Australasian Biotechnology (backfiles) AusBiotech ISSN: 1036-7128 Vol. 8, Num. 2, 1998

Apical Membrane Antigen 1: A Leading Malaria Vaccine Candidate

Robin F. Anders, Pauline E. Crewther and Anthony N. Hodder,

The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Victoria, 3050

Code Number:AU98017
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The development of a malaria vaccine remains one of the world's major public health priorities. Numerous proteins in several different life cycle stages have been identified as potential vaccine components. In the asexual blood stages of Plasmodium falciparum two antigens, merozoite surface protein 1 (MSP1) and apical membrane antigen 1 (AMA1) stand out as the leading vaccine candidates because of the extensive assessment of vaccines containing these antigens in animal models of the human disease. Studies in monkeys and mice have shown that AMA1 can induce antibody-mediated protective immune responses. Diversity in AMA1 exists but whether this limits the efficacy of an AMA1 vaccine will need to be examined in clinical trials carried out in areas where malaria is endemic.

Introduction

Malaria is one of the world's major public health problems with approximately 40% of the world's population at some risk of infection (WHO, 1997). Infection with Plasmodium falciparum causes severe morbidity in individuals lacking immunity and without effective treatment there is a risk of death, usually from severe anaemia or cerebral malaria. The treatment of malaria has become more difficult because of the emergence of drug resisitant parasites and the control of transmission has been made more difficult by the emergence of insecticide resistant mosquito vectors. Because of this situation new tools are urgently required to combat malaria and, therefore, the development of a vaccine against malaria is a major priority of the World Health Organisation and other organisations addressing the health needs of developing countries (WHO, 1996).

The complicated life cycle of the malaria parasite provides numerous points at which induced immune responses could prevent or limit the development of malaria parasites in their human host. Consequently, groups involved in malaria vaccine development are focusing on antigens expressed in sporozoites, exoerythrocytic (liver) stages, asexual blood stages, gametes and zygotes (Engers and Godal, 1997). Numerous potential vaccine candidates have been identified in one or other of these life-cycle stages but because there is a large number of vaccine candidates it is difficult to find the resources to test them all in clinical trials where the subjects are exposed to natural challenge. The development of a malaria vaccine is further complicated by the numerous alternative strategies that now might be adopted to make a vaccine. These different approaches: recombinant proteins, synthetic peptides, live recombinant virus or bacteria, and naked DNA, are all being investigated by groups involved in malaria vaccine development (Engers and Godal, 1997).

A Vaccine Against the Asexual Blood-Stages of P. falciparum

The symptoms of malaria are caused by the synchronous development of the asexual blood stages of the parasite within host erythrocytes. Individuals who have developed immunity to malaria as a consequence of naturally-acquired infections have circulating antibodies which limit the development of the disease-causing asexual blood stages (Cohen et al. 1961) Although studies in animal models suggest that both T- and B-cell responses are necessary for the elimination of parasites, the reduction of parasitaemias by antibodies is likely to reduce morbitity and mortality associated with P. falciparum infections. For these reasons we have been attempting to develop a recombinant protein vaccine which induces an antibody response that limits development of the asexual blood stages of P. falciparum.

The asexual blood stages of malaria parasites are antigenically complex and it has been assumed in the past that only a small number of the protein antigens that characterize these life cycle stages are capable of inducing protective immune responses. The identification of numerous antigens, either on the merozoite surface or within merozoite secretory organelles, the mutation pattern in some of these antigens, and the evidence from a variety of model systems indicate that the number of antigens capable of inducing protective immune responses is larger than once thought.

Two asexual blood stage antigens, merozoite surface protein 1 (MSP1) and apical membrane antigen 1 (AMA1), stand out as leading vaccine candidates because they have been extensively studied in animal models of the human infection. The identification of the genes for MSP1 and AMA1 in many simian and rodent parasites has allowed vaccine trials to be carried out in monkeys and mice using the antigen from simian and rodent parasites, respectively. MSP1 is a large (~200kDa) GPI-anchored membrane protein which undergoes post-translational processing into a number of fragments (Holder and Blackman, 1994). Because it has been difficult to express full-length MSP1 as a recombinant protein, preclinical assessment of the vaccine efficacy of MSP1 has focussed on fragments, particularly MSP119, a naturally occurring C-terminal fragment which contains two "EGF-like" domains (Anders, 1997).

AMA1 is a type 1 integral membrane protein of approximately 80 kDa but like MSP1 it is post-translationally processed to smaller fragments. The sites of cleavage in AMA1 have not been mapped accurately however, and for the preclinical assessment of this antigen in rodents and monkeys recombinant proteins corresponding to either the full-length protein or the presumed ectodomain of the protein have been used. Recombinant AMA1 is currently being prepared for a Phase I clinical trial in Brisbane and here we will briefly review the preclinical data on which the vaccine candidacy of AMA1 is based.

Testing an AMA1 Vaccine in Animal Models

AMA1 was first identified on the surface of P. knowlesi merozoites with a monoclonal antibody that inhibited the invasion of rhesus erythrocytes (Deans et al.. 1982; Deans et al. 1984). The parasite antigen (~ 66kDa), isolated by affinity chromatography from infected rhesus erythrocytes, induced partial protection when used to immunize rhesus monkeys (Deans et al. 1988). The results of these early studies in the P. knowlesi/monkey model, which first identified AMA1 as a potential vaccine component, are now supported by results from vaccine trials using two other animal models: P. fragile in the Saimiri monkey and P. chabaudi in the mouse (Table 1).

Table 1: Evidence That AMA1 Can Induce Protective Immune Response

In a Saimiri monkey trial in which the animals were immunized with full length recombinant P. fragile AMA1 produced using the baculovirus (BV) expression system, four of the five immunized monkeys recovered without treatment whereas all control monkeys required treatment (Collins et al. 1994) Consistent with the earlier studies in P. knowlesi, which indicated that protection may be mediated by anti-AMA1 antibodies, the outcome in the immunized group of Saimiri monkeys appeared to correlate with the titre of AMA1 antibodies induced by immunization. All immunized monkeys developed patent parasitaemias and, although the parasitaemias were controlled without chemotherapy, parasitaemias persisted at a low level. The AMA1 genes for these breakthrough parasitaemias were sequenced and found to be identical to that of the challenge inoculum.

Further preclinical assessment of AMA1 has been carried out in the P. chabaudi/mouse model. This more convenient model has allowed more trials to be carried out with larger numbers of animals in the vaccine and placebo groups. Recombinant P. chabaudi AMA1 produced using baculovirus expression protected immunized mice in one of two experiments, but more consistent protection was obtained in mice by immunizing with the ectodomain of AMA1 expressed in E. coli and refolded in vitro (Anders et al. 1998). The AMA1 ectodomain was expressed with an N-terminal hexa-his tag which allowed chelate chromatography on Ni-NTA resin to be used as a first step in the purification procedure. The P. chabaudi AMA1 ectodomain formed inclusion bodies in E. coli and these were solubilized in 6M guanidine-HCl without any reducing agent prior to the Ni-chelate chromatography. The protein eluted at pH 4.5 from the Ni-NTA resin was refolded by dilution to ~50 mg/ml in a buffer containing oxidized and reduced glutathione to generate intramolecular disulphide bonds which stabilise the native protein. Following refolding, the protein was further purified by anion exchange chromatography.

In three trials, two using inbred mice and one using outbred mice, immunization with the refolded ectodomain of P. c. adami DS strain almost abolished mortality (Table 2) and reduced average peak parasitaemias from ~50% to 1-3% (Anders et al. 1998). Although some immunized mice in each experiment did not develop patent parasitaemias most developed parasitaemias which resolved without the severe morbidity usually seen when mice are infected with the DS strain of P. c. adami. Immunization with the refolded AMA1, which was usually formulated in the SEPPIC adjuvant ISA720, induced very high antibody titres to the vaccinating antigen and, as seen in the earlier P. fragile/Saimiri studies (Collins et al. 1994), the titre of this response correlated with the degree of protection.

In all the AMA1 sequences that have been determined to date there are 16 conserved cysteine residues which form eight intramolecular disulphide bonds (Hodder et al. 1996). The results of extensive analyses indicated that the majority, if not all, of these disulphide bonds were in place in the refolded P. chabaudi AMA1 used in these mouse vaccine trials (Hodder et al. unpublished results). The protection induced by immunizing with the refolded AMA1 was dependent on the disulphide-bonded structure as there was no reduction in peak parasitaemias in mice immunized with reduced and alkylated antigen(Anders et al. 1998). When the data from all of the trials of AMA1 in mice are combined, immunization with the reduced and alkylated AMA1 does appear to have been associated with reduced mortality (Table 2). It may be that T-cell priming by the reduced and alkylated antigen enhances protective AMA1 antibody responses induced by the developing parasitaemia following challenge (Amante et al. 1997).

Table 2: Effect of Immunization with AMA1 on Mortality in P. chabaudi-Infected Mice⁺

+See Anders et al. 1998 for details of these trials

*The reduction and alkylation of AMA1 irreversibly destroys the intramolecular disulphide bonds which are important for stabilizing the native conformation of AMAl.

Although AMA1 lacks the major polymorphisms that characterize a number of the other merozoite surface antigens, numerous point mutations have occurred in the AMA1 genes of various Plasmodium species. In P. falciparum the majority of these mutations are non-synonymous, resulting in amino acid substitutions at more than 50 positions in the sequence (Marshall et al. 1996). The bias towards non-synonymous mutations, clustering of the mutations, and the radical nature of the majority of the amino acid substitutions indicates that selection, possibly by protective antibodies, has played a role in the emergence of this sequence diversity (Crewther et al. 1996).

One reason P. chabaudi was chosen as the parasite for use in the vaccine trials in mice was the availability of two subspecies and several different parasite isolates. This allows testing of an AMA1 vaccine for efficacy against challenge with heterologous as well as homologous parasites. When mice immunized with recombinant AMA1 from the DS strain were challenged with the 556KA strain of P. c. adami there was no protection (Crewther et al. 1996). The DS and 556KA AMA1 sequences differ at 36 positions which is slightly more different than the two most different P. falciparum sequences (Marshall et al. 1994). Antibodies raised in rabbits to the DS AMA1 cross-reacted with the 556KA antigen but on passive transfer into mice, these antibodies had no effect on a 556KA parasitaemia whereas they caused a marked reduction in DS parasitaemias.

Preparing For a Human Trial With P. falciparum AMA1

These preclinical studies in monkeys and mice have provided the justification for testing a recombinant AMA1 vaccine in human volunteers, but the studies in mice with heterologous challenge indicate that diversity may limit the efficacy of an
AMA1 vaccine. We have been encouraged to find that antibodies raised in rabbits to refolded recombinant AMA1 from the 3D7 line of P. falciparum are inhibitory in vitro for all three lines of P. falciparum so far examined (Hodder et al. unpublished results). The AMA1 of one of these lines, HB3, has 24 amino acid differences from the 3D7 sequence. Ultimately, the efficacy of AMA1 and the other leading vaccine candidates must be tested in field trials where the subjects will be challenged with an enormous diversity of parasite genotypes. In an endemic setting, the majority of these parasites cause no disease and this will provide an opportunity for the immune responses primed by immunization to be boosted by natural infections. It is possible that such boosting will increase the titre and broaden the specificity of the protective antibody response so that there is efficacy against infections due to parasites of diverse genotypes.

Acknowledgements

The research on AMA1 at The Walter & Eliza Hall Institute of Medical Research has been funded by the National Health and Medical Research Council (Australia), the Cooperative Research Centre for Vaccine Technology and the United Nations Development Programme/World Bank/World Health Organisation Special Programme for Research and Training in Tropical Diseases.

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