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Not All Antigens Are Created Equally: Progress, Challenges, and Lessons Associated with Developing a Vaccine for Leishmaniasis

ABSTRACT From experimental models and the analyses of patients, it is well documented that antigen-specific T cells are critical for protection against Leishmania infection. Effective vaccines require both targeting to the pathogen and an immune stimulant to induce maturation of appropriate immune responses. While a great number of antigens have been examined as vaccine candidates against various Leishmania species, few have advanced to human or canine clinical trials. With emphasis on antigen expression, in this minireview we discuss some of the vaccine platforms that are currently being explored for the development of Leishmania vaccines. It is clear that the vaccine platform of choice can have a significant impact upon the level of protection induced by particular antigens, and we provide and highlight some examples for which the vaccine system used has impacted the protective efficacy imparted.

with L. donovani, while in the Mediterranean, the Middle East, Latin America, and other parts of Asia, it results from infection with L. infantum.
Protection from, and clearance of, Leishmania infection is strongly associated with the generation of antigen-specific Th1 responses, knowledge that provides a clear goal for immunization. To target and appropriately skew the anti-Leishmania response, vaccines require both pathogen-specific antigens and immune-stimulating molecules to be rendered effective. Whole and genetically attenuated parasites, such as L. donovani centrin-deleted parasites (LdCen1 Ϫ/Ϫ ), have undergone trials in both dogs and humans (4)(5)(6)(7)(8). Dendritic cell (DC)-based vaccines represent a more refined strategy and have been used in preclinical models. Although financial constraints and the likely difficulties in delivering such vaccines in regions of Leishmania endemicity seem to preclude their use in all but a very limited subset of individuals, DC-based vaccines provide further proof of concept supporting the use of more defined vaccines (9).
Our understanding of pattern recognition receptors and adjuvants that can be used in conjunction with recombinant proteins within defined subunit vaccines continues to expand (10)(11)(12). A wide variety of adjuvants have been used in Leishmania research programs, and the importance of adjuvants within vaccines has been the subject of numerous review articles (13)(14)(15)(16). Simultaneous with the progress in adjuvant technologies, advances in bioinformatics and molecular techniques have expanded the number of potential Leishmania-specific targets for use within a vaccine (17)(18)(19). A considerable number of Leishmania antigens have been tested as subunit protein or DNA vaccine candidates against various Leishmania species, but variable results have meant that very few antigens have advanced to human or canine clinical trials (20). Factors such as the Leishmania source of the target gene, the Leishmania species involved in the challenge, the quantity of antigen/vector used, and the frequency and numbers of immunizations all impact our ability to interpret the data beyond their immediate application. In addition, once targets are selected, there are also now a plethora of strategies available with which to ready them for use within a vaccine (21). In this minireview, we discuss the current status of vaccine development for leishmaniasis with emphasis on the impact of the expression/vaccine platforms.

PURIFIED RECOMBINANT PROTEINS
Native Leishmania proteins have been used in either a crude or a purified manner to elicit protective immune responses. Escherichia coli and yeast are among the most widely used systems for the production of recombinant proteins (22)(23)(24), and bioinformatics has facilitated the production of a variety of recombinant proteins that have been investigated as Leishmania vaccine antigen candidates in animal models (15,20). Key to the use of recombinant proteins is the cost-effectiveness of production, an important consideration when modeling has shown that a cost of $2 or less per dose for a preventive vaccine would be economically advantageous over the currently available leishmaniasis treatments (25). In addition, recombinant methods also provide the opportunity to manipulate and combine complementary proteins/epitopes in a single gene product.
Our first defined vaccine against leishmaniasis came with the use of E. coli-expressed Leish-111f (L111f; LEISH-F1) that joined the L. major homologue of eukaryotic thiolspecific antioxidant (TSA), the L. major stress-inducible protein-1 (LmSTI1) and the L. braziliensis elongation and initiation factor (LeIF) into a chimeric fusion protein. Leish-111f protects mice, hamsters, and rhesus macaques when appropriately formulated with adjuvant, and LEISH-F1ϩ monophosphoryl lipid A (MPL)-SE was the first defined Leishmania vaccine to enter clinical trials, where it demonstrated an excellent safety profile and induced antigen-specific responses (26)(27)(28)(29). A second, distinct E. coli-derived fusion, LEISH-F3, that combines two components that can protect in animal models of VL, nucleoside hydrolase (NH) and a sterol 24-c-methyltransferase (SMT), has also progressed through phase I clinical trial (30).
The cost-effectiveness of recombinant proteins can also be enhanced by the selection of particular expression platforms. In some instances, yeast (Saccharomyces cerevi-siae or Pichia pastoris) expression systems provide benefits over E. coli expression systems, such as variable folding, glycosylation, or direct secretion into the fermentation media of the target protein as a soluble and stable protein (31,32). An E. coli-expressed L. donovani nucleoside hydrolase (LdNH36) surface protein has provided partial protection against L. donovani infection in mice (33). Expression of LdNH36 in P. pastoris produced LdNH36-Y-WT that was of significantly higher molecular weight than the E. coli expressed protein (34). Gel analyses suggested that expression in yeast resulted in several high-mannose glycoforms, whose presence was first reduced by mutating N-linked glycosylation sites located outside the major immunogenic domain from asparagines to serines (LdNH36-dg) and then removed by mutating asparagines to glutamines. The LdNH36-dg2 product was similar in size to the nonglycosylated E. coli expressed protein, but the glutamine mutations resulted in increased expression levels. Mice immunized with LdNH36-dg2 did have high levels of antibodies that inhibited the hydrolase activity of the wild-type LdNH36 (34), although challenge experiments have not been reported.

DNA VACCINE CONSTRUCTS
Besides traditional recombinant expression approaches, many antigens have also been investigated in DNA vaccines (20). VL patients possess antibodies against the hemoglobin receptor (HbR) of Leishmania, and this protein is conserved across various Leishmania strains. Immunization with HbR-DNA generated multifunctional CD4 and CD8 T cells and was reported to provide complete (sterile) protection against L. donovani infection in both mice and hamsters (35). Given these findings, we produced recombinant HbR in E. coli to examine compatibility with our own vaccine candidates. Surprisingly, unlike our previous data indicating various candidates that retained their protective properties when expressed in E. coli (36), we did not observe protection in mice that were immunized with HbR in conjunction with the glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) (Fig. 1). Furthermore, adding HbR as a component within fusion proteins disrupted their ability to confer protection. We still do not understand why these differences arose, but it is possible that differential processing of epitopes may occur, or some DNA vaccines may target different antigen-presenting cells than protein/adjuvant. While subtle, such changes may be enough to lead to critical variations in the protective efficacy of the respective vaccines.
Fused targets can also be created within DNA vaccines, and seven Leishmania genes (H2A, H2B, H3, H4, A2, KMP11, and HSP70) are encoded within the HisAK70 DNAvaccine (37). In the VL model, HisAK70-immunized mice exhibited many sterile hepatic granulomas associated with the resolution of hepatic parasite burdens, and in the CL model, spread of parasites to the viscera was completely inhibited in HisAK70immunized mice. The results suggest that immunization with the HisAK70 DNA may provide a rapid, suitable, and efficient vaccination strategy to confer cross-protective immunity against VL and CL.

BACTERIAL AND VIRAL VECTORS
In terms of bacterial vectors, Listeria monocytogenes, Mycobacterium bovis BCG, and Salmonella enterica serovar Typhimurium have all been used as vehicles to deliver Leishmania targets (20). As an example of this approach, novel Leishmania parasite antigens were selected by an in silico proteomic approach and expressed in the vector Salmonella Typhimurium SL3261. Immunization of mice with individual Salmonella vaccine strains expressing the antigens LinJ08.1190 and LinJ23.0410, or a mixture of these bacilli, significantly delayed the progression of L. major and enhanced systemic resistance against L. donovani (38). These data identify Salmonella as another valid vaccine carrier for inducing protection against VL. Given that 2 antigens that were also selected and expressed did not protect, however, this Salmonella system may not be appropriate for all antigens.
With regard to viral vectors, the most advanced is a nonreplicative adenovirus vector encoding the A2 antigen (Ad5-A2) that has progressed to L. infantum challenge in macaques (39). Using a variety of prime boost schemes involving Ad5-A2 as well as recombinant protein and a plasmid DNA-encoding A2 gene (DNA-A2), parasite burdens in the liver were lower in immunized macaques and correlated with hepatic granuloma resolution and the reduction of clinical symptoms. Considering the importance of both CD4 and CD8 T cells in the control of Leishmania infection, we recently generated an adenoviral vector expressing the F3 protein (Ad5-F3) to complement the use of recombinant F3 and investigated immunization strategies with the promise of generating combined protective T-cell responses (40). Overall, a F3ϩGLA-SE prime/Ad5-F3 boost strategy induced the best combined CD4 and CD8 T cell memory response, although we also found that simultaneous immunization with Ad5-F3 and F3ϩGLA-SE in a single injection was potent enough to provide protection. As an alternative, although only limited experimental information regarding the effectiveness for use against human pathogens is available, the NYVAC poxvirus vector has also recently been explored (41). Using DNA prime/virus boost protocols, a replication-competent NYVAC expressing LACK (Leishmania homologue of mammalian RACKs, the receptors for activated C kinase) (NYVAC-LACK-C7L) induced more polyfunctional CD4 and CD8 T cell responses and greater protection against L. major than nonreplicating NYVAC-LACK.

CLOSING REMARKS
A great number of defined antigens, delivered as recombinant protein in adjuvant, as plasmid DNA, or within vector systems, have provided protection in animal models of leishmaniasis. In an attempt to consolidate information generated across many platforms, we recently selected and expressed 7 candidates as recombinant proteins in E. coli. Each recombinant protein was recognized by VL patients and, when formulated with the Th1-potentiating adjuvant GLA-SE, retained its protective efficacy against the challenge of mice with L. donovani (36). This indicates the possibility of combining candidates that have advanced in diverse vaccine platforms. It is clear, however, that the vaccine platform of choice can have a significant impact upon the level of protection induced and in some instances can negate protection altogether. Thus, without actually generating independent results, vaccine developers must be cautious when navigating through the existing data and between the platforms (Table 1). On the positive side, as is seen most clearly with viral vectors, exploring interactions between these technologies has the potential to yield regimens that augment, complement, or synergize each other to yield a robust protective regimen.

ACKNOWLEDGMENTS
This work was funded by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award R01AI025038. Additional program support came from the Bill and Melinda Gates Foundation under grant 631.
The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Malcolm S. Duthie and Steven G. Reed are coinventors of a patent for leishmaniasis vaccine development.