Introduction Leishmania are obligate-intracellular protozoan parasites that establish infection in mammalian hosts following transmission to the skin by the bite of an infected Phlebotomine sand fly [1]. Different Leishmania species are associated with a spectrum of clinical outcomes in humans, including fatal, disseminated infection of the spleen and liver following infection with L. donovani, and self-curing cutaneous lesions associated with L. major and other cutaneous strains. Healed cutaneous lesions often result in a permanent scar that has been shown to harbor low numbers of parasites over the long term [2]. While this chronic, sub-clinical state can serve as a long-term reservoir for disease, it also maintains powerful protective immunity for the host, as individuals with healed primary lesions are highly resistant to re-infection, and complete elimination of a primary infection in animal models results in susceptibility to reinfection [3],[4]. Deliberate needle inoculation with viable parasites in a selected site, referred to as “leishmanization,” has been employed extensively as a live “vaccine” in people for generations, and is highly effective against natural exposure [5],[6],[7],[8]. However, due to reports of adverse reactions at the site of inoculation, quality control issues, and concerns over causing serious disease in immuno-compromised individuals, leishmanization has fallen out of favor [8],[9]. Employing the mouse model of L. major infection, numerous non-living [10],[11],[12],[13],[14],[15] and live-attenuated [13],[16],[17], or DNA-based [10],[18] vaccine formulations have been developed as alternatives to leishmanization, which in many cases have conferred relatively long-term protection against experimental needle challenge [10],[11],[12],[18]. In contrast, non-living vaccines, including formulations similar to those shown to work effectively in mice against needle challenge [11],[13], have yet to confer significant protection against natural exposure in people, despite the generation of measurable cell-mediated immunity [9],[19],[20],[21],[22],[23],[24],[25],[26],[27],[28]. This contradiction between the results in humans and animal trials suggests that the correlates of vaccine efficacy developed mainly from the mouse model, namely the generation of Th1 responses and the reduction of lesion size and/or parasite number following needle challenge, may not adequately define the requirements for protection against natural transmission. Observations by Rogers et al. [29], in which vaccination with soluble leishmanial antigen plus IL-12 delayed the onset of progressive lesions following needle, but not infected sand fly challenge in BALB/c mice, support this suggestion. In addition to the delivery of infectious stage parasites into the dermis, sand flies also deposit pharmacologically active saliva, which aids in blood feeding, and egest parasite-released glycoconjugates, which accumulate behind the mouthparts in infected flies and form a promastigote secretory gel (PSG). These molecules have been shown to enhance the severity of disease when co-administered with infectious stage parasites [30],[31],[32],[33]. We have recently reported that sand fly transmission induces a qualitatively unique inflammatory response at the localized bite site that includes a dynamic recruitment of neutrophils, and that these neutrophils markedly enhance the ability of parasites to establish primary infection [34]. Thus, an analysis of the influence of sand fly transmission on vaccine efficacy is likely to be highly relevant to the generation of a Leishmania vaccine that is effective in people. Results Healed primary infection protects against infected sand fly challenge Healed primary L. major infection initiated by needle inoculation of mice has been extensively employed as a model that mimics the clinical practice of leishmanization. Mice with resolved primary lesions harbor L. major specific CD4 T cells that simultaneously produce IFN-γ, TNF-α, and IL-2 effector cytokines and mount powerful protective immunity at a site of needle re-challenge, resulting in the rapid control of parasite growth [13],[35]. In order to characterize the protective immune response following natural transmission, 4 P. duboscqi sand flies, infected with L. major (L.m.-SF), were allowed to feed on the ears of C57BL/6 mice with a healed primary lesion in the footpad. Under these conditions, a median of 2 flies will show evidence of blood engorgement, thereby ensuring parasite transmission to a sufficient number of ears to conduct the experiment, while at the same time more faithfully replicating natural transmission, which likely occurs following exposure to a single infected fly. At 1 and 3 days following exposure to the infected flies, a slight but significant increase in infiltrating CD4 T cells was found in the ears of healed mice relative to fly challenged, naïve, age-matched controls (AMC) ( Figure 1A ). At 7 days post-challenge, the number of infiltrating CD4 cells in the healed mice was dramatically increased relative to controls. In order to determine if parasite antigen was required to mediate this recruitment, healed mice were also exposed to uninfected sand fly bites (SF). Both infected or uninfected bites recruited equivalent numbers of T cells at day 3 post-bite, however, parasite antigen appeared necessary for the dramatic increase observed on day 7 ( Figure 1A ). Remarkably, Ag re-stimulation of dermal derived cells revealed Leishmania-specific IFN-γ producing CD4+ T cells at the challenge site within 24 hours, a response that gradually increased to 17% of the total CD4 T cell population at 7 days ( Figure 1B ), correlating with a >100 fold reduction in parasite numbers in the skin ( Figure 1C ). Antigen re-stimulation of T cells from the ears of healed mice exposed to uninfected sand fly bites also revealed the presence of L.m.-specific IFN-γ producing CD4+ T cells ( Figure 1B ), suggesting that a functional property of these effector cells is their ability to rapidly migrate to sites of tissue inflammation whether antigen is present or not. 10.1371/journal.ppat.1000484.g001 Figure 1 Mice with healed primary infections mount robust immunity and control parasite growth following transmission of L. major by infected sand fly bite. Ears of naïve, age matched control mice (AMC), or healed mice infected by needle inoculation s.c. in the footpad with 104 L.m. metacyclic promastigotes 22 weeks previously, were exposed to the bites of 4 uninfected (SF) or L.m.-infected P. duboscqi sand flies (L.m.-SF). Ear derived cells were analyzed at the indicated times following exposure to sand flies. (A and B) Total number of TcRβ+CD4+ T cells per ear as determined by flow cytometric analysis of duplicate samples of pooled ears (A); or frequency (total number per ear in brackets) of IFN-γ+/TcRβ+CD4+ T cells following in-vitro re-stimulation of pooled ears with BMDC plus L. major-antigen (DC+Ag) (B). (*) or (‡) in 1A indicates a significant difference (0.021
