A vaccine that protects mice against needle challenge of L. major
fails when challenged by sand fly–transmitted parasites (Rogers et al., 2004
; Peters et al., 2009
). This highlights the virulence of vector-initiated Leishmania
infections and the need to identify new approaches or antigens to combat this neglected disease (Rogers et al., 2004
; Bethony et al., 2011
). Here, we demonstrate that immunization with a defined salivary protein from Lu. longipalpis
, LJM11, in the absence of adjuvant modulates the host immune response to Leishmania
parasites, resulting in protection against challenge with infected sand flies.
Throughout the study, we used a recent field isolate of L. major
transmitted by vector bites, a highly virulent challenge. This highlights both the potency of the protection observed in rLJM11-immunized mice and the unusual severity of lesions in control groups. The potency of rLJM11 immunity is underscored by ulcer-free protection from CL observed in mice challenged up to 5 months following the last immunization. As a result of the lesion severity in control groups, mice were killed 5 weeks after infection. At this time point, live parasites were still observed in rLJM11-immunized mice despite the absence of ulcers. Further studies with longer follow-up periods are needed to assess the duration of LJM11-mediated protection from CL. Reassuringly, mice vaccinated with complementary DNA encoding LJM11 and challenged by needle injection of parasite were protected up to 12 weeks after infection (Xu et al., 2011
). Although immunization with rLJM11 may not mimic the outcome of vaccination with LJM11 DNA, we previously demonstrated that immunization with the sand fly salivary gland homogenate, in which the dominant Th1-inducing protein is LJM11, in the absence of adjuvant, produced a sustained protection against CL 12 weeks after challenge (Xu et al., 2011
). This argues in favor of long-lasting immunity by rLJM11-induced immunity in the absence of adjuvant.
The cornerstone of LJM11-mediated immunity is cellular, where CD4+
T cells producing IFN-γ or both IFN-γ and tumor necrosis factor-α were prominent in the ears and draining LN of rLJM11-immunized mice challenged with infected sand flies. Induction of multifunctional T cells that have been implicated in enhanced immunity to CL (Darrah et al., 2007
) further emphasizes the significance of anti-saliva immunity in protection from leishmaniasis. The vital role of CD4+
T cells in LJM11-mediated protection is underlined by the abrogation of protection on depletion of these cells. Despite the fact that we cannot distinguish saliva-specific from Leishmania
T cells in this study, we can conclude that the mechanism of saliva-mediated protection is not independent from CD4+
T cells. If anything, this finding suggests that saliva-mediated protection is intricately connected to the development of Leishmania
-specific immunity. It is noteworthy that in the absence of CD4+
T cells CD8+
T cells do not have a major role in saliva-mediated protection from CL. Furthermore, antibodies are not required, suggesting that the protective effect is not related to antibody neutralization of LJM11.
Importantly, this protection was observed following immunization with a small amount (500
ng) of rLJM11 in the absence of adjuvant. LJM11 belongs to the Yellow family of proteins found exclusively in insects and only from sand fly saliva in its secreted form. The crystal structure of LJM11 displays a strong positive charge on one of its surfaces (Xu et al., 2011
). The induction of long-term immunity by this protein in the absence of adjuvant is remarkable and represents an important feature in its consideration as a vaccine candidate. Part of the immunogenicity of this molecule may be due to its exclusive presence in sand fly saliva, making it quite foreign to the mammalian immune system. In addition, this molecule may be acting as a self-adjuvanting protein via its positively charged face that may promote interaction with cells of the innate immune system.
BSA-immunized mice challenged 2 weeks after the last immunization showed severe lesions that contained a large number of viable parasites despite the presence of a high level of Leishmania-specific IFN-γ. The failure to control CL in the presence of such an elevated level of IFN-γ remains unexplained particularly in light of the low level of detectable IL-4. We cannot fully exclude the fact that the observed low levels of IL-4 may have contributed to disease progression. Alternatively, other regulatory molecules could be protecting the parasite and promoting disease. In comparison, the ulcer-free protection resulting from immunity to LJM11 in mice challenged 2 weeks after the last immunization is striking. The immune response in LNs 2 weeks after infection in these mice reflects a Th1 environment characterized by the induction of LJM11-specific IFN-γ in the absence of IL-4. We believe that, in contrast to the BSA group, parasite replication is controlled in rLJM11-immunized mice by a moderate induction of LJM11-specific IFN-γ. In a separate experiment, rLJM11-immunized mice challenged 5 months after the last vaccination maintained powerful protection from CL. Importantly, these mice demonstrated a robust Leishmania-specific immunity 5 weeks after infection. This is important for long-lasting protection of rLJM11-immunized mice.
Leishmanization—the inoculation of virulent parasites—results in strong and durable immunity (Handman, 2001
). This involves the development of patent disease at the chosen site of inoculation followed by cure. Leishmanization was discontinued owing to several factors, including the development of adverse reactions in a proportion of “leishmanized” individuals and lack of reproducibility (Handman, 2001
). Thus far, leishmanization, the gold standard for protection in humans, is represented by mice that have healed their lesions (Peters et al., 2009
). Immunization with rLJM11 provides a better alternative in which rLJM11-immunized mice challenged by vector transmission of virulent L. major
-specific immunity characterized by persistence of live parasites in the absence of CL lesions.
In conclusion, this work reveals several new aspects pertinent to understanding the potency and mechanism of sand fly saliva–mediated protection from leishmaniasis. We demonstrate that (i) immunity to a single salivary molecule confers protection against vector-transmitted CL; (ii) adjuvantless immunity generated by such a molecule is long lasting, conferring undiminished protection in mice challenged 5 months after the last immunization; (iii) protection from CL as a result of saliva-induced immunity is cell mediated and dependent on CD4+ T cells; and (iv) anti-saliva immunity leads to the development of a controlled ulcer-free Leishmania-specific immunity.