Epitope length, genospecies dependency, and serum panel effect in the IR6 enzyme-linked immunosorbent assay for detection of antibodies to Borrelia burgdorferi.

In the absence of erythema migrans, the basis for diagnosis of Lyme disease is the demonstration of an antibody response against Borrelia burgdorferi in an appropriate clinical setting. The C6 enzyme-linked immunosorbent assay, based on the IR6 region of VlsE, has become widely used in both the United States and Europe. We mapped the antigenic epitopes of IR6 to a shorter sequence that is equivalent in sensitivity and specificity to the full-length IR6 25-residue peptide. In addition, we observed significant differences in sensitivity between serum panels (60 to 100%), indicating that the selection of the serum panels can shape the apparent overall sensitivity of the assay. Contrary to prior reports, the assay sensitivity is greater when the IR6 peptide is derived from the sequence of the same infecting Borrelia genospecies. Using our North American panels and the two panels obtained from European Lyme disease patients, we determined that the IR6 assay that is based on a single genospecies of Borrelia spp. is not optimal for use as a universal diagnostic assay for Lyme disease.

Oral vaccine that breaks the transmission cycle of the Lyme disease spirochete can be delivered via bait.

Borrelia burgdorferi causes Lyme disease, a potentially debilitating human disease for which no vaccine is currently available. We developed an oral bait delivery system for an anti-B. burgdorferi vaccine based in OspA. Mice were immunized orally via gavage and bait feeding. Challenge was performed via Ixodes scapularis field nymphs carrying multiple B. burgdorferi strains. Vaccination protected 89% of the mice and the systemic immune response was skewed toward IgG2a/2b production. Moreover, this oral vaccine reduced the pathogen in the tick vector by eight-fold. We conclude that this oral vaccine induces a protective systemic immune response against a variety of infectious B. burgdorferi strains found in nature and therefore it can eliminate this zoonotic pathogen from its major host reservoirs. Because we observed elimination of the spirochete from the tick vector, a broad delivery of this oral vaccine to wildlife reservoirs is likely to disrupt the transmission cycle of this pathogen.

LcrV capture enzyme-linked immunosorbent assay for detection of Yersinia pestis from human samples.

In the United States, there is currently a major gap in the diagnostic capabilities with regard to plague. To address this, we developed an antigen capture assay using an essential virulence factor secreted by Yersinia spp., LcrV, as the target antigen. We generated anti-LcrV monoclonal antibodies (MAbs) and screened them for the ability to bind bacterially secreted native Yersinia pestis LcrV. Anti-LcrV MAb 19.31 was used as a capture antibody, and biotinylated MAb 40.1 was used for detection. The detection limit of this highly sensitive Yersinia LcrV capture enzyme-linked immunosorbent assay is 0.1 ng/ml. The assay detected LcrV from human sputum and blood samples treated with concentrations as low as 0.5 ng/ml of bacterially secreted native Y. pestis LcrV. This assay could be used as a tool to help confirm the diagnosis of plague in patients presenting with pneumonia.

IFNgamma production in peripheral blood of early Lyme disease patients to hLFAalphaL (aa326-345).

BACKGROUND: It has been proposed that outer surface protein A (OspA) of Borrelia burgdorferi sensu stricto contains a T helper 1 (Th1) cell epitope that could play a role in an autoimmune response to hLFA1. METHODS: We used two peptides, hLFAalphaL (aa326-345) and Borrelia burgdorferi OspAB31 (aa164-183), as stimulating antigens to measure Th1 proinflammatory IFNgamma cytokine production in peripheral blood of Lyme disease patients presenting with EM without history of arthritis, as well as in peripheral blood of healthy individuals. RESULTS: IFNgamma responses to hLFA1 peptide were observed in 11 of 19 Lyme disease patients and in 3 of 15 healthy controls. In contrast, only 2 of 19 of the Lyme disease patients and none of the controls responded to the homologous OspAB31 peptide. CONCLUSIONS: IFNgamma was produced in response to stimulation with peptide hLFAalphaL (aa326-345) in peripheral blood of 58% of patients with early Lyme disease without signs of arthritis, as well as in peripheral blood of 20% of healthy individuals, but not in response to stimulation with the homologous OspAB31 (aa164-183) peptide (p < 0.05). Our results suggest that reactivity to the hLFA1 peptide in peripheral blood may be the result of T cell degeneracy.

Recombinant assay for serodiagnosis of Lyme disease regardless of OspA vaccination status.

All current seroassays using cultured Borrelia burgdorferi as their antigen source have been rendered obsolete by the recombinant OspA Lyme disease vaccine. OspA is the major outer surface protein expressed in cultured B. burgdorferi, and any seroassay that uses whole organisms as its antigen source cannot differentiate between subjects who received the vaccine and those who were naturally infected. We developed a new sensitive and specific enzyme-linked immunosorbent assay (ELISA) utilizing recombinant chimeric borrelia proteins devoid of OspA (rNon-OspA) that can be used to detect antibodies to diagnostically important B. burgdorferi antigens in both OspA-vaccinated and nonvaccinated individuals. We tested sera from patients with Lyme disease and with conditions associated with false-positive serologies, OspA-vaccinated individuals, and healthy high-risk workers from an area of endemicity and normal sera from individuals from areas of nonendemicity. The rNon-OspA test was compared with two commercially available whole-cell immunoassays. The rNon-OspA assay is as sensitive and specific as the whole-cell assay (P > 0.05) for detection of anti-B. burgdorferi antibodies. However, the rNon-OspA assay can differentiate between populations comprised of naturally infected and OspA-vaccinated individuals (P < 0.05). Our data demonstrate that this new sensitive rNon-OspA ELISA can be used for the laboratory detection of B. burgdorferi antibodies regardless of vaccination status and could replace existing serologic assays for Lyme disease.