IMMUNE REGULATION AND VACCINE DEVELOPMENT IN LEISHMANIASIS

已经开发了许多实验性疫苗，在皮肤或内脏形式利什曼病的动物模型中显示出希望，但仍然不存在有效的人类疫苗。在一系列III期临床试验中，在野外暴露于受感染的沙蝇叮咬的个体中测试了利什曼原虫灭活疫苗，但没有观察到任何保护作用。值得注意的是，在动物模型中测试的候选疫苗都没有在实验条件下使用受感染的沙蝇进行评估，我们认为临床试验中的疫苗失败可能反映了天然沙蝇挑战的更严格条件。我们比较了针刺和感染沙蝇攻击，以评估目前临床试验中唯一确定的候选疫苗的有效性，该疫苗由多蛋白（KSAC或L110f）组成，含有利什曼原虫的多个抗原表位，用稳定的乳剂（SE）（油包水）和glucopyranosyl脂质a （GLA）递送，GLA是一种适合于人类使用的TLR4激动剂。在注射L. major后2周，接种多蛋白疫苗的小鼠CD4+ IFNg+ T细胞数量比对照动物增加60倍，这与寄生虫负荷减少100倍相关。相比之下，在受感染的沙蝇叮咬攻击后，接种多蛋白疫苗的动物与未接种疫苗的对照组相比，寄生虫载量相当，攻击部位的中性粒细胞数量更多，CD4+IFNg+/IL-17+比率降低。重要的是，原发感染愈合的小鼠对受感染的沙蝇攻击有坚实的保护，这与效应细胞在攻击部位出现的速度有关，提供了可能需要的效应细胞因子的立即爆发，以抵消沙蝇叮咬引起的高度局部，中性粒细胞主导的反应所产生的下调环境。我们使用一种T-bet调节的、gpi锚定的表面糖蛋白Ly6C，表达在T-bethi CD4+ T细胞上，对快速募集的细胞进行表型分析，发现预先存在的、短期的CD44+CD62L- T-bet+Ly6C+效应（TEFF）细胞，而不是记忆或记忆来源的细胞，介导愈合小鼠的伴随免疫。在过继性转移和挑战后，非分裂Ly6C+ TEFF细胞优先归巢到皮肤，释放IFNg并给予保护。尽管Ly6C+ TEFF细胞寿命很短，但在原发感染愈合的小鼠中，Ly6C+ TEFF细胞的频率很高，这可能是这些小鼠持续感染的结果。缺乏有效的利什曼病疫苗可能是因为对这些感染的保护需要预先存在的TEFF细胞介导的伴随免疫，而不是记忆细胞，因此不适合传统的、记忆诱导的疫苗接种策略。

Many experimental vaccines have been developed that show promise in animal models of cutaneous or visceral forms of leishmaniasis, but an effective human vaccine still does not exist. In a series of phase III clinical trials in which killed Leishmania vaccines were tested in individuals exposed to infected sand fly bites in the field, no protection was observed. Remarkably, none of the candidate vaccines tested in animal models have been evaluated under experimental conditions using infected sand flies, and we addressed the possibility that vaccine failure in clinical trials may reflect the more stringent conditions of natural sand fly challenge. We compared needle and infected sand fly challenge to evaluate the efficacy of the only defined candidate vaccine currently in clinical trial, comprised of polyproteins (either KSAC or L110f), containing multiple antigenic epitopes from Leishmania delivered in a stable emulsion (SE) (water in oil) with glucopyranosyl lipid A (GLA), a TLR4 agonist suitable for use in people. Polyprotein-vaccinated mice had a 60-fold increase in CD4+ IFNg+ T cell numbers versus control animals at 2 wk postneedle inoculation of L. major, and this correlated with a 100-fold reduction in parasite load. By contrast, following challenge by infected sand fly bite, polyprotein-vaccinated animals had comparable parasite loads, greater numbers of neutrophils at the challenge site, and reduced CD4+IFNg+/IL-17+ ratios versus non-vaccinated controls. Importantly, mice with a healed primary infection were solidly protected against infected sand fly challenge that was associated with the speed with which effector cells appeared at a site of challenge, providing an immediate burst of effector cytokines that may be required to counteract the down-modulatory environment created by the highly localized, neutrophil-dominated, response to sand fly bite. We employed Ly6C, a T-bet regulated, GPI-anchored, surface glycoprotein expressed on T-bethi CD4+ T cells, to phenotype the rapidly recruited cells, and found that pre-existing, short-lived, CD44+CD62L- T-bet+Ly6C+ effector (TEFF) cells, not memory or memory-derived cells, mediate concomitant immunity in healed mice. Upon adoptive transfer and challenge, non-dividing Ly6C+ TEFF cells preferentially homed to the skin, released IFNg and conferred protection. Despite being short-lived, Ly6C+ TEFF cells were maintained at high frequencies in the mice with healed primary infection, presumably as a consequence of the persistent infection in these mice. The lack of effective vaccines against Leishmaniasis may be because protection against these infections requires concomitant immunity mediated by pre-existing TEFF cells, not memory cells, and is therefore not amenable to conventional, memory inducing, vaccination strategies. As indicated above, primary L. major infection typically produces cutaneous lesions that heal but that harbor persistent parasites. While the opposing roles of CD4+ T cell-derived IFNg and IL-10 in promoting parasite killing and persistence have been well established, how these responses develop from nave precursors has not been directly monitored throughout the course of infection. We used peptide:Major Histocompatibility Complex II (pMHCII) tetramers to investigate the endogenous, parasite-specific primary CD4+ T cell response to L. major in mice resistant to infection, and applied this approach to enumerate the expansion, contraction, tissue distribution, and function of parasite-specific CD4+ T cells throughout the course of the infection. Maximal frequencies of IFNg+ CD4+ T cells were observed in the spleen and infected ears within a month after infection and were maintained into the chronic phase. In contrast, peak frequencies of IL-10+CD4+ T cells emerged within 2 weeks of infection, persisted into the chronic phase, and accumulated in the infected ears but not the spleen, via a process that depended on local antigen presentation. T helper type-1 (Th1) cells, not Foxp3+ regulatory T cells, were the chief producers of IL-10 and were not exhausted. Therefore, tracking antigen-specific CD4+ T cells revealed that IL-10 production by Th1 cells is not due to persistent T cell antigen receptor stimulation, but rather driven early and sustained locally by antigen encounter at the site of infection. In addition to studying the mechanisms underlying parasite persistence in healed mice, mouse models are also being used to study non-healing forms of disease. Infection of C57BL/6 mice with most L. major strains results in a healing lesion with minimal pathology at the site of inoculation in the skin. By contrast, using a strain of L. major (Lm Sd) isolated from a patient with chronic cutaneous lesions, C57BL/6 mice also fail to heal their dermal lesions or effectively control tissue parasite burden despite a strong and polarized Th1 response. In studies designed to identify the earliest cells and mediators that precede and promote the severe pathology, we detected elevated levels of IL-1b mRNA and IL-1b+ cells in the inoculation site 3-4 weeks post-infection, followed by a neutrophil infiltrate that persisted until the onset of the pathology. Whereas no phenotype was observed in IL-17 deficient mice, IL-1R deficient mice, as well as IL-1b, ASC, and caspase-1/11 deficient mice, each showed minimal pathology and healed their Lm Sd infection. These studies are unique in the innate immunity field in identifying inflammasome dependent IL-1b as preventing rather than promoting host defense against a microbial pathogen. The mechanisms underlying the failure to control the growth and systemic spread of Leishmania parasites in human visceral leishmaniasis (VL) are not well understood. A key immunological feature of VL is the inability of peripheral blood mononuclear cells (PBMCs) to proliferate or to produce IFNg in response to leishmanial antigens. IL-10 has been implicated in the suppression of antigen-specific T cell responses in human VL based on the elevated levels of IL-10 observed in plasma and lesional tissue, and its role in preventing clearance of L. donovani in murine models of VL. In unexpected findings from our recent studies we were able to show that in sharp contrast to assays employing PBMCs, a cytokine release assay involving L. donovani antigen-stimulated whole-blood cells was able to detect the secretion of IFNg by cells from the majority of patients with active VL. In 35 patients with active VL, 80% secreted high levels of IFNg as compared to 85% of cured VL patients, and 24% of EHCs with presumed subclinical infections. The findings do not support a severe Th1 response defect in kala-azar. Importantly, only the whole blood cells from patients with active VL also secreted IL-10, which better reflects the response that distinguishes individuals with active disease from cured or subclinically infected, immune individuals. More generally, the findings reveal that PBMCs may not accurately reflect the immune competency of peripheral blood cells.