Detection of IFN-γ Secretion in Blood Samples Collected Before and After Treatment of Varying Stages of Lyme Disease.

BACKGROUND: QuantiFERON enzyme-linked immunosorbent assay (ELISA; Qiagen) with Borrelia burgdorferi peptide antigens was previously shown to reliably detect interferon-γ (IFN-γ) in blood samples from adult patients with early Lyme disease and the response disappeared rapidly after treatment. We evaluated the response before and after appropriate antibiotic therapy in adolescent and adult subjects with more diverse stages of the illness. METHODS: Blood was obtained from patients with clinician-identified Lyme disease with constitutional complaints, erythema migrans, nerve palsy, cardiac abnormality, or arthritis before (n = 68) and 6 weeks (n = 46) and 6 months (n = 45) after therapy. The sera were tested for Lyme disease by standard 2-tiered testing (STTT) and anti-C6 antibodies by ELISA and the levels of IFN-γ in the blood samples were detected by QuantiFERON ELISA. RESULTS: A positive STTT result supported the clinical diagnosis of 37 (54%) subjects and anti-C6 antibodies were detected in 45 (66%) subjects, including 36 (97%) STTT-positive subjects, and the responses often persisted or expanded after antibiotic therapy. IFN-γ was detected in 49 (72%) subjects prior to treatment and the response most often significantly decreased 6 weeks (P = .007) or 6 months (P = .001) after treatment. CONCLUSIONS: The QuantiFERON ELISA reliably detected IFN-γ in blood samples from adult and adolescent patients with varying stages of Lyme disease and the response disappeared rapidly after treatment. Additional studies to more critically evaluate clinical utility as a laboratory test for diagnosis and confirmation of effective therapy are warranted.

Single-dose intranasal subunit vaccine rapidly clears secondary sepsis in a high-dose pneumonic plague infection.

Yersinia pestis, the causative agent of plague, has killed millions throughout human history. Though public health initiatives have reduced the number of plague cases, it remains endemic in many areas of the world. It also remains a significant threat for use as a biological weapon. Naturally occurring multi-drug antibiotic resistance has been observed in Y. pestis, and resistant strains have been engineered for use as a biological weapon. Vaccines represent our best means of protection against the threat of antibiotic resistant plague. We have developed a vaccine consisting of two Y. pestis virulence factors, LcrV (V) and F1, conjugated to Tobacco Mosaic Virus (TMV), a safe, non-replicating plant virus that can be administered mucosally, providing complete protection against pneumonic plague, the deadliest form of the disease and the one most likely to be seen in a biological attack. A single intranasal (i.n.) dose of TMV-F1 + TMV-V (TMV-F1/V) protected 88% of mice against lethal challenge with 100 LD50 of Y. pestis CO92pgm-, while immunization with rF1 + rV without TMV was not protective. Serum and tissues were collected at various timepoints after challenge to assess bacterial clearance, histopathology, cytokine production, and antibody production. Overall, TMV-F1/V immunized mice showed a significant reduction in histopathology, bacterial burden, and inflammatory cytokine production following challenge compared to rF1 + rV vaccinated and unvaccinated mice. Pneumonic challenge resulted in systemic dissemination of the bacteria in all groups, but only TMV-F1/V immunized mice rapidly cleared bacteria from the spleen and liver. There was a direct correlation between pre-challenge serum F1 titers and recovery in all immunized mice, strongly suggesting a role for antibody in the neutralization and/or opsonization of Y. pestis in this model. Mucosal administration of a single dose of a Y. pestis TMV-based subunit vaccine, without any additional adjuvant, can effectively protect mice from lethal infection.

Borrelia burgdorferi-specific IgA in Lyme Disease.

The laboratory diagnosis of Lyme disease is currently dependent on the detection of IgM and IgG antibodies against Borrelia burgdorferi, the causative agent of the disease. The significance of serum IgA against B. burgdorferi remains unclear. The production of intrathecal IgA has been noted in patients with the late Lyme disease manifestation, neuroborreliosis, but production of antigen-specific IgA during early disease has not been evaluated. In the current study, we assessed serum IgA binding to the B. burgdorferi peptide antigens, C6, the target of the FDA-cleared C6 EIA, and FlaB(211-223)-modVlsE(275-291), a peptide containing a Borrelia flagellin epitope linked to a modified VlsE sequence, in patients with early and late Lyme disease. Specific IgA was detected in 59 of 152 serum samples (38.8%) from early Lyme disease patients. Approximately 50% of early Lyme disease patients who were seropositive for peptide-specific IgM and/or IgG were also seropositive for peptide-specific IgA. In a subpopulation of patients, high peptide-specific IgA could be correlated with disseminated disease, defined as multiple erythema migrans lesions, and neurological disease complications. These results suggest that there may be an association between elevated levels of antigen-specific IgA and particular disease manifestations in some patients with early Lyme disease.

Intranasal delivery of a protein subunit vaccine using a Tobacco Mosaic Virus platform protects against pneumonic plague.

Yersinia pestis, one of history's deadliest pathogens, has killed millions over the course of human history. It has attributes that make it an ideal choice to produce mass casualties and is a prime candidate for use as a biological weapon. When aerosolized, Y. pestis causes pneumonic plague, a pneumonia that is 100% lethal if not promptly treated with effective antibiotics. Currently, there is no FDA approved plague vaccine. The current lead vaccine candidate, a parenterally administered protein subunit vaccine comprised of the Y. pestis virulence factors, F1 and LcrV, demonstrated variable levels of protection in primate pneumonic plague models. As the most likely mode of exposure in biological attack with Y. pestis is by aerosol, this raises a question of whether this parenteral vaccine will adequately protect humans against pneumonic plague. In the present study we evaluated two distinct mucosal delivery platforms for the intranasal (IN) administration of LcrV and F1 vaccine proteins, a live bacterial vector, Lactobacillus plantarum, and a Tobacco Mosaic Virus (TMV) based delivery platform. IN administration of L. plantarum expressing LcrV, or TMV-conjugated to LcrV and F1 (TMV-LcrV+TMV-F1) resulted in the similar induction of high titers of IgG antibodies and evidence of proinflammatory cytokine secretion. However, only the TMV-conjugate delivery platform protected against subsequent lethal challenge with Y. pestis. TMV-LcrV+TMV-F1 co-vaccinated mice had no discernable morbidity and no mortality, while mice vaccinated with L. plantarum expressing LcrV or rLcrV+rF1 without TMV succumbed to infection or were only partially protected. Thus, TMV is a suitable mucosal delivery platform for an F1-LcrV subunit vaccine that induces complete protection against pneumonic infection with a lethal dose of Y. pestis in mice.