SUMMARY


That humans in endemic areas become immune to malaria offers encouragement to the idea of developing protective vaccines However natural immunity is relatively inefficient, being bought at the cost of substantial childhood mortality, and current vaccines are only partially protective Understanding potential targets and mechanisms of protective immunity is important in the development and evaluation of future vaccines Some of the problems in identifying such targets and mechanisms in humans naturally exposed to malaria may stem from conceptual and methodological issues related to defining who in a population is susceptible, problems in defining immune responsiveness at single time points and issues related to antigenic polymorphism, as well as the failure of many current approaches to examine functional aspects of the immune response Protective immune responses may be directed to the pre erythrocytic parasite, to the free merozoite of the blood stage parasite or to new antigens induced on the infected red cell surface Tackling the methodological issues of defining protection and immune response, together with studies that combine functional assays with new approaches such as allelic exchange and gene knock out offer opportunities for better defining key targets and mechanisms

Immune effector mechanisms in malaria.

Plasmodium falciparum infections in malaria-naive individuals can lead to severe morbidity, which may be life threatening if untreated. Continued exposure to infection leads to a degree of immunity, and consequently, older children and adults living in areas of endemicity are protected from the severe clinical consequences of infection with P. falciparum. The effector mechanisms that mediate naturally acquired immunity to malaria are not completely understood, but antibodies to the asexual blood stage parasites play a role. This has been most clearly demonstrated by the reduction of parasitemias following the passive immunization of children with clinical malaria with immunoglobulin G (IgG) from malaria-immune adults (6, 7, 32).

Antigens recognized by antibodies that are active in passive-immunization experiments are prime candidates for testing in a vaccine. Studies with an assay for antibody-dependent cellular inhibition have identified antibodies to MSP3 as an active component of passively transferred human IgG (28), but much evidence indicates that other merozoite antigens are capable of inducing antibodies that limit parasite development (13–15, 17, 26, 29, 33, 34).

One of the prime candidate antigens for inclusion in a malaria vaccine is apical membrane antigen 1 (AMA1). AMA1 is an 83-kDa antigen that is synthesized in mature stages of the parasite and is initially localized in the necks of the rhoptry organelles (9, 30). At about the time of merozoite release, the full-length 83-kDa molecule is localized at the apical pole, and an N-terminally processed form of 66 kDa can be detected distributed around the merozoite surface (27, 30). Although the biological function of AMA1 is unknown, its location and stage specificity suggest that it may be involved in the process of erythrocyte invasion.

AMA1 is one of only a few asexual blood stage antigens that have been identified in all Plasmodium species examined (42), and this has enabled the vaccine potential of AMA1 to be investigated using various animal models. Active immunization of monkeys or mice with either native (11) or recombinant (2, 8) forms of AMA1 has protected these animals against simian and rodent parasites, respectively. Much evidence indicates that anti-AMA1 antibodies mediate protection. Monoclonal antibodies raised against P. falciparum AMA1 and against PK66, the Plasmodium knowlesi homologue of AMA1, inhibit merozoite invasion in vitro (20, 35). Furthermore, passive immunization of AMA1-specific polyclonal antibodies into Plasmodium chabaudi-infected mice prevented lethal parasitemias (2). These protective antibodies react with conformational epitopes stabilized by disulfide bonds, as immunization with the reduced and alkylated AMA1 failed to protect mice against challenge with P. chabaudi (10).

The sequence of AMA1 is relatively conserved among various Plasmodium spp., with the level of amino acid sequence identity exceeding 50% in pairwise comparisons among all known sequences (5, 12, 24, 25, 31, 42). AMA1 lacks the sequence repeats and marked polymorphisms found in other malaria antigens, such as the merozoite surface antigens MSP1 and MSP2 (3). However, some sequence variation, resulting from point mutations, is observed among alleles of AMA1 in P. falciparum (25, 30, 36), P. knowlesi (43), Plasmodium vivax (5), and P. chabaudi (10), and studies with the P. chabaudi-mouse model indicate that this variation is immunologically significant. Mice immunized with AMA1 or receiving passively transferred anti-AMA1 antibodies were not protected from a heterologous strain of P. chabaudi parasites, indicating that the protective antibodies recognized strain-specific epitopes.

Early clinical trials with AMA1 have commenced, and it is important to determine the effect of sequence diversity on the efficacy of the recombinant AMA1 as a vaccine against P. falciparum. In this study, we demonstrate that immunization of rabbits with the refolded P. falciparum AMA1 ectodomain (the vaccine molecule) induces antibodies that inhibit merozoite invasion in vitro. The refolded antigen has also been used to affinity purify AMA1-specific antibodies from the plasma of individuals who have been exposed to chronic malaria infections. These naturally occurring human antibodies were also able to inhibit the invasion of erythrocytes by P. falciparum merozoites.

Specificity of the Protective Antibody Response to Apical Membrane Antigen 1

Background
One of the criteria to objectively prioritize merozoite antigens for malaria vaccine development is the demonstration that naturally acquired antibodies are associated with protection from malaria. However, published evidence of the protective effect of these antibodies is conflicting.

/pdf/the-relationship-between-anti-merozoite-antibodies-and-yie2jikhq9.pdf

The Relationship between Anti-merozoite Antibodies and Incidence of Plasmodium falciparum Malaria: A Systematic Review and Meta-analysis

Malaria accounts for an enormous burden of disease globally, with Plasmodium falciparum accounting for the majority of malaria, and P. vivax being a second important cause, especially in Asia, the Americas and the Pacific. During infection with Plasmodium spp., the merozoite form of the parasite invades red blood cells and replicates inside them. It is during the blood-stage of infection that malaria disease occurs and, therefore, understanding merozoite invasion, host immune responses to merozoite surface antigens, and targeting merozoite surface proteins and invasion ligands by novel vaccines and therapeutics have been important areas of research. Merozoite invasion involves multiple interactions and events, and substantial processing of merozoite surface proteins occurs before, during and after invasion. The merozoite surface is highly complex, presenting a multitude of antigens to the immune system. This complexity has proved challenging to our efforts to understand merozoite invasion and malaria immunity, and to developing merozoite antigens as malaria vaccines. In recent years, there has been major progress in this field, and several merozoite surface proteins show strong potential as malaria vaccines. Our current knowledge on this topic is reviewed, highlighting recent advances and research priorities.

https://minerva-access.unimelb.edu.au/bitstream/handle/11343/258332/PMC4852283.pdf

Merozoite surface proteins in red blood cell invasion, immunity and vaccines against malaria

Background Surrogate markers of protective immunity to malaria in humans are needed to rationalize malaria vaccine discovery and development. In an effort to identify such markers, and thereby provide a clue to the complex equation malaria vaccine development is facing, we investigated the relationship between protection acquired through exposure in the field with naturally occurring immune responses (i.e., induced by the parasite) to molecules that are considered as valuable vaccine candidates.

/pdf/long-term-clinical-protection-from-falciparum-malaria-is-3g7iqc04ks.pdf

Long-term clinical protection from falciparum malaria is strongly associated with IgG3 antibodies to merozoite surface protein 3.

Monocyte-dependent as well as direct inhibitory effects of antimalarial antibodies point toward antigens accessible at the time of merozoite release as targets for biologically active antibodies capable of mediating protection against Plasmodium falciparum. The glutamate-rich protein (GLURP), being an antigen associated with mature schizont-infected erythrocytes, was therefore the object of the present investigation, in which we analyzed whether anti-GLURP antibodies can either interfere directly with merozoite invasion or act indirectly by promoting a monocyte-dependent growth inhibition, antibody-dependent cellular inhibition. GLURP-specific human immunoglobulin G (IgG) antibodies, from pooled IgG of healthy Liberian adults who were clinically immune to malaria, were purified by affinity chromatography on columns containing R0 (N-terminal nonrepetitive region of GLURP) or R2 (C-terminal repetitive region of GLURP) recombinant protein or synthetic peptides as ligands. Analysis of the pattern of reactivity of highly purified anti-GLURP antibodies led to the definition of at least four B-cell epitopes. One epitope was specific for R0, two were specific for R2, and the fourth displayed cross-reactivity between R0 and R2. None of the purified IgG antibodies had direct invasion-inhibitory effects, even at high concentrations. In contrast, when allowed to cooperate with monocytes, all anti-GLURP IgG preparations mediated a strong monocyte-dependent parasite growth inhibition in a dose-dependent manner.

https://iai.asm.org/content/iai/66/1/11.full.pdf

The Glutamate-Rich Protein (GLURP) of Plasmodium falciparum Is a Target for Antibody-Dependent Monocyte-Mediated Inhibition of Parasite Growth In Vitro

Identification of a major B-cell epitope of the Plasmodium falciparum glutamate-rich protein (GLURP), targeted by human antibodies mediating parasite killing.

Steffen Danielsen

Papers

The Glutamate-Rich Protein (GLURP) of Plasmodium falciparum Is a Target for Antibody-Dependent Monocyte-Mediated Inhibition of Parasite Growth In Vitro

Identification of a major B-cell epitope of the Plasmodium falciparum glutamate-rich protein (GLURP), targeted by human antibodies mediating parasite killing.