This study demonstrates in two different mouse models of L. major infection that CpG ODN provides long-term protection when used as a vaccine adjuvant with either ALM or recombinant leishmanial protein. In addition, similar to our previous studies with DNA vaccines, CpG ODN conferred more potent and durable protection when compared with IL-12 protein as an adjuvant with ALM. These data strongly suggest that a heat-killed Ag and/or a recombinant protein vaccine can be useful for inducing durable immunity for diseases requiring cellular immune responses depending on the adjuvant used.
Our data demonstrating that ALM or Trifusion protein plus CpG ODN provides long-term protection are in contrast to previous studies using CpG ODN as a vaccine adjuvant with other leishmanial Ag preparations. In the study by Stacey and Blackwell (24
), partial protection was demonstrated in BALB/c mice infected 6 wk or 6 mo after vaccination, but the quality of protection at any time after vaccination was moderate because all of the mice developed significant footpad swelling. Similarly, Walker et al. (25
) demonstrated that the effects of CpG ODN as an adjuvant were partial and limited, as protection was demonstrated in only 40% of BALB/c mice challenged 3 wk after vaccination. The greater efficacy of the leishmanial protein plus CpG ODN in our study may be due to several factors. First, the leishmanial Ag used in the other studies was a preparation of SLA, which might be less immunogenic than ALM, a clinical grade Ag that has been used in human vaccine trials against L. major
). Second, the parasite dose and strain differed in our studies compared with the other studies. Third, the CpG ODN sequence we used was different than that used in one of the previous studies showing partial long-term immunity. Nevertheless, the results presented here showing that CpG ODN provide long-term protection have been repeated multiple times in both BALB/c and B6 mouse models of leishmania infection with consistent results and little variability.
With regard to the mechanism by which CpG ODN mediate their function in vivo and why they are better than IL-12 protein as a vaccine adjuvant, their role in enhancing APC function needs to be considered. At the APC level, CpG ODN through toll-like receptor 9, augments both the activation and maturation of DC as well as the induction of proinflammatory cytokines (35
). Thus, in comparison to the relatively short-lived effect of exogenous IL-12 protein as an adjuvant, the endogenous production of IL-12, IL-18, and other soluble mediators from activated DC induced by CpG ODN are likely to result in a more physiologic cognate interaction between the DC and T cell, resulting in both a qualitatively and quantitatively different type of CD4+
T cell response. These data highlight the potential importance of targeting DC, especially for vaccines against diseases requiring cellular immune responses.
In terms of immune correlates of protection, it is well established that CpG ODN are potent inducers of Th1 responses, which is consistent with our findings that mice vaccinated with ALM plus CpG ODN had striking enhancement in the frequency and production of IFN-γ from CD4+
T cells 6 mo after vaccination in B6 mice () and after infection in BALB/c mice, respectively (). Moreover, an additional possibility relating to the effectiveness of CpG ODN as a vaccine adjuvant compared with IL-12 protein in the BALB/c and B6 models is the role of CD8+
T cells. As previously mentioned, CD8+
T cell responses are not seen in vaccination with recombinant leishmanial protein plus IL-12 but have an important role in maintaining the protection elicited by DNA vaccination (7
). In this regard, CpG ODN have been shown to elicit CD8+
T cell responses in mice when given with a protein Ag such as OVA (20
). Thus, our findings that there were significant frequencies of Ag-specific CD8+
cells in B6 mice 6 mo after vaccination with ALM plus CpG ODN and that the depletion of CD8+
T cells at the time of vaccination enhances the size of the lesions and the parasite load in B6 mice, provide compelling evidence for the importance of CD8+
T cells in mediating long-term immunity. Furthermore, the data showing that lesion development is altered in B6 mice immunized with a recombinant leishmanial protein plus CpG ODN depleted of CD8+
T cells at the time of vaccination provides additional support for CpG ODN inducing CD8+
T cell responses with a defined parasitic protein Ag. Finally, although lesion size in leishmanial Ag plus CpG ODN–vaccinated mice depleted of CD8+
T cells was increased, these mice had demonstrably smaller lesions than control vaccinated mice. This is consistent with the induction of both CD4+
T cells by such a vaccine. Together, these data suggest that vaccines that elicit both CD4+
T cell responses are sufficient to confer long-term protection against L. major
Although the vaccine approach described here potentially has immediate clinical use because ALM is already a widely tested vaccine Ag in humans, it remains to be determined whether the effects of CpG ODN on the cellular immune response in humans will be comparable to those observed in mice. One encouraging recent report related to this issue showed that there was a decrease in lesion size after infectious challenge with L. major
in primates vaccinated with a killed preparation of leishmanial Ag and a specific type of CpG ODN compared with vaccination with Ag alone (36
). To conclude, it is worth noting that the historical standard for effective vaccination against L. major
in humans is leishmaniazation (attenuated live infection). Moreover, the fact that previous infection to L. major
confers life-long protection in humans and that there is evidence for the persistence of parasitic Ag raises the critical issue of whether the induction of potent CD4+
T cell responses induced after DNA or a killed and/or recombinant leishmanial Ag plus CpG ODN will be sufficient to confer life-long protection in humans. This distinction between the apparent lack of an Ag requirement to maintain cellular immune memory for CD4+
T cell responses for nonliving vaccines versus the potential requirement of Ag for mediating biologic protection will be the critical determinant in whether specific vaccines will be successful over the course of a lifetime against many infections requiring cellular immunity in humans.