Date Published: April 16, 2008
Publisher: Public Library of Science
Author(s): Fabiano Oliveira, Phillip G. Lawyer, Shaden Kamhawi, Jesus G. Valenzuela, Mike J. Lehane
Abstract: BackgroundLeishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects biologically active salivary components that favorably change the environment at the feeding site. Exposure to bites or to salivary proteins results in immunity specific to these components. Mice immunized with Phlebotomus papatasi salivary gland homogenate (SGH) or pre-exposed to uninfected bites were protected against Leishmania major infection delivered by needle inoculation with SGH or by infected sand fly bites. Immunization with individual salivary proteins of two sand fly species protected mice from L. major infection. Here, we analyze the immune response to distinct salivary proteins from P. papatasi that produced contrasting outcomes of L. major infection.Methodology/Principal FindingsDNA immunization with distinct DTH-inducing salivary proteins from P. papatasi modulates L. major infection. PpSP15-immunized mice (PpSP15-mice) show lasting protection while PpSP44-immunized mice (PpSP44-mice) aggravate the infection, suggesting that immunization with these distinct molecules alters the course of anti-Leishmania immunity. Two weeks post-infection, 31.5% of CD4+ T cells produced IFN-γ in PpSP15-mice compared to 7.1% in PpSP44-mice. Moreover, IL-4-producing cells were 3-fold higher in PpSP44-mice. At an earlier time point of two hours after challenge with SGH and L. major, the expression profile of PpSP15-mice showed over 3-fold higher IFN-γ and IL-12-Rβ2 and 20-fold lower IL-4 expression relative to PpSP44-mice, suggesting that salivary proteins differentially prime anti-Leishmania immunity. This immune response is inducible by sand fly bites where PpSP15-mice showed a 3-fold higher IFN-γ and a 5-fold lower IL-4 expression compared with PpSP44-mice.Conclusions/SignificanceImmunization with two salivary proteins from P. papatasi, PpSP15 and PpSP44, produced distinct immune profiles that correlated with resistance or susceptibility to Leishmania infection. The demonstration for the first time that immunity to a defined salivary protein (PpSP44) results in disease enhancement stresses the importance of the proper selection of vector-based vaccine candidates.
Partial Text: In leishmaniasis, phlebotomine sand flies transmit Leishmania parasites to a mammalian host by depositing the parasite in the skin during probing and feeding. Together with the parasite, sand flies deposit a repertoire of salivary components that assist the sand fly in getting a blood meal . Some of these salivary proteins are immunogenic in humans, canids and mice –. Repeated exposure to sand fly salivary gland homogenate (SGH) or sand fly bites have been shown to protect mice to subsequent challenge with Leishmania major and SGH  or L. major infected sand flies .
It is established that a Th1 immune response and the production of IFN-γ are correlated with protection from L. major infection in C57BL/6 mice . Conversely, a Th2 immune response is associated with susceptibility . Earlier studies have demonstrated the potential of immunity to sand fly saliva in the control of Leishmania infection ,,,. More information is needed to define the immune profile induced by distinct salivary proteins and its specific effect on the outcome of disease. In this work, we demonstrate that DTH-inducing P. papatasi sand fly salivary molecules are not universally protective against L. major infection and that immunity to some can result in its exacerbation. Mice immunized with PpSP15 controlled the infection and had significantly lower parasite load compared to naïve mice, as previously reported . In contrast, mice immunized with PpSP44 exacerbated the infection showing lesions with severe tissue erosion and maintaining a high number of parasites up to 11 weeks post-infection. This is the first account in which an immune response to a defined sand fly salivary protein results in disease exacerbation. Protection in PpSP15-immunized mice and exacerbation in PpSP44-immunized mice were correlated with an anti-Leishmania Th1 and Th2 immune response, respectively (Fig. 4). The anti-Leishmania immune response was characterized by a considerable increase in IFN-γ producing CD4+ T cells in PpSP15-immunized mice (over four-fold higher compared to control DNA- and PpSP44-immunized mice) and over three-fold lower IL-4 producing CD4+ T cells compared to PpSP44-immunized mice (Fig. 4). At this time point a small increase in the percent of CD4+ T cells producing IL-4 in PpSP44-immunized mice was detected compared to controls. Nevertheless, there is clear exacerbation both in lesion size and tissue pathology in PpSP44-immunized mice (Fig. 2, 3). We propose that the polarization of anti-Leishmania immunity towards a Th1 or Th2 response in these mice is the result of their prior immunization with DNA encoding the respective salivary proteins. Earlier studies have hypothesized that anti-saliva immunity leads to protection from L. major by the creation of a hostile environment that kills the parasite, acceleration and priming of the anti-Leishmania immunity, or a combination of both ,. Indeed, mice protected from L. major infection through pre-exposure to sand fly bites showed an increase in the frequency of ear epidermal cells producing IFN-γ and IL-12 six hours after challenge . This rapid production of IFN-γ prompted us to investigate the expression profile of pro-inflammatory cytokines induced by PpSP15 and PpSP44 at an early time point (two hours) following challenge with SGH-LM. Macroarray results validated by real-time PCR showed that mice immunized with PpSP15 selectively induced transcripts associated with a Th1 immune response (IFN-γ and IL-12rβ2) and downregulated Th2 associated transcripts (IL-4). IL-12rβ2 is expressed on both activated Th1 CD4+ cells and NK cells –. Recently, it has been shown that NK cells could play a role in adaptive immunity  and may be the source of the early IFN-γ expression seen in PpSP15-immunized mice. Alternately, we cannot exclude the possibility that the up-regulation of IFN-γ expression is by specific CD4 memory T cells that are rapidly recruited to the site of infection. The cells that are responsible for the expression of IFN-γ at this early time point is currently under investigation. PpSP44- immunized mice that exacerbated L. major infection selectively induced IL-4 (a marker of Th2 differentiation) and did not upregulate IFN-γ showing the specificity of the observed immune responses to each of the salivary proteins. It should be noted that neither IFN-γ nor IL-4 were induced in the CTL DNA-immunized mice. Enhancement of Leishmania infection in mice pre-exposed to sand fly saliva was recently demonstrated for Lu. intermedia and L. braziliensis in a BALB/c model of infection . Mice immunized with SGH of Lu. intermedia showed a low IFN-γ to IL-4 ratio that correlated with an enhanced disease profile . It is possible that the immunodominant protein in the salivary repertoire of Lu. intermedia induces an immune response similar to that of PpSP44 resulting in the exacerbation of L. braziliensis infection in BALB/c mice. This is in contrast to the protection from L. major infection observed in BALB/c mice pre-exposed to P. papatasi bites or SGH ,. Interestingly, the molecular weight of a strongly antigenic salivary protein of Lu. intermedia is 45 kDa  corresponding to the molecular weight of PpSP44. This raises the question whether immuno-dominance of salivary proteins vary in different sand fly species. Saliva is composed of a repertoire of proteins and their overall effect is likely influenced by the sand fly species, the Leishmania species and the mammalian host resulting in an overriding exacerbative or protective immune profile. Stimulatory and suppressive immune responses to salivary molecules have been previously described in ticks . Lymphocytes from tick resistant donors proliferated in response to tick salivary gland antigens demonstrating antigen-specific stimulation. However, their non-specific PHA-induced proliferation was significantly suppressed .