Salp25D, an Ixodes scapularis antioxidant, is 1 of 14 immunodominant antigens in engorged tick salivary glands.

Rabbits or guinea pigs infested with Ixodes scapularis acquire resistance to tick bites, a phenomenon, known as tick immunity, that is partially mediated by antibody. To determine the salivary gland antigens that elicit antibodies in the host, an I. scapularis salivary gland cDNA expression library was probed with serum from tick-immune rabbits. Sera from sensitized rabbits strongly recognized 47 of 100,000 library clones in an antibody-screening assay. These 47 clones encoded 14 different I. scapularis genes, including a glutathione peroxidase homologue. Expression of these 14 genes in engorged tick salivary glands was confirmed by reverse-transcription polymerase chain reaction. The I. scapularis glutathione peroxidase homologue, named salp25D, was expressed in both unfed and fed nymphal salivary glands. Recombinant Salp25D was able to catalyze the reduction of hydrogen peroxide in the presence of reduced glutathione and glutathione reductase. These results categorize the prominent salivary gland proteins in I. scapularis and demonstrate the presence of a potent antioxidant in tick saliva.

SALP16, a gene induced in Ixodes scapularis salivary glands during tick feeding.

Guinea pigs infested with Ixodes scapularis acquire antibody-mediated resistance to tick bites, a phenomenon known as tick-immunity. An I. scapularis salivary gland cDNA expression library was therefore probed with sera from tick-immune guinea pigs to identify antigens that elicit humoral responses in the host. Sera from sensitized guinea pigs strongly recognized 3 of 4,500 library clones in an initial screening. The open reading frames of all 3 clones encoded a putative 16.4-kD acidic protein, designated Salp16, with an N-terminal signal sequence and signal peptidase cleavage sites specific for secretory proteins. The salp16 mRNA and Salp16 protein were detected in the salivary glands of engorged, but not unfed, nymphal and adult ticks, and Salp16 was also found in the saliva of engorged ticks. Immunization with recombinant Salp16 induced high antibody titers in guinea pigs, but did not elicit tick-immunity. Salp16 is the first feeding inducible gene that has been cloned from L. scapularis. Molecular characterization of I. scapularis salivary antigens that are induced upon tick feeding should help to facilitate our understanding of tick-host interactions.

Prevention of Borrelia burgdorferi transmission in guinea pigs by tick immunity.

We examined the effect of repeated infestation of guinea pigs with Ixodes scapularis on the capacity of ticks to transmit Borrelia burgdorferi infection. Repeated challenges with nymphs or larvae lead to a reduction in duration of nymphal tick attachment and weight of recovered ticks consistent with the development of tick immunity. Only one of 18 I. scapularis-immune guinea pigs challenged with B. burgdorferi-infected nymphal ticks became infected, whereas 10 of 18 naive guinea pigs similarly challenged became infected. We conclude that tick immunity interferes with borrelial transmission.

Granulocytic ehrlichiosis in tick-immune guinea pigs.

We investigated whether Ixodes scapularis-mediated host immunity interrupts transmission of the agent of human granulocytic ehrlichiosis (aoHGE) to guinea pigs. Ticks infected with aoHGE readily transmitted aoHGE to tick-immune guinea pigs, despite incomplete tick engorgement and host attachment. Although tick immunity can prevent Lyme borreliosis, protection is not afforded against granulocytic ehrlichiosis.

Immune evasion by tickborne and host-adapted Borrelia burgdorferi.

Immune sera from mice infected with the Lyme disease spirochete, Borrelia burgdorferi, have strong biologic activity against spirochetes cultured in vitro. Recent studies with rodents and ticks infected with B. burgdorferi indicate that spirochetes undergo major changes in protein expression as they adapt to the diverse environments encountered by a vectorborne pathogen. The purpose of this study was to explore the susceptibility of three different adaptive forms of B. burgdorferi (in vitro cultured, host-derived, and tickborne) to immune sera. Passive transfer of immune sera protected mice when they were challenged with spirochetes cultured in vitro. Immune sera did not protect mice from tickborne spirochetes or spirochetes derived from infected mice. These results indicate that spirochetes that have adapted within either the feeding tick or host are relatively invulnerable to the protective effects of immune sera, unlike spirochetes grown in vitro, which are highly susceptible.

Identification of a Borrelia burgdorferi OspA T cell epitope that promotes anti-OspA IgG in mice.

Lyme disease, due to infection with the tick-borne spirochete Borrelia burgdorferi, is a multisystem disorder that can lead to chronic disabling symptoms. Abs to the outer surface protein A (OspA) of B. burgdorferi provide protection against infection, and OspA is now the basis of a first generation recombinant vaccine undergoing phase III efficacy studies. Recent studies have suggested that T cells reactive with N-terminal epitopes in OspA could contribute to the development of treatment-resistant Lyme arthritis. In the present studies, we use the murine model of Lyme borreliosis to define an OspA T cell epitope located in the carboxyl terminus that accelerates anti-OspA IgG production after infection. In addition, we show that this T cell epitope is elicited by immunization with rOspA or with a truncated form of OspA that contains the B cell epitope targeted by protective OspA mAb. Polyclonal antisera to the truncated OspA fragment can protect mice from challenge infection. These results are the first demonstration of a B. burgdorferi-specific peptide that elicits a biologically important T cell response in vivo and have implications for the design of a second generation OspA-based subunit vaccine.

Novel Borrelia burgdorferi isolates from Ixodes scapularis and Ixodes dentatus ticks feeding on humans.

Seven cultures of Borrelia burgdorferi differing from strains B31 and ZS7 were identified from among 99 isolates from Ixodes scapularis ticks and from white-footed mice (Peromyscus leucopus) and 1 isolate from an Ixodes dentatus tick. Five of the six novel isolates from I. scapularis and the isolate from I. dentatus were from ticks feeding on humans. The six isolates from I. scapularis lacked OspA and OspB, four possessed an OspD band, and two reacted with an anti-OspC monoclonal antibody. Restriction fragment length polymorphisms of HindIII-digested DNAs from six OspA-negative isolates did not hybridize with radiolabeled ospA or LA88 DNA, and only isolate 46047 hybridized with the pG gene. Fragments similar to those recorded for the standard B. burgdorferi sensu stricto strains B31 and ZS7 were obtained with the fla and the HSP70 genes. Pulsed-field gel electrophoresis patterns of DNA digested with MluI included the specific B. burgdorferi sensu stricto band at 135 kbp for the five OspA-negative isolates from I. scapularis ticks. The six novel isolates apparently lack the 55-kbp plasmid encoding OspA. The pG-containing plasmid may be missing from all but isolate 46047. The isolate from the I. dentatus tick was similar to previous isolates from I. dentatus ticks feeding on rabbits. None of the isolates could be recovered from inoculated C3H/HeNCrlBR or white-footed mice. All isolates reacted with sera from humans with early or late Lyme disease. Our studies demonstrate that these borreliae occur in ticks feeding on humans, and therefore, at least some humans in the northeastern United States are likely being exposed to borreliae other than the classic B31-type strains that have thus far been isolated from humans.

Efficacy of human Lyme disease vaccine formulations in a mouse model.

Although immunization with recombinant outer surface protein A (OspA) appears to protect mice against infection by the agent of Lyme disease, all reported experiments have involved formulations that would not be suitable for use in humans or have not used realistic challenges. This study was designed to determine whether vaccines prepared and used in a phase I human trial, including one currently being used for a phase II trial in sites with endemic Borrelia burgdorferi, conferred protection in the C3H/HeJ mouse model. The challenge was ticks collected from a major site of the trial. None of the vaccinated mice became infected or developed disease, whereas 60% of unvaccinated mice became infected. Spirochetes were destroyed within the guts of virtually all recovered challenge ticks. These preparations of recombinant OspA effectively induced immunity to protect mice from Lyme disease when bitten by ticks collected from a field trial site.

Vaccination against Lyme disease caused by diverse Borrelia burgdorferi.

Diversity and mutations in the genes for outer surface proteins (Osps) A and B of Borrelia burgdorferi sensu lato (B. burgdorferi), the spirochetal agent of Lyme disease, suggests that a monovalent OspA or OspB vaccine may not provide protection against antigenically variable naturally occurring B. burgdorferi. We now show that OspA or OspB immunizations protect mice from tick-borne infection with heterogeneous B. burgdorferi from different geographic regions. This result is in distinct contrast to in vitro killing analyses and in vivo protection studies using syringe injections of B. burgdorferi as the challenge inoculum. Evaluations of vaccine efficacy against Lyme disease and other vector-borne infections should use the natural mode of transmission and not be predicated on classification systems or assays that do not rely upon the vector to transmit infection.

Disarming Lyme disease.

Antibiotics are usually curative. A vaccine is in clinical trials. Next on the research agenda: how to help people suffering from chronic symptoms.

Outer surface proteins E and F of Borrelia burgdorferi, the agent of Lyme disease.

We report the cloning and characterization of two outer surface proteins (Osps), designated OspE and OspF, from strain N40 of Borrelia burgdorferi, the spirochetal agent of Lyme disease. The ospE and ospF genes are structurally arranged in tandem as one transcriptional unit under the control of a common promoter. The ospE gene, located at the 5' end of the operon, is 513 nucleotides in length and encodes a 171-amino-acid protein with a calculated molecular mass of 19.2 kDa. The ospF gene, located 27 bp downstream of the stop codon of the ospE gene, consists of 690 nucleotides and encodes a protein of 230 amino acids with a calculated molecular mass of 26.1 kDa. Pulsed-field gel electrophoresis showed that the ospE and ospF genes are located on a 45-kb plasmid. Comparison of the leader sequences of OspE and OspF with those of the four known B. burgdorferi Osps (OspA, OspB, OspC, and OspD) reveals a hydrophobic domain and a consensus cleavage sequence (L-X-Y-C) recognized by signal peptidase II, and [3H]palmitate labeling shows that OspE and OspF are lipoproteins. Immunofluorescence studies demonstrated that both the OspE and OspF proteins are surface exposed. These features are consistent with the finding that OspE and OspF are B. burgdorferi surface lipoproteins.

Inability of truncated recombinant Osp A proteins to elicit protective immunity to Borrelia burgdorferi in mice.

The murine immune response to Borrelia burgdorferi (Bb), the etiologic agent of Lyme disease, is characterized by the development of antibodies reactive with the outer surface protein (Osp) A. It has been demonstrated that passive immunization of mice with at least some Osp A antibodies, including an Osp A mAb (CIII.78) that binds to a conformational epitope in the carboxyl-terminus of Osp A, provides protection against Bb challenge. Active immunization of mice with Osp A also confers protection, making Osp A a candidate for a vaccine Ag. To determine the regions of the Osp A protein that can elicit protective immunity, we immunized boosted mice with overlapping recombinant truncated fragments of Osp A, then challenged them with Bb. All groups of mice developed IgG Osp A antibodies detectable by immunoblotting with sera diluted at least 5000-fold. As expected, vaccination with full-length recombinant Osp A protected mice from challenge infection. In contrast, none of the mice vaccinated with the truncated Osp A proteins demonstrated immunity, even those immunized with an Osp A fragment binding the neutralizing mAb CIII.78. Osp A antibodies contained in the truncated Osp A antisera also failed to immunoprecipitate in vitro translated full-length Osp A and did not bind, as demonstrated by indirect immunofluorescence, to live or acetone-fixed Bb. Taken together, these results suggest that neutralizing Osp A antibodies are induced by vaccination with the full-length recombinant Osp A protein but not by vaccination with recombinant fragments.

Borrelia burgdorferi infection and immunity in mice deficient in the fifth component of complement.

When immunocompetent mice are inoculated with Borrelia burgdorferi, they develop acute arthritis and carditis that undergo spontaneous regression despite the persistence of infection. Specific T- and/or B-cell immunity appears to be necessary for resolution of disease manifestations. Humoral immune responses to B. burgdorferi are also important in prevention of B. burgdorferi infection, in that passive transfer of immune sera or protective monoclonal antibodies prevents the spirochete from establishing infection. It has previously been suggested that complement is necessary for effective antibody-mediated host responses against B. burgdorferi. To investigate the role of complement in the pathogenesis and prevention of Lyme disease, we compared the responses to B. burgdorferi challenge inoculation of mice genetically deficient in the fifth component of complement (C5) with those of C5-sufficient mice. All C5-deficient strains tested were susceptible to B. burgdorferi infection, and disease manifestations underwent regression in a similar time-course to those of complement-sufficient mice. Moreover, passive immunization of C5-deficient mice with either immune rabbit sera or neutralizing monoclonal antibody protected them from challenge infection. These results demonstrate that the expression of Lyme disease is not altered in mice deficient in C5 and that C5-mediated complement activation is not necessary for antibody-mediated protection from infection.

Evasion of protective immunity by Borrelia burgdorferi by truncation of outer surface protein B.

We analyzed variability in outer surface protein B (OspB) from Borrelia burgdorferi (Bb), the causative agent of Lyme disease, to determine how Bb escapes immune destruction. We have shown that vaccination with OspB from Bb strain B31 protected mice from infection with Bb B31 but not against Bb N40. The present study demonstrates that Bb N40 spirochetes which evade vaccination immunity to OspB have a truncated form of OspB, due to a TAA stop codon at nucleotide 577. In contrast, Bb N40 spirochetes that express full-length OspB are unable to infect mice immunized with OspB, analogous to our previous studies with Bb B31. Mapping of the OspB antibody response shows that epitopes in the C terminus of OspB are surface-exposed and bind protective monoclonal and polyclonal antibodies. This suggests that the C terminus of OspB is important for eliciting a protective immune response to OspB. Truncation or modification of outer surface proteins that do not bind protective antibody may be a means by which Bb evades host defenses.

Protective immunity in lyme borreliosis.

Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common vector-borne illness in the USA. Although early infection can be treated with antibiotics, the initial diagnosis is difficult and late disease may be recalcitrant to therapy. A vaccine against Lyme disease is therefore needed, and murine models of Lyme borreliosis have facilitated its development. In this review, Erol Fikrig, Fred Kantor, Stephen Barthold and Richard Flavell focus on the use of Borrelia surface antigens as vaccine candidates for Lyme disease.

Elimination of Borrelia burgdorferi from vector ticks feeding on OspA-immunized mice.

Although recombinant outer surface protein A (OspA) of Borrelia burgdorferi protects mice against injected Lyme disease spirochetes, the mode of protection has not yet been explored. Indeed, the efficacy of vaccine-induced immunity against a realistic vector-mediated challenge remains unexplored. Accordingly, we determined whether this immunogen protects mice against spirochetes delivered by nymphal Ixodes dammini ticks. Following challenge by tick bite, no spirochetes could be cultured from immunized mice, and no characteristic histopathology was found. The spirochete was not detected in ticks that fed on immunized animals and was present in virtually all ticks that fed on nonimmunized mice. We conclude that OspA-immunized mice are protected from spirochetal infection, at least in part, because the spirochete is destroyed in the infecting tick.

Borrelia burgdorferi strain 25015: characterization of outer surface protein A and vaccination against infection.

Mice vaccinated with outer surface protein A (OspA) from Borrelia burgdorferi strain N40 are protected from challenge with an intradermal syringe inoculum of B. burgdorferi strains N40, B31, and CD16. Vaccination experiments were done to determine if protection extended to strains 297 and 25015. We now show that OspA-N40 immunized mice are protected against challenge with strain 297, isolated from the cerebrospinal fluid of a patient with neuroborreliosis, but not against challenge with strain 25015, isolated from a tick in Millbrook, NY. The OspA gene from strain 25015 was therefore cloned and sequenced. The deduced OspA-25015 protein sequence differs from OspA-N40 at 40 of 273 amino acids. Furthermore, mice vaccinated with rOspA-25015 are protected from challenge with strain 25015 but not against strain N40. The results extend the usefulness of OspA as a vaccine candidate, but indicate that OspA can vary among strains of B. burgdorferi and that vaccination of mice with OspA-N40 does not protect against intradermal challenge with an inoculum of 10(4) strain 25015 spirochetes.

Long-term protection of mice from Lyme disease by vaccination with OspA.

Mice vaccinated with recombinant outer surface protein A (OspA) have been shown to be protected from infection with Borrelia burgdorferi, the agent of Lyme disease, when sacrificed 14 days after challenge with an intradermal inoculum of the spirochete. To determine whether infection was not merely delayed and that protection was long-lasting, we sacrificed vaccinated mice 60, 120, and 180 days after challenge; and to determine whether vaccinated mice retained their immune state over long periods, we challenged mice with B. burgdorferi 60, 90, 120, and 150 days after vaccination. The results of both groups of experiments show that the mice remained free from infection and disease and extend the usefulness of OspA as a vaccine candidate for Lyme borreliosis.

Roles of OspA, OspB, and flagellin in protective immunity to Lyme borreliosis in laboratory mice.

Vaccination with recombinant outer surface protein A (OspA) has been shown to protect mice from infection with Borrelia burgdorferi, the Lyme disease agent. To determine whether antibodies to B. burgdorferi proteins other than OspA are involved in protective immunity, antibodies to OspA were removed from protective anti-B. burgdorferi serum; the residual serum was still protective. Absorption of OspA and OspB antibodies from anti-B. burgdorferi serum eliminated the protective effect. Therefore, active immunization experiments were performed to determine the roles of OspB and flagellin in protective immunity and to determine whether protective immunity induced by OspA is dose dependent. Active immunization with recombinant OspA protected mice from infection with an inoculum of 10(4) spirochetes, but this protection could be overcome with a challenge of 10(7) spirochetes; OspB protected mice from infection with an inoculum of 10(3) spirochetes but was insufficient to fully protect against 10(4) organisms; and immunization with flagellin had no protective effect. These studies suggest that OspA and OspB, but not flagellin, play roles in protective immunity to spirochete infection.

Protection of mice from Lyme borreliosis by oral vaccination with Escherichia coli expressing OspA.

Mice immunized with recombinant outer surface protein A (OspA) in Freund's adjuvant or with intraperitoneal injections of live Escherichia coli expressing OspA have been shown to be protected from infection with Borrelia burgdorferi. To investigate the efficacy of oral vaccination, C3H/He mice were inoculated with 10(8) live E. coli expressing recombinant OspA by gavage and boosted in a similar manner on days 10, 20, 30, and 40. The animals developed serum IgG antibodies to OspA by immunoblot and were protected from infection when challenged with 10(4) B. burgdorferi intradermally 14 days after the last boost. Control mice did not develop antibodies to OspA and were not protected against challenge infection. These results suggest that an oral preparation of recombinant OspA could potentially be used for vaccination.