Leishmaniasis causes human suffering on a global scale, threatening approximately 350 million people in endemic areas, with an estimated 12 million current cases and 2 million additional cases annually (222
). Several species of protozoan parasites of the genus Leishmania
are transmitted by the bite of infected sand flies and cause human infections ranging from disfiguring cutaneous lesions to potentially fatal VL. Available chemotherapeutics are largely effective, though often toxic, and drug-resistance is an issue (223
). Even if ideal drugs were available, elimination of leishmaniasis can only be achieved through vaccination as humans are the main reservoir for many Leishmania
spp., and elimination of insect vectors is not an alternative. For these reasons, we at the Infectious Diseases Research Institute (IDRI) have been focused on vaccine development.
Leishmania parasites reside mainly within macrophages, and therefore vaccines that stimulate cellular immune responses are required for control of intracellular replication. Appropriate CD4+
T-cell responses correlate with protection against leishmaniasis in humans and in animal models (224
). The discovery of the Th1/Th2 separation of CD4 response based on cytokine production was aided largely by studies using resistant and susceptible inbred mouse strains (225
). Using crude or defined antigens with appropriate adjuvants, protection against visceral and cutaneous disease has been achieved in mice, hamsters, dogs, and non-human primates (59
). Protection studies, particularly in mice, have corroborated the Th1-dependence of effective immunity against Leishmania
. Thus, understanding how to induce protective immune responses against Leishmania
has broad relevance to the development of T-cell vaccines and vaccines against intracellular organisms.
Partial clinical efficacy has been obtained using first generation vaccines, primarily for cutaneous leishmaniasis (CL) (236
), though results have been inconsistent. These studies involved the use of crude preparations that cannot be standardized or be optimally formulated to induce desired immune responses while avoiding undesirable immune responses. Defined antigens delivered as plasmid DNA, vectored DNA, or as recombinant protein have advantages in this regard, and have proven to be effective in animal models. Of these platform technologies, only recombinant proteins have advanced to licensure in human vaccines, while both protein- and DNA-based vaccines have advanced as veterinary products. While recombinant proteins provide a versatile, scalable, and cost-effective approach for vaccine development, they generally induce only weak T-cell responses. However, this can be overcome with the inclusion of adjuvants. We have optimized adjuvants for vaccine targets requiring potent CD4+
T-cell responses, including leishmaniasis. For our human vaccine development we are emphasizing recombinant protein/adjuvant, while our canine vaccine program includes evaluation of nucleic acid as well as protein vaccine constructs.
The development of a safe, effective, and practical vaccine against leishmaniasis involves: (i) identification of effective antigens and (ii) delivering antigens in formulations that induce effective T-cell responses. Although partial efficacy has been demonstrated with crude first generation vaccines, attempts to turn such preparations into sustainable products have been unsuccessful. To date, there has been no licensure of effective T-cell vaccines, although several are in development (e.g. tuberculosis, malaria, HIV, leishmaniasis), as well as immune-therapeutics for cancer. Our approach has been to identify protein antigens, create polyprotein fusions, optimize the proteins for maximum immunogenicity, and develop antigen delivery platforms, including adjuvant formulations that promote appropriate T-cell responses and protection in animal models. Criteria for antigen selection have also included conservation among Leishmania species, potentially allowing the development of a vaccine effective against both VL and CL.
Turning antigens into effective immunogens requires understanding of the nature of the desired immune response and selection of delivery platforms capable of inducing such a response. Our first defined vaccine against leishmaniasis consisted of a combination of four recombinant antigens formulated with GM-CSF (used before the availability of other adjuvants). This vaccine was successfully used to treat drug refractory mucosal leishmaniasis (ML) caused by Leishmania braziliensis
) and was the first example of a defined vaccine being successfully used for immunochemotherapy for this disease, providing proof-of-concept for this approach.
A major breakthrough in the development of vaccine candidates against leishmaniasis, as well as other diseases requiring potent and directed T-cell responses, occurred with the identification of adjuvants capable of inducing Th1 responses. The discovery that properly formulated Toll-like receptor (TLR) agonists can stimulate Th1 immune responses has profoundly impacted vaccine development against intracellular pathogens such as Leishmania
. In particular, the extensive experience with monophosphoryl lipid A (MPL), a TLR4 agonist obtained from the cell wall of Salmonella
, and MPL’s approval in vaccines for hepatitis B and human papilloma virus have demonstrated the safety and efficacy of engaging TLR4. MPL is the only TLR agonist in approved vaccines and thus has an extensive history of safety and efficacy. We have used MPL in preclinical models of leishmaniasis and have demonstrated efficacy in several species. The next generation vaccine candidate consisted of a poly-protein, designed and produced to be more cost effective than came with the use of recombinant fusion protein Leish-111f (L111f) together with MPL formulated in an oil-in-water emulsion (MPL-SE) (59
). This vaccine antigen () was shown to protect mice, hamsters, and rhesus macaques, when formulated with an effective adjuvant or as DNA (230
), and has subsequently been used in multiple clinical trials. L111f was shown to be safe and immunogenic as well as to have therapeutic efficacy in humans (240
) and in dogs (233
). The antigens were chosen based on their ability to protect mice or, in the case of LeIF, to act as an adjuvant through the stimulation of IL-12, as well as on their conservation among Leishmania
Leish-111f (L111f): a tandemly linked protein of three subunits
The primary patient (and reservoir) populations for leishmaniasis are humans and dogs. Dogs are a natural host for VL and represent both a disease protection model as well as a target for epidemiological intervention of disease transmission. In endemic regions of the Mediterranean and Latin America, dogs are the most important reservoir of Leishmania infantum
. Humans are the VL reservoir in the Indian subcontinent and parts of Africa. Relatively little is known with regard to the generation of protective T-cell responses in dogs, although we (233
) and others (234
) have demonstrated partial efficacy in both prophylactic and therapeutic vaccine approaches in canine leishmaniasis. Thus, canine VL studies have provided important proof of concept for the use of vaccines to effectively treat fatal VL. Clinical trials to evaluate this concept in human VL will begin next year.
We have performed several clinical trials using recombinant Leishmania antigens formulated in granulocyte macrophage colony stimulating factor (GM-CSF) or monophosphoryl lipid A-squalene (MPL-SE). Several interesting observations have emerged from our clinical studies. Individuals with active CL or ML have strong anti-Leishmania immune responses, including high antibody levels and significant T-cell responses. Thus, treating these individuals with vaccine may seem counterintuitive. However, we have found that infected individuals responded poorly to vaccine antigens prior to immunization, but responded to the vaccine antigens with both specific antibody () and T cells () following immunization. Thus, infected individuals can be immunized with properly formulated antigens, which often times are not well recognized by the infected individual, resulting in the generation of vaccine antigen-specific T cells which seems to correlate with disease resolution ().
Induction of specific antibody following immunization of active cutaneous leishmaniasis patients
(A). Induction of specific cellular immune responses following immunization of active cutaneous leishmaniasis patients using Fluorescence Activated Cell Sorting (FACS)
In a recent study in CL patients in Brazil, a significantly faster cure rate was observed in patients who received vaccine in addition to chemotherapy, as opposed to chemotherapy alone (). Cumulatively, our clinical results have demonstrated safety and partial efficacy of therapeutic vaccination and point to the possibility of using this approach in patients who fail chemotherapy, as well as potentially devising protocols involving reduced doses of drug in combination with vaccination. Further innovation has been in the area of adjuvant development. Because of the effectiveness of MPL-based adjuvants in animal models of leishmaniasis, as well as in humans, we have focused on improving MPL. Although we have a license to MPL from GSK, there are several reasons for emphasizing the development of synthetic molecules based on MPL. For one thing, continued cost effective access to the molecule cannot be guaranteed. In addition, we developed structures with increased potency over MPL, allowing the use of comparatively smaller doses. Using information from the crystal structure of the human TLR4, we selected one molecule for further development, based on the ability of this molecule to fit in the human MD2 structure. We have developed formulations of this novel synthetic TLR4 agonist glucopyranosyl lipid A (GLA), which is more potent than MPL in in vitro studies with human cells, and have shown that GLA is an effective adjuvant in models of CL and VL. Furthermore, GLA can be synthesized in large amounts (we currently have nearly 1 million human doses in inventory) and is independent from control by pharmaceutical companies. In addition to being an effective adjuvant molecule, GLA can synergize with ligands of other TLRs.
Beneficial effects of vaccination of cutaneous leishmaniasis patients receiving antimony chemotherapy; decreased time to cure
It is evident that solid protection can be achieved in experimental models using recombinant proteins properly formulated in safe and effective adjuvants. The fact that certain protective antigens are highly shared between Leishmania species, that protection can be achieved with an adjuvant approved in vaccines in over 100 countries, that VL vaccine development can be pursued in both dogs and humans, and that vaccine products can be pursued for both prophylactic and therapeutic applications are all advantages for targeting Leishmania for vaccine development. IDRI has completed or has ongoing clinical trials in several countries, including USA, Brazil, Peru, Colombia, Venezuela, India, and Sudan. It is hoped and expected that information from these trials will lead to one or more safe and effective vaccines for human and canine leishmaniasis.