Immuno Technologies (United States)

Vaccines targeting outer surface protein A (OspA) ofBorrelia burgdorferiprotect against Lyme disease by inducing antibodies in the host that neutralize spirochetes in theIxodes scapularistick midgut during engorgement before transmission occurs. We evaluated whether heterologous vaccination enhances protection compared to homologous delivery of the immunogen. C3H-HeN mice were immunized with a parainfluenza virus 5 vector (PIV5) containing a modified OspA protein (OspABPBPk) using three prime-boost immunization regimens: homologous PIV5 intranasal/intranasal (IN/IN), homologous rOspABPBPk(protein) subcutaneous/subcutaneous (SC/SC), or heterologous intranasal PIV5-ABPBPk/subcutaneous rOspABPBPk(IN/SC). Immunized mice were then challenged with nymphalI. scapularisticks infected with 19 strains ofB. burgdorferithree months post-prime vaccination. Three weeks after the last day of tick challenge, blood and tissues were collected from euthanized mice. All OspA-containing regimens elicited strong systemic IgG antibody responses that exceeded established protective thresholds. Vaccination markedly reducedB. burgdorferiloads in engorged nymphal ticks. Homologous IN/IN and SC/SC regimens produced the lowest geometric meanflaBburdens in nymphs (2.6 × 103and 1.8 × 103copies, respectively), corresponding to ∼1.8–2.0 log10reductions relative to controls; the heterologous IN/SC regimen produced a more modest reduction (∼1.7 log10; p = 0.0071 vs IN/IN, p = 0.0003 vs SC/SC). Across all vaccinated groups, no systemic infection withB. burgdorferiwas observed as evidenced by absence of motile spirochetes in cultures from tissues, although one mouse (1/9, 11%) in the heterologous IN/SC regimen, had evidence of increased pepVF seroconversion and low-levelflaBDNA in culture. Thus, homologous regimens yielded more consistent protective immunity with absent of signs ofB. burgdorferidissemination, suggesting that high systemic anti-OspABPBPkIgG antibody titers, rather than alternate immunization routes, were associated with the most consistent protection outcomes. PIV5-ABPBPkis a promising vaccine candidate for development of next-generation homologous or heterologous human Lyme disease vaccines.

The order of switching between the immunoglobulin heavy chain (Fc) is time dependent, progressing from IgM/D to IgG3/IgG1/IgA1/IgG2/IgG4 and later to IgE/IgA2. In this study, we show that B. burgdorferi-VlsE-specific antibody switching proceeds in a predictable sequence between class (Ig M/G/A) and IgG isotype (IgG 1/2/3/4) as Lyme disease progresses from early to late stage and that antibody class and isotype may be more helpful to distinguish the early stages of Lyme disease. This study advances our understanding of the tempo and structure of the humoral immune response to B. burgdorferi and is applicable to the development of new diagnostic assays for Lyme disease.

There are no assays for detecting B. burgdorferi antigen in blood of infected Lyme disease individuals. Here, we provide proof-of-principle evidence that we can quantify B. burgdorferi antigen in spiked blood using a portable smartphone-based fluorescence microscope that measures immunoagglutination on a paper microfluidic chip. We targeted B. burgdorferi OspA to develop a working prototype and added examples of two antigens (OspC and VlsE) that have diagnostic value for discrimination of Lyme disease stage. Using an extensively validated monoclonal antibody to OspA (LA-2), detection of OspA antigen had a broad linear range up to 100 pg/mL in 1% blood and the limit of detection (LOD) was 100 fg/mL (= 10 pg/mL in undiluted blood), which was 1000 times lower than our target of 10 ng/mL. Analysis of the two other targets was done using polyclonal and monoclonal antibodies. OspC antigen was detected at LOD 100 pg/mL (= 10 ng/mL of undiluted blood) and VlsE antigen was detected at LOD 1–10 pg/mL (= 0.1–1 ng/mL of undiluted blood). The method is accurate and was performed in 20 min from sample to answer. When optimized for detecting several B. burgdorferi antigens, this assay may differentiate active from past infections and facilitate diagnosis of Lyme disease in the initial weeks of infection, when antibody presence is typically below the threshold to be detected by serologic methods.

ABSTRACTLeptospirosis is a widespread zoonotic disease caused by pathogenic spirochetes of the genus Leptospira which affect both humans and animals. A somewhat contradictory published body of evidence suggests that sex impacts severity outcomes of human Leptospirosis. In this study, we used an acute animal model of disease to analyze how male and female hamsters infected side-by-side with low but increasing doses of L. interrogans under the same exposure conditions develop Leptospirosis. We found that female hamsters were considerably more resistant to Leptospirosis given that 87.5% survived infection; male hamsters did not gain weight and 93.7% succumbed to infection with the same infectious doses. Analysis of bacterial burden in kidney of male hamsters showed that infection with the lowest dose (103) resulted in a 4Log increase of L. interrogans, whereas females infected with the same dose had a reduction of ~1Log, after 28 days of infection. Non-surviving hamsters had signs of compromised renal function (higher levels of creatinine in blood), as well as increased levels of anti-inflammatory IL-10 and innate response pro-inflammatory CCL3, CxCL10 and TNF-α in kidney, as well as ColA1 which is a marker of kidney fibrosis. In endemic areas, humans are more often exposed to the lower infection doses which were lethal to males, than the higher doses which were lethal to males and females. It is possible that the frequency of lower infectious doses in nature and increased biological susceptibility both contribute for aggravated outcomes of leptospirosis in males.

Although Leptospira can infect a wide range of mammalian species, most studies have been conducted in golden Syrian hamsters, a species particularly sensitive to acute disease. Chronic disease has been well characterized in the rat, one of the natural reservoir hosts. Studies in another asymptomatic reservoir host, the mouse, have occasionally been done and have limited infection to mice younger than 6 weeks of age. We analyzed the outcome of sublethal infection of C3H/HeJ mice older than age 10 weeks with Leptospira interrogans serovar Copenhageni. Infection led to bloodstream dissemination of Leptospira, which was followed by urinary shedding, body weight loss, hypothermia, and colonization of the kidney by live spirochetes 2 weeks after infection. In addition, Leptospira dissemination triggered inflammation in the kidney but not in the liver or lung, as determined by increased levels of mRNA transcripts for the keratinocyte-derived chemokine, RANTES, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-1β, inducible nitric oxide synthase, interleukin-6, and gamma interferon in kidney tissue. The acquired humoral response to Leptospira infection led to the production of IgG mainly of the IgG1 subtype. Flow cytometric analysis of splenocytes from infected mice revealed that cellular expansion was primarily due to an increase in the levels of CD4(+) and double-negative T cells (not CD8(+) cells) and that CD4(+) T cells acquired a CD44(high) CD62L(low) effector phenotype not accompanied by increases in memory T cells. A mouse model for sublethal Leptospira infection allows understanding of the bacterial and host factors that lead to immune evasion, which can result in acute or chronic disease or resistance to infection (protection).

Eight symptomatic individuals chronically exposed to indoor formaldehyde (HCHO) at low concentrations (0.07-0.55 ppm) were compared to 8 nonexposed subjects with respect to: (1) presence of IgG and IgE antibodies to HCHO conjugated to human serum albumin (F-HSA); (2) the percentage of venous blood T and B cells by E and EAC-rosetting; and (3) the ability of T and B cells to undergo mitogen (PHA, PWM) stimulated blastogenesis as measured by the incorporation of tritiated thymidine. Anti-F-HSA IgG, but no IgE, antibodies were detected in the sera of the 8 exposed subjects; none were found in 7 of the unexposed controls. T lymphocytes were decreased in the exposed (48 +/- 11.5%) compared to the control (65.9 +/- 4.97%) subjects (p greater than .001 less than .01). B cells were 12.6 +/- 1.6% (HCHO group) and 14.75 +/- 2.1% (controls) (p greater than .02 less than .05). The incorporation of labeled thymidine by T cells (PHA) was decreased: 17,882 +/- 2,293 cpm (HCHO group) and 28,576 +/- 3,807 cpm (p greater than .001 less than .01). T and B cell blastogenesis (PWM) was 9,698 +/- 1,441 cpm (HCHO group) and 11,279 +/- 1,711 (controls) (p greater than .05 less than .1). Exposure to HCHO appears to stimulate IgG antibodies to F-HSA and decrease the proportion of peripheral T cells.