Still seeking an effective "one-two" malaria vaccine punch.

Malaria is a mosquito-borne infectious disease that has been estimated to occur in more than 200 million people, resulting in about 1 million deaths per year, including those of many children in Africa. Vaccine development has been complicated by the biology of the eukaryotic malaria parasite that causes chronic or repeated infections and has evolved sophisticated immune evasion mechanisms during its multistage life cycle. In this issue of Molecular Therapy, Sheehy et al. report on a clinical trial evaluating the efficacy of a viral-vectored subunit vaccine to protect against the malaria-causing parasite Plasmodium falciparum.1 Despite showing evidence of a robust T-cell response to the vaccine antigens and a slowing of progression to the blood stage of the disease, only 1 of 36 vaccinated volunteers showed complete protection from a malaria-infected mosquito bite. This study highlights the value of the human malaria challenge model to test vaccine concepts as well as the challenges for the development of an effective malaria vaccine. Which components of the immune system must be recruited for a successful vaccine? Which antigen(s) should be targeted? Also, not trivially, what adjuvant and delivery method will best evoke the desired immune response? Trial outcomes suggest that neither T cells nor antibody-producing B cells alone are sufficient for complete protection against such a complex organism. Furthermore, there remains much work to be done in identifying the proper delivery system and proteins to target if there is to be a subunit vaccine. After all, the malaria parasite is antigenically diverse both throughout its life cycle and among the distinct Plasmodium parasite strains and species that cause malaria. The malaria life cycle is characterized by a succession of briefly extracellular and longer lasting intracellular parasite stages, and includes pre-erythrocytic (liver stage) and blood stages of infection (Figure 1). This enormous complexity poses challenges to the immune system and malaria vaccine researchers. To date, one of the few methods of eliciting sterile protection from experimental human malaria infection has been immunization with radiation-attenuated sporozoites that arrest during development in the liver.2,3 Unfortunately, this strategy requires over 1000 mosquito bites from infected, irradiated mosquitoes, which is neither scalable nor deployable. Although immunization with late liver stage–arresting, genetically attenuated sporozoites confers superior immunity in rodent models, optimizing sporozoite vaccines for intradermal or subcutaneous delivery, as well as improvements in scaling and delivery of live sporozoites remain key issues.4,5,6 Recombinant protein–adjuvant formulations provide an alternative approach, but the challenge has been identifying suitable targets. Results from the first phase III clinical trial of a malaria subunit vaccine (RTS,S) were reported recently. This vaccine targets the circumsporozoite protein (CSP) found on the surface of the infectious sporozoite stage. Despite evidence for strong antibody- and cell-mediated immunity, efficacy of only 35–50% was achieved, and the effect was not long-lasting.7 Thus, targeting only one antigen of one stage of the parasite may not be ideal, and new approaches are needed to deliver a more effective “one–two” vaccine punch. Ideally, this vaccine would stimulate both antibody and T-cell responses and target more than one parasite stage. However, finding a vaccine formulation that accomplishes all these tasks remains immensely challenging. Figure 1 The malaria life cycle. The cycle consists of the following life stages: mosquito, pre-erythrocytic/liver, and blood. Plasmodium is primarily an intracellular parasite (in the liver and red blood cell, RBC), and the malaria parasite is only briefly extracellular ... Sheehy et al.1 evaluated a new vaccine strategy in humans to induce humoral and cellular immunity and improve cross-stage immunity. The prime-boost approach consisted of viral vectors expressing either a single or multiple antigens that target the predominantly blood-stage antigens (MSP1 and AMA1) or the pre-erythrocytic antigen (multiple-epitope–thrombospondin-related adhesion protein, ME-TRAP). Although recombinant MSP1 and AMA1 immunogens have so far failed in human vaccine trials, previous efforts focused on eliciting high-titer neutralizing antibodies against blood stages. In a mouse model of malaria, a similar dual-virus prime-boost approach showed protection from blood-stage challenge.8 In this case, blood-stage protection was due almost exclusively to high-titer antibodies. Conversely, a subsequent study using the mosquito-bite challenge showed good efficacy with MSP1 viral vaccines, with evidence for multistage immunity that appeared to require both CD8 and CD4 T cells.9 Other work showed that immunization with ultra low-dose blood-stage parasites induced cross-stage immunity against late liver stages mediated by CD4 and CD8 T cells.10,11 Together, these findings set the stage for the current human trial to evaluate whether a vaccine strategy designed to confer cross-stage immunity would be more effective than blood-stage immunity alone. Unfortunately, despite eliciting a strong cellular immune response against the vaccine antigens in both single- and double-antigen–expressing vectors, this may have come at the expense of a robust antibody response and a resulting failure to protect immunized individuals from mosquito-bite challenge. There was some evidence of a delay in parasite progression from the liver to blood, which could help reduce blood-stage burden. However, the absence of high-titer blood-stage antibodies capable of slowing parasite growth prevented effective control of infection. This finding is reminiscent of another recent adenovirus 5–vectored trial targeting a combination of AMA1 and CSP antigens that ultimately was unable to protect any of the volunteers.12 Another important aspect of the new study is evaluation of immune interference and immune dominance in a multiantigen vaccine. Mouse studies from the same group, using a similar vaccine strategy, showed that the MSP1–CSP combination reduced the CD8 T-cell response but not the antibody or CD4 T-cell response and thus resulted in less protection from sporozoite challenge.13 This difference was largely mitigated in mice by administering MSP1 and CSP at separate sites, a strategy replicated in the clinical study reported here.13 However, the authors still saw significant antigenic interference from MSP1, as it reduced AMA1-specific but not MSP1 antibodies and T-cell responses. This effect of MSP1 on AMA1 was also suggested from an earlier study in rhesus macaques.14 Therefore, antigen choice will not depend solely on protective ability but also on the ability to complement, not interfere with, other antigens. Does this result mean that T cell–inducing blood-stage malaria vaccines are not viable? Antigens that lead to cross-stage immunity following blood-stage immunization regimens remain undefined.10,11 In mouse models, immunization with individual defined T-cell epitopes from MSP119 did not confer protection, yet other MSP1 epitopes as well as the enzyme hypoxanthine guanine xanthine phosphoribosyl transferase have been suggested to be a major target for cell-mediated immunity.15 Thus, more work is needed to define protective T-cell antigen targets, and their protective ability could depend on antigen density or other peculiarities of class I and class II antigen processing and cross-presentation from infected hepatocytes and infected red blood cells. Sterile protection is a challenging vaccine goal for an organism to which most individuals never develop sterile immunity, even after repeated infections. Subunit vaccine approaches have struggled to achieve this level of protection, and whole-parasite vaccine approaches are still being optimized. However, even a partially effective malaria vaccine may still be beneficial if it can achieve enough control of the blood-stage parasite replication to prevent disease and lead to natural boosting through natural infections. Identification of better blood-stage antigens that are less polymorphic and are more susceptible to cell-mediated or humoral responses may be crucial.16 Another priority should be to identify additional antibody targets on the sporozoite, as the pre-erythrocytic stage of infection is under much less immune pressure than blood stage. Results from whole-parasite blood-stage or sporozoite immunization protocols should be instructive in addressing these issues as we begin to understand the immunological markers and antigenic targets that define an effective vaccine.