Exposure to live saprophytic Leptospira before challenge with a pathogenic serovar prevents severe leptospirosis and promotes kidney homeostasis.

Previous studies demonstrated that Leptospira biflexa, a saprophytic species, triggers innate immune responses in the host during early infection. This raised the question of whether these responses could suppress a subsequent challenge with pathogenic Leptospira. We inoculated male C3H/HeJ mice with a single or a double dose of L. biflexa before challenge with a pathogenic serovar, L. interrogans ser. Copenhageni FioCruz (LIC). Pre-challenge exposure to L. biflexa did not prevent LIC dissemination and colonization of the kidney. However, it rescued weight loss and mouse survival thereby mitigating disease severity. Unexpectedly, there was correlation between rescue of overall health (weight gain, higher survival, lower kidney fibrosis) and higher shedding of LIC in urine. This stood in stark contrast to the L. biflexa unexposed LIC challenged control. Immune responses were dominated by increased frequency of B cells and effector T helper (CD4+) cells in spleen, as well as significant increases in serologic IgG2a. Our findings suggest that exposure to live saprophytic Leptospira primes the host to develop Th1 biased immune responses that prevent severe disease induced by a subsequent challenge with a pathogenic species. Thus, hosts exposed to live saprophytic Leptospira before challenge with a pathogenic serovar may withstand LIC infection with far better outcomes. Furthermore, a status of homeostasis may have been reached after kidney colonization that helps LIC complete its enzootic cycle.

Intranasal vaccine for Lyme disease provides protection against tick transmitted Borrelia burgdorferi beyond one year.

Strategies for disease control are necessary to reduce incidence of Lyme Disease (LD) including development of safe vaccines for human use. Parainfluenza virus 5 (PIV5) vector has an excellent safety record in animals and PIV5-vectored vaccines are currently under clinical development. We constructed PIV5-vectored LD vaccine candidates expressing OspA from B. burgdorferi (OspAB31) and a chimeric protein containing sequences from B. burgdorferi and B. afzelii (OspABPBPk). Immunogenicity and vaccine efficacy were analyzed in C3H-HeN mice after prime-boost intranasal vaccination with live PIV5-OspAB31 or PIV5-OspABPBPk, subcutaneous (s.c.) vaccination with rOspAB31+Alum, and the respective controls. Mice vaccinated intranasally with live PIV5-AB31 or PIV5-ABPBPk had higher endpoint titers of serum antibody against OspAB31 at 6- and 12- months post vaccination, compared to mice vaccinated s.c. with rOspAB31. Neutralization activity of antibody was maintained up to 18-months post-immunization, with the response greater in live PIV5-delivered OspA vaccines, than that induced by s.c. rOspAB31. Challenge with infected ticks carrying 10-19 strains of B. burgdorferi performed at 4-, 9- or 15-months post-immunization showed increased breakthrough infections in mice vaccinated with s.c. rOspAB31 compared to intranasal PIV5-AB31 or PIV5-ABPBPk at 9- and 15-months, as determined by quantification of serologic antibodies to B. burgdorferi proteins as well as flaB DNA in tissues, and by visualization of motile B. burgdorferi in culture of tissues under dark field microscope. These findings indicate that immunization of mice with PIV5 delivered OspA generates immune responses that produce longer-lasting protection ( > 1 year) against tick-transmitted B. burgdorferi than a parenteral recombinant OspA vaccine.

A parainfluenza virus 5 (PIV5)-vectored intranasal vaccine for Lyme disease provides long-lasting protection against tick transmitted Borrelia burgdorferi in mice.

Lyme disease (LD) is the most prevalent vector borne disease in North America and Europe and its geographic range continues to expand. Strategies for disease control are necessary to effectively reduce incidence of LD including development of safe vaccines for human use. Parainfluenza virus 5 (PIV5) vector has an excellent safety record in animals and PIV5-vectored COVID-19 and RSV vaccines are currently under clinical development. We constructed PIV5-vectored LD vaccine candidates expressing OspA from B. burgdorferi sensu stricto (OspAB31) and a chimeric protein containing sequences from B. burgdorferi and B. afzelii (OspABPBPk). Immunogenicity and vaccine efficacy were analyzed in C3H-HeN mice after prime-boost intranasal (IN) vaccination with PIV5-OspAB31 and PIV5-OspABPBPk, subcutaneous (SC) vaccination with rOspAB31+Alum as well as the respective controls. Mice vaccinated with either PIV5-AB31 or PIV5-ABPBPk intranasally had high endpoint titers of serum antibody against OspA antigen beyond 1 year post vaccination, similar to levels detected in mice vaccinated SC with rOspAB31. Flowcytometric analysis of spleen cells at 9-months post-immunization demonstrated that immunization with the intranasal PIV5 vaccine candidates led to an overall increase in the number of memory B cells, cytotoxic T and cytotoxic effector T cells compared to SC groups. Borreliacidal activity measured by neutralization assay was maintained up to 18 months post-immunization, with the response greater in intranasal PIV5-delivered OspA vaccines, than that induced by SC rOspAB31. Challenge with infected ticks (10-19 strains of B. burgdorferi) performed at 4-, 9- or 15-months post-immunization showed increased breakthrough infections in mice vaccinated with SC rOspAB31 compared to IN PIV5-AB31 or IN PIV5-ABPBPk at 9- and 15-months, as determined by qPCR of B. burgdorferi in tissues, culture of B. burgdorferi from tissues, and antibodies against B. burgdorferi protein VIsE. These data demonstrate that intranasal PIV5-based immunization is superior to parenteral immunization with the same recombinant protein and provides long-lasting protection (> 1 year) against Lyme disease.

A portable immunosensor provides sensitive and rapid detection of Borrelia burgdorferi antigen in spiked blood.

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.

Deployment of a Reservoir-Targeted Vaccine Against Borrelia burgdorferi Reduces the Prevalence of Babesia microti Coinfection in Ixodes scapularis Ticks.

In the Northeast and upper Midwest of the United States, Babesia microti and Borrelia burgdorferi use Ixodes scapularis ticks as vector and Peromyscus leucopus mice as major reservoir host. We previously established, in a 5-year field trial, that a reservoir-targeted outer surface protein A vaccine reduces the prevalence of B. burgdorferi-infected ticks. We accessed ticks and mouse blood samples collected during the trial, extracted total DNA, and amplified the B. microti 18S rRNA gene. Vaccine deployment reduced the prevalence of ticks coinfected with B. microti and that of mice infected with B. microti. Breaking the enzootic cycle of B. burgdorferi may reduce the incidence of babesiosis.

Infected Ixodes scapularis Nymphs Maintained in Prolonged Questing under Optimal Environmental Conditions for One Year Can Transmit Borrelia burgdorferi (Borreliella genus novum) to Uninfected Hosts.

In recent decades, Lyme disease has been expanding to previous nonendemic areas. We hypothesized that infected I. scapularis nymphs that retain host-seeking behavior under optimal environmental conditions are fit to fulfil their transmission role in the enzootic cycle of B. burgdorferi. We produced nymphal ticks in the laboratory under controlled temperature (22-25°C), humidity (80-90%), and natural daylight cycle conditions to allow them to retain host-seeking/questing behavior for 1 year. We then analyzed differences in B. burgdorferi infection prevalence in questing and diapause nymphs at 6 weeks postmolting (prime questing) as well as differences in infection prevalence of questing nymphs maintained under prolonged environmental induced questing over 12 months (prolonged questing). Lastly, we analyzed the fitness of nymphal ticks subjected to prolonged questing in transmission of B. burgdorferi to naive mice over the course of the year. B. burgdorferi infected unfed I. scapularis nymphal ticks maintained under optimal environmental conditions in the laboratory not only survived for a year in a developmental state of prolonged questing (host-seeking), but they retained an infection prevalence sufficient to effectively fulfil transmission of B. burgdorferi to uninfected mice after tick challenge. Our study is important for understanding and modeling Lyme disease expansion into former nonendemic regions due to climate change. IMPORTANCE Lyme disease is rapidly spreading from its usual endemic areas in the Northeast, Midwest, and Midatlantic states into neighboring areas, which could be due to changing climate patterns. Our study shows that unfed I. scapularis nymphal ticks kept under optimal environmental conditions in the laboratory survived for a year while exhibiting aggressive host-seeking behavior, and they maintained a B. burgdorferi infection prevalence which was sufficient to infect naive reservoir hosts after tick challenge. Our study raises important questions regarding prolonged survival of B. burgdorferi infected host-seeking nymphal I. scapularis ticks that can potentially increase the risk of Lyme disease incidence, if conditions of temperature and humidity become amenable to the enzootic cycle of B. burgdorferi in regions currently classified as nonendemic.

Necroptosis Contributes to Persistent Inflammation During Acute Leptospirosis.

Leptospirosis is an emerging infectious disease. Recently, canine and human leptospirosis outbreaks were reported in California and New York, respectively. In this study we evaluated the role that cell death processes play in the inflammatory response to Leptospira. Groups of male C3H/HeJ mice were infected with pathogenic L. interrogans and non-pathogenic L. biflexa for 24 and 72 hours; inflammatory processes were characterized for apoptosis and necroptosis by flowcytometry of spleen cells and were further assessed for expression of biomarkers of necroptosis by western blot. We found that pathogenic L. interrogans promotes apoptosis in myeloid neutrophils and monocytes at 24h and 72h post-infection, whereas L. biflexa promotes apoptosis of myeloid monocytes only at 24h post-infection. It is interesting that the immune cells undergoing the common programmed cell death pathway (apoptosis) are the cell types which were not increased in frequency in spleen of mice infected with L. interrogans (neutrophils) and L. biflexa (monocytes) in our previous study. The same trend was observed with pathogenic L. interrogans inducing necroptosis of myeloid neutrophils in addition to monocytes and macrophages at 24h and/or 72h post-infection, whereas L. biflexa promoted this pro-inflammatory cell death process in monocytes and macrophages only at 24h post-infection. Thus, early apoptosis and necroptosis of these cell types may explain its absence in frequency in spleen. Furthermore, at 24h and 72h, expression of the necroptosis molecular biomarkers p-MLKL, p-RIP1 and p-RIP3 was increased post infection with pathogenic L. interrogans. These data suggest that the underlying cell death processes involved in immune responses to pathogenic Leptospira contribute directly to persistent inflammation during the early stages of leptospirosis.

Transient Presence of Live Leptospira interrogans in Murine Testes.

Analysis of Leptospira dissemination and colonization of sex organs in rodents is of significant value as it queries the possibility of mammal-to-mammal venereal transmission. The aim of our study was to evaluate the presence and viability of Leptospira interrogans in testes of mice using models of infection that we previously developed. Using sublethal and lethal doses of bioluminescent strains of L. interrogans serovars Manilae and Copenhageni, we visualized the presence of leptospires in testes of C57BL/6 mice as early as 30 min and up to days 3-4 postinfection. This was confirmed by qPCR for the Copenhageni serovar after lethal infection of C3H/HeJ mice. In this model, no histopathological changes were noticed in testis. We further studied persistence of serovar Copenhageni in C3H/HeJ testes after lethal and sublethal infection, with different doses of leptospires. No viable leptospires were recovered from testes of lethally infected mice. However, we found live culturable Leptospira in testes of 19/19 (100%) sublethally infected mice at the acute phase but not at 15 days postinfection, which corresponds to the chronic phase of renal colonization. The data suggest that colonization of testes with live and potentially infectious leptospires is transient and limited to the spirochetemic phase of infection. Further studies are necessary to evaluate if presence of Leptospira in testes of mice leads to excretion in semen and to venereal transmission to female mice. IMPORTANCE Analysis of venereal transmission of Leptospira is important to determine if direct animal to animal transmission occurs, which could impact measures to prevent and treat leptospirosis. The goal of this study was to determine if live Leptospira colonize mouse testes. We found that colonization of mouse testes with live Leptospira was transient and limited to the acute spirochetemic phase of infection and that transient colonization of the testes was insufficient to cause histopathological changes.

Enzyme immunoassays (EIA) for serodiagnosis of human leptospirosis: specific IgG3/IgG1 isotyping may further inform diagnosis of acute disease.

The laborious microscopic agglutination test (MAT) is the gold standard serologic test for laboratory diagnosis of leptospirosis. We developed EIA based serologic assays using recombinant proteins (rLigA, rLigB, rLipL32) and whole-cell extracts from eight Leptospira serovars as antigen and assessed the diagnostic performance of the new assay within each class, against MAT positive (MAT+) human sera panels from Portugal/PT (n = 143) and Angola/AO (n = 100). We found that a combination of recombinant proteins rLigA, rLigB and rLipL32 correctly identified antigen-specific IgG from patients with clinical and laboratory confirmed leptospirosis (MAT+) with 92% sensitivity and ~ 97% specificity (AUC 0.974) in serum from the provinces of Luanda (LDA) and Huambo (HBO) in Angola. A combination of whole cell extracts of L. interrogans sv Copenhageni (LiC), L. kirschneri Mozdok (LkM), L. borgpetersenii Arborea (LbA) and L. biflexa Patoc (LbP) accurately identified patients with clinical and laboratory confirmed leptospirosis (MAT+) with 100% sensitivity and ~ 98% specificity for all provinces of Angola and Portugal (AUC: 0.997 for AO/LDA/HBO, 1.000 for AO/HLA, 0.999 for PT/AZ and 1.000 for PT/LIS). Interestingly, we found that MAT+ IgG+ serum from Angola had a significantly higher presence of IgD and that IgG3/IgG1 isotypes were significantly increased in the MAT+ IgG+ serum from Portugal. Given that IgM/IgD class and IgG3/IgG1 specific isotypes are produced in the earliest course of infection, immunoglobulin G isotyping may be used to inform diagnosis of acute leptospirosis. The speed, ease of use and accuracy of EIA tests make them excellent alternatives to the laborious and expensive MAT for screening acute infection in areas where circulating serovars of pathogenic Leptospira are well defined.

The year that shaped the outcome of the OspA vaccine for human Lyme disease.

The expansion of Lyme borreliosis endemic areas and the corresponding increase of disease incidence have opened the possibility for greater acceptance of a vaccine. In this perspective article, we discuss the discovery of outer surface protein A (OspA) of B. burgdorferi, and the subsequent pre-clinical testing and clinical trials of a recombinant OspA vaccine for human Lyme disease. We also discuss in detail the open public hearings of the FDA Lyme disease vaccine advisory panel held in 1998 where concerns of molecular mimicry induced autoimmunity to native OspA were raised, the limitations of those studies, and the current modifications of recombinant OspA to develop a multivalent subunit vaccine for Lyme disease.

Maximum antigen diversification in a lyme bacterial population and evolutionary strategies to overcome pathogen diversity.

Natural populations of pathogens and their hosts are engaged in an arms race in which the pathogens diversify to escape host immunity while the hosts evolve novel immunity. This co-evolutionary process poses a fundamental challenge to the development of broadly effective vaccines and diagnostics against a diversifying pathogen. Based on surveys of natural allele frequencies and experimental immunization of mice, we show high antigenic specificities of natural variants of the outer surface protein C (OspC), a dominant antigen of a Lyme Disease-causing bacterium (Borrelia burgdorferi). To overcome the challenge of OspC antigenic diversity to clinical development of preventive measures, we implemented a number of evolution-informed strategies to broaden OspC antigenic reactivity. In particular, the centroid algorithm-a genetic algorithm to generate sequences that minimize amino-acid differences with natural variants-generated synthetic OspC analogs with the greatest promise as diagnostic and vaccine candidates against diverse Lyme pathogen strains co-existing in the Northeast United States. Mechanistically, we propose a model of maximum antigen diversification (MAD) mediated by amino-acid variations distributed across the hypervariable regions on the OspC molecule. Under the MAD hypothesis, evolutionary centroids display broad cross-reactivity by occupying the central void in the antigenic space excavated by diversifying natural variants. In contrast to vaccine designs based on concatenated epitopes, the evolutionary algorithms generate analogs of natural antigens and are automated. The novel centroid algorithm and the evolutionary antigen designs based on consensus and ancestral sequences have broad implications for combating diversifying pathogens driven by pathogen-host co-evolution.

Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) Infection Prevalence and Host Associations of Ticks Found on Peromyscus spp. in Maryland.

Lyme disease, caused by Borrelia burgdorferi sensu stricto and most commonly transmitted by Ixodes scapularis Say (Ixodida: Ixodidae), is the most common tick-borne disease in Maryland. Because B. burgdorferi s.s. is maintained in enzootic cycles among wild mice (Peromyscus spp) and Ixodes spp ticks, differing patterns of parasitism of ticks on mice could impact the infection prevalence with B. burgdorferi. We determined the infection prevalence of Peromyscus spp as well as questing and partially engorged nymphal ticks collected at six sites on private land in five counties in Maryland from May to August 2020. Questing nymph infection prevalence (NIP) was 14%. We trapped 1258 mice and collected 554 ticks and 413 ear tissue samples. The prevalence of infested Peromyscus spp varied based on host age and sex, with older and male mice more likely to be infested. We detected a significant difference amongst the proportion of attached Ixodes and the location of trapping. Similarly, the prevalence of B. burgdorferi infected Peromyscus spp mice varied between locations (average mouse infection prevalence was 40%), with the highest prevalence in locations where Ixodes were the most commonly found ticks. The B. burgdorferi infection prevalence in partially engorged I. scapularis nymphs retrieved from Peromyscus spp was ~36% which lends further support to the host infection prevalence. Local differences in distribution of infected vectors and reservoirs are important factors to consider when planning interventions to reduce Lyme disease risk.

Fatal Multisystem Inflammatory Syndrome in Adult after SARS-CoV-2 Natural Infection and COVID-19 Vaccination.

We describe a fatal case of multisystem inflammatory syndrome in an adult with onset 22 days after a second dose of mRNA coronavirus disease vaccine. Serologic and clinical findings indicated severe acute respiratory syndrome coronavirus 2 infection occurred before vaccination. The immunopathology of this syndrome, regardless of vaccination status, remains poorly understood.

Inflammatory Signatures of Pathogenic and Non-Pathogenic Leptospira Infection in Susceptible C3H-HeJ Mice.

The exact global impact of leptospirosis is unknown due to inadequate surveillance systems in place in most low-income countries. In this study, we analyzed the differences in mouse inflammatory signatures involved in pathogenic versus non-pathogenic Leptospira recognition at 24h and 72h post infection. Injection of C3H-HeJ mice with non-pathogenic L. biflexa increased circulation of a few chemokines (5/21, 24%) without secretion of cytokines in blood that resulted in engagement of resident macrophages, dendritic cells, neutrophils and NK cells without engagement of T cells. In contrast, pathogenic L. interrogans induced circulation of a much higher panel of chemokines (18/21, 86%) and pro- and anti-inflammatory cytokines (11/19, 58%) in blood with a resulting signaling cascade leading to engagement of macrophages, dendritic cells, monocytes, NK cells and T cells without engagement of neutrophils. Although neutrophils do not appear to be engaged, a considerable number of chemokines that recruit other granulocytes such as eosinophils and basophils were also increased at 72h post infection with L. interrogans. Overall, the data suggest that prevention of dissemination of L. biflexa is associated with an early engagement of the innate immune response characterized by upregulation of a few chemokines that results in an efficacious phagocytic response without an overwhelming increase of pro-inflammatory cytokines. However, when macrophages fail to clear a pathogenic serovar such as L. interrogans, the adaptive response (T cells) is engaged to help out, but the resulting chemo-cytokine storm mediates a robust but non-resolving inflammatory response to pathogenic Leptospira that results in dissemination, kidney colonization, pathology and disease.

Maternal transfer of neutralizing antibodies to B. burgdorferi OspA after oral vaccination of the rodent reservoir.

Lyme Disease presents unique challenges for public health. Transfer of protective antibodies between mothers and offspring should occur after vaccination of mice. We present new evidence for maternal transfer of oral vaccine induced neutralizing anti-OspA IgG antibodies to mouse pups mainly through ingestion of colostrum. We found a strong statistical correlation of antibody transfer between mothers that produced the most robust IgG response to OspA and their respective pups. OspA-specific antibody was detected as early as 24 h after birth and protective levels of antibodies lasted until ~5 weeks of age in the majority of pups but persisted in some mice until 9 weeks. This was further supported by detection of neutralizing antibodies in serum of all pups at 2-3 weeks after birth and in some offspring adult mice at 9 weeks of age. A clear association was found between robust antibody responses in mothers and the length of time antibody persisted in the respective pups using a novel longitudinal Bayesian model. These factors are likely to impact the enzootic cycle of B. burgdorferi if reservoir targeted OspA-based vaccination interventions are implemented.

A Mouse Model of Sublethal Leptospirosis: Protocols for Infection with Leptospira Through Natural Transmission Routes, for Monitoring Clinical and Molecular Scores of Disease, and for Evaluation of the Host Immune Response.

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira species that are maintained in sylvatic and domestic environments by transmission among rodents and other carriers. Humans become infected after contact of breached skin or mucosa with contaminated water or soil. Understanding persistent or sublethal infection in a host is critical for controlling human risk of exposure to pathogenic Leptospira. Animal models that recapitulate disease progression after infection via natural transmission routes are more appropriate for validation of vaccines and therapeutics. Furthermore, the ability to measure shedding of live Leptospira in urine of reservoir and carrier hosts can be used to develop new diagnostic assays and sensors to evaluate human risk of exposure. We developed inbred mouse models of Leptospirosis, that bypass survival as a criterion, in which we can analyze both pathogen and host factors affecting sublethal infection (<1 month), including shedding of Leptospira in urine. Mice are infected with pathogenic Leptospira using a physiologic route, and the clinical, histological, and molecular scores of disease are measured. Furthermore, the host immune response to Leptospira is evaluated. This mouse model also provides a tool in which to test fundamental hypotheses related to host-pathogen interactions and the immune mechanisms engaged in protective and pathogenic immune responses. © 2020 Wiley Periodicals LLC Basic Protocol 1: Culture and maintenance of virulent Leptospira Basic Protocol 2: Infection of mice through a physiologic route and collection of clinical scores and biological samples Basic Protocol 3: Analysis of pathogenesis after Leptospira infection.

The route of infection with Leptospira interrogans serovar Copenhageni affects the kinetics of bacterial dissemination and kidney colonization.

The goal of this study was to characterize how natural routes of infection affect the kinetics of pathogenic Leptospira dissemination to blood and kidney. C3H/HeJ mice were sublethally infected with L. interrogans serovar Copenhageni FioCruz L1-130 (Leptospira) through exposure of a dermis wound and through oral and nasal mucosa, in comparison to uninfected mice and to mice infected via standard intraperitoneal inoculation. In striking contrast to oral mucosa inoculation, transdermal and nasal mucosa infections led to weight loss, renal colonization and inflammation, as previously observed for conjunctival and intraperitoneal infections. However, the timing at which Leptospira gained access to blood, as well as Leptospira' colonization of the kidney and shedding in urine, differed from intraperitoneal infection. Furthermore, a comparative analysis of transcription of pro-inflammatory mediators in kidney and total immunoglobulin isotyping in serum from infected mice, showed increased innate immune response markers (KC, MIP-2, TNF-α) and lower Th1 associated IFN-γ in kidney, as well as lower Th1 associated IgG2a in mice infected through the nasal mucosa as compared to intraperitoneal infection. We conclude that the route of infection affects the timing at which Leptospira gains access to blood for dissemination, as well as the dynamics of colonization and inflammation of the kidney.

Role of TLR4 in Persistent Leptospira interrogans Infection: A Comparative In Vivo Study in Mice.

Toll-Like Receptor (TLR) 4, the LPS receptor, plays a central role in the control of leptospirosis and absence of TLR4 results in lethal infection in mice. Because human TLR4 does not sense the atypical leptospiral-LPS, we hypothesized that TLR4/MD-2 humanized transgenic mice (huTLR4) may be more susceptible to leptospirosis than wild-type mice, and thus may constitute a model of acute human leptospirosis. We infected huTLR4 mice, which express human TLR4 but not murine TLR4, with a high dose of L. interrogans serovar Copenhageni FioCruz (Leptospira) in comparison to C57BL/6J wild-type (WT) and, as a control, a congenic strain in which the tlr4 coding sequences are deleted (muTLR4Lps-del). We show that the huTLR4 gene is fully functional in the murine background. We found that dissemination of Leptospira in blood, shedding in urine, colonization of the kidney and overall kinetics of leptospirosis progression is equivalent between WT and huTLR4 C57BL/6J mice. Furthermore, inflammation of the kidney appeared to be subdued in huTLR4 compared to WT mice in that we observed less infiltrates of mononuclear lymphocytes, less innate immune markers and no relevant differences in fibrosis markers. Thus, huTLR4 mice showed less inflammation and kidney pathology, and are not more susceptible to leptospirosis than WT mice. This study is significant as it indicates that one intact TLR4 gene, be it mouse or human, is necessary to control acute leptospirosis.

Protective Immunity and New Vaccines for Lyme Disease.

Lyme disease, caused by some Borrelia burgdorferi sensu lato, is the most common tick-borne illness in the Northern Hemisphere and the number of cases, and geographic spread, continue to grow. Previously identified B. burgdorferi proteins, lipid immunogens, and live mutants lead the design of canonical vaccines aimed at disrupting infection in the host. Discovery of the mechanism of action of the first vaccine catalyzed the development of new strategies to control Lyme disease that bypassed direct vaccination of the human host. Thus, novel prevention concepts center on proteins produced by B. burgdorferi during tick transit and on tick proteins that mediate feeding and pathogen transmission. A burgeoning area of research is tick immunity as it can unlock mechanistic pathways that could be targeted for disruption. Studies that shed light on the mammalian immune pathways engaged during tick-transmitted B. burgdorferi infection would further development of vaccination strategies against Lyme disease.

A Multiplexed Serologic Test for Diagnosis of Lyme Disease for Point-of-Care Use.

Single multiplexed assays could replace the standard 2-tiered (STT) algorithm recommended for the laboratory diagnosis of Lyme disease if they perform with a specificity and a sensitivity superior or equal to those of the STT algorithm. We used human serum rigorously characterized to be sera from patients with acute- and convalescent-phase early Lyme disease, Lyme arthritis, and posttreatment Lyme disease syndrome, as well as the necessary controls (n = 241 samples), to select the best of 12 Borrelia burgdorferi proteins to improve our microfluidic assay (mChip-Ld). We then evaluated its serodiagnostic performance in comparison to that of a first-tier enzyme immunoassay and the STT algorithm. We observed that more antigens became positive as Lyme disease progressed from early to late stages. We selected three antigens (3Ag) to include in the mChip-Ld: VlsE and a proprietary synthetic 33-mer peptide (PepVF) to capture sensitivity in all disease stages and OspC for early Lyme disease. With the specificity set at 95%, the sensitivity of the mChip-Ld with 3Ag ranged from 80% (95% confidence interval [CI], 56% to 94%) and 85% (95% CI, 74% to 96%) for two panels of serum from patients with early Lyme disease and was 100% (95% CI, 83% to 100%) for serum from patients with Lyme arthritis; the STT algorithm detected early Lyme disease in the same two panels of serum from patients with early Lyme disease with a sensitivity of 48.5% and 75% and Lyme arthritis in serum from patients with Lyme arthritis with a sensitivity of 100%, and the specificity was 97.5% to 100%. The mChip-Ld platform outperformed the STT algorithm according to sensitivity. These results open the door for the development of a single, rapid, multiplexed diagnostic test for point-of-care use that can be designed to identify the Lyme disease stage.