The Current Status of Malaria Vaccines

BioDrugs 1998 Aug; 10 (2): 123-136 IMMUNOLOGY-BASED AGENTS 1173-8804/98/0008-0123/$07.00/0

José A. Stoute and W. Ripley Ballou Department of Immunology, Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Washington DC, USA

Contents Abstract ...... 123 1. Vaccines Targeting the Pre-Erythrocytic Stage ...... 124 1.1 Vaccines That Target the Sporozoite ...... 124 1.2 Other Pre-Erythrocytic Stage Vaccines ...... 127 2. Vaccines Targeting the Asexual Erythrocytic Stage ...... 127 3. Transmission-Blocking Vaccines ...... 129 4. Major Roadblocks to a Successful Malaria Vaccine ...... 130 5. New Approaches to Malaria Vaccine Development ...... 131 5.1 Multistage, Multicomponent Vaccines ...... 131 5.2 DNA Vaccines ...... 132

Abstract A vaccine against Plasmodium falciparum malaria is needed now more than ever due the resurgence of the parasite and the increase in drug resistance. How- ever, success in developing an effective malaria vaccine has been elusive. Among pre-erythrocytic antigens, the major antigen coating the surface of the sporozoite, the circumsporozoite protein (CS), has been, and continues to be, the major target for vaccine development. Despite initial limited success with CS- based vaccines, the use of new adjuvant formulations has led to the development of a promising candidate (the RTS,S vaccine) which has shown significant efficacy in a preliminary trial. In addition to CS, many other malaria antigens have been identified that play an important role in the parasite life cycle which are being considered for, or are currently undergoing, clinical trials. Among the blood stage antigens, the merozoite surface protein 1 (MSP-1) is the most promising vaccine candidate. New approaches to immunisation against malaria being considered include the use of multistage, multicomponent vaccines in attenuated viral vectors (NYVAC-Pf7), or in a combination DNA vaccine. While there is reason to be optimistic about the prospects for an effective vaccine, many challenges lie ahead that still have to be overcome. Among these are the antigenic polymorphism exhibited by wild parasite strains and the genetic restriction of immune responses.

Malaria is one of the world’s deadliest infec- parasite, a vaccine seems now more than ever the tious diseases responsible for 1.5 to 2.7 million only hope for controlling this disease. However, deaths per year, mostly in children. Due the contin- success in achieving a malaria vaccine has been ued development of drug-resistant strains of this elusive. The objective of this article is to summa- 124 Stoute & Ballou

rise the most important developments in the field will induce immune responses capable of eliminat- of malaria vaccine research. For this purpose, we ing sporozoites from the circulation and/or capable will review the most promising vaccine candidate of eliminating infected hepatocytes leading to ster- antigens within the context of the portion of the life ile immunity. This is especially important if it is to cycle they are intended to disrupt. This review is be used in nonimmune populations that have no limited to Plasmodium falciparum, the deadliest of pre-existing immunity against blood stage para- all human malarias. sites. In order to better understand the approaches to malaria vaccine development it is necessary to 1.1 Vaccines That Target the Sporozoite have some familiarity with the life cycle of the parasite (fig. 1). The malaria life cycle is complex. The One of the first malaria genes to be fully char- sporozoite form of the parasite is transmitted to acterised was the major surface protein of the ma- man by the bite of the female Anopheles mosquito. laria sporozoite, referred to as the circumsporozo- Sporozoites travel through the bloodstream and in- ite protein (CS).[1,2] The CS of all the malaria vade liver cells where they multiply asexually as species share a number of features. They all contain pre-erythrocytic stage parasites. P. falciparum pre- a central segment of amino acid repeats flanked by erythrocytic forms mature in 5 to 7 days, releasing two nonrepeat regions. In the case of P. falciparum thousands of tissue merozoites which invade eryth- malaria, the central portion contains 36 to 43 repeats rocytes and initiate the erythrocytic or blood stage of the amino acids asparagine-alanine-asparagine- phase of the infection. During the erythrocytic proline, or NANP, and 3 to 4 asparagine-valine- phase, free merozoites invade erythrocytes, un- aspartic acid-proline (NVDP) repeats.[3] The cen- dergo asexual maturation into trophozoites and tral repeating unit is the immunodominant portion schizonts, and ultimately rupture the erythrocyte of the molecule, since most of the humoral immune releasing new merozoites. It is this phase of the life responses of animals and humans against sporozo- cycle, with the cyclic destruction of erythrocytes, ites seem to be directed against this central re- that leads to the clinical disease known as malaria. peat.[4] Because antibodies against P. falciparum A small portion of merozoites that invade erythro- and P. berghei repeats were shown to inhibit spo- cytes do not multiply, but instead differentiate into rozoite invasion in vitro and protected rodents sexual forms, gametocytes. When ingested by an- against sporozoite challenge respectively,[5,6] the other female Anopheles, female and male gametes first malaria vaccines tested were based solely on unite in the mosquito midgut to form a zygote some version of this repeating amino acid unit. which subsequently develops into a motile ookinete. Since its initial identification, CS has been a ma- After invading the basal lamina of the mosquito jor focus of malaria vaccine development. This em- midgut epithelium, the ookinete develops into phasis on CS is justified by numerous studies in an oocyst containing hundreds of sporozoites. animal and human models as well as in several clin- Oocysts then mature and release sporozoites which ical trials, some of which are summarised below, migrate to the mosquito salivary glands, thus com- that have identified CS as the target of protective pleting the life cycle. immune responses. Soon after the discovery of the CS gene 2 1. Vaccines Targeting the NANP-based vaccines were tested in humans. One Pre-Erythrocytic Stage of these early vaccines consisted of a recombinant protein expressed in Escherichia coli containing 30 The pre-erythrocytic portion of the malaria life NANP repeats and 2 NVDP repeats in fusion with cycle includes events from the entry of sporozoites 32 amino acids of the out-of-frame tetracycline-re- [7] in the blood circulation to the release of merozoites sistant gene (R32tet32). The other vaccine con- from the liver. The ideal pre-erythrocytic vaccine sisted of the synthetic peptide (NANP)3 conjugated

Sporozoite antigens CS TRAP Liver stage antigens LSA-1 Exp-1

Pre-erythrocytic stage ▼

Blood stage Merozoite

Merozoite antigens

MSP-1 RESA AMA-1 EBA-175

Schizont

Trophozoite Exp-1 SERA

Gametocyte Pfs230 Pfs48/45

Oocysts Gametes

Mosquito midgut Ookinete Pfs25 Pfs28

Fig. 1. Life cycle of malaria. AMA-1 = apical membrane antigen 1; CS = circumsporozoite protein; EBA-175 = erythrocyte-binding antigen 175; Exp-1 = Exported Protein 1; LSA-1 = liver stage antigen 1; MSP-1 = merozoite surface protein 1; Pfs = surface antigen; RESA = ring-infected erythrocyte surface antigen; SERA = serine repeat antigen. to tetanus toxoid.[8] The results of both studies meagre and one volunteer from each group with were surprisingly similar despite the use of very relatively high anti-repeat antibody levels was pro- different antigenic constructs. In both studies, hu- tected against sporozoite challenge. Although the moral responses against the NANP repeat were demonstrated efficacy was poor, these studies

served to reaffirm that sterile immunity against ma- to a portion of the hepatitis B surface antigen.[25] laria can be induced by immunisation with a syn- The development of a dual malaria/hepatitis B vac- thetic subunit vaccine. Since protection seemed to cine made sense from the standpoint that these 2 correlate with high antibody levels, it was felt that diseases are frequently endemic in the same geo- the development of more immunogenic versions of graphic areas of the world and are the cause of these vaccines would lead to better efficacy. Sub- much morbidity and mortality. In addition, it was sequent sporozoite vaccines, based on the NANP hoped that the use of the hepatitis B virus surface repeat, contained modifications conducive to the antigen as a carrier would improve the immunoge- enhancement of immune responses. Modifications nicity of the malaria CS protein. In a preliminary such as the addition of Pseudomonas aeruginosa trial, this vaccine was shown to be safe and immu- toxin A,[9] encapsulation in liposomes containing nogenic.[25] monophosphoryl lipid A (MPL),[10,11] and inclu- A subsequent generation of this vaccine in- sion of a mixture of MPL, mycobacterial cell wall cluded the nonrepeat C-terminal portions of CS in skeleton and squalene,[12] generally resulted in addition to 19 NANP repeats (RTS,S).[23] The higher antibody levels, but contrary to expectations RTS,S antigen was initially tested as two formula- did not significantly improve efficacy. tions adsorbed onto alum with or without the lipid Studies in rodents and humans immunised A derivative 3-deacyl-monophosphoryl lipid A.[23] against malaria with irradiation-attenuated sporo- Although the anti-repeat antibody levels were zoites suggested that CD4+ T helper and CD8+ T modest, 2 of 8 volunteers in the MPL plus alum cytotoxic (CTL) T cell responses were important group were protected and had CTL responses to components of the protective immune responses peptides from the C-terminus of CS. More recently, against malaria.[13,14] The target epitopes of these 3 formulations of this antigen were tested contain- T cells were subsequently identified and found ing as adjuvants either MPL plus alum (SmithKline mostly in the C-terminal variant regions of the CS Beecham Adjuvant System 4, SBAS4) [vaccine protein.[15-20] Another small region of the C-termi- number 1], a proprietary oil-in-water emulsion nus of CS was also implicated in the binding of (SBAS3, vaccine number 2) or MPL, the saponin sporozoites to hepatocytes, Region II.[21] QS21[26] and SBAS3 (SBAS2, vaccine number The recognition of peptide sequences outside 3).[24] These new adjuvant systems were chosen on the CS repeat of importance for induction of cellu- the basis of preliminary studies in rodents and Rhe- lar immunity and for hepatocyte invasion led to the sus monkeys that demonstrated that SBAS3 and development of CS vaccine constructs which in- SBAS2 induced optimal humoral and/or cellular cluded non-repeat portions of the molecule with, or responses in combination with this antigen (DG without, the immunodominant repeat epitopes.[22- Heppner, personal communication). 24] One vaccine construct was designed completely Volunteers immunised with RTS,S plus SBAS3 devoid of repeat epitopes with the rationale in mind and SBAS2 had the highest anti-repeat antibody that repeat epitopes may serve as an immunological responses ever induced by any sporozoite-based smoke screen, diverting most of the humoral re- vaccine. Among the volunteers who received vac- sponses away from more important regions of cine number 1 and vaccine number 2, 1 out of 8 and CS.[22] This repeatless construct was tested in hu- 2 out of 7 volunteers, respectively, were protected. mans and was found to induce antibody responses By contrast, in the SBAS2 group, 6 out of 7 volun- that recognised sporozoites.[22] However, none of teers were protected.[24] Cellular responses are pre- 11 volunteers challenged were protected and no de- sumed to be an important component of protection lay in the prepatent period was observed. in the vaccine containing SBAS2, and indeed pro- A different approach to malaria vaccine devel- liferation and release of cytokines such as inter- opment came about by the fusion of the CS repeats feron-γ (IFN-γ) and interleukin 4 (IL-4) in response

to RTS,S have been found in vitro. However, none Exp-1, also known as QF116 or circumsporozo- of these responses correlated with protection (U ite-related antigen (CRA), is a 23kD antigen lo- Krzych, personal communication). cated in the parasitophorous vacuole membrane, The sporozoite surface protein 2 (SSP2), also the membrane surrounding the parasite when in- known as thrombospondin anonymous protein side the host cell.[38-41] This antigen has been de- (TRAP), is a second protein that has been identi- tected both in infected erythrocytes and hepato- fied on the surface of sporozoites. The protein was cytes.[38,42] Its role in the survival of the parasite initially identified in asexual blood stage parasites has not been elucidated. The amino acid sequence but it was not until later that it was recognised as a of this antigen contains areas of homology with the sporozoite surface protein.[27,28] SSP2 has been CS protein, including one copy of the NANP re- identified as the target of CD4+ and CD8+ protec- peat, which explains why antibodies raised against tive T cell responses in mice and humans[29-32] and this antigen also recognise CS by immunoblot is therefore, an important target for vaccine devel- and sporozoites by immunofluorescence assay opment. This idea is supported by studies in ro- (IFA).[38,42] This immunological cross-reactivity dents that have shown that immunisation with P. suggests that Exp-1 could serve as a multistage yoelii SSP2 and CS confers sterile immunity vaccine against sporozoites, liver stages and in- against sporozoite challenge.[33] SSP2 was infected erythrocytes. Evidence this antigen may be cluded in a recent trial of the multistage vaccine an important target for vaccine development comes NYVAC-Pf7 and will be included in future DNA from work done with its P. yoelii homologue vaccine trials (see section 5.2). PyHEP17.[43,44] Immunisation of a variety of in- bred mouse strains with plasmid DNA constructs encoding PyHEP17 induced sterile immunity in 33 1.2 Other Pre-Erythrocytic Stage Vaccines to 85% of the mice, depending on the strain.[43] This immunity was shown to be dependent on the In addition to sporozoite antigens, 2 other anti- presence of CD8+ cytotoxic T cells.[43,44] A recom- gens have been identified that are expressed in the binant protein consisting of part of the Exp-1 pro- developing liver schizont and are considered po- tein in fusion with the sequence (NANP)19 was tential candidates for vaccine development, Liver tested for safety, immunogenicity and efficacy in a Stage Antigen 1 (LSA-1) and Exported Protein 1 small number of volunteers.[45] While immuno- (Exp-1). genic, this product failed to induce protective re- LSA-1 is a 200kD antigen that contains a central sponses. The potential of Exp-1 as a vaccine can- 17-amino-acid repeat region.[34] The evidence sup- didate could not be assessed effectively in this porting the inclusion of LSA-1 as a vaccine candi- study, since only one volunteer developed antibod- date has been mostly circumstantial. Hill and col- ies against blood stage parasites by IFA. leagues[35] demonstrated an association between the class I molecule HLA-B53 and relative resis- 2. Vaccines Targeting the Asexual tance to severe malaria in a population with re- Erythrocytic Stage peated exposure to malaria in The Gambia, West Africa. Subsequent studies revealed an epitope The erythrocytic or blood stage portion of the within LSA-1 that is presented by HLA-B53 class life cycle can be divided into asexual and sexual I molecules and is the target of CD8+ cytotoxic T stages. The asexual stage includes the trophozoites cell responses.[36] More recently, T and B cell and schizonts developing within red cells. The sex- epitopes have been identified within the C-termi- ual erythrocytic stage constitutes the male and fe- nal nonrepetitive region recognised by serum and male gametes. peripheral blood mononuclear cells (PBMC) from Vaccines that target the asexual erythrocytic individuals living in malaria endemic areas.[37] stage of the malaria life cycle are intended to elim-

inate the infected erythrocytes or to prevent free induce protection.[53] Lack of protection in some merozoites from invading red cells. An effective cases could be due to use of antigenic constructs erythrocytic vaccine targeting the asexual stage of that do not have a proper conformational structure, malaria would induce sterile immunity in the host since this region of MSP-1 is believed to contain or, more realistically, lead to a reduction in the conformational, constrained structures. MSP-1 is number and/or severity of clinical episodes of ma- also the target of CD4+ T cells and several T cell laria. The outcome of decreased disease severity is epitopes have been identified.[54,55] more difficult to measure than disease incidence. A second merozoite antigen that has been imp- The full potential of these vaccines will only be licated in the process of erythrocyte invasion is determined by large randomised field trials. the erythrocyte-binding antigen 175 (EBA-175). The merozoite stage of the parasite, like the spo- EBA-175 was initially identified by examining rozoite, is a logical target for a malaria vaccine parasite antigens released into the culture superna- since a blockade of erythrocyte invasion would tant that were able to bind to the surface of red completely prevent clinical disease. Therefore, the cells.[56] Later, it was demonstrated that antibodies identification of the molecular mechanisms of mer- to a region of this protein were able to inhibit merozoite invasion of red blood cells has been an active ozoite invasion.[57] The surface receptor for EBA- area of investigation in the field of malaria vaccine 175 on the erythrocyte is glycophorin A.[58,59] Fur- research. ther work on this molecule has been hampered by Several antigens have been identified that have the unavailability of recombinant proteins to fur- the potential to inhibit merozoite invasion of red ther study its potential as a vaccine in animal mod- cells. The most studied of these antigens, and also els. However, Daugherty et al.[60] recently have re- the most promising blood stage vaccine candidate, ported the successful expression of recombinant is the merozoite surface protein 1 (MSP-1). MSP-1 products of this molecule in insect cells. is a 195kD antigen found on the surface of mero- Other merozoite antigens have been identified zoites. It undergoes processing by proteolytic whose role in the process of red cell invasion is less cleavage to a 19kD fragment that has been impli- completely understood but that are known to elicit cated in the invasion of erythrocytes by merozo- invasion-inhibitory antibodies. One of these mero- ites.[46] zoite antigens is the apical membrane antigen 1 Several lines of evidence lead to the conclusion (AMA-1).[61] This antigen was initially identified that MSP-1 is a promising vaccine candidate. An- as a 66kD antigen in P. knowlsei which was the tibodies directed against portions of MSP-1, in par- target of growth-inhibitory monoclonal antibod- ticular against the 42 kD and its conserved 19kD ies.[62,63] In a recent study, monkeys were im- processing C-terminal fragment, inhibit erythro- munised with the P. fragile analogue of AMA-1 cyte invasion.[46,47] At least one field study has followed by challenge with P. fragile-infected demonstrated an association between the existence erythrocytes. Although all the immunised monkeys of antibodies against MSP-1 and resistance to clin- developed infection, 4 of 5 immunised monkeys ical malaria.[48] Immunisation with recombinant exhibited marked prolongation of the prepatent pe- fragments of this molecule also protects monkeys riod and decreased parasitaemia.[64] against P. falciparum[49] when used with Complete The ring-infected erythrocyte surface antigen Freund’s Adjuvant, and mice against P. yoelii.[50,51] (RESA) is a 155kD protein deposited on the sur- Passive transfer of immune sera in mice also con- face of red cells upon entry by merozoites and is fers protection.[52] found within mature merozoites prior to inva- Although the weight of the evidence indicates sion.[65] It is also the target of invasion inhibitory that antibodies against the C-terminal fragment of antibodies.[66] Monkeys immunised with recombi- MSP-1 are protective, not all C-terminal constructs nant fragments of this molecule experienced lower

parasitaemia and spontaneous remissions.[67] Sev- constructs were tested in humans, at least 3 out of eral epidemiological studies have also suggested 5 individuals immunised with the polymer SPf66 that antibodies to this molecule may be important demonstrated partial immunity against a blood in controlling parasitaemia.[68-71] stage challenge.[83] The serine repeat antigen (SERA) was identi- Today we know that the 83kD peptide sequence fied by screening a malaria cDNA library with par- is part of the sequence of MSP-1. The identity of asite inhibitory monoclonal antibodies.[71,72] This the other source antigens remains unclear. Scepti- protein is unique in that it has a very high content cism arose in the research community regarding (11%) of the amino acid serine, including one 35- the usefulness of this vaccine when at least one amino-acid stretch of serine residues.[73] The func- study failed to reproduce the results previously ob- tion of this protein has not been identified. How- served in immunisations of Aotus monkeys.[84] ever, by homology with other serine-rich antigens, Early field trials of the SPf66 vaccine in endemic it is possible this protein may function as a parasite areas of Colombia nonetheless showed that this protease.[74] It is believed to be abundant within the formulation was safe, immunogenic and of prom- [85-88] parasitophorous vacuole space of trophozoites and ising efficacy in the range of 33 to 82%. How- schizonts and proteolytic fragments of it are found ever, some of these early studies had serious meth- in the media following rupture of schizonts.[75] odology flaws, making comparability of results Several studies have demonstrated that immunisa- difficult. Subsequently, 3 well-designed placebo- tion with recombinant fragments of SERA is able controlled randomised trials, 2 in Africa and 1 in to induce protective immune responses against ex- Thailand, gave conflicting estimates of efficacy perimental blood stage challenge in Aotus mon- compared to those reported from South Amer- [89-91] keys.[76-78] ica. The reasons for these discrepant results are not clear, but it is possible that differences in A more empirical approach to the development the intensity of transmission between testing sites of a malaria vaccine was followed by a Colombian and in the genetic makeup of the target populations team led by Manuel Patarroyo. This effort resulted may have contributed to these differences. At pres- in the development of the synthetic peptide SPf66. ent, the future of SPf66 as a malaria vaccine is SPf66 is a hybrid synthetic polymeric peptide that uncertain. contains sequences from 3 putative asexual blood stage antigens of approximate molecular weights 83, 55 and 35kD, interlinked by the CS sequence 3. Transmission-Blocking Vaccines NANP.[79] The rationale behind the design of this molecule is based on the initial observation that Unlike the vaccines reviewed above, transmis- immunisation with proteins of molecular weight sion-blocking vaccines are not intended to disrupt 155, 83, 55 and 35kD obtained from a lysate of the life cycle of the malaria parasite in the human asexual stage parasites protected monkeys against host but rather, the life cycle in the mosquito vec- an experimental blood stage challenge.[80] In sub- tor. Antibodies directed against antigens found in sequent studies, peptides were synthesised based the gametocytes or in the developing zygotes can on the N-terminal amino acid sequences of these be ingested during a blood meal and are effective proteins. Immunisation of Aotus monkeys with in blocking parasite development in mosquitoes. If combinations of peptides from the 83, 55 and 35kD most of the population in an area of malaria trans- proteins, but not from the 155kD protein, induced mission was immunised with an effective transmis- partial protection against blood stage chal- sion-blocking vaccine, malaria transmission, in lenge.[81,82] The peptide motifs that were most ef- theory, would be effectively reduced or eliminated. fective in the monkey studies were included in the Since immunisation with transmission-blocking design of several polymeric peptides. When these vaccines does not confer protection against malaria

to the host, these vaccines will need to be used in rected against it also recognise cross-reactive carefully supervised campaigns. epitopes in other gametocyte antigens.[100] Several antigens have been identified as candi- As opposed to immunity induced by gametocyte dates for inclusion in a transmission-blocking vac- antigens, immunity induced by the ookinete anti- cine. These transmission-blocking antigens can be gens would not be boosted by natural infection be- categorised into two groups: firstly, those ex- cause these antigens are only expressed in the mos- pressed during gametocytogenesis in the host and quito. Pfs25 is the major surface antigen found on to some extent in the mosquito midgut (Pfs230 and zygotes and ookinetes. It has also been shown to be Pfs48/Pfs45) and secondly, those expressed exclu- the target of transmission-blocking monoclonal an- sively in the developmental stages of the parasite tibodies and is able to induce antibodies that have in the mosquito midgut (Pfs25 and Pfs28). Both transmission-blocking activity in animals.[101-103] Pfs230 and Pfs48/45 are found on the surface of Recently, a second ookinete surface antigen, Pfs28, gametes upon emerging from red cells in mosqui- has been identified and its transmission-blocking toes.[92,93] They are both targets of transmission- activity seems to be synergistic with that of [104] blocking monoclonal antibodies and are able to in- Pfs25. Of all the transmission-blocking anti- duce transmission-blocking antibodies in animal gens, Pfs25 is at the most advanced stage of devel- models.[94] opment and is currently undergoing safety and im- [105] The presence of transmission-blocking activity munogenicity trials in human volunteers. in serum from individuals living in endemic areas has been correlated with the presence of antibodies 4. Major Roadblocks to a Successful against both antigens.[95,96] However, in another Malaria Vaccine study, transmission-blocking activity only corre- While significant advances in malaria vaccine lated with antibodies against gamete surface anti- research have been made, major obstacles still lie gens measured by IFA, suggesting perhaps there ahead. Most of these obstacles are developmental are other gamete antigens that are the target of [97] adaptations of the parasite designed to evade the transmission-blocking immunity. Because Pfs230 host immune system. They include the following and Pfs48/45 are expressed in the host, immunisa- points: (i) the fact the organism is intracellular for tion with these antigens has the advantage that nat- most of its life cycle means malaria antigens are ural infection should boost immune responses. not as readily available to the host immune system; Two other gametocyte antigens have been iden- (ii) the developmental regulation of the parasite life tified, Pfs2400 and Pfg27/25. Pfs2400 is a mega- cycle, where most antigens are only expressed at dalton gametocyte antigen that has also been pro- specific stages and not throughout, implies an im- posed for consideration as a transmission-blocking mune response against one antigen expressed dur- vaccine. However, one monoclonal antibody that ing a limited portion of the life cycle will have no recognises this antigen, mAb1A1, was shown to effect on other portions of the life cycle; (iii) the significantly decrease the number of oocysts in genetic restriction of immune responses means no mosquitoes but not the number of infected mosqui- single antigen is uniformly immunogenic in all toes.[98] The mechanism of this transmission- populations. This is due to the requirement for blocking activity is not clear, since Pfs2400 is only stimulation of CD4+ or CD8+ T cells by peptide accessible on the surface briefly on emergence epitopes presented in the context of major histo- from an erythrocyte. compatibility complex (MHC) class II or class I A fourth gametocyte antigen of potential trans- molecules respectively. These epitopes are present mission-blocking activity has been identified, in some malaria antigens for individuals of some Pfg27/25.[99] It is not clear whether it induces trans- MHC type but not for others; and (iv) wild strains mission-blocking immunity since antibodies di- of malaria exhibit antigenic polymorphism, in

which the amino acid sequence for one protein can infecting strain of the parasite will likely express differ from one strain to another, meaning that im- at least some antigens that are similar in sequence mune responses against an antigen found in one to those contained in the vaccine. strain may not be effective against the same antigen Two approaches to multicomponent immunisa- in another strain. tion have been explored: (i) immunisation with a Antigenic polymorphism is of particular rele- viral vector able to express cloned parasite anti- vance in the testing of CS-based vaccines such as gens; and (ii) immunisation with a mixture of plas- RTS,S. In CS, this polymorphism seems to be con- mid DNA constructs that are able to express differ- centrated in regions that contain T cell epi- ent parasite antigens. topes.[106] This observation suggests these variations have been brought about by immunological pres- sure on the parasite. Since the human challenges 5.1 Multistage, Multicomponent Vaccines carried out today have consisted of a single strain of the parasite expressing the exact same amino A multistage, multicomponent vaccine could be acid sequence (homologous challenge) as the vac- achieved by simply combining several antigens in cine antigen, it is not known what the outcome will one formulation. However, current limitations on be on immunisation of a population exposed to the expression and purification of many malaria widely divergent isolates (heterologous chal- antigens prohibit this approach, or at least make it lenge). Not only is efficacy an important outcome very costly. Therefore, investigators have turned to to measure, but also the effect of the vaccine-in- utilising attenuated viral vectors, such as vaccinia, duced immunity on the prevalence of different par- as vehicles for the delivery of multiple antigens of asite strains. Field trials contemplated for the fu- P. falciparum. ture will attempt to answer some of these An initial attempt at using attenuated vaccinia questions. virus as a vector expressing P. berghei CS failed to induce protective immune responses by intra- 5. New Approaches to Malaria peritoneal immunisation in mice.[107] However, a Vaccine Development subsequent study utilising the attenuated vaccinia Due to some of the reasons stated in section 4, NYVAC expressing the P. berghei CS gene and the many investigators feel that immunisation with a same route of immunisation led to protection in single antigen is unlikely to induce 100% protec- >80% of mice.[108] A NYVAC containing 7 genes tion under natural exposure. Therefore, immunisa- of P. falciparum pre-erythrocytic and erythrocytic tion with multiple antigens at multiple stages of the stages (CS, LSA-1, SERA, MSP1, SSP2, AMA-1 parasite life cycle has been explored. The theoret- and Pfs25; NYVAC-Pf7) was subsequently engi- ical advantage of a multistage, multicomponent neered and administered to human volunteers in a vaccine is that any parasites that escape elimina- preliminary Phase I/IIa clinical trial. This vaccine tion by the host immune system upon entry may was found to be moderately immunogenic and still be eliminated by immune responses directed while no significant protection against sporozoite against subsequent stages. Use of multiple anti- challenge was achieved, a delay in the number of gens would also overcome, to some extent, the ge- [109] netic restriction observed in immunisations with days to patent infection was observed. Strate- single antigens. Therefore, in a multicomponent gies for enhancing the immunogenicity of this vac- vaccine, individuals that cannot mount a response cine including, in particular, use of prime im- against one antigen may be able to mount a re- munisations with vaccinia followed by boosts with sponse against other antigens. Antigenic polymor- recombinant products are currently under consid- phism would also be less of a problem since the eration.

5.2 DNA Vaccines purification is obviated. For these reasons, DNA vaccines may be ideal for multistage, multi- The immunisation strategies discussed above component immunisation as long as they are able involve the use of recombinant proteins, synthetic to induce protective responses in humans. peptides or viral vectors for the induction of an In addition to questions about immunogenicity, immune response. Recently, immunisation with safety questions about DNA vaccines need to be plasmid DNA that codes for a specific protein has addressed, such as the possibility of integration of been shown to be equally effective in inducing an the foreign DNA into the host genome. A small immune response against an antigen.[110] Since this phase I clinical trial is currently underway with a initial demonstration, DNA immunisations have PfCS plasmid construct. It is hoped that this pre- been used to elicit immune responses against a liminary study will lead to further studies of the wide variety of organisms.[111] Although the mech- safety, immunogenicity and efficacy of multigene anisms by which DNA immunisations work are not DNA vaccines in humans. completely understood, it is thought that DNA in- jected directly into the tissue is taken up by cells Acknowledgements and the encoded proteins expressed by the normal cell machinery. The subsequent fate of the antigen We are grateful to Dr Jeff Lyon for providing us with a is not clear, but is probably taken up by profes- draft of the life cycle figure. The views of the authors do not sional antigen-presenting cells (APC) and taken to purport to reflect the position of the Department of the Army or the Department of Defence. The US Government has the the regional lymph nodes where the primary im- right to retain a nonexclusive, royalty-free license in and to [112] mune response takes place. Recent experi- any copyright covering this paper. ments have demonstrated that mice can be partially protected against challenge with P. yoelii sporozo- References ites by immunisation with plasmid DNA coding for 1. Dame JB, Williams JL, McCutchan TF, et al. Structure of the the P. yoelii CS gene.[113] Furthermore, immunisa- gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium tion with a combination of DNA plasmids encod- falciparum. Science 1984 Aug 10; 225 (4662): 593-9 ing for two different pre-erythrocytic antigens of P. 2. Enea V, Ellis J, Zavala F, et al. DNA cloning of Plasmodium yoelii induced a higher degree of protection in mice falciparum circumsporozoite gene: amino acid sequence of repetitive epitope. Science 1984 Aug 10; 225 (4662): 628-30 of diverse genetic background than either vaccine 3. Caspers P, Gentz R, Matile H, et al. The circumsporozoite gene alone.[43] from NF54, a Plasmodium falciparum isolate used in malaria These results suggest that a cocktail of plasmids vaccine trials. Mol Biochem Parasitol 1989; 35: 185-9 4. Burkot TR, Da ZW, Geysen HM, et al. Fine specificities of with coding for different antigens may be more ef- monoclonal antibodies against the Plasmodium falciparum fective at inducing protection in a population of circumsporozoite protein: recognition of both repetitive and nonrepetitive regions. Parasite Immunol 1991; 13: 161-70 diverse genetic background. DNA immunisation, 5. Potocnjak P, Yoshida N, Nussezweig R, et al. Monovalent frag- therefore, is another potential way in which multi- ments (Fab) of monoclonal antibodies to a sporozoite surface stage, multicomponent vaccines could be delivered antigen (Pb44) protect mice against malarial infection. J Exp Med 1980 Jun; 151: 1504-13 effectively. 6. Yoshida N, Nussezweig RS, Potocnjak P, et al. Hybridoma pro- DNA vaccines have several advantages over duces protective antibodies directed against the sporozoite routine immunisations using peptide antigens: stage of malaria parasite. Science 1980; 207: 71 7. Ballou WR, Sherwood JA, Neva FA, et al. Safety and efficacy (i) DNA is relatively inexpensive to produce and of a recombinant DNA Plasmodium falciparum sporozoite purify; (ii) there may be no requirement for an ad- vaccine. Lancet 1987 Jun 6; 1277-81 8. Herrington DA, Clyde DF, Losonsky G, et al. Safety and immu- juvant; and (iii) DNA is more stable than peptide nogenicity in man of a synthetic peptide malaria vaccine antigens. DNA vaccines are more appealing than against Plasmodium falciparum sporozoites. Nature 1987 Jul recombinant vaccines for inducing immune re- 16; 328: 257-9 9. Fries LF, Gordon DM, Schneider I, et al. Safety, immunogenic- sponses against multiple antigens, since the need ity, and efficacy of a Plasmodium falciparum vaccine com- for expensive and time-consuming expression and prising a circumsporozoite protein repeat region peptide

conjugated to Pseudomonas aeruginosa toxin A. Infect Im- pled to hepatitis B surface antigen. Am J Trop Med Hyg 1991 mun 1992 May; 60 (5): 1834-9 Nov; 45 (5): 533-8 10. Ribi E, Cantrell J, Feldner T, et al. Biological activities of 26. Bomford R, Stapleton M, Winsor S, et al. Adjuvanticity and monophosphoryl lipid A. In: Levine L, Bonventre PF, Morello ISCOM formation by structurally diverse saponins. Vaccine JA, et al., editors. Microbiology. Washington, DC: American 1992; 10 (9): 572-7 Society for Microbiology, 1986 27. Rogers WO, Malik A, Mellouk S, et al. Characterization of 11. Fries LF, Gordon DM, Richards RL, et al. Liposomal malaria Plasmodium falciparum sporozoite surface protein 2. Proc vaccine in humans: a safe and potent adjuvant strategy. Proc Natl Acad Sci USA 1992 Oct 1; 89 (19): 9176-80 Natl Acad Sci USA 1992 Jan; 89: 358-62 28. Robson KJH, Hall JRS, Jennings MW, et al. A highly conserved 12. Hoffman SL, Edelman R, Bryan JP, et al. Safety, immunogenic- amino-acid sequence in thrombospondin, properdin and in ity, and efficacy of a malaria sporozoite vaccine administered proteins from sporozoites and blood stages of a human ma- with monophosphoryl lipid A, cell wall skeleton of laria parasite. Nature 1988 Sep; 335: 79-82 mycobacteria, and squalene as adjuvant. Am J Trop Med Hyg 29. Wizel B, Rogers WO, Houghten RA, et al. Induction of mu- 1994; 51 (5): 603-12 rine cytotoxic T lymphocytes against Plasmodium falci- 13. Weiss WR, Sedegah M, Beaudoin R, et al. CD8+ T cells (cyto- parum sporozoite surface protein 2. Eur J Immunol 1994; toxic/suppressors) are required for protection in mice immu- 24: 1487-95 nized with malaria sporozoites. Proc Natl Acad Sci USA 1987 30. Wizel B, Houghten RA, Parker KC, et al. Irradiated sporozoite Jan; 85: 573-6 vaccine induces HLA-B8-restricted cytotoxic T lymphocyte 14. Schofield L, Villaquiran J, Ferreira A, et al. γInterferon, CD8+ T cells and antibodies required for immunity to malaria spo- responses against two overlapping epitopes of the Plasmo- rozoites. Nature 1987 Dec; 330: 664-6 dium falciparum sporozoite surface protein 2. J Exp Med 15. Sinigaglia F, Matile H, Pink JRL. Plasmodium falciparum- 1995 Nov; 182: 1435-45 specific human T cell clones: evidence for helper and cyto- 31. Wizel B, Houghten R, Church P, et al. HLA-A2-restricted cytotoxic activities. Eur J Immunol 1987; 17: 187-92 toxic T lymphocyte responses to multiple Plasmodium 16. Good MF, Pombo D, Quakyi IA, et al. Human T-cell recognition falciparum sporozoite surface protein 2 epitopes in sporozo- of the circumsporozoite protein of Plasmodium falciparum: ite-immunized volunteers. J Immunol 1995; 155: 766-75 immunodominant T-cell domains map to the polymorphic re- 32. Wang R, Charoenvit Y, Corradin G, et al. Protection against gions of the molecule. Proc Natl Acad Sci USA 1988 Feb; 85: malaria by Plasmodium yoelii sporozoite surface protein 2 1199-203 linear peptide induction of CD4+ T cell and IFN-γ-dependent 17. Kumar S, Miller LH, Quakyi IA, et al. Cytotoxic T cells specific elimination of infected hepatocytes. J Immunol 1996; 157: for the circumsporozoite protein of Plasmodium falciparum. 4061-7 Nature 1988 Jul; 334: 258-60 33. Khusmith S, Charoenvit Y, Kumar S, et al. Protection against 18. Sinigaglia F, Guttinger M, Kilgus J, et al. A malaria T-cell malaria by vaccination with sporozoite surface protein 2 plus epitope recognized in association with most mouse and hu- CS protein. Science 1991 May 3; 252 (5006): 715-8 man MHC class II molecules. Nature 1988 Dec; 336: 778-80 34. Guerin-Marchand C, Druilhe P, Galey B, et al. A liver-stage- 19. Moreno A, Clavijo P, Edelman R, et al. CD4+ T cell clones specific antigen of Plasmodium falciparum characterized by obtained from Plasmodium falciparum sporozoite-immu- gene cloning. Nature 1987 Sep; 329: 164-7 nized volunteers recognize polymorphic sequences of the cir- 35. Hill AVS, Allsopp CEM, Kwiatkowski D, et al. Common west cumsporozoite protein. J Immunol 1993 Jul; 151: 489-99 African HLA antigens are associated with protection from 20. Malik A, Egan JE, Houghten RA, et al. Human cytotoxic T severe malaria. Nature 1991 Aug 15; 352: 595-600 lymphocytes against the Plasmodium falciparum cir- 36. Hill AVS, Elvin J, Willis AC, et al. Molecular analysis of the cumsporozoite protein. Proc Natl Acad Sci USA 1991 Apr 15; association of HLA-B53 and resistance to severe malaria. 88 (8): 3300-4 Nature 1992 Dec; 360: 434-9 21. Cerami C, Frevert U, Sinnis P, et al. The basolateral domain of 37. Fidock DA, Gras-Masse H, Lepers JP, et al. Plasmodium the hepatocyte plasma membrane bears receptors for the cir- falciparum liver stage antigen-1 is well conserved and con- cumsporozoite protein of Plasmodium falciparum sporozo- tains potent B and T cell determinants. J Immunol 1994; 53: ites. Cell 1992 Sep; 70: 1021-33 190-204 22. Heppner DG, Gordon DM, Gross M, et al. Safety, immunoge- 38. Coppel RL, Favaloro JM, Crewther PE, et al. A blood stage nicity, and efficacy of Plasmodium falciparum repeatless cir- antigen of Plasmodium falciparum shares determinants with cumsporozoite protein vaccine encapsulated in liposomes. J Infect Dis 1996; 174: 361-6 the sporozoite coat protein. Proc Natl Acad Sci USA 1985 23. Gordon DM, McGovern TW, Krzych U, et al. Safety, immu- Aug; 82 (15): 5121-5 nogenicity, and efficacy of a recombinantly produced Plas- 39. Hope I, Mackay M, Hyde J, et al. The gene for an exported modium falciparum circumsporozoite protein-hepatitis B antigen of the malaria parasite Plasmodium falciparum surface antigen subunit vaccine. J Infect Dis 1995; 171: cloned and expressed in Escherichia coli. Nucleic Acids Res 1576-85 1985; 13: 369-79 24. Stoute JA, Slaoui M, Heppner DG, et al. A preliminary evalu- 40. Gunther K, Tummler M, Hans-Henning A, et al. An exported ation of a recombinant circumsporozoite protein vaccine protein of Plasmodium falciparum is synthesized as an inte- against Plasmodium falciparum malaria. N Engl J Med 1997; gral membrane protein. Mol Biochem Parasitol 1991; 46: 336: 86-91 149-58 25. Vreden SGS, Verhave JP, Oettinger T, et al. Phase I clinical trial 41. Simmons D, Woollett G, Bergin-Cartwright M, et al. A malaria of a recombinant malaria vaccine consisting of the cir- protein exported into a new compartment within the host cumsporozoite repeat region of Plasmodium falciparum cou- erythrocyte. EMBO J 1987; 6 (2): 485-91

42. Sanchez GI, Rogers WO, Mellouk S, et al. Plasmodium dium falciparum recognizes the terminal Neu5Ac(α2-3)Gal- falciparum: Exported Protein-1, a blood stage antigen, is ex- Sequences of glycophorin A. J Cell Biol 1992 Feb; 116 (4): pressed in liver stage parasites. Exp Parasitol 1994; 79: 59-62 901-9 43. Doolan DL, Sedegah M, Hedstrom RC, et al. Circumventing 59. Sim BKL, Chitnis CE, Wasniowska K, et al. Receptor and genetic restriction of protection against malaria with multi- ligand domains for invasion of erythrocytes by Plasmodium gene DNA immunization: CD8+ T cell-, interferon γ-, and falciparum. Science 1994 Jun; 264: 1941-4 nitric oxide-dependent immunity. J Exp Med 1996 Apr; 183: 60. Daugherty JD, Murphy CI, Doros-Richert LA, et al. Bac- 1739-46 ulovirus-mediated expression of Plasmodium falciparum 44. Doolan DL, Hedstrom RC, Rogers WO, et al. Identification and erythrocyte binding antigen-175 (EBA-175) polypeptides and characterization of the protective Hepatocyte Erythrocyte their recognition by human antibodies. Infect Immun. In press Protein 17 kDa gene of Plasmodium yoelii, homolog of Plas- 61. Perrin LH, Merkli B, Loche M, et al. Antimalarial immunity in modium falciparum Exported Protein 1. J Biol Chem 1996 Saimiri monkeys. Immunization with surface components of Jul; 271 (30): 17861-8 asexual blood stages. J Exp Med 1984 Aug; 160: 441-51 45. Stürchler D, Just M, Berger R, et al. Evaluation of 5.1- 62. Deans JA, Alderson T, Thomas AW, et al. Rat monoclonal anti- [NANP]19, a recombinant Plasmodium falciparum vaccine bodies which inhibit the in vitro multiplication of Plasmodium candidate, in adults. Trop Geogr Med 1992; 44: 9-14 knowlesi. Clin Exp Immunol 1982; 49: 297-309 46. Blackman MJ, Heidrich HG, Donachie S, et al. A single frag- 63. Thomas AW, Deans JA, Mitchell GH, et al. The Fab fragments ment of a malaria merozoite surface protein remains on the of monoclonal IgG to a merozoite surface antigen inhibit parasite during red cell invasion and is the target of invasion- Plasmodium knowlesi invasion of erythrocytes. Mol Biochem inhibiting antibodies. J Exp Med 1990 July; 172: 379-82 Parasitol 1984; 13: 187-99 47. Chang SP, Gibsoon HL, Lee-Ng CT, et al. A carboxyl-terminal 64. Collins WE, Pye D, Crewther PE, et al. Protective immunity fragment of Plasmodium falciparum gp195 expressed by a induced in Squirrel monkeys with recombinant apical mem- recombinant baculovirus induces antibodies that completely brane antigen-1 of Plasmodium fragile. Am J Trop Med Hyg inhibit parasite growth. J Immunol Jul 1992; 149 (2): 548-55 1994; 51 (6): 711-9 48. Egan AF, Morris J, Barnish G, et al. Clinical immunity to Plas- 65. Brown GV, Culvenor JG, Crewther PE, et al. Localization of the modium falciparum malaria is associated with serum antibod- ring-infected erythrocyte surface antigen (RESA) of Plasmo- ies to the 19-kDa c-terminal fragment of the merozoite surface dium falciparum in merozoites and ring-infected erythro- antigen, PfMSP-1. J Infect Dis March 1996; 173: 765-9 cytes. J Exp Med 1985 Aug; 162: 774-9 49. Chang SP, Case SE, Gosnell WL, et al. A recombinant bac- 66. Berzins K, Perlmann H, Whalin B, et al. Rabbit and human ulovirus 42-kilodalton c-terminal fragment of Plasmodium antibodies to a repeated amino acid sequence of a Plasmodium falciparum merozoite surface protein 1 protects Aotus mon- falciparum antigen, Pf 155, react with the native protein and keys against malaria. Infect Immun 1996 Jan; 64 (1): 253-61 inhibit merozoite invasion. Proc Natl Acad Sci USA 1986 50. Ling IT, Ogun SA, Holder AA. Immunization against malaria Feb; 83: 1065-9 with a recombinant protein. Parasite Immunol 1994; 16: 63-7 51. Daly TM, Long CA. A recombinant 15-kilodalton carboxy-ter- 67. Collins WE, Anders RF, Pappaioanou M, et al. Immunization minal fragment of Plasmodium yoelii 17XL merozoite surface of Aotus monkeys with recombinant proteins of an erythro- protein 1 induces a protective immune response in mice. In- cyte surface antigen of Plasmodium falciparum. Nature 1986 fect Immun 1993 Jun; 61 (6): 2462-7 Sep; 323: 259-62 52. Daly T, Long CA. Humoral response to a carboxy-terminal re- 68. Chirakalwasan N, Kamol-Ratanakul P, Lermaharit S, et al. A gion of the merozoite surface protein-1 plays a predominant longitudinal study of seroreactivities to a major blood stage role in controlling blood-stage infection in rodent malaria. J antigen (Pf155/RESA) of the malaria parasite Plasmodium Immunol 1995; 155: 236-43 falciparum in an endemic area of Thailand. Southeast Asian 53. Burghaus PA, Wellde BT, Hall T, et al. Immunization of Aotus J Trop Med Public Health March 1994; 25 (1): 25-31 nancymai with recombinant C terminus of Plasmodium 69. Al-Yaman F, Genton B, Anders R, et al. Assessment of the role falciparum merozoite surface protein 1 in liposomes and alum of the humoral response to Plasmodium falciparum MSP2 adjuvant does not induce protection against a challenge infec- compared to RESA and Spf66 in protecting Papua New Guin- tion. Infect Immun 1996 Sep; 64 (9): 3614-9 ean children from clinical malaria. Parasite Immunol 1995; 54. Crisanti A, Müller HM, Hilbich C, et al. Epitopes recognized 17: 493-501 by human T cells map within the conserved part of the GP190 70. Rooth I, Perlmann H, Anders B. Plasmodium falciparum rein- of P. falciparum. Science 1988 Jun; 240: 1324-6 fection in children from a holoendemic area in relation to 55. Rzepczyk CM, Ramasamy R, Mutch DA, et al. Analysis of seroreactivities against oligopeptides from different malaria human T cell response to two Plasmodium falciparum mero- antigens. Am J Trop Med Hyg 1991; 45 (3): 309-18 zoite surface antigens. Eur J Immunol 1989; 19: 1797-802 71. Banyal HS, Inselburg J. Isolation and characterization of para- 56. Camus D, Hadley TJ. A Plasmodium falciparum antigen that site-inhibitory Plasmodium falciparum monoclonal antibod- binds to host erythrocytes and merozoites. Science 1985 Nov; ies. Am J Trop Med Hyg 1985; 34: 1055-64 230: 553-6 72. Horii T, Bzik DJ, Inselburg J. Characterization of antigen-ex- 57. Sim KL, Orlandi PA, Haynes JD, et al. Primary structure of the pressing Plasmodium falciparum cDNA clones that are reac- 175K Plasmodium falciparum erythrocyte binding antigen tive with parasite inhibitory antibodies. Mol Biochem and identification of a peptide which elicits antibodies that Parasitol 1988; 30: 9-18 inhibit malaria merozoite invasion. J Cell Biol 1990 Nov; 111: 73. Bzik DJ, Li W, Horii T, et al. Amino acid sequence of the ser- 1877-84 ine-repeat antigen (SERA) of Plasmodium falciparum deter- 58. Orlandi PA, Klotz FW, Haynes JD. A malaria invasion receptor, mined from cloned cDNA. Mol Biochem Parasitol 1988; 30: the 175-kilodalton erythrocyte binding antigen of Plasmo- 279-88

74. Higgins DG, McDonnell DJ. Malarial proteinase? Nature 1989 children in northwestern Thailand. Lancet 1996 Sept; 348: Aug; 340: 604 701-7 75. Delplace P, Fortier B, Tronchin G, et al. Localization, biosyn- 92. Quakyi IA, Carter R, Rener J, et al. The 230-kDa gamete sur- thesis, processing and isolation of a major 126 kDa antigen face protein of Plasmodium falciparum is also a target for of the parasitophorous vacuole of Plasmodium falciparum. transmission-blocking antibodies. J Immunol 1987 Dec; 139: Mol Biochem Parasitol 1987; 23: 193-201 4213-7 76. Inselburg J, Bzik DJ, Li W, et al. Protective immunity induced 93. Vermeulen AN, Meuwissen JHET. Sequential expression of an- in Aotus monkeys by recombinant SERA proteins of Plasmo- tigens on sexual stages of P. falciparum accessible to trans- dium falciparum. Infect Immun 1991 Apr; 59 (4): 1247-50 mission blocking antibodies in the mosquito. J Exp Med 77. Inselburg J, Bathurst IC, Kansopon J, et al. Protective immunity 1985; 162: 1460-76 induced in Aotus monkeys by a recombinant SERA protein 94. Williamson KC, Keister DB, Muratova O, et al. Recombinant of Plasmodium falciparum: adjuvant effects on induction of Pfs230, a Plasmodium falciparum gametocyte protein, in- protective immunity. Infect Immun 1993 May; 61 (5): 2041-7 duces antisera that reduce the infectivity of Plasmodium 78. Inselburg J, Bathurst, Kansopon J, et al. Protective immunity falciparum to mosquitoes. Mol Biochem Parasitol 1995; 75: induced in Aotus monkeys by a recombinant SERA protein 33-42 of Plasmodium falciparum: further studies using SERA 1 and 95. Graves PM, Carter R, Burkot TR, et al. Antibodies to Plasmo- MF75.2 adjuvant. Infect Immun 1993 May; 61 (5): 2048-52 dium falciparum gamete surface antigens in Papua New 79. Lopez MC, Silva Y, Thomas MC, et al. Characterization of Guinea sera. Parasite Immunol 1988; 10: 209-18 SPf(66)n: a chimeric molecule used as a malaria vaccine. 96. Roeffen W, Mulde R, Teelen K, et al. Association between anti- Vaccine 1994; 12 (7): 585-91 Pfs48/45 reactivity and P. falciparum transmission-blocking 80. Patarroyo ME, Romero P, Torres ML, et al. Protective synthetic activity in sera from Cameroon. Parasite Immunol 1996; 18: peptides against experimental Plasmodium falciparum-in- 103-9 duced malaria. In: Brown F, Chanock R, Lerner R, editors. 97. Premawansa S, Gamage-Mendis A, Perera L, et al. Plasmodium Vaccines 87. Cold Spring Harbor Laboratory, 1987: 117-24 falciparum malaria transmission-blocking immunity under 81. Patarroyo ME, Romero P, Torres ML, et al. Induction of pro- conditions of low endemicity as in Sri Lanka. Parasite Im- tective immunity against experimental infection with malaria munol 1994; 16: 35-42 using synthetic peptides. Nature 1987 Aug; 328: 629-32 98. Feng Z, Hoffmann RN, Nussenzweig RS, et al. Pfs2400 can 82. Rodriguez R, Moreno A, Guzman F, et al. Studies in owl mon- mediate antibody-dependent malaria transmission inhibition keys leading to the development of a synthetic vaccine against and may be the Plasmodium falciparum 11.1 gene product. J the asexual blood stages of Plasmodium falciparum. Am J Exp Med 1993 Feb; 177: 273-81 Trop Med Hyg 1999; 43 (4): 339-54 99. Carter R, Graves PM, Creasy A, et al. Plasmodium falciparum: 85. Patarroyo ME, Amador R, Clavijo P, et al. A synthetic vaccine an abundant stage-specific protein expressed during early ga- protects humans against challenge with asexual blood stages metocyte development. Exp Parasitol 1989; 69: 140-9 of Plasmodium falciparum malaria. Nature 1988 Mar; 332: 100. Wizel B, Kumar N. Identification of a continuous and cross-re- 158-61 acting epitope for Plasmodium falciparum transmission- 84. Ruebush TK, Campbell GH, Moreno A, et al. Immunization of blocking immunity. Proc Natl Acad Sci USA 1991 Nov; 88: owl monkeys with a combination of Plasmodium falciparum 9533-7 asexual blood-stage synthetic peptide antigens. Am J Trop Med Hyg 1990; 43 (4): 355-66 101. Kaslow DC, Quaki IA, Syin C, et al. A vaccine candidate from 85. Amador R, Moreno A, Valero V, et al. The first field trials of the sexual stage of human malaria that contains EGF-like the chemically synthesized malaria vaccine SPf66: safety, im- domains. Nature 1988 May; 333: 74-6 munogenicity and protectivity. Vaccine 1992; 10 (3): 179-84 102. Barr PJ, Green KM, Gibson HL, et al. Recombinant Pfs25 pro- 86. Valero MV, Amador LR, Galindo C, et al. Vaccination with tein of Plasmodium falciparum elicits malaria transmission- SPf66, a chemically synthesized vaccine, against Plasmo- blocking immunity in experimental animals. J Exp Med 1991 dium falciparum malaria in Colombia. Lancet 1993 Mar; 341: Nov; 174: 1203-8 705-10 103. Kaslow DC, Bathurst IC, Lensen T, et al. Saccharomyces cere- 87. Noya O, Berti YG, Alarcon de Noya B, et al. A population-based visiae recombinant Pfs25 adsorbed to alum elicits antibodies clinical trial with the SPf66 synthetic Plasmodium falciparum that block transmission of Plasmodium falciparum. Infect Im- malaria vaccine in Venezuela. J Infect Dis 1994; 170: 396-402 mun 1994 Dec; 62 (12): 5576-80 88. Sempertegui F, Estrella B, Moscoso J, et al. Safety, immunoge- 104. Duffy P, Kaslow DC. A novel malaria protein, Pfs28, and Pfs25 nicity and protective effect of the SPf66 malaria synthetic are genetically linked and synergistic as falciparum malaria vaccine against Plasmodium falciparum infection in a ran- transmission-blocking vaccines. Infect Immun 1997 Mar; 65 domized double-blind placebo-controlled field trial in an en- (3): 1109-13 demic area of Ecuador. Vaccine 1994; 12 (4): 337-42 105. Kaslow DC. Transmission-blocking vaccines: uses and current 89. Alonso PL, Smith T, Armstrong JRM, et al. Randomised trial status of development. Int J Parasitol 1997; 27 (2): 183-9 of efficacy of SPf66 vaccine against Plasmodium falciparum 106. Lockyer MJ, Marsh K, Newbold CI. Wild isolates of Plasmo- malaria in children in southern Tanzania. Lancet 1994 Oct; dium falciparum show extensive polymorphism in T cell 344: 1175-81 epitopes of the circumsporozoite protein. Mol Biochem 90. D’Alessandro U, Leach A, Drakeley CJ, et al. Efficacy trial of Parasitol 1989 Dec; 37 (2): 275-80 malaria vaccine SPf66 in Gambian infants. Lancet 1995 Aug; 107. Satchidanandam V, Zavala F, Moss B. Studies using a recom- 346: 462-7 binant vaccinia virus expressing the circumsporozoite protein 91. Nosten F, Luxemburger C, Kyle DE, et al. Randomised double- of Plasmodium berghei. Mol Biochem Parasitol 1991; 48: blind placebo-controlled trial of SPf66 malaria vaccine in 89-100

108. Lanar DE, Tine JA, de Taisne C, et al. Attenuated vaccinia 112. Mor G, Klinman DM, Shapiro S, et al. Complexity of the cytok- virus-circumsporozoite protein recombinants confer pro- ine and antibody response elicited by immunizing mice with tection against rodent malaria. Infect Immun 1996 May; 64 Plasmodium yoelii circumsporozoite protein plasmid DNA. J (5): 1666-71 Immunol 1995; 155: 2039-46 109. Ockenhouse CF, Sun PF, Lanar DE, et al. Phase I/IIa safety, 113. Sedegah M, Hedstrom R, Hobart P, et al. Protection against malaria by immunization with plasmid DNA encoding cir- immunogenicity and efficacy trial of NYVAC-Pf7, a pox- cumsporozoite protein. Proc Natl Acad Sci USA 1994 Oct; vectored, multiantigen, multistage vaccine candidate for Plas- 91: 9866-70 modium falciparum malaria. J Infect Dis 1998 Jun; 177 (6): 1664-73 110. Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection against influenza by injection of DNA encoding a viral Correspondence and reprints: Dr José A. Stoute, Department protein (see comments). Science 1993 Mar 19; 259: 1745-9 of Immunology, Bldg. 40, Walter Reed Army Institute of 111. Liu MA, Hilleman, MR, Kurth R. DNA vaccines: a new era in Research, Washington, DC 20307, USA. vaccinology. Ann NY Acad Sci 1995; 72: 1-294 E-mail: [email protected]