Outer surface protein C (OspC) of the Lyme disease spirochetes is an important virulence factor that has potential utility for vaccine development. Of the 21 OspC types that have been identified, it has been postulated that types A, B, I, and K are specifically associated with invasive infections. Through an analysis of isolates collected from patients in Maryland we found that OspC types C, D, and N are also associated with invasive infections. This observation suggests that there is greater diversity in the group of OspC types associated with invasive infection than has been previously suggested. Detailed knowledge of the antigenic structure of OspC is essential for vaccine development. To determine if the antibody response to OspC is type specific, recombinant proteins of several different OspC types were immunoblotted and screened with sera from mice infected with isolates having known OspC types. These analyses revealed a high degree of specificity in the antibody response and suggested that the immunodominant epitopes of OspC reside in the variable domains of the protein. To localize these epitopes, OspC fragments were generated and screened with serum collected from infected mice. These analyses led to identification of previously uncharacterized epitopes that define the type specificity of the OspC antibody response. These analyses provide important insight into the antigenic structure of OspC and also provide a basis for understanding the variable nature of the antibody response to this important virulence factor of the Lyme disease spirochetes.

Maintenance of Borrelia burgdorferi within its enzootic cycle requires a complex regulatory pathway involving the alternative σ factors RpoN and RpoS and two ancillary trans -acting factors, BosR and Rrp2. Activation of this pathway occurs within ticks during the nymphal blood meal when RpoS, the effector σ factor, transcribes genes required for tick transmission and mammalian infection. RpoS also exerts a ‘gatekeeper’ function by repressing σ 70 -dependent tick phase genes (e.g., ospA , lp6.6 ). Herein, we undertook a broad examination of RpoS functionality throughout the enzootic cycle, beginning with modeling to confirm that this alternative σ factor is a ‘genuine’ RpoS homolog. Using a novel dual color reporter system, we established at the single spirochete level that ospA is expressed in nymphal midguts throughout transmission and is not downregulated until spirochetes have been transmitted to a naïve host. Although it is well established that rpoS /RpoS is expressed throughout infection, its requirement for persistent infection has not been demonstrated. Plasmid retention studies using a trans -complemented Δ rpoS mutant demonstrated that (i) RpoS is required for maximal fitness throughout the mammalian phase and (ii) RpoS represses tick phase genes until spirochetes are acquired by a naïve vector. By transposon mutant screening, we established that bba34/oppA5 , the only OppA oligopeptide-binding protein controlled by RpoS, is a bona fide persistence gene. Lastly, comparison of the strain 297 and B31 RpoS DMC regulons identified two cohorts of RpoS-regulated genes. The first consists of highly conserved syntenic genes that are similarly regulated by RpoS in both strains and likely required for maintenance of B. burgdorferi sensu stricto strains in the wild. The second includes RpoS-regulated plasmid-encoded variable surface lipoproteins ospC , dbpA and members of the ospE/ospF/elp , mlp , revA , and Pfam54 paralogous gene families, all of which have evolved via inter- and intra-strain recombination. Thus, while the RpoN/RpoS pathway regulates a ‘core’ group of orthologous genes, diversity within RpoS regulons of different strains could be an important determinant of reservoir host range as well as spirochete virulence.

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ABSTRACTLyme disease in the United States is caused byBorrelia burgdorferisensu stricto, which is transmitted to mammals by infected ticks.Borreliaspirochetes differentially express immunogenic outer surface proteins (Osp). Our aim was to evaluate antibody responses to Osp antigens to aid the diagnosis of early infection and the management of Lyme disease. We analyzed antibody responses during the first 3 months after the experimental infection of dogs using a novel multiplex assay. Results were compared to those obtained with two commercial assays detecting C6 antigen. Multiplex analysis identified antibodies to OspC and C6 as early as 3 weeks postinfection (p.i.) and those to OspF by 5 weeks p.i. Antibodies to C6 and OspF increased throughout the study, while antibodies to OspC peaked between 7 and 11 weeks p.i. and declined thereafter. A short-term antibody response to OspA was observed in 3/8 experimentally infected dogs on day 21 p.i. Quant C6 enzyme-linked immunosorbent assay (ELISA) results matched multiplex results during the first 7 weeks p.i.; however, antibody levels subsequently declined by up to 29%. Immune responses then were analyzed in sera from 125 client-owned dogs and revealed high agreement between antibodies to OspF and C6 as robust markers for infection. Results from canine patient sera supported that OspC is an early infection marker and antibodies to OspC decline over time. The onset and decline of antibody responses toB. burgdorferiOsp antigens and C6 reflect their differential expression during infection. They provide valuable tools to determine the stage of infection, treatment outcomes, and vaccination status in dogs.

SummaryBorrelia burgdorferi outer surface protein C (ospC) is required for the establishment of infection in mammals. However, its precise function remains controversial. The biologically active form of OspC appears to be a homodimer. Alpha helix 1 and 1Ј of the apposing monomers form a solvent-accessible pocket at the dimeric interface that presents a putative ligand-binding domain (LBD1). Here we employ site-directed and allelic-exchange mutagenesis to test the hypothesis that LBD1 is a determinant of OspC function in the mammalian environment. Substitution of residues K60, E61 and E63 which line LBD1 resulted in the loss of infectivity or influenced dissemination. Analyses of the corresponding recombinant proteins demonstrated that the loss of function was not due to structural perturbation, impaired dimer formation or the loss of plasminogen binding. This study is the first to assess the involvement of individual residues and domains of OspC in its in vivo function. The data support the hypothesis that OspC interacts with a mammalian derived ligand that is critical for survival during early infection. These results shed new light on the structure-functions relationships of OspC and challenge existing hypotheses regarding OspC function in mammals.

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.

Using available Borrelia outer surface protein C (OspC) sequences, a phylogenetic analysis was undertaken to delineate the number of antigenic domains required for inclusion in a broadly protective, chimeric, OspC-based Lyme disease vaccine. The data indicate that approximately 34 would be required and that an OspC-based vaccinogen is feasible.

Botulinum neurotoxins (BoNT) are some of the most toxic proteins known, with a human LD50 of ~1 ng/kg. Equine antitoxin has a half-life in circulation of less than 1 day and is limited to a treatment rather than a prevention indication. The development of monoclonal antibodies (mAbs) may represent an alternative therapeutic option that can be produced at high quantities and of high quality and with half-lives of >10 days. Two different three mAb combinations are being developed that specifically neutralize BoNT serotypes A (BoNT/A) and B (BoNT/B). We investigated the pharmacokinetics of the anti-BoNT/A and anti-BoNT/B antibodies in guinea pigs (Cavia porcellus) and their ability to protect guinea pigs against an aerosol challenge of BoNT/A1 or BoNT/B1. Each antibody exhibited dose-dependent exposure and reached maximum circulating concentrations within 48 h post intraperitoneal or intramuscular injection. A single intramuscular dose of the three mAb combination protected guinea pigs against an aerosol challenge dose of 93 LD50 of BoNT/A1 and 116 LD50 of BoNT/B1 at 48 h post antibody administration. These mAbs are effective in preventing botulism after an aerosol challenge of BoNT/A1 and BoNT/B1 and may represent an alternative to vaccination to prevent type A or B botulism in those at risk of BoNT exposure.