Vaccines against tropical parasitic diseases: A persisting answer to a persisting problem

Abstract

Live and live-attenuated whole organism vaccines against Plasmodium falciparum malaria and cutaneous leishmaniasis due to Leishmania major remain the most uniformly effective vaccines against human parasitic diseases. These vaccines are discussed in terms of the nature of the T cell populations that mediate the strong and durable localized immunity to these infections, and the requirement for persisting antigen to generate and maintain the protective response. The difficulties in developing subunit vaccines that fulfill this requirement argue that despite their own formidable problems in manufacture and delivery, live and live- attenuated whole organism vaccines against human parasitic diseases should be vigorously pursued.

Tropical infectious diseases caused by parasites are major causes of illness in the poorest countries of Asia, Africa and Latin America. Amongst the highest burden tropical diseases commonly referred to as ‘neglected’, eleven are caused by helminthic and protozoan parasites, that along with malaria affect more than 1 billion people and cause more than 1 million deaths annually¹. The greater impact of these infections may be the chronic disabilities that they produce, such as malnutrition, anemia, cognitive defects, and disfigurement, and the economic hardships that result from the cost of treatment and loss of worker productivity².

The measures currently available to reduce the burden of tropical parasitic diseases are confined to drug treatment programs, and/or to vector control. These interventions have selected for both resistant parasites and vectors, which along with their high cost and low sustainability, have reinforced the need for preventive vaccines. Unfortunately, there is as yet no safe, uniformly effective vaccine against any human parasitic infection. The development of what Hotez and Ferris have referred to as anti-poverty vaccines², must be considered one of the major unachieved goals of modern immunology. The absence of a commercial market remains a serious disincentive for industry to take on this effort, but even when product development partnerships have existed to oversee vaccine development through to proper human trials, the goal of producing a highly effective vaccine has still not been met. The greater impediments to vaccine development may be the gaps in our knowledge about the biology of these eukaryotic pathogens, their complexity as immunologic targets and their remarkable adaptability to immunologic pressure.

The hallmark of parasitic infections is their chronicity, which implies a certain capacity to avoid or delay sterilizing immunity. The adaptive strategies that protozoan and metazoan parasites use to evade immunity - e.g. antigenic variation, sequestration, immunosuppression - are driven in many parasites by their need to prolong their survival in the mammalian host in order to counteract their relatively low transmissibility to the arthropod vector in which their cyclical development depends. Thus, for a given anti-parasite vaccine to succeed, it will have to outperform the immune response to natural, primary infection. This is fundamentally different from most licensed vaccines, which are designed to mimic the sterilizing response to natural infection without producing disease. It will be especially difficult for a vaccine to contend with protective antigens that display extensive allelic or somatic polymorphisms. Such targets would include the variant surface glycoprotein of African trypanosomes, the merozoite surface and infected erythrocyte surface proteins of malaria blood stages, and the transialidase surface antigens of T. cruzi³.

There are, nonetheless, the rare experiences with anti-parasite vaccines in humans that are remarkable for their success. Two vaccines in particular, whole sporozoite approaches that protect against P. falciparum malaria, and ‘leishmanization’ that protects against cutaneous leishmaniasis, have established themselves as the gold standards of acquired resistance against their respective diseases. In each case, live or live-attenuated organisms have been used. While one clear advantage of whole cell vaccines is their breadth of coverage against a multiplicity of antigens to better contend with parasite strain polymorphisms and host genetic restrictions, the more critical character of the two vaccines, and the focus of this commentary, is antigen persistence.

Nearly 40 years ago it was observed that sterilizing immunity against P. falciparum could be achieved by exposing human volunteers to the bites of irradiated mosquitoes carrying sporozoites in their salivary glands⁴. The radiation-attenuated parasites were unable to develop beyond their liver stages. These trials followed closely on the ground breaking studies in the mouse by Nussenzweig and colleagues using intravenous inoculation of irradiated P. bergei sporozoites⁵. In both mice and humans the complete protection against infectious sporozoite challenge was dependent on the parasites being metabolically active, and on a high dose exposure (> 1000 bites were needed to achieve protection in people). Subsequent studies revealed that a few volunteers were still protected 23 – 42 weeks after their primary or secondary immunization⁶. Early on it seemed clear, however, that the inability to grow sporozoites in culture would preclude their use as a practical approach to vaccination. An era of subunit, pre-erythrocytic stage vaccine development ensued, both antibody and T cell approaches, and culminating with the recent phase III trials of the RTS,S vaccine, a circumsporozoite protein (CS) - based subunit vaccine expressed in a hepatitis-B like particle⁷. In no case has the efficacy of the sub-unit vaccines approached the potency of the protection conferred by live or live-attenuated whole organism vaccines. Starting around 10 years ago, a team led by Steve Hoffman revisited the feasibility of developing a live, attenuated vaccine using irradiated, asceptic, cryopreserved sporozoites manually dissected from mosquito salivary glands, and delivered intravenously by needle⁶. The most recent studies employing this vaccine revealed that high doses (>600,000) completely protected 6 out of 6 volunteers against infectious sporozoite challenge⁸. While these numbers are small, and the high dose and route of inoculation pose substantial challenges moving forward, this trial still represents the most efficacious outcome in humans of a bone fide malaria vaccine.

The individuals receiving the highest dose of sporozoites and who were most protected had higher liver-stage specific antibody and T cell responses, both CD4 and CD8. The tissue infiltrating or resident memory cells in the liver that are apt to be more relevant to the immune status of these individuals could not be studied. Studies in non-human primates and mice immunized with irradiated sporozoites have confirmed that high numbers of liver-stage specific CD8+ T cells and found predominantly in the liver are required for sterile immunity⁹. The mouse studies have further revealed a requirement for CD4+ T cells and antigen persistence to generate and maintain the protective CD8+ T cell response. Thus, primaquine treatment completely abolished the long lived protection conferred by irradiated or genetically attenuated sporozoites¹⁰. In some studies, an effect of primaquine on immunity was not observed, but antigen persistence was still concluded based on the finding that CS - specific CD8+ T cells could be primed when transferred in mice that were immunized up to 60 days previously¹¹. In mice immunized with genetically attenuated sporozoites¹², the population present prior to challenge that was associated with the rapid clearance of infected hepatocytes, had the phenotype of short-lived effector CD8+ T cells (KLRG1+CD127−), and would presumably require periodic renewal by persisting antigen.

If antigen persistence is necessary to maintain the sterilizing immunity that is the remarkable feature of attenuated sporozoite vaccines, and if immunological memory as conventionally defined is maintained by long lived memory cells in the absence of antigen, then what, if any, is the role of memory cells in this vaccine? Employing the markers available to distinguish between effector memory and fully differentiated effector cells may not resolve this question, and seems a semantic exercise in any case since some effector memory cells, especially CD4+ T cells, may need continual reminding¹³. Operationally, the critical behavior is how quickly the protective response decays in the absence of antigen. It is not yet known if the long-lived protection elicited by irradiated sporozoites is dependent on persisting antigen. An alternative live, whole organism approach has been shown to provide long lasting sterile immunity in volunteers¹⁴, and as it involves exposure to P. falciparum infected mosquitoes under cover of chloroquine treatment that kills blood-stage parasites, it might be concluded that the long-lived protection is not antigen dependent. However, just as antigen presentation and immunity remained intact in primaquine treated mice immunized with irradiated sporozoites¹¹, immunity might have been sustained by persisting antigen long after chemoprophylaxis in these infected individuals.

In the mouse, the requirement for antigen persistence can be more carefully addressed by adoptive transfer of the protective cells into naïve recipients and delaying the time of challenge, which has so far been extended to only a few days using the protective cells generated by sporozoite vaccines. Using his approach, it was established unequivocally that long-lived, memory CD8+ T cells could mediate protection against lymphocytic choriomenigitis virus (LCMV), as both the numbers and protective function of the virus-specific cells were maintained when challenge was delayed for up to 18 months following transfer¹⁵. The closest that any malaria vaccine has come to reproducing the LCMV experience is the long-lived, sterile immunity achieved using a prime-boost strategy involving CS-peptide coated dendritic cells followed by Listeria monocytogenes expressing the same peptide¹⁶. While the life-span of the protective CD8+ T cells was not defined by adoptive transfer, the nature of the vaccine would suggest that the immunity did not depend on persisting antigen. This vaccine, similar to LCMV but distinct from attenuated sporozoites, and due to its strength of signal, clonality, or independence from CD4⁺ T cell help, may bypass the need for antigen persistence to generate and maintain the enormous expansion CD8⁺ T cells required for protection. It is not clear how relevant or practical this prime-boost, epitope-based approach is to human vaccination. A heterologous prime-boost vaccine designed to generate high numbers of CD8+ T cells specific for the liver-stage antigen ME-TRAP induced some protection in over half, but sterile immunity in only 3 of 14 volunteers¹⁷.

For cell-mediated immunity against pathogens that reside in phagosomes, e.g. Mycobacterium, Salmonella, and Leishmania spp., the need to recognize peptides that are generated in endosomal compartments dictates that CD4⁺ T cells will be a critical component of the protective response. There is clear evidence that antigen persistence is necessary to maintain this response¹⁸, and is the essential quality of the other anti-parasite vaccine that provides strong and long lasting protection in people. Deliberate needle inoculation with viable L. major parasites in a selected site, referred to as “leishmanization,” has been used for generations as a live “vaccine” in people living in regions endemic for cutaneous forms of leishmaniasis, and provides long lasting protection against natural exposure¹⁹. Since the live organisms used have not been deliberately attenuated, this is not a vaccine in the conventional sense. Nonetheless, the inoculum is effectively attenuated since it contains cultured organisms that are less virulent than the forms inoculated by the sand fly vector, and especially as they are delivered without factors co-egested by the fly that are known to promote infection. That said, the practice has been largely discontinued, in part because of the severity of the primary lesions experienced by some vaccinees. Many attempts to reproduce this protection using whole cell killed vaccines, including formulations shown to work effectively as immunotherapy, have failed in multiple phase III trials to confer significant protection against natural exposure²⁰. Thus, leishmanization remains the only effective prophylactic vaccine in people, and irradiated or genetically attenuated organisms could well deal with the safety concerns.

In mice, as in people, a primary infection with L. major that heals, represents the gold standard of acquired resistance, unequaled by a variety of experimental whole cell killed and subunit vaccine that have been evaluated, some of which have failed entirely when tested under the more stringent conditions of infected sand fly challenge²¹. The acquired resistance in healed mice is sometimes referred to as concomitant immunity, meaning that the protection against reinfection coincides with the persistence of the primary infection. Despite this persistence, the cells mediating resistance to reinfection do not become exhausted; to the contrary, long-lived protection from secondary challenge is lost if the original infection is cleared²².

The rapid recruitment of IFN-γ⁺CD4⁺ Th cells to the cutaneous site of infected sand fly bite or needle injection is the strongest correlate of protection against re-infection with L. major in healed mice²¹. Adoptive transfer of polyclonal, antigen experienced T cell populations from healed mice into naïve recipients have confirmed that CD4⁺ Th1 cells, and present within both the central and effector memory pools, are sufficient to transfer protection against needle challenge²³. In more recent studies, only cells bearing markers of terminally differentiated Th1 effector cells (Teff) (CD44⁺CD62L⁻T-bet⁺Ly6C⁺) were rapidly recruited to the site of cutaneous challenge, and these cells were required to fully reconstitute the protective response (Peters, N. et al., manuscript submitted). Most importantly, if L. major challenge was delayed by only 14 days after transfer, no recruitment was observed, the Teff could not be found, and immunity was lost. Thus, concomitant immunity is mediated by a population of pre-existing and short-lived Teff. A stable population of Teff is nonetheless maintained by the persisting infection, continuously renewed by the activation of recent thymic emigrants, and/or by the periodic restimulation and differentiation of memory cells.

Concomitant immunity is thus a dynamic state, and memory cells undoubtedly contribute to its maintenance. But their generation is not a sufficient condition for protection; Zinkernagel’s edict²⁴, memory ≠ protection. Just as the best correlate of protection against extracellular infections is the level of pre-existing neutralizing antibody, the counterpart for infections like Leishmania and malaria intracellular stages, is the number of Teff present at the time of exposure. Like antibody, Teff perform critical peripheral immune surveillance functions. They would be expected to have a decided advantage in localized pathogen control over central memory cells whose immune functions are delayed by the time it takes to undergo antigen-stimulated expansion, differentiation, and trafficking. In this context, tissue resident memory cells would be expected to possess the most immediate capacity for localized control of infection, and they function in persistent viral infections to limit the establishment of new infections, particularly in the skin²⁵. It is still not clear, however, if persisting infection is required to maintain their long term tissue residence, whether memory CD4⁺ T cells establish tissue residence, or more generally, if resident memory cells contribute to concomitant immunity in chronic parasitic infections.

The difficulties associated with live, whole organism vaccines are clear and present, and continued priority should be given to defined, subunit vaccines against human parasitic diseases, especially those already in the development pipeline. But the exceptional outcomes of live, whole organism vaccines against human malaria and Leishmaniasis, and the experimental models that have revealed the requirement for persisting antigen to drive the breadth, size, and rapidity of the localized protective response, suggest that there may be no adequate replacement for live, and live-attenuated, whole organism approaches. So rather than endure the long wait for the outcome of clinical trials of sub-unit vaccines to be known, and so long as safety concerns are fully met, these approaches should be pursued as a parallel effort at the least.