Malaria vaccines

Malaria vaccines

Guest editorial

Alberto Moreno and Manuel E Patarroyo

Universidad Nacional de Colombia, Santaf~ de Bogota, DC Colombia

Current Opinion in Immunology 1995, 7:607-611

Introduction

Malaria is by far the most important human parasitic disease. The World Health Organization has estimated that around 270 million clinical cases are reported annually, with an overall mortality of 3 million people per year, mainly in sub-Saharan Africa [1]. Several efforts have been made to develop effective controls such as anti- vector and anti-parasite measures. The most relevant approach in terms of epidemiological impact, however, is the design of effective and inexpensive vaccines.

Malaria vaccine development has a long research history. In the 1970s, successful induction of protective immune responses against Plasmodium falciparum and P. vivax was shown using irradiated sporozoites as immunogens [2]. Further improvements to this classical approach of bi- ological vaccines have been impeded by the extreme complexity of the parasite life cycle, and by biosafety and logistical constrains.

In theory, vaccines must be targeted at the different stages of the parasite cycle and must contain both B- and T-cell epitopes. The aim of modern vaccines is to induce, in addition to a protective immunity, a boosting effect by natural exposure. This is particularly important in highly endemic areas where the most susceptible pop- ulations are children under five years of age [3]. Modern technologies must be used to design a safe multicompo- nent and multistage product; these include recombinant and synthetic systems with subunit components [4].

Pre-erythrocytic vaccines are targeted to block the first step of malarial infection: the invasion of the hepatocytes by the parasites introduced into the body during the mosquito bite [5]. Considering the fluctuation in the size of the inoculum and the high rate of division of the parasites within the hepatocyte, pre-erythrocytic vaccines need to reduce substantially the development of the liver stage parasites. Their effectiveness must be at a range well above 90% in order to prevent progression of the disease.

Blood stage vaccines aim to decrease the level of par- asitaemia and thus reduce disease severity [6]. The decrease in parasite multiplication must modify the mortality and severity of the disease. Even partial immunity

to the asexual blood stage parasites could be useful, as disease severity is generally related to the level of para- sitaemia [7].

Sexual blood stage or transmission-blocking vaccines have to be designed to impede the sexual reproduction of the parasite. These vaccines could destroy gametocytes, interfere with fertilization or prevent the development of the parasite within the mosquito. Blocking vaccines attempt to control the disease in communities rather than protect individuals [8].

A novel approach that is under development is the use of anti-disease vaccines. These factors are not aimed at modifying the parasite multiplication, but at inhibiting the sequestration [9], or decreasing the production of cytokines, such as tumor necrosis factor, released in response to parasite components. [10,11].

Several conceptual problems must be considered in the design of subunit vaccines. These factors are associated with the parasite, the host and the environment. Par- asite proteins show, amongst other characteristics, stage specificity and a high degree of antigenic variation and polymorphism [12]. In addition, strategies designed to block the invasion process have to deal with a complex phenomenon that involves more than a single protein, some of them not exposed on the parasite's surface [13]. Several parasite proteins have been involved in the thymus-independent activation of polyclonal antibody. This reactivity, termed the smokescreen phenomenon, seems to involve a CD5 + B-ceU population and is related to evasion mechanisms [10].

Different effector mechanisms involved in the protective immune response against malaria have been suggested [14--16]. Nevertheless, it is not feasible to predict protection in a vaccinated human population (on the basis of the traditional approach of characterizing the antibody or cellular response) because of the tremendous complexity of the parasite and its host interactions. Such limitations complicate the follow up of vaccinees in endemic areas.

A global malaria control program will require combined strategies, that, in addition to an effective vaccine, must include the implementation of bed nets, chemotherapy, early diagnosis, vector control and sanitation improve- lnents.

Abbreviations CS~circumsporozoite; LSA--liver stage antigen.

© Current Biology Ltd ISSN 0952-7915 607

608 Guest editorial

Pre-erythrocytic vaccines

The rational approach used to develop a sporozoite vaccine was based on the capacity of antibodies to neutralize the process of the invasion ofhepatocytes by sporozoites. Polyclonal or monoclonal antibodies have been shown to be able to inhibit the infectivity of sporozoites [17]. These antibodies recognize a major surface antigen, the circumsporozoite (CS) protein. Although several B-cell epitopes have been described on the CS protein [18], the most prominent response has been identified to be directed against the central repeat sequences. Neverthe- less, there is no correlation between protection and sero- logical responses against the repeat sequence in endemic areas. The reactivity has been suggested to be useful to evaluate contact [19].

Clinical trials with recombinant and synthetic vaccines were done in the late 1980s. The vaccines were safe and able to induce a dose-dependent immune response, but with poor protective effcacy [20]. On the basis of these results, several groups have been involved in the design of new constructs that include T- and B-cell epitopes. Since then, potential T-ceU epitopes have been described [16], that are relevant to the induction of an anamnestic immune response or directly involved in the protective response as effector cells [21].

The basis of modern subunit malaria vaccines is the combination of T- and B-cell epitopes. The strategy of production includes recombinant cocktails, multiple antigen peptides (MAPs) or complex peptides able to present monomeric epitopes in polymeric form. Re- cently, preclinical trials with MAP constructs and recombinant products, which include sporozoite fragments, have been shown to be promising for future developments.

The comprehension of the invasion process of the hepatocytes by the sporozoite has opened the possibility of new developments in malaria sporozoite vaccine research [22]. Putative ligands on the carboxy-terminal portion of the CS protein have been described that bind to heparan sulfate proteoglycans on the membrane on hepatocytes [23]. Nevertheless, intriguing results have shown that the CS protein is released from the parasite before the invasion process. This finding suggests that the invasion process seems to be a multistep process mediated by different structures.

Protective immunity against the hepatic forms of the parasite includes anti-parasite cytokines and effector T cells able to destroy the parasitized hepatocytes [15,24]. Three liver stage antigens (LSAs) have been described. LSA-1, the best characterized protein of this group, is synthe- sized during schizogony [25]. This antigen, containing repetitive and nonrepetitive regions, is well conserved between the two isolates described. Humoral and cellular responses have been characterized in donors liv- ing in endemic areas [26] and have shown that LSA-1 is highly immunogenic in natural conditions. The intriguing manner in which the host recognizes a hidden

antigen must be explored in order to define the mecha- nism involved in the protective immune response against exo-erythrocytic forms,

Asexual blood stage vaccines

An effective vaccine to the erythrocytic stage, targeted to block the phase where the parasite acquires clinical significance, is the major goal for implementation in highly endemic areas. The vaccine must use the natural boosting effect to increase its effcacy. Immunity, which under natural conditions is acquired after several threatening in- fections, could be achieved by vaccination [14].

Several proteins have been characterized as specific for the asexual blood stages. Their significance in vaccine development has been considered on the basis of their expression on the surface, the significance for the parasite in terms of invasion, their immunogenicity in donors from endemic areas by natural exposure, the ability of specific antibodies to induce inhibition of the invasion process, and their protective efficacy after challenge in experimental models.

The major surface antigen PfMSA1, which has a molecular mass of 180-200 kDa, is the best studied protein that is expressed at the merozoite stage and is the precur- sor for several fragments (83 kDa, 38 kDa, 30kDa and 42 kDa) from the amino-terminal. Secondary process- ing of the 42 kDa fragment gives rise to PflVISA-119, the only fragment expressed on the surface of the merozoite during the invasion process. Several isolates have been sequenced and, on the basis of their structural diversity, the protein has been divided in 17 blocks of variable, semiconserved or dimorphic, and conserved fragments in the different isolates [13].

PflVISA-2 was isolated from detergent extracts of metabolically-labeled parasites. The protein corres- ponded to a surface antigen with a molecular mass of 46 kDa and was not related to PflVISA-1. The diversity of the genes for PflVISA-2 found on different isolates per- mits the assignment of variable sequences to two major aUelic forms. Wild isolates are extremely heterogeneous in terms of the expression of the protein: polymerase chain reaction amphfications of clinical parasites have shown diverse amplification patterns in a single isolate [27].

Erythrocyte-binding proteins have been described to be involved in the invasion of P. falcipansm and P. vivax. The EBA-175 and the Duffy antigen-binding proteins contain conserved adhesion ligands associated with the recognition of surface receptors on the erythrocytes: sialic acid and Duffy blood group, respectively [28,29]. The antigens have been localized in micronemes, apical structures of the merozoites [30]. Conservation in the sequence among this family of genes has permitted the identification of domains required for binding [31].

Several other proteins of the asexual, as well as the sexual, blood stages have been well characterized [8] including,

Malaria vaccines Moreno and Patarroyo 609

glycophorin binding protein (GBP-130), ring-infected erythrocyte surface antigen (R_ESA), serine-rich protein (SERP), apical merozoite antigen (AMA-1), histidine- rich protein 2 (HRP-2), rhoptry-associated proteins (RAP-1 and RAP-2), Pfs230 [32], Pfs40 [33] and Pfs25 [34,35]. The latter protein is the most promising sexual blood stage antigen as antibodies directed against this zygote surface antigen are able to inhibit the development of the ookinete and the production of sporozoites in the mosquito. Recently, an enzyme with a chitinase activity was described. As chitinase is essential for invasion of the gut wall in the mosquito after the blood meal, this enzyme has been suggested as a potential target for a blocking vaccine [36].

Anti-disease vaccines

The pattern of clinical malaria and virulence factors has been shown to be complex and variable. In endemic areas of Africa, a defined spectrum between mild and virulent disease has been established [37]. The expression of cerebral or severe malaria anemia versus un- complicated disease depends on the balance between the different phenotypes of the parasite involved during the infection. Virulent phenotypes are associated with the adhesion, rosetting phenomenon and cytokine secretion capacity.

The adhesion of infected erythrocytes has been cor- related with the sequestration of the parasite in the postcapillary venules. This phenomenon confers several advantages to the parasite: it simplifies the invasion process in compartments where the blood flow has been slowed, interferes with the clearance mechanisms mediated by the spleen, and facilitates asexual reproduction in a relatively hipoxemic environment [38].

The parasite-derived proteins involved in sequestration belong to an extremely heterogeneous fanfily of proteins that have been termed erythrocyte membrane proteins 1 (Pt[EMP1). The members of this family express a molecular mass of 200-350kDa [39]. Their heterogeneity, associated with antigenic variation, has been shown in both in viw, and in vitro assays [40]. Recently, a fanfily of genes termed var have been described and shown to encode proteins of the P ~ M P 1 fanfily [41]. The genes encode a large repertoire of proteins including proteins with binding domains homologous with proteins involved with ligand-receptor interaction in the red blood cells (EBA-175 and the Dully binding proteins).

A heterogeneous group of receptors are recognized by infected erythrocytes. Wild isolates have been shown to bind differentially to CD36, trombospondin, intercellu- lar adhesion molecule (ICAM)-I, endothelial-leukocyte adhesion molecule (ELAM) and vascular cell adhesion nmlecule (V-CAM) [42]. Some of these ligands are up- regulated by different host cytokines, in particular tumor necrosis factor [43]. The characterization of binding proteins, ligands and cytokines involved in the seques-

tration process led to the idea of developing an effective inhibitor or vaccine with anti-disease characteristics [44].

SPf66 malaria vaccine

The rationale for the developnlent of a synthetic malaria subunit vaccine was based on the suggestion that nmlti- ple antigens were likely to be involved in the invasion of red blood cells. The chimeric molecule consists of three epitopes consisting of anfino acid sequences derived from three blood stage proteins, intercalated by PNANP sequences derived from the repeat domain of the circumsporozoite protein of P. falcipamm [45,46].

The results of the Phase I clinical trial of SPf66 showed that the vaccine was immunogenic and was well tol- erated, with only minor local reactions. The calcu- lated efficacy of the vaccine was 75% after experimental challenge [47]. Several Phase lib and III trials to assure safety, efficacy and formulation of the vaccine were done with volunteers subject themselves to natural exposure to malaria. The efficacy of the vaccine against P. falcipamm ranged between 38.8-60.2%, with a significant differ- ence among distinct age groups in a low transmission areas [47-50].

Two clinical trials have been reported outside Latin America [51,52]. The first trial in Africa was conducted in Tanzania, where intense perennial malaria transmission occurs [51]. This randomized trial compronfised 586 children aged one to five years years who received SPf66 vaccine or tetanus toxoid as placebo. Morbid- ity was determined over a one year follow-up period. The trial confirmed that SPf66 is safe, imnmnogenic, and reduces the risk of clinical malaria. The estimated vaccine efffcacy against malaria was 31%.

Recently, a randomized trail in The Gambia was published [52]. 630 children aged 6-11 months were immunized with three doses of either SPf66 or injected polio vaccine. In this special group, the vaccine had no significant protective affect against clinical episodes of malaria. This result should not be compared with earlier clinical trials for several reasons. The follow-up period of three and a half months coincided with the rainy season, which is the period of greatest malaria transmission in The Gambia. In contrast, parasite prevalence in Tanzania shows no seasonality. In the Gambia trial, the children were younger and the immune responses, as measured by IgG, were not protective. It is well known that the imnmne system takes some time to mature. Very young children may not respond well to vaccines that work well in older age [53]. Finally, in The Gambia trial, the children were followed for only three and half months and this period may be too short to assess vaccine efficacy. A longer follow-up period might help to more accurately determine protection.

Perspectives

SPf66 is the first recognized malaria vaccine [54], and the first synthetic vaccine able to induce a protective

610 Guest editorial

immune response to an infectious disease [55]. Never- theless, a second generation of subunit malaria vaccine should be developed to improve the efficacy shown by SPf66. Our group is currently working on the characterization of a number of proteins from the malaria parasite. The approach uses assays to characterize receptor-ligand interaction in an effort to dissect the molecular events involved in the invasion process. Preliminary data have permitted us to identify several domains, from different malaria antigens, involved in the invasion of sporozoite and merozoite to the host cell, and several peptides have been chosen for inclusion in the design of new synthetic vaccines.

To overcome the problem of antigenic variation, poten- tially useful vaccines must protect against the different variants by including either conserved protective sequences or protective sequences from each variant. Nevertheless, if several variants are included in a novel construct, epitopic suppression or epitopic compe- tition may result in a decrease in vaccine efficacy. In our approach, peptides are tested in mice, rabbits and three different species of Aotus monkeys, in order to determine the feasibility of epitope combinations.

The final issue that must be addressed is the poor immunogenicity of deposit-based adjuvants. Several new adjuvants able to enhance the uptake and presentation of antigens by antigen-presenting cells, stimulate lym- phokine production and modulate T helper and cyto- toxic T lymphocyte responses, are currently being developed [56]. The implementation of novel adjuvants with the second generation of malaria vaccines should lead to the improvement of their efficacy [57].

Note added in proof

Readers of the accompanying editorial from Dr Riley should take into account the following additional points. First, preclinical data concerning the immunogenicity and induction of protection in Aotus monkeys with SPf66 were published by us in A m J Trop Hyg 1990, 43:339-354. Second, our 1987 paper [46] reported results obtained with a combination of three synthetic peptides, not with SPf66, and showed partial or complete protection of five out of eight monkeys that were immunized twice, and clear evidence of protection of a similar number of monkeys immunized five times in a second group. Third, in our first clinical trial [47], three out of five volunteers immunized with SPf66 were protected; the other volunteers were immunized with SPf105. Fourth, before vaccinating 18000 people [11], we performed safety and immunogenicity trials on increas- ing numbers of Columbian soldier volunteers (Vaccine 1992, 10:179-183; Parasite Immunol 1992, 14:95-109) and children [48]. Fifth, in our double-blind, randomized placebo phase Ill trial carried out for one year in La Tola, there were 169 cases of P falciparum malaria in the vaccinated group and 297 in the placebo group [50]. And finally, WHO, through the different T D R committees supports some of the field trials scientifi- cally and, sometimes, economically. Also, W H O has friendly and very close connections with our institute to improve the development of the SPf66 vaccine, so it is likely to sanction further field trials.

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A Moreno and ME Patarroyo, Instituto de lnmunologia, Hospital San Juan de ])los, Universidad Nacional de Colombia, Avenida 1 10-01, Santaf~ de Bogota, ])C Colombia.

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