British Medical Bulletin

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British Medical Bulletin 62:59-72 (2002)
© 2002 The British Council

Malaria vaccines

Vasee Moorthy and Adrian V S Hill

Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK
MRC Laboratories, Fajara, The Gambia

	Abstract

Top Abstract Introduction Life-cycle Natural versus vaccine immunity Development of malaria subunit... Pre-erythrocytic vaccines Blood stage vaccines Sexual stage vaccines Discussion References

 
Malaria kills one child in Africa every 30 s. After summarising the burden of malaria, the life-cycle of this parasite in humans and female Anopheles mosquitoes is outlined. Important differences between natural immunity and that induced by current candidate vaccines are discussed. In the main part of the review, the recent rapid expansion in evaluation of candidate malaria vaccines in clinical trials across the world is discussed. Subunit vaccine technologies are progressing rapidly with new delivery systems, vectors and antigens under evaluation as well as new polyepitope approaches. Combination vaccination regimens, improved adjuvants and genetic engineering of antigens are all improving the immunogenicity of candidate vaccines. We also discuss particular difficulties in vaccination against malaria, the conduct of field trials of malaria vaccines in non-industrialised countries and the need for even greater co-operation between researchers. Finally, the important concept of iterative vaccine development is raised and the prospects for effective malaria vaccination are discussed.

	Introduction

Top Abstract Introduction Life-cycle Natural versus vaccine immunity Development of malaria subunit... Pre-erythrocytic vaccines Blood stage vaccines Sexual stage vaccines Discussion References

 
Malaria causes more deaths than any other parasitic disease and is probably the most important pathogen for children under 5 years of age in sub-Saharan Africa. Every 30 seconds one child in Africa dies from malaria¹. There are over 2 billion people living in exposed regions, 300–500 million clinical cases of malaria each year and 1–2 million deaths annually^2,3. These numbers, however, do not convey the full impact of the disease. In much of sub-Saharan Africa, malaria contributes to a great extent to high rates of maternal and infant mortality. It plays a critical role in sustaining the cycle of ill health and poverty. The socio-economic prospects for malaria-endemic countries may be linked closely to the disease burden of malaria⁴. The estimated loss to the African economy in 1997 alone was $2 billion⁵. The World Bank rates malaria as the single biggest component of total disease burden in Africa. In recent years, malaria has returned to areas from which it had been previously eradicated and transmission has spread into areas where it had never been documented in the past⁶ and the disease burden has increased in many endemic countries⁷. The following factors contribute to the resurgence of malaria: (i) increasing insecticide-resistance in the vector; (ii) increasing and often multiple drug resistance in the parasite; (iii) increasing instability in many areas leading to large and unpredictable population migrations; (iv) atmospheric carbon accumulation increasing the proportion of the world climatically suitable for transmission; and (v) increasing tourism between non-endemic and endemic countries and deteriorating public health systems in some endemic countries which had previously controlled disease. Advances have been made in malaria control in recent years, particularly demonstration of the effectiveness of insecticide-treated bednets^8,9 and the clinical development of dapsone/chlorproguanil, a new, cheap antimalarial suitable for wide-spread use in research-poor settings¹⁰. Combining dapsone/chlorproguanil with artemesinin derivatives, if subsidies are available, should delay or prevent the emergence of drug resistance. Nevertheless, these control measures are unlikely to be the answer to the malaria problem. Africa waits for an effective vaccine.

We shall review recent advances in the understanding of protective immune responses to malaria, appraise promising candidate vaccines (Table 1) in preclinical and clinical development, and signpost likely future directions and pitfalls in malaria vaccine development. The focus is on Plasmodium falciparum as this species accounts for the vast majority of deaths from malaria.

View this table: [in this window] [in a new window]   Table 1 Candidate malaria vaccines in phase 1/phase 2 clinical trials

 

	Life-cycle

Top Abstract Introduction Life-cycle Natural versus vaccine immunity Development of malaria subunit... Pre-erythrocytic vaccines Blood stage vaccines Sexual stage vaccines Discussion References

 
Each bite of a P. falciparum infected female anopheline mosquito inoculates about 5–20 sporozoites^11,12. These migrate to and enter hepatocytes within minutes. Over 7–14 asymptomatic days, each sporozoite multiplies and differentiates intracellularly into a liver-stage trophozoite and ultimately schizonts which lyse the hepatocyte releasing 10,000–30,000 merozoites into the systemic circulation. From this point onwards, a cycle of red cell invasion, about 10-fold multiplication, and lysis continues until the death of the human host or control of the infection by the immune system. Some merozoites responding to unknown stimuli differentiate into male or female gametocytes, which can persist for years within the host. When a male and female gametocyte are ingested by a female anopheline mosquito, thermal and pH signalling trigger exflagellation of the male gametocyte and zygote formation. Migration through the mosquito midgut epithelium occurs with differentiation into an adherent oocyst. When this oocyst lyses, sporozoites are released which swim to the salivary glands to complete the life-cycle.

	Natural versus vaccine immunity

Top Abstract Introduction Life-cycle Natural versus vaccine immunity Development of malaria subunit... Pre-erythrocytic vaccines Blood stage vaccines Sexual stage vaccines Discussion References

 
Repeated infection with malaria causes gradual acquisition of strain-specific, short-lived, antibody- and cell-mediated immunity to blood stage malaria with prevention of: first, complications and death, then disease and, in areas of intense stable transmission, ultimately suppression of parasitaemia to low or undetectable levels. The degree to which pre-erythrocytic immunity contributes to overall malaria immunity is unclear. Thus the crux of the difficulty in developing a malaria vaccine, vaccine-induced immunity must be superior to natural immunity in several ways – rapid acquisition, greater duration, and broad-strain transcendence. Vaccination studies have shown that immunity to a malarial antigen from one stage of the parasite's life-cycle is confined to that stage. This stage-specificity of malarial immunity is a further hindrance to vaccine development. With sporozoite inoculation rates greater than 1 per day in parts of Africa (reviewed by Hay et al¹³), an effective vaccine would benefit from boosting by natural exposure.

	Development of malaria subunit vaccines

Top Abstract Introduction Life-cycle Natural versus vaccine immunity Development of malaria subunit... Pre-erythrocytic vaccines Blood stage vaccines Sexual stage vaccines Discussion References

 
Until the 1980s, all vaccines in use were either live, attenuated or inactivated whole organism vaccines mostly to viral pathogens. The development of live, attenuated or killed malaria parasites for vaccination is not practical at the present time. Subunit vaccines, a product of recombinant DNA technology, are a major advance. Protein subunit vaccines are derived from whole or partial genes expressed in bacterial, baculovirus or plasmid systems or synthetically and purified as protein products. The hepatitis B vaccine widely administered today is an example of an effective vaccine developed in this way. A drawback of this method is the lack of inflammatory cytokines induced by most protein subunits and the consequent need for adjuvants for immunogenicity. Effective adjuvants with minimal reactogenicity are necessary. When immunogenic, this approach tends to lead to a Th2 bias with good antibody induction but limited cellular responses. Addition of strong T helper epitopes is likely to improve such immunogenicity. Such vaccines could not target the liver-stage malaria parasite.

The latest generation of vaccines are DNA and recombinant viral subunit vaccines, which have emerged clinically over the last few years. The DNA sequence for a protein or series of epitopes is inserted into either a circular Escherichia coli derived purified plasmid DNA molecule or the genome of a double-stranded DNA virus such as vaccinia. These lead to intracellular synthesis, processing and presentation of class I and II T-cell epitopes by HLA molecules for strong CD8⁺ and CD4⁺ T-cell immunogenicity. These vaccines are attractive as candidates for liver-stage malaria vaccines as well as, for example, HIV and tuberculosis. As well as use with whole antigens, these vaccines are particularly well suited to the use of strings of minimal T-cell epitopes known to be the targets of HLA-restricted T-cell responses in individuals naturally exposed to malaria.

Finding the correct answer to two key questions will determine the efficacy of such subunit vaccines in malaria: (i) which delivery system is best for stage-specific immunity; and (ii) more challengingly, which antigen or combination of antigens will provide the desired protection? There are two approaches to identifying antigens for inclusion in subunit vaccines. The first approach is identification of immunological correlates of protection from both vaccine- and naturally-induced immune responses. An HLA association with disease severity for malaria in The Gambia is one example of such a correlate¹⁴. The enormous heterogeneity of both vaccine-induced and natural protective immunity has rendered the search for such correlates extremely challenging. Recent progress has been made in this area thanks to advances in immunoassays available to analyse T-cell responses. A CD4⁺ response to a conserved epitope from the circumsporozoite protein (CS) correlated recently with protection from P. falciparum malaria infection in Gambian adults¹⁵. CS is the best characterised sporozoite protein^16,17. Re-assessment of this correlate in infants and differing transmission settings and populations would be of value. If vaccine design could be tailored to amplify such immunological correlates of protection, this would provide a particularly focused and non-empirical approach to vaccine development.

Running in parallel to this approach is the activity of antigen identification. Sequencing of the entire P. falciparum genome, estimated to include over 5000 genes, is scheduled for completion very soon. A proteomics exercise will follow to identify the full complement of proteins expressed by the sporozoite, by the liver-stage parasite, on the surface of merozoites, infected red cells, gametocytes and ookinetes. Characterisation of immune responses to this new array of potential target antigens will need to be required. Any protein expressed in the liver-stage might be the target of a protective immune response irrespective of function or location within the parasite. In contrast, only a subset of antigens and epitopes expressed by blood-stage parasites are likely to be of value for antibody-mediated protection.

	Pre-erythrocytic vaccines

Top Abstract Introduction Life-cycle Natural versus vaccine immunity Development of malaria subunit... Pre-erythrocytic vaccines Blood stage vaccines Sexual stage vaccines Discussion References

 
A pre-erythrocytic vaccine would either prevent invasion of hepatocytes by sporozoites or kill parasites within infected hepatocytes. For 30 years it has been known that immunisation of mice¹⁸ and humans¹⁹ with radiation-attenuated sporozoites, which abort their development at the liver-stage, confers sterile protective immunity for up to 9 months²⁰. This form of immunisation is impractical for wide-spread use. The finding that immunisation with more highly attenuated sporozoites (exposed to higher doses of radiation and thus rendered unable to invade hepatocytes) is not protective implies that protection in this model is primarily cell-mediated against the liver-stage parasite. Nevertheless, much attention has been focused on antibody induction to the NANP repeat region of CS. Initial human studies with recombinant²¹ and synthetic²² CS protein vaccines were disappointing. A recombinant fusion protein of the C-terminus of CS and hepatitis B surface antigen, known as RTS,S²³, when administered with the proprietary adjuvant AS02 including monophosphoryl lipid A and QS-21, provides partial protection against laboratory challenge with the homologous strain of P. falciparum. Efficacy in this model is 30–50%²⁴, the highest of any non-sporozoite vaccine to date. RTS,S/AS02 induces high-titre IgG to the CS repeat, to a lesser extent to the flanking CS regions, and high-titre antibodies to the hepatitis B surface antigen as well as strong proliferative T lymphocyte responses to RTS,S. Effector T-cell responses (as assayed by an ex vivo IFN- enzyme-linked immunospot [ELISPOT] assay) are comparatively low after RTS,S/AS02 vaccination and no CD8⁺ T-cells specific for CS peptides have been detected in vaccinees²⁵. In a field efficacy trial of RTS,S/AS02 in Gambian semi-immune adults, there was 71% efficacy to 9 weeks (95% confidence interval [CI] 46–85%) with marked waning of efficacy thereafter and no significant difference in incidence of parasitaemia at the end of the 15 week surveillance period²⁶. Whilst an association between IgG titre to the CS repeat and protection has been seen in efficacy trials with RTS,S/AS02, it remains unclear which immune effector mechanism causes the protection afforded by this vaccine. Phase I trials of RTS,S/AS02 in children occurred in 2001 and contingent on acceptable reactogenicity, a field efficacy trial is planned in children aged 1–5 years in 2002/2003.

In an elegant minimal epitope approach, vaccination in alum/QS-21 adjuvant with a synthetic multiple antigen peptide construct containing only a single human class II epitope from CS and three NANP repeats has been shown to induce high-titre antibody to CS, antibodies which reacted with sporozoites²⁷. However, volunteers were selected for possession of HLA alleles known to bind the class II epitope and there were problematic immediate hypersensitivity reactions in some volunteers. In a later study, a related synthetic construct, including a universal class II epitope, was safe and immunogenic in 7/10 volunteers²⁸. The major advantages of use of class II rather than class I epitopes are 2-fold. Some class II epitopes can bind to a number of HLA types and so population coverage is possible with a limited number of epitopes. Second, class II epitopes induce CD4⁺ T-cells which can kill intracellular parasites by secreting IFN-²⁹ as well as providing help to B-cells for antibody production. However CD4⁺ T-cells may be less effective in eliminating liver-stage parasites than CD8⁺ T-cells³⁰, because human infected hepatocytes show limited HLA class II expression.

In 1994, protection from malaria in a mouse model was demonstrated with a DNA vaccine encoding Plasmodium yoelii CS³¹. A phase I clinical trial reported safety and immunogenicity of a DNA vaccine encoding P. falciparum CS in 1998³². Improvement of murine immunogenicity occurs with co-administration of a plasmid encoding GM-CSF³³. Clinical trials were recently undertaken by a US Navy group with a combination of five plasmid DNAs each encoding a different P. falciparum pre-erythrocytic antigen together with a plasmid encoding human GM-CSF³⁴, but little evidence of efficacy was observed in this initial challenge study.

Researchers in Oxford discovered that immunisation of mice or non-human primates with a DNA vaccine followed by immunisation with a recombinant virus encoding the same antigen amplified CD8⁺ T-cell responses 10–100-fold in malaria and HIV models. The viral vector which gave highest immunogenicity in this system was modified vaccinia virus Ankara (MVA)^35–38. No amplification of the response was seen when immunisations with MVA were followed by DNA. In a mouse model of malaria, complete protection against challenge was obtained in some experiments and was correlated with secretion of IFN- by freshly isolated spleen cells stimulated by a 9 amino acid T-cell epitope in an ELISPOT assay³⁹. Recently, protection was demonstrated with a DNA/MVA polyprotein regimen from mucosal challenge in a HIV model in macaques, confirming that the principle transfers well to primate immunity⁴⁰.

Since 1999, clinical trials of this DNA/MVA so-called prime/boost approach designed to maximise T-cell immunogenicity against liver-stage parasites have been on-going in Oxford. The two vaccines encode the identical malarial DNA sequence, consisting of a string of T- and B-cell epitopes from P. falciparum pre-erythrocytic antigens, known as the ME (multiple epitope) string fused to the entire sequence of thrombospondin-related adhesion protein (TRAP).⁴¹ TRAP is essential for sporozoite motility⁴² and T-cell responses in naturally exposed Gambians are present to several conserved regions⁴³ in contrast to the highly polymorphic region on which T-cell responses to CS are focused⁴⁴.

MVA has the advantage as a candidate viral vector that it was developed specifically for a high safety profile and given preferentially as a smallpox vaccine to high-risk individuals, such as the very young or immunocompromised, during the 1970s⁴⁵. Safety and tolerability of both DNA ME-TRAP and MVA ME-TRAP has been very good with no serious adverse events⁴⁶. Immunogenicity of either vaccine alone was moderate but, as expected, sequential immunisation with DNA followed by MVA vaccine produced much greater T-cell responses. Recently, encouraging preliminary evidence of efficacy against heterologous strain sporozoite challenge has been observed with this immunisation regimen⁴⁷.

A phase I clinical trial of these two vaccines in semi-immune adults has been completed in The Gambia. In this trial, there were no tolerability problems as in malaria-naive adults from the UK. Higher T-cell responses were seen in individuals who received MVA whether with or without prior DNA vaccination compared to the UK, and there was far greater cross-reactivity of the T-cell responses to a non-vaccine strain of TRAP than in UK adults⁴⁸.

Three new clinical vaccines are in production by the Oxford group. Firstly FP9 ME-TRAP, which is a recombinant fowlpox encoding the identical fusion protein, entered clinical trials in 2001 in the UK and in early 2002 in The Gambia. In humans, FP9 (fowlpox strain 9) is replication-deficient and is not known to cause disease. FP9 can be substituted for DNA as a priming agent leading to greater immunogenicity in pre-clinical studies (R Anderson et al, unpublished data). Further such heterologous prime-boost studies are planned using vaccines expressing the CS protein. This will include testing MVA expressing this antigen as a boosting agent for RTS,S.

Studies of expression, function and natural immune responses highlight two further pre-erythrocytic antigens as promising candidate antigens. Expression of liver-stage antigen 1 (LSA-1) begins within the infected hepatocyte and several studies have shown associations between LSA-1 specific immune responses and protection from severe disease or parasitaemia⁴⁹. The lack of a known homologue in species of malaria which infect mice or non-human primates has retarded vaccine development.

Liver-stage antigen 3 (LSA-3) is less characterised than CS, TRAP or LSA-1. A vaccination study showed some protection using lipopeptide and recombinant protein LSA-3 constructs, but the expression of this antigen by blood-stage parasites complicates interpretation of this result^50,51.

	Blood stage vaccines

Top Abstract Introduction Life-cycle Natural versus vaccine immunity Development of malaria subunit... Pre-erythrocytic vaccines Blood stage vaccines Sexual stage vaccines Discussion References

 
Effective vaccination with a blood stage vaccine would either prevent invasion of red cells or prevent complications, such as cerebral malaria, malarial anaemia, renal failure and all the manifestations of severe malaria in pregnancy. These complications, which occur only in P. falciparum infection, are likely due to cyto-adherence of PfEMP-1 (erythrocyte membrane protein-1), expressed on the surface of infected red cells, to CD36 or intercellular adhesion molecule-1 (ICAM-1) on microvascular endothelial cells or chondroitin sulphate A or other glycosaminoglycans in the placenta. Sequestered infected red cells may cause organ dysfunction through microvascular obstruction and release of systemic cytokines such as TNF- and local mediators such as nitric oxide. Although PfEMP1, which was cloned in 1995⁵², is the prime target for an 'anti-complication' vaccine, creating such a vaccine is a very difficult challenge due to its extraordinary variation and clonal switching, the characterisation of which has now begun⁵³.

Therefore, most blood stage vaccine development has focused on targeting the antigens responsible for invasion of the red cell. The best known is MSP-1, the major surface protein of the merozoite. Antibodies to the C-terminus of MSP-1 are associated with protection from high parasitaemia and clinical disease⁵⁴ and have been shown to inhibit parasite growth in vitro⁵⁵ and prevent red cell invasion⁵⁶. These antibodies probably inhibit parasite entry by preventing a proteolytic cleavage of the MSP-1 protein to a smaller fragment, which is essential for successful invasion⁵⁷. A phase I trial in Washington DC with a recombinant protein form of the C-terminus of MSP-1 fused to T helper epitopes from tetanus toxoid produced seroconversion in 9 out of 16 volunteers immunised with the higher dose, but with hypersensitivity problems after the third dose⁵⁸. Different formulations will be necessary to improve safety and immunogenicity. Trials are underway by the Walter Reed group in the US combining RTS,S and MSP-1.

Two phase I trials of a mixture of 3 recombinant blood stage antigens have occurred in Brisbane and Papua New Guinea^59,60. Encouragingly, no competition was seen between antigens for induction of immune responses, but immunogenicity was modest, perhaps due to the adjuvant chosen and/or the lack of a carrier or fusion protein.

A difficulty with blood stage vaccine trials is the choice of end-point to use for efficacy. Can blood stage vaccines be expected to increase time to detectable parasitaemia given that their target is the blood stage parasite? Delay of treatment to compare rates of rise of parasitaemia between control and vaccine groups has occurred in the past, but is fraught with safety problems. In the future, blood stage candidates will probably progress directly from phase I in malaria-naives to phase I in malaria-endemic areas without progressing through the small scale efficacy trials in malaria-naives favoured for pre-erythrocytic vaccines. In field efficacy trials, the more relevant end-point of clinical disease could be used or a parasite density end-point could be used safely in semi-immune populations.

	Sexual stage vaccines

Top Abstract Introduction Life-cycle Natural versus vaccine immunity Development of malaria subunit... Pre-erythrocytic vaccines Blood stage vaccines Sexual stage vaccines Discussion References

 
Interestingly, immunisation with sexual stage parasite antigens induces antibodies which prevent fertilisation within the mosquito. The leading candidate vaccines are Pfs25 and Pfs28. Neither is expressed while the parasite is in humans and they are, therefore, under no immune selection pressure. Whilst this implies that they will be more conserved than antigens expressed during human stages, it also means that vaccinated individuals will not be boosted by natural exposure. Thus such vaccines will need a long duration of vaccine-induced immunity to be useful. A recombinant fusion protein of these two antigens known as TBV25-28 is being developed for clinical trials^61,62. It is immunogenic for transmission-blocking antibodies in mice and rabbits when administered with alum and QS-21 as adjuvant.

Two gametocyte antigens, Pfs230 and Pfs48/45, have also been characterised. Natural immune responses to these occur although association with prevention of transmission in vivo is unclear. High-titre antibodies to either antigen have been shown to prevent transmission ex vivo⁶³.

Transmission-blocking vaccines are an unattractive financial proposition for the private sector as they would have no place in the market for tourists from industrialised countries. However, an effective transmission-blocking vaccine could be an excellent method of malaria control particularly if combined with other measures. The political will needs to be mobilised to secure public sector funding for this enterprise. One potential advantage for development of a transmission blocking vaccine is the ability to determine efficacy without human infection with malaria. An ex vivo assay has been developed which may be a good indicator of transmission blocking malaria immunity. Mosquitoes are membrane-fed gametocytes with or without addition of human serum.

	Discussion

Top Abstract Introduction Life-cycle Natural versus vaccine immunity Development of malaria subunit... Pre-erythrocytic vaccines Blood stage vaccines Sexual stage vaccines Discussion References

 
Scepticism is warranted in any estimation of the future rate of progress in malaria vaccine development. Forty-five years after the publication by a respected malariologist of a book entitled Man's Mastery of Malaria⁶⁴, malaria continues to cause human suffering on a vast scale. We make no guess here at the time-scale of development of an effective malaria vaccine. Nevertheless, a few points are clear. A new generation of vaccines has emerged for clinical testing, i.e. DNA and recombinant viral vaccines. Molecular characterisation of target antigens will continue to accelerate. Funding remains an important limiting factor, but funding allocations have increased over recent years largely due to the commitment of the Wellcome Trust of Great Britain and the advent of the Malaria Vaccine Initiative at PATH (the Program for Appropriate Technology in Health), which is in turn funded by the Bill and Melinda Gates Foundation. Nonetheless, chronic underfunding of this field remains an important problem. The following factors could lead to an exponential increase in candidate vaccination regimens: many new candidate antigens, new delivery systems, novel administration routes, polyprotein constructs, 'cocktails' of vaccines with each antigen encoded individually and the need to test prime-boost regimens involving sequential immunisations with combinations of delivery systems. This high number of candidate regimens coupled with limited funding will make the selection of vaccines for efficacy testing problematic in coming years. Continuing improvements in immunoassay techniques in tandem with an expansion in the number of malaria vaccine field efficacy trials are likely to increase our knowledge of correlates of protection. A robust system of immunological correlates would allow the empiricism, currently unavoidable in selection for efficacy trials, to be superseded by a rational iterative approach.

An unresolved question is how and where field studies should best be conducted. Small-scale efficacy trials in industrialised countries are possible for pre-erythrocytic vaccines, but correlation with field efficacy has yet to be satisfactorily proved. Furthermore, the subjects of such studies are genetically and immunologically distinct from the target population. No such generally accepted controlled efficacy model is available for blood stage or sexual stage vaccines. It is likely that field efficacy trials in African children will speed identification of relevant correlates and effective vaccine development. Clearly, certain criteria must be met before a candidate should be tested in the field, but what are they? Should field trials occur in adults before progression to children? Our view is that if efficacy is unclear, then adult efficacy trials in the field can be a useful tool. But if efficacy has already been proven, safety assessments should be the primary determinants of the speed of progression to field trials in children.

The current practcse of pharmaceutical company management of vaccine development is not necessarily the most efficient for malaria. Development of a malaria vaccine for African children is, without external support, not a tenable activity commercially. Therefore, sole development of vaccines that will not be widely used by travellers by companies whose primary interest is profit is unlikely. Large-scale public sector and charitable funding or a public–private partnership are probably more suitable approaches. Once an effective vaccine is available, funding and political will must be mobilised for a major new vaccination programme.

Despite these obstacles remarkable progress, particularly in the area of pre-erythrocytic vaccines, has been achieved in recent years and malaria may be one of the first diseases to benefit from the application of advances made over the last decade in cellular immunology.

	Footnotes

 
Correspondence to: Prof. Adrian V S Hill, Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK

	References

Top Abstract Introduction Life-cycle Natural versus vaccine immunity Development of malaria subunit... Pre-erythrocytic vaccines Blood stage vaccines Sexual stage vaccines Discussion References

 

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