antigen alone or fused to OspA and under the transcriptional control of the human elongation factor 1alpha promoter were evaluated for their protective and/or curative potential. In contrast to ospA-containing plasmids, none of the six constructs with ospC alone were immunogenic in vivo, independent of whether they contained promoter or leader sequences from ospA and/or ospC, or alternatively, the signal sequence of the human tissue plasminogen activator. Solely, a DNA vaccine encoding an OspA-OspC fusion product led to expression of the respective polypeptide chain in transfected cells in vitro and to the induction of OspA- and OspC-specific antibodies in vivo. Immune sera raised against the OspA-OspC fusion product conveyed full protection against subsequent infection, most probably via OspA-specific antibodies, but were unable to resolve infection.
Subject(s)
Antigens, Bacterial , Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines/immunology , Borrelia burgdorferi Group/immunology , Lipoproteins , Lyme Disease Vaccines/immunology , Lyme Disease/prevention & control , Vaccines, DNA/immunology , Animals , Antigens, Surface/genetics , Bacterial Outer Membrane Proteins/genetics , Humans , Immunization , Lyme Disease Vaccines/genetics , Mice , Mice, Inbred BALB C , Mice, SCID , Tumor Cells, CulturedABSTRACT
Piscirickettsia salmonis is the aetiological agent of salmonid rickettsial septicaemia, an economically devastating rickettsial disease of farmed salmonids. Infected salmonids respond poorly to antibiotic treatment and no effective vaccine is available for the control of P. salmonis. Bacterin preparations of P. salmonis were found to elicit a dose-dependent response in coho salmon (Oncorhynchus kisutch), which varied from inadequate protection to exacerbation of the disease. However, an outer surface lipoprotein of P. salmonis, OspA, recombinantly produced in Escherichia coli elicited a high level of protection in vaccinated coho salmon with a relative percent survival as high as 59% for this single antigen. In an effort to further improve the efficacy of the OspA recombinant vaccine, T cell epitopes (TCE's) from tetanus toxin and measles virus fusion protein, that are universally immunogenic in mammalian immune systems, were incorporated tandemly into an OspA fusion protein. Addition of these TCE's dramatically enhanced the efficacy of the OspA vaccine, reflected by a three-fold increase in vaccine efficacy. These results represent a highly effective monovalent recombinant subunit vaccine for a rickettsia-like pathogen, P. salmonis, and for the first time demonstrate the immunostimulatory effect of mammalian TCE's in the salmonid immune model. These results may also be particularly pertinent to salmonid aquaculture in which the various subspecies are outbred and of heterologous haplotypes.
Subject(s)
Bacterial Vaccines/pharmacology , Gammaproteobacteria/immunology , Lipoproteins , Salmonidae/microbiology , Amino Acid Sequence , Animals , Antigens, Surface/genetics , Antigens, Surface/immunology , Bacterial Infections/immunology , Bacterial Infections/prevention & control , Bacterial Infections/veterinary , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines/genetics , Bacterial Vaccines/immunology , Fish Diseases/immunology , Fish Diseases/prevention & control , Fisheries , Gammaproteobacteria/genetics , Gammaproteobacteria/pathogenicity , Lyme Disease Vaccines/genetics , Lyme Disease Vaccines/immunology , Molecular Sequence Data , Oncorhynchus kisutch , T-Lymphocytes/immunology , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Vaccines, Subunit/pharmacologyABSTRACT
The genome of Borrelia burgdorferi encodes a large number of lipoproteins, many of which are expressed only at certain stages of the spirochete's life cycle. In the current study we describe the B. burgdorferi population structure with respect to the production of two lipoproteins [outer surface protein A (OspA) and outer surface protein C (OspC)] during transmission from the tick vector to the mammalian host. Before the blood meal, the bacteria in the tick were a homogeneous population that mainly produced OspA only. During the blood meal, the population became more heterogeneous; many bacteria produced both OspA and OspC, whereas others produced only a single Osp and a few produced neither Osp. From the heterogeneous spirochetal population in the gut, a subset depleted of OspA entered the salivary glands and stably infected the host at time points >53 hr into the blood meal. We also examined genetic heterogeneity at the B. burgdorferi vlsE locus before and during the blood meal. In unfed ticks, the vlsE locus was stable and one predominant and two minor alleles were detected. During the blood meal, multiple vlsE alleles were observed in the tick. Tick feeding may increase recombination at the vlsE locus or selectively amplify rare vlsE alleles present in unfed ticks. On the basis of our data we propose a model, which is different from the established model for B. burgdorferi transmission. Implicit in our model is the concept that tick transmission converts a homogeneous spirochete population into a heterogeneous population that is poised to infect the mammalian host.