Hiding in plain sight: an epitope-based strategy for a subunit malaria vaccine

Abstract

Recent data suggest that approaches to developing a subunit blood stage malaria vaccine may be mis-directed. While antigenic polymorphism is recognized as a challenge, efforts to counter this have primarily involved enhancing the quantity and quality of antibody with potent adjuvants, identifying conserved target proteins, or combining multiple antigens to broaden the immune response. However, paradoxically, evidence has emerged that narrowing, rather than broadening the immune response may be required to obtain an immune response protective against multiple Plasmodium strains. Non-immunodominant, conserved epitopes are crucial. The evidence comes from studying the immune response to red cell surface-expressed antigens but should also be applicable to merozoite surface antigens. Strategies to define the targets of these highly focused immune responses are provided.

Vaccines: the unmet need

Malaria remains a global curse on world health. The latest WHO World Malaria Report [1] documents that an estimated 619,000 people, mostly young children, succumbed to the disease in 2021. While this number is significantly less than what was reported at the turn of the century (896,000 deaths), there has not been a decline in mortality over the last 4 years as increasing levels of insecticide resistance [2] and the pandemic have derailed further progress. The spread of parasite resistance to artemisinin in Asia and Africa presents a further challenge moving forward [3,4], as does the spread of the highly competent peri-urban vector, Anopheles stephensi, to Africa [5], and parasite mutations affecting the performance of rapid diagnostic tests. It is widely accepted that to reduce the number of deaths any further and to move towards elimination, a successful vaccine will be required.

To date, most efforts to develop a vaccine have been put into those that either stop sporozoites from entering the liver or kill infected hepatocytes (‘pre-erythrocytic’ vaccines). RTS,S, based on a hepatitis B surface antigen fusion with the circumsporozoite protein of P. falciparum, is in Phase IV trials and is the most advanced (reviewed in [6]). WHO recently commented on the post-licensure Phase IV trials of RTS,S noting 30% protection against severe disease over a 2-year period (‘Full evidence report on the RTS,S/AS01 malaria vaccine’, in SAGE Yellow Book for October 2021; 5.1_Malaria). In the months following vaccination, immunity gives only 30–50% protection and this wanes rapidly after 6 months as antibody levels decline and exposure to malaria sporozoites fails to boost antibody levels [7]. The other major pre-erythrocytic candidates are the whole parasite sporozoite vaccines, (PfSPZ [8,9] and PfSPZ-CVac [10]), and R21 [11]. Vaccines against the sexual stages of malaria (gametocytes) that could prevent transmission from one infected person to another are also in development [12]. For the blood stages - the stage responsible for all the clinical manifestations of malaria - many different subunit antigens have been tested in pre-clinical and early-stage clinical trials but none have progressed beyond Phase II trials. This article will focus on this stage in the life cycle and present an hypothesis for why progress towards a vaccine has been limited, and then present a novel strategic approach.

Evolution of the current subunit vaccine strategy

Malaria antigens were first cloned in 1983 and there was an expectation that a successful vaccine would soon be developed [13] [14]. The optimism did not seem unreasonable given that in one of the studies [13] an expression library was screened using serum from malaria-immune adults to identify antigens. The theory was that since antibodies from immune adults could dramatically cure malaria infections through passive transfer to sick children [15,16], those antibodies should be able to identify the critical antigen or antigens responsible for inducing such immunity. Candidate malaria vaccine antigens were identified and by 1999 a combination of fragments of three different merozoite recombinant proteins (RESA, MSP1 and MSP2, referred to as ‘Combination B’) was tested for safety and efficacy in Phase I and II trials [17,18]. The trial reported limited efficacy [18]. Since then, over 100 vaccine trials of different subunit blood stage malaria vaccines have been listed on ClinicalTrials.gov. However, WHO reported in November 2022 that only five antigens are still under active investigation, and none having progressed to Phase III clinical trials (WHO review of malaria vaccine clinical development, November 2022). Analysis of the results of the Phase II trials to date shows zero to minimal protection.

The disappointing results of these trials are attributed primarily to the quantity and quality of the antibody response, leading to the testing of novel adjuvants to enhance immunity [19]. The hope is that by selecting potent adjuvants, vaccine antigens that induced limited antibody responses and partial protection would now induce much stronger protection [20]. Antigenic polymorphism, while well recognized as a feature of parasite surface proteins [21], was not considered an insurmountable hurdle because of the view that a recombinant protein will express multiple epitopes (see Glossary), many of which will be found on all strains of Plasmodium [22]. Nevertheless, the evidence that polymorphism is a major contributor to lack of vaccine efficacy was clearly shown in vaccine trials. In the ‘Combination B’ trial in Papua New Guinea [18], genotypic analysis of the parasites found in vaccinated individuals (that had evaded immunity) showed that they predominantly carried different alleles of the msp2 gene to that on which the vaccine was based. The partial protection observed in an MSP3 vaccine trial in Burkina Faso ([23]) may have been due to the known polymorphism within the subunit vaccine ([24]). In the ‘PRIMVAC’ trial, based on the red cell surface-expressed PfEMP1 protein, VAR2CSA, and which aimed to protect against placental malaria [25], the vaccine antigen was from the NF54 strain. Sera from vaccinated individuals induced functional antibodies to the NF54 strain of parasites and could significantly inhibit binding of erythrocytes infected with the NF54 strain to the ligand, chondroitin sulphate A (CSA). However, the sera could not inhibit binding of two other parasite strains (FCR3 and 7G8). A similar result was reported in a different VAR2CSA vaccine trial using the ‘PAMVAC’ vaccine antigen [26]. In an AMA1 (a merozoite surface protein) trial [27], there was also evidence that the vaccine induced strain-specific protection only. The data thus suggest that although recombinant proteins contain multiple potential epitopes, the immune response tends to focus on a limited number of epitopes, which happen to be polymorphic.

The antigens, MSP2, MSP3, VAR2CSA and AMA1 are considered ‘leading’ candidates for a vaccine and their failure as single recombinant proteins is now driving new approaches with these and other antigens. For example, the results have prompted significant interest in: (i) identifying antigenically more conserved protein targets (such as PfRH5 [28]); (ii) combining multiple allelic strains (such as the AMA1-based vaccine, ‘DiCO’ [29]; (iii) partnering AMA1 with its parasite-encoded receptor, RON2 [30,31]; and (iv) exploring different adjuvants, such as Matrix-M, which showed promise in an ‘R21’ sporozoite vaccine trial [11]. These studies are currently at the pre-clinical, non-human primate level or Phase I level of investigation. While all these trials are using large recombinant fragments of the candidate antigens, attempts to use conserved epitopes (the minimal structures recognized by B-cells and often defined by small peptides) have been ignored.

Evidence supporting an epitope-based strategy

The first evidence that an epitope-based vaccine for malaria might be successful can be seen in data published in 1986. Marsh and Howard [32] demonstrated that convalescent serum from children in The Gambia could agglutinate parasitized erythrocytes (pE) of the clinical isolate with which the child was infected, whereas their serum taken at the time of clinical presentation could not. The presence of agglutinating antibodies had long been regarded as a very good correlate of protection against malaria [33]. The data thus suggested that infection had given rise to protective antibodies against the infecting strain. However, looking at many different children, they observed that convalescent serum could only agglutinate the isolate from that particular child; it did not agglutinate isolates from other sick children. The antibodies in these sera were thus targeting variant antigens on the pE, most likely PfEMP1. They then examined serum from immune adults living in the same village as the children and observed that these adult sera could agglutinate the parasites from all the children. These data suggested that immune adults contained either agglutinating antibodies of multiple specificities or that they contained antibodies to a conserved epitope(s) present on all strains (or both). To test this, they took serum from one immune adult and adsorbed it on one of the children’s clinical isolates. The antibodies were then eluted from the isolate and were shown to agglutinate multiple clinical isolates, demonstrating that the serum contained antibodies to a conserved epitope. Furthermore, the antibodies remaining after adsorption were not able to agglutinate any isolate. Thus, not only did the immune adult serum contain antibodies to a conserved agglutinating epitope, but it did not contain any variant-specific antibodies – similar in this last respect to serum from children, which did not contain agglutinating antibodies to other children’s isolates. While other studies also demonstrated that immune adult serum can contain cross-reactive agglutinating antibodies [34], the prevalence of this form of immunity in adults is not known. It is likely that immunity in adults is due to other factors as well, including the presence of antibodies to various merozoite surface proteins [35,36] and to cellular immunity [37,38]. However, the fact that at least some adults do contain cross-reactive agglutinating antibodies provides an important clue that could benefit subunit vaccine design.

These homologous agglutinating red cell data from children and the heterologous agglutinating red cell data from at least some adults are consistent with a model in which an infection leads to short-lived antibodies to a variant-specific epitope but that over many years of exposure, functional antibodies develop to an invariant epitope (Figure 1). Periodic exposure to different parasite strains then boosts the cross-reactive memory B-cells and maintains a level of protective antibodies. Leaving an endemic area would prevent on-going boosting and lead to a drop in the level of these antibodies leaving the individual susceptible to a future infection. This reflects the clinical observation that adults lose their immunity when they leave an endemic area [39,40].

Figure 1. Cartoon depicting antigenic variation on the surface of the infected red cell.

The conserved sub-dominant epitope is in yellow and is present on all strains of the parasite. The other surface colours represent the immunodominant antigens, which are strain specific. It takes many years of exposure before the immune system recognizes the sub-dominant epitope. This is not unique to malaria, but is found in other organisms as well, including Streptococcus pyogenes. Based on data presented in various published papers including [32,34,41,44,45,50]. Created by Pulp Studios Inc., Edmonton.

The data are also consistent with recent studies of antibodies in multigravid women to different recombinant VAR2CSA proteins [41]. Primigravid women are susceptible to placental malaria due to Plasmodium falciparum (even if they were previously immune to non-placental malaria) due to lack of antibodies to VAR2CSA, which specifically binds to chondroitin sulphate proteoglycans uniquely expressed on the placenta. However, after a number of pregnancies while living in a malaria-endemic region, multigravid women become immune to placental malaria and their serum can now inhibit multiple strains of placental malaria parasites from binding to CSA in vitro (a surrogate of protection). Doritchamou et al [41] purified IgG from multigravid women that bound to individual recombinant (rec) VAR2CSA proteins and demonstrated that those antibodies could bind to multiple recVAR2CSA variant proteins and inhibit the relevant P. falciparum strains from binding to CSA. Those data demonstrate that sera from multigravid women contain functional antibodies that recognize a conserved epitope(s) expressed on VAR2CSA. However, as mentioned above [25], vaccination with recVAR2CSA does not lead to antibodies to the conserved epitope(s). These data are very similar to the observations of Marsh and Howard [32] showing that prolonged exposure to different strains of P. falciparum can lead to induction of antibody responses to an invariant epitope(s). The important point is that the exposure must be prolonged to give rise to these cross-reactive antibodies. Exposure resulting from vaccination with recVAR2CSA will not do this; rather, it will induce antibodies to immunodominant epitopes, which are polymorphic. The data of Doritchamou et al [41] and of the PRIMVAC trial [25] thus demonstrate important aspects of the immunobiology of VAR2CSA, which may apply to various malaria antigens: there are two classes of antibodies that bind the protein – those recognizing immunodominant epitope(s) and those recognizing conserved but subdominant epitope(s). Both can be inhibitory to the parasite; however, the former recognize homologous parasites only while the latter recognize heterologous parasites. It is critical that vaccines induce the latter.

Conserved functional epitopes are not unique to malaria

This situation in malaria is very similar to the observations with a different organism, Streptococcus pyogenes (group A Strep, or ‘Strep A’) ([42]). There are hundreds of different Strep A strains, which differ predominantly at the amino terminal end of their surface M-protein, a major virulence factor of the organism that inhibits phagocytosis. Exposure to one strain leads to antibodies to the amino terminus of the M-protein of that strain that can protect against future infection with that strain, but not other strains [43]. After many years of exposure, adults living in highly Strep A-endemic localities develop opsonic antibodies to a highly conserved epitope at the C-terminal region of the protein [44,45]. These antibodies can opsonize all strains of Strep A. This area is not immunodominant and young children from endemic areas do not have antibodies to this sub-dominant/cryptic epitope. However, vaccination with the epitope (conjugated to CRM [a carrier protein used in different vaccines to provide T-cell help]) can protect mice from all strains. The vaccine is now undergoing a Phase I clinical trial. Even though infection of children [45] or naïve mice [46] with Strep A does not lead to induction of antibodies to the conserved epitope, prolonged exposure in an endemic area does eventually give rise to these antibodies [45]. Furthermore, infection of mice that were vaccinated with the epitope leads to significant boosting of the antibody response to the epitope [46] and an enhanced level of protection. It is hypothesized that this is due to the increased number and avidity of epitope-specific memory B-cells, compared to epitope-specific naïve B-cells in an unvaccinated animal.

Why do malaria or streptococcal infections of children lead to variant-specific antibody responses and not responses to cryptic or sub-dominant epitopes? In the case of the M-protein, the N-terminal region, which is the most exposed and therefore most likely to be recognized by B-cells, is the polymorphic region. The amino acid sequences there do not have a specific biological function for the organism other than to attract the antibody response and prevent B-cells targeting conserved regions of the protein, which could lead to immunity to all strains.

Identifying protective epitopes

A vaccine lesson from Strep A is that even though the conserved regions of proteins may be cryptic or subdominant and not recognized during infection, antibodies targeted to these regions by vaccination can bind the organism. But how can they be identified? In Strep A they were identified by making a series of synthetic overlapping peptides spanning the conserved region of the M-protein and vaccinating mice with each of them to ask whether antibodies that were induced to any of them could opsonize the organism. A related approach for malaria would be to make a series of conserved peptides representing surface antigens present on the infected red cell and generate a panel of peptide-specific sera in mice, which could be tested in functional assays. Alternatively, antibodies from immune adults could be eluted from recombinant malaria proteins or parasitized erythrocytes (pE) and screened against libraries of peptides made synthetically or in phage or other systems. Identified epitopes could then be tested for their ability to induce strain-transcending functional antibodies. In one study, monoclonal antibodies were generated in mice to the CIDR1 region of PfEMP1 and two were shown to agglutinate multiple strains of pE [47]. However, attempts to identify the minimal epitope were unsuccessful suggesting that the epitope was non-linear. Others have used high density linear arrays to identify multiple B-cell epitopes in malaria [48] and some of these could progress to clinical trial.

Yet a different approach to identify subdominant epitopes is being taken in placental malaria. Sera from men and children who had been exposed to P. vivax in South America were shown to inhibit the binding of P. falciparum infected red cells to CSA [49]. This was unexpected since inhibition of binding of P. falciparum to CSA was only known to be associated with antibodies to VAR2CSA and which had arisen following multiple placental infections with P. falciparum. Subsequently, a monoclonal antibody raised to the P. vivax protein, PvDBP, was shown to inhibit the binding of three different placental strains of P. falciparum pE to CSA regardless of polymorphisms in VAR2CSA [50]. Thus, the data suggest that there is a cryptic protective epitope in VAR2CSA that is similar to the epitope in P. vivax that gave rise to the PvDBP-specific mAb. The biological explanation for this is that both P. falciparum VAR2CSA and P. vivax DBP contain Duffy binding domains with similar structure and may share a conformational epitope. Most epitopes in nature are conformational ([51]) and identifying and recapitulating conformational epitopes is challenging but has been achieved using cyclic peptides that incorporate scaffolds to generate a variety of secondary structures [52]. Indeed, a conformationally-constrained peptide of subdomain 1 from PvDBP elicited cross-reactive antibodies to VAR2CSA in mice and rabbits [53]. Identifying this and other conserved epitopes [54] via linear peptide arrays, mimotopes [55] or cyclic peptides, or other technologies could enable the design of a vaccine that would induce functional antibodies against multiple strains of P. falciparum responsible for placental malaria.

Concluding remarks

The strategies proposed here are based primarily on observed immune responses to variant red cell surface antigens, following natural infections and vaccine studies. Identifying conserved target epitopes provides a novel unexploited strategy for blood stage vaccine development. Although there are less comparable data on immune responses to conserved epitopes on merozoite proteins, highly conserved proteins (such as PfRH5 and ABRA [also known as MSP9]) have been studied and a short peptide epitope from ABRA induced significant growth inhibitory antibodies in rabbits[56], but this peptide has not been tested in a human trial.

Short peptides have not yet been licensed for use in any human vaccine. However, they may find their niche in fighting organisms that escape immunity by presenting immunodominant epitopes, thus evading exposure of highly sensitive conserved cryptic epitopes (see Outstanding questions).

Outstanding Questions.

• Do antibodies to conserved sub-dominant epitopes contribute to immunity in all adults, or in just some adults?

• Are sub-dominant epitopes also found on merozoite surface proteins and would they also induce protective responses?

• How common will linear sub-dominant epitopes be in malaria? Linear epitopes will be easier to define.

• Will malaria infection boost the antibody response to a vaccine-induced sub-dominant epitope, as streptococcal infections do to a sub-dominant protective epitope on the M-protein?

Highlights.

• Antigenic polymorphism is the major obstacle to sub-unit vaccine development for malaria

• PfEMP1 is a major variant antigen on the surface of infected erythrocytes

• Serum antibodies from adults, but not children, in malaria-endemic areas recognize multiple strains of infected erythrocytes and recombinant PfEMP1s

• However, vaccination with the pregnancy-associated PfEMP1 variant, VAR2CSA, induces only strain-specific antibodies

• Thus, years of repeated exposure are required to develop antibodies to conserved epitopes that can be targets of protective immunity

• Other organisms, including Streptococcus pyogenes also have a conserved cryptic epitope on their major virulence factor, the M-protein; vaccination with this epitope can induce protection against multiple strains of S. pyogenes

• Conserved cryptic epitopes could be defined for P. falciparum - representing a novel vaccine strategy

Glossary

Epitope

the smallest determinant on an antigen recognized by an antibody

Heterologous

of a different strain

Homologous

of the same strain

Mimotope

a synthetic or recombinant fragment designed to mimic a conformational epitope

Multigravid woman

a woman who has had multiple pregnancies

Primigravid woman

a woman during her first pregnancy

Subunit vaccine

a vaccine consisting of onlyone or a few antigens from the organisms