Despite the fact that the majority of vaccines licensed for clinical use against VL remain live, attenuated, or killed crude preparations [2, 3], much effort has been devoted to identify new Leishmania subunit/adjuvant combinations that are clinically efficacious. However, there are only few suitable adjuvants that have been licensed for human and veterinary vaccine use. Thus, a successful anti-leishmanial subunit vaccine will need to be assessed with human-compatible adjuvants. In our laboratory we have identified LAg as a potential candidate antigen, which was efficacious when associated with liposomes and vaccinated intraperitonealy in mice and hamsters [4, 5]. However, In contrast to other reports utilizing differential liposomal formulations and administered subcutaneously [22, 23], comparative evaluation of intraperitoneal and subcutaneous vaccination with LAg entrapped in our liposomal composition failed to protect against challenge infection through subcutaneous route . Alum remains the most widely used adjuvant in human vaccines, and saponin is one of the promising adjuvant that has more recently been licensed for human use [7, 12]. To facilitate broad clinical applicability, the preferred route of delivery is the minimally invasive subcutaneous route. Thus in an attempt to overcome the failure of subcutaneous vaccination with LAg in liposomes, this study investigated the protective ability of LAg in formulation with two widely used human-compatible adjuvants when injected subcutaneously.
Alum has been conventionally used as a clinical adjuvant for a wide range of vaccines that target a humoral immune response. However, the use of alum as an adjuvant for vaccination against the intracellular pathogen Leishmania has also been tested previously. In L. major, a vaccine containing killed parasites and IL-12 adjuvant was found to be prophylactically ineffective , however this antigen along with alum and IL-12 did induce protection in primates . Moreover, encouraging results following vaccination in a primate model with combinations of alum-precipitated ALM and either BCG  or IL-12  formed the basis of a human trial for a potential vaccine against VL. Safety and immunogenicity studies conducted under field conditions including healthy volunteers , as well as children who are at high risk of VL , indicated that the vaccine containing alum-precipitated ALM with BCG was safe and well tolerated. Again, the observation that the vaccine was highly immunogenic and could induce a strong Th1 response [10, 26] led to the use of the formulation as an immunological stimulus for the successful treatment of patients with persistent PKDL . Despite these satisfactory results, to our knowledge, such a formulation has not been examined for its efficacy in trials against VL. Herein we observed that alum + LAg failed to protect BALB/c mice against challenge with L. donovani. We therefore envisage that inclusion of a second Th1 promoting adjuvant such as IL-12 or BCG with alum will be necessary for an alum containing vaccine to be clinically successful against both CL and VL [8, 9]. Nonetheless, it must be considered that failure of alum-ALM + BCG to protect susceptible BALB/c against L. major raises some concern about the similar use of such an adjuvant in humans.
Saponin remains the immunopotentiator of choice in many cancer and infectious disease vaccine trials, such as malaria, HIV, hepatitis and tuberculosis . In experimental VL FML or the immunodominant leishmanial antigen (NH36) formulated with saponin was found to be effective when administered prophylactically [13, 28], and furthermore such formulations were also found to be efficacious when utilized immunotherapeutically [14, 16]. These results facilitated the development of the currently licensed vaccine Leishmune®, composed of FML with increased amounts of saponin for field trials against canine VL. Indeed, Leishmune® has been recently shown immunotherapeutic potential for vaccination against canine VL . In contrast to these reports, our study showed that saponin + LAg immunization not only failed to reduce parasite burden in liver of L. donovani challenged mice but also caused exacerbation of infection in spleen. These findings are partly in keeping with those of Grenfell et al., who observed that antigenic extracts of L. amazonensis or L. braziliensis in association with saponin conferred only partial protection against L. chagasi. Thus, the efficacy of saponin with leishmanial antigens other than FML may vary, and such observations warrant further pre-clinical studies to establish the potential of saponin to adjuvant vaccines against leishmaniasis.
Hypergammaglobulinemia and non-specific polyclonal antibody responses are hallmarks of VL. However, vaccine-induced antigen specific humoral response and their isotype profiles are often used as convenient surrogate markers of Th1 and Th2 response . Evidence from both human patients and mice indicate that B-cell activation and production of polyclonal IgG may contribute to disease pathogenesis, leading to exacerbation of disease [19, 20]. The absence of a detectable non-specific IgG response in mice immunized with alum + LAg and saponin + LAg suggests that polyclonal antibody responses do not contribute to the failure of protection in our system. Conversely, isotypic analysis revealed high levels of IgG1, IgG2a and IgG2b in both groups and demonstrate a mixed Th1/Th2 response. With infection the alum + LAg group failed to maintain the levels of IgG2a and IgG2b but nonetheless exhibited elevation of IgG1, reflecting a dominance of Th2, which correlates with the failure of protection in this group. In contrast, saponin + LAg immunized mice showed levels of IgG2a, IgG2b and IgG1 comparable with controls. Nevertheless, an increased IgG2a:IgG1 in the saponin + LAg condition is suggestive of a subtle Th1 bias, but it remains unclear how this may relate to the exacerbation of challenge infection in the spleen. Mice immunized with lip + LAg induced high levels of both IgG2a and IgG2b revealing that strong Th1 dominance is a correlate of protection in this group.
In an effort to further define the mechanism/s underlying protection induced by intraperitoneal lip + LAg versus the inability of subcutaneous immunization with alum + LAg or saponin + LAg to induce protection, we finally analyzed cytokine production by vaccinated cohorts in response to re-stimulation with LAg in vitro. Analysis of cytokines from splenocytes ex vivo revealed that animals vaccinated with lip + LAg produced high levels of both IL-12 and IFN-γ. Specifically we found that CD4+ and CD8+ T cells both contributed to this cytokine production, and may play an essential role in inducing resistance versus L. donovani[5, 6, 18]. Immunization with lip + LAg also enhanced the production of IL-4 and thus substantiated earlier observations from our lab and others suggesting that low levels of IL-4 at early time points are not detrimental and may even be beneficial in promoting Th1 differentiation, both maintaining IFN-γ production and priming IL-12 production in VL [5, 18, 30–32].
In contrast, mice vaccinated with alum + LAg produced low but nevertheless detectable levels of IFN-γ derived mainly from CD8+ T cells, whereas we also observed a robust IL-4 response from CD4+ T cells in these conditions. It is well established that alum promotes Th2 responses , but recently Serre et al. found that alum-precipitated proteins can also induce CD8+ T cells to produce Th1-associated IFN-γ . In L. major, susceptibility to infection is related with the Th1/Th2 balance, and in particular IL-4 expression has been implicated as playing a role. Protective efficacy of vaccine formulations in CL is related not only with induction of Th1 responses but also the prevention of a Th2 response. Th2 responses have been suggested to override and thus abrogate even a strong Th1 effector function . The higher levels of IL-4 induced by alum + LAg immunization in comparison to other vaccinated groups may therefore hinder the protective efficacy in this group. Thus, the failure of protection in alum + LAg immunized mice may be a direct result of the strong IL-4-driven Th2 response that predominated.
Interestingly, we observed that saponin + LAg immunized mice produced high levels of IL-12 and IFN-γ from both CD4+ and CD8+ T cells suggesting an overriding Th1-skewed response in this group. Such effects were also paralleled with significantly elevated Th2 cytokine production, namely IL-4 and IL-10, that was predominantly CD4+ T cell dependent. Several authors have shown an ability of saponin to upregulate the production of IFN-γ [12, 13, 28]. However, to our knowledge, our report represents the first observation that a saponin adjuvanted vaccine can induce robust IL-4. On the contrary, Greenfell et al., reported that vaccination with antigenic extracts of L. braziliensis and L. amazonensis associated with saponin resulted in reduced production of IL-4 . There are few reports of low levels of IL-10 production  and a low ratio of IFN-γ/IL-10 producing T cells  with vaccination of FML antigen or its component formulated with saponin in mice. However, most of the studies with these formulations have not been investigated for the stimulation of IL-10 production. In contrast, strong IL-10 as well as IL-4 responses was observed following immunization of Trypanosoma cruzi lysate adjuvanted with saponin . Studies in humans , in mice with genetic ablation of IL-10 , or in conjunction with IL-10 receptor blockade , established that IL-10 is the major immunosuppressive cytokine in VL. The generalized negative regulatory role of IL-10 in vaccine failure is indeed well established . Interestingly, exacerbation of L. major infection was associated with higher levels of both IL-4 and IL-10 relative to IFN-γ . Consistent with this study, our results suggest that IL-10 is a major determinant of L. donovani disease progression in saponin + LAg vaccinated mice, and moreover IL-10 may collude with IL-4, to override the proinflammatory functions of IFN-γ.
L. donovani infection is characterized by distinct organ-specific pathogen/immune interactions, whereby the liver is the site of infectious resolution, whereas the spleen represents the site of parasitic persistence. In the liver, IFN-γ produced by both NK cells and T cells functions to resolve L. donovani infection . In keeping with these findings, saponin + LAg immunized mice induced robust IFN-γ leading to specific protection in the liver at an early stage of infection (2 months). Infection models have produced unequivocal evidence that IL-10 is responsible for pathogen persistence [42, 43] and thus, neutralization of IL-10 resulted in more effective clearance of Leishmania from the splenic compartment . Thus, simultaneous production of high IL-4 and IL-10 may be the mechanistic determinant of the exacerbated infection observed in the spleen of saponin + LAg immunized mice.
Taken together, our study highlights the difficulties underlying the search for a highly efficacious leishmanial subunit vaccine in a clinical setting. The results herein support a model whereby efficacious subcutaneous vaccine formulations will be predicted to target both robust IFN-γ production and a strong Th1 response, but must minimally induce the immunosuppressive cytokines IL-4 and IL-10.