Vaccines Against Parasites

1.1

Naturally Acquired Immunity and Immune Evasion Mechanisms

In endemic regions, young children are highly susceptible to deaths due to malaria. With exposure, older children rarely die due to malaria, and may become immune to severe disease after only a few malaria episodes. Importantly, older children, despite years of repeated exposures, do not have sterile immunity and can still develop mild febrile illness. Immunity to infection, therefore, is slow to develop, but the reduced prevalence of symptomatic infection with age and lower rates of severe disease indicate that natural immunity does occur. Understanding the nature of this slow developing immunity is thought to be key to vaccine design. Naturally acquired resistance seems not to depend on an immune response directed at the preerythrocytic stages because immune adults are still protected against symptoms when liver-stage infection is bypassed by direct challenge using blood-stage parasites. The absence of a strong protective response directed against the liver stages is thought to be due to the low numbers of transmitted sporozoites, that is, the form injected by the mosquito, that results in too few hepatocytes becoming infected to induce an immune response, or to be detected quickly enough by effector T cells to prevent their release of merozoites into the blood, which infect and damage RBC ( Fig. 17.1 ).

The acquired resistance that does develop in older children after repeated exposure is mainly against blood-stages, directed to antigens that are expressed on the merozoite surface, or on the surface of infected RBC. In each case the target antigens are highly polymorphic due to both allelic and somatic gene variation, and the prevailing view is that development of immunity requires the accumulated exposure to a large number of strains circulating in a community so as to cover the diverse repertoire of blood-stage antigens. The importance of antibody in naturally acquired immunity was directly demonstrated long ago by the passive transfer of gamma-globulin from immune adults into semiimmune individuals that conferred stronger protection against blood-stage infection. Antibodies can function by blocking merozoite invasion of RBCs, or by promoting the effective clearance of parasitized RBCs by the spleen. The best characterized of the variant surface antigens is P. falciparum erythrocyte membrane protein-1 (PfEMP-1), which the parasite exports to the red cell surface to mediate binding to vascular endothelial receptors, allowing the parasite to sequester in peripheral tissues and avoid being cleared in the spleen. Adhesion by infected RBCs to the endothelial cells lining blood vessels and capillaries clogs the microvasculature, triggers local inflammation, and gives rise to the cerebral, respiratory, and renal symptoms associated with severe malaria. Antibodies against PfEMP-1 block adherence or sequestration, and the parasite employs a system of clonal antigenic variation to produce chronic infection. There are more than 60 copies of the gene for PfEMP-1 or var genes per parasite, tightly regulated at the transcriptional level and only 1 gene is expressed at a time. PfEMP1 has been implicated as the key target antigen involved in naturally acquired immunity and the extensive antigenic variation of PfEMP-1 poses the most serious obstacle to the development of a blood-stage vaccine.

Antibodies targeting merozoite proteins involved in erythrocyte invasion can also contribute to acquired immunity. Malaria parasites possess apical organelles that contain hydrolytic enzymes required for cell invasion. Several P. falciparum proteins have been shown to be involved in erythrocyte invasion, including merozoite surface protein-1 (MSP-1) involved in the initial attachment, merozoite apical membrane antigen-1 (AMA-1) that mediates the reorientation of merozoites, and erythrocyte binding ligands (EBLs) that bind to sialic acid containing glycoproteins on the erythrocyte membrane to create the tight junction that allows invasion to proceed. The genes encoding each of these proteins display extensive different allelic forms or polymorphisms, and antibody-mediated inhibition of the invasion process is generally strain-specific.

Last, a consideration of immune memory will be critical to the design of any malaria vaccine. Based largely on anecdotal evidence, it seems that immunity to malaria is rapidly lost if an individual leaves an endemic region and then returns, suggesting that there is poor immunological memory, and that continued exposure to malarial antigens is required to maintain the populations of memory and/or effectors cells necessary for protection. Consistent with this continuous exposure requirement, in young children living in low transmission areas, antibody responses to merozoite antigens appear to be short-lived. With respect to memory T cells, Th1 cells producing IFNγ gradually declined in the absence of continued malaria exposure, and rodent models also indicate a rapid decay in the frequency of memory CD4+ T cells in the absence of infection. The requirement for continuous exposure or persisting infection to maintain the threshold number of memory and/or effector cells required for protection has crucial implications for the design of an effective vaccine.

1.2

Preerythrocytic Stage Vaccines

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 glands. These trials were inspired by the groundbreaking studies in mice using intravenous inoculation of irradiated Plasmodium berghei sporozoites. In each case the radiation-attenuated parasites were unable to develop beyond their liver stages, and live, metabolically active sporozoites were required for the protection. Protection also required a high dose exposure to the sporozoites, with more than 1000 mosquito bites needed to achieve sterile immunity. Studies in the mouse revealed that CD8+ T cells are paramount for protection, and the high-dose immunization, by generating greater numbers of C8+ T cells to more effectively survey the extremely low numbers of infected hepatocytes following natural transmission, likely explains why irradiated sporozoites can achieve far better protection than natural infection.

The inability to grow sporozoites in culture posed a major obstacle to their wide application as live, attenuated vaccines. The cloning of the gene for the major surface coat on sporozoites, called circumsporozoite protein (CSP), and its identification as a major target of the antibody and T-cell response in vaccinated mice and people, led to a number of clinical trials involving recombinant protein- or DNA-based CSP vaccines. The initial trials, however, showed disappointing efficacy against sporozoite challenge. A newer formulation of the P. falciparum CSP, called RTS,S, is the most clinically advanced and encouraging malaria vaccine candidate to date. The vaccine is produced by GlaxoSmithKline (GSK) and incorporates the CSP repeat region (R) and T-cell epitopes (T) as fusion proteins with hepatitis B surface antigen (S) that along with unfused hepatitis B surface antigen (S), spontaneously assemble into virus-like particles. The vaccine is given with a new adjuvant system, AS02 _A , which contains monophosphoryl lipid A (MPL) and saponin in an oil-in-water emulsion. An important private/public partnership between GSK and PATH malaria vaccine initiative (MVI) has carried out the pediatric development of RTS,S. The main evidence for its efficacy derives from a large clinical trial conducted in seven African countries showing modest protection against P. falciparum malaria in 56% of children aged 5–17 months, and in 31% of children aged 6–12 weeks, without significant protection from severe malaria after 18 months. Despite these modest results, the benefits of vaccination to reduce mortality among children in high-transmission areas was considered important enough that its use has recently been recommended by a regulatory agency, the European Medicines Agency, an important step toward eventual licensure.

Because irradiated sporozoite immunization still represents the gold standard for sterile protection, there remains a concerted effort to understand and reproduce the essential features of the immunity conferred by this whole sporozoite vaccine. The most direct and ambitious approach has been to scale up the production of irradiated, asceptic, cryopreserved sporozoites manually dissected from mosquito salivary glands, and deliver them by needle. The most recent studies employing this vaccine found that high i.v. doses (>600,000 sporozoites) completely protected six out of six volunteers against infectious sporozoite challenge. Despite the clear disadvantages associated with the need for high doses and i.v. administration, the approach still represents the first innocuous vaccine produced under current good manufacturing practice (cGMP) standards, and delivered by needle to confer sterile immunity against malaria. Further studies are needed to determine if the vaccine confers long term heterologous protection against other strains, and to define the immune correlates of protection.

In the meantime, additional approaches employing whole organism sporozoite vaccines are being pursued, including genetically modified parasites that are arrested at a late stage in liver-stage development, and chemoprophylaxis with infectious sporozoites. The advantage of these approaches compared to nonreplicating, irradiated sporozoites is that a greater repertoire and quantity of liver stage antigens is expressed. Sterile immunity was reported in the clinical trial of 10 volunteers immunized by repeated exposures to infectious mosquitoes under chemoprophylaxis with chloroquine that kills the merozoites as they emerge from the liver. Importantly, all were completely protected against blood-stage infections following sporozoite challenge, some volunteers for as long as 2 years, and some even against heterologous challenge, suggesting that liver stage immunity may not be strain-specific. While the requirement for large numbers of infected mosquitoes delivering high doses of sporozoites is clearly not practical for large scale vaccination, recent progress in cryopreservation of purified, infectious sporozoites for needle inoculation should enhance the feasibility of this approach.

Because the sterile immunity conferred by whole sporozoite vaccines is associated with strong CD8+ T-cell responses, a number of prime-boost strategies vaccines have been tried to induce CD8+ T cells targeting liver stage antigens in people. In the most recent trial, a heterologous prime-boost vaccine employing a replication-deficient chimpanzee adenovirus vector followed by a modified Vaccinia virus Ankara booster induced high frequencies of CD8+ T cells specific for the liver stage antigen ME-TRAP, and lower blood stage parasitemia was observed in 8 of 14 volunteers following controlled human malaria infection (CHMI), but sterile immunity in only three of them. The same vectors encoding CSP showed even lower efficacy then ME-TRAP. Thus, a strong CD8+ T-cell response that targets a single liver stage antigen does not appear to be a sufficient condition to confer sterile immunity, and other attributes of whole sporozoite vaccines, notably the multiplicity and persistence of the protective antigens that they present, may be essential qualities that can only be accommodated by live, and live-attenuated vaccination strategies. The next few years should yield critical new information regarding the immunogenicity of these vaccines in infants and semiimmune children, and the strain transcendence and duration of the immunity induced.

1.3

Blood Stage Vaccines

The rationale for the development of a blood-stage vaccine is based on the naturally acquired immunity that is directed against blood stages of the parasite, and the fact that sterilizing immunity is not necessary to achieve a reduction in the severity of illness or the transmissibility of infection. Unfortunately, despite a number of candidate vaccines progressing to clinical testing, in no case have Phase II trials demonstrated significant efficacy to justify further evaluation in a Phase III clinical trial. The main vaccine candidates, including AMA-1 and MSP1 were selected based on in vitro assays showing that they are targets of antibody responses that inhibit merozoite invasion of erythrocytes. In one of the few trials to show some efficacy, analysis of breakthrough infections showed that the protection was strain specific, with the monoallelic MSP2 component of the vaccine selecting for patent infections with a strain expressing another MSP2 allele. A recombinant AMA1 vaccine appeared to confer a similar strain-specific immunity, and the short-lived protection conferred by a recombinant vaccine in Kenya was thought to be due to polymorphisms in the region of MSP3 covered by the vaccine.

Because sterile immunity is unlikely to be achieved by any blood-stage vaccine, there is widespread agreement that a more achievable vaccination goal is to reduce the incidence of severe disease, including death. The fact that immunity to severe disease develops more rapidly than immunity to mild disease suggests that severe disease might be associated with a restricted set of PfEMP-1 variants, and there is recent evidence to support this possibility. The identification of a particular PfEMP1 subset associated with severe malaria that binds endothelial protein receptor C (EPCR), and the characterization of broadly inhibitory antibodies that bind to a conserved EPCR binding structure on PfEMP1, offer a sound rationale for development of a recombinant vaccine targeting this conserved structure. Similarly, a particular PfEMP1 variant, known as var2csa, binds selectively to chondroitin sulfate A (CSA) and mediates attachment of infected erythrocytes to the syncytiotrophoblasts of the placenta. Pregnancy-associated malaria produces high rates of fetal mortality and accounts for a very high risk of maternal death as well. Naturally acquired immunity to pregnancy malaria is indicated by the reduced incidence of placental infections that is directly correlated with the number of pregnancies and the increased levels of maternal antibodies against var2csa. Thus, var2csa is considered a leading candidate to prevent malaria in pregnancy.

1.4

Transmission-Blocking Vaccines

Transmission-blocking vaccines (TBV) are designed to target antigens expressed on gametocytes or ookinetes, the rare sexual forms of malaria parasites that are acquired from blood meals by mosquitoes. Antibodies have been shown to reduce oocyst development in the mosquito below the number required to produce a transmissible infection. The vaccinated individuals are intended to be the source of both the sexual stage parasites and the transmission-blocking antibodies, Transmission-blocking antibodies are sometimes referred to as “altruistic vaccines” because although the vaccinees might not be directly protected, they would contribute a potentially important indirect benefit by helping to reduce the level of malaria transmission in their community. One advantage of TBVs is that the target antigens are under little if any immune selection pressure and thus tend to be conserved. This is particularly true of antigens confined to mosquito stages of the parasite, for example, ookinetes. The disadvantage is the absence of natural boosting. Because nonsterilizing immunity induced by other vaccines may not reduce the number of infection reservoirs in a community, TBVs may be an important component of any multipronged effort to control malaria, especially if malaria eradication is the ultimate goal. To date, the only TBV that has progressed to clinical testing is Pfs25 and its ortholog in Plasmodium vivax , Pvs25, each of which is expressed on zygote and ookinete stages. A Phase Ia trial involving recombinant Pvs25 given intramuscularly in combination with hydrogel as adjuvant produced modest antibody titers and modest transmission-blocking activity. Efforts to improve the immunogenicity of Pfs25 include fusion with heterologous proteins and expression by viral vectors or as protein nanoparticles, each of which elicited high anti-Pfs25 titers in mice. Clinical trials of these vaccines have been initiated.