Molecular approaches to understand vector-host and vector-pathogen interactions

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

The accomplishment of the section are: 1. We developed a novel biomarker for sand fly exposure in humans using two recombinant salivary proteins from the sand fly Phlebotomus argentipes. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the Indian subcontinent, as immunodominant proteins in humans. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The receiver operating characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts after visceral leishmaniasis elimination. 2. We demonstrated that Leishmania major-Infected sand fly bites enhance mast cell degranulation. Leishmania parasites infect mammalian hosts through the bites of sand fly vectors. The response by mast cells (MC) to the parasite and vector-derived factors, delivered by sand fly bites, has not been characterized. MC were found at the bite sites of infective and non-infected sand flies throughout 48 h, showing the release of granules with intense TNF-, histamine, and tryptase staining. At 30 min and 48 h, the MC numbers were significantly higher (p < 0.001) in infected as compared to non-infected bites or controls. Our data show that MC orchestrate an early inflammatory response after infected and non-infected sand fly bites, leading to neutrophilic recruitment, which potentially provides a safe passage for the parasite within the mammalian host. 3. We demonstrate that the sand fly salivary protein PpSP32 exerts immunomodulatory effects on human monocytes, macrophages, and lymphocytes. We aimed to study the role of PpSP32 by screening of several immunomodulatory activities either on lymphocytes or on monocytes/macrophages. Our data showed that PpSP32 down-modulated the expression of activation markers in LPS-stimulated monocytes and THP1-derived macrophages. This protein negatively modulated the secretion of Th1 and Th2 cytokines by human lymphocytes as well as pro-inflammatory cytokines by monocytes, and THP1-derived macrophages. PpSP32 treatment led to a dose-dependent reduction of IB phosphorylation. Our data indicates that PpSP32 induces a potent immunomodulatory effect on monocytes and THP-1-derived macrophages. This inhibition could be mediated, among others, by the modulation of the NF-kB signaling pathway. 4. We evaluated the human skin immune response to mosquito bites from subjects living in a disease endemic area. Initial viral inoculation occurs in the skin via the mosquito 'bite', eliciting immune responses that shape the establishment of infection and pathogenesis. Here we assess the cutaneous innate and adaptive immune responses to controlled Aedes aegypti feedings in humans living in Aedes-endemic areas. In this single-arm, cross-sectional interventional study (trial registration #NCT04350905), we enroll 30 healthy adult participants aged 18 to 45 years of age from Cambodia between October 2020 and January 2021. We perform 3-mm skin biopsies at baseline as well as 30 min, 4 h, and 48 h after a controlled feeding by uninfected Aedes aegypti mosquitos. The primary endpoints are measurement of changes in early and late innate responses in bitten vs unbitten skin by gene expression profiling, immunophenotyping, and cytokine profiling. The results reveal induction of neutrophil degranulation and recruitment of skin-resident dendritic cells and M2 macrophages. As the immune reaction progresses T cell priming and regulatory pathways are upregulated along with a shift to Th2-driven responses and CD8+ T cell activation. These results identify key immune genes, cell types, and pathways in the human response to mosquito bites and can be leveraged to inform and develop novel therapeutics and vector-targeted vaccine candidates to interfere with vector-mediated disease. 5. We performed a Safety and immunogenicity of AGS-v PLUS, a mosquito saliva peptide vaccine against arboviral diseases: A randomized, double-blind, placebo-controlled Phase 1 trial. Immunity to mosquito salivary proteins could provide protection against multiple mosquito-borne diseases and significantly impact public health. We evaluated the safety and immunogenicity of AGS-v PLUS, a mosquito salivary peptide vaccine, in healthy adults 18-50 years old. Primary endpoints were safety and antibody and cytokine responses. Participants experienced no treatment-emergent or serious adverse events. Only injection site pain was more common in vaccine groups (15/51 after dose 1 and 11/51 after dose 2) versus placebo. Compared to placebo, all vaccine groups had significantly greater fold change in anti-AGS-v PLUS IgG and IFN- from baseline. AGS-v PLUS had favorable safety profile and induced robust immune responses. Next steps will determine if findings translate into clinical efficacy against mosquito-borne diseases. 6. We demonstrated that Centrin-deficient Leishmania mexicana confers protection against Old World visceral leishmaniasis. In this study, we tested whether LmexCen-/- parasites can protect against visceral leishmaniasis caused by L. donovani. We showed that immunization with LmexCen-/- parasites is safe and does not cause lesions. Furthermore, such immunization conferred protection against visceral leishmaniasis caused by a needle-initiated L. donovani challenge, as indicated by a significant reduction in the parasite burdens in the spleen and liver as well as reduced mortality. Similar control of parasite burden was also observed against a sand fly mediated L. donovani challenge. Importantly, immunization with LmexCen-/- down-regulated the disease promoting cytokines IL-10 and IL-4 and increased pro-inflammatory cytokine IFN- resulting in higher IFN-/IL-10 and IFN-/IL4 ratios compared to non-immunized groups. LmexCen-/- immunization also resulted in long-lasting protection against L. donovani infection. Taken together, our study demonstrates that immunization with LmexCen-/- parasites is safe and efficacious against the Old World visceral leishmaniasis. 7. We identified and characterized a novel bispecific inhibitor of complement and coagulation that blocks activation in complementopathy models. Surface plasmon resonance experiments show that lufaxin stabilizes the binding of serine protease factor B (FB) to C3b but does not detectably bind either C3b or FB alone. The crystal structure of the inhibitor reveals a novel all -sheet fold containing 2 domains. A structure of the lufaxin-C3bB complex obtained via cryo-electron microscopy (EM) shows that lufaxin binds via its N-terminal domain at an interface containing elements of both C3b and FB. By occupying this spot, the inhibitor locks FB into a closed conformation in which proteolytic activation of FB by FD cannot occur. C3bB-bound lufaxin binds fXa at a separate site in its C-terminal domain. In the cryo-EM structure of a C3bB-lufaxin-fXa complex, the inhibitor binds to both targets simultaneously, and lufaxin inhibits fXa through substrate-like binding of a C-terminal peptide at the active site as well as other interactions in this region. Lufaxin inhibits complement activation in ex vivo models of atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH) as well as thrombin generation in plasma, providing a rationale for the development of a bispecific inhibitor to treat complement-related diseases in which thrombosis is a prominent manifestation.