One of the greatest challenges of medicine has been the development of an effective and safe vaccine against malaria. Despite the persistent efforts for more than a century, spending hundreds of millions of dollars and lifelong sacrifice from dedicated physicians and scientists, the malaria vaccine has remained elusive. Although many promising experimental vaccines have been developed, the confident promise of researchers in the mid-1980s that an effective vaccine against P. falciparum would be available within a decade has not been realized yet.[1-3] A Malaria Vaccine Technology Roadmap, developed by more than 230 experts representing 100 organizations from 35 countries, has set out a strategic goal to develop a malaria vaccine by 2025 that would have a protective efficacy of more than 80% against clinical disease and would provide protection for more than 4 years. In the interim, it seeks to develop and license a first generation malaria vaccine by 2015 that has a protective efficacy of more than 50% against severe disease and death, and lasts for at least 1 year.
Development of a vaccine for malaria has turned out to be a highly complex exercise owing to a multitude of difficulties. A natural malaria infection does not induce much immune protection: after repeated and prolonged exposure to malaria parasite over several years, only partially effective immunity is acquired, which is short-lived and is highly stage- and strain-specific.[2,3] This immunity is unable to eradicate all parasites nor does it provide complete protection against future challenge. Instead, it only results in a mild, sometimes asymptomatic infection with the persistence of parasites.
This kind of a partial immune response is due to the complex biology of the Plasmodium parasite, its extensive antigenic diversity, and its immune evasion strategies, and all these factors make vaccine development against malaria challenging. Several vaccine candidates have been tested over the years, but without much success. As many as 80 malaria vaccine candidates are at the preclinical development stage, out of which more than 30 have entered clinical testing and at least 3 have gone as far as Phase IIb trials or beyond.[6-7] Updated information on malaria vaccine candidates is available on the WHO,[6,7] MVI, and Clinical Trials websites. These candidates have been chosen on the basis of their ability to elicit some form of inhibitory immune response against the parasite, and all these candidates have predated the publication of the complete genome sequence of Plasmodium. Following the sequencing of the entire genome complement of 5300–5500 genes of P. falciparum, 579 of annotatedfalciparum genes have been predicted to encode proteins containing signal peptides. Only 197 of these proteins that are expressed in the life-cycle stages are targeted for vaccine development: 43 in gametocytes, 57 in sporozoites, and 94 in either the young or late schizont or merozoite stages. Additional post-genome analyses have helped to pinpoint potential candidate proteins that are associated with surface/rhoptry transport structures called Maurer’s clefts in the parasite-infected erythrocytes or with schizont/merozoite lipid rafts enriched with GPI anchored proteins. These studies have also helped in validating the existing vaccine candidates such as the apical membrane antigen 1 (AMA-1) and in identifying certain multistage vaccine candidates like the erythrocyte binding ligand MAEBL.
The candidate malaria vaccines target the different phases of the parasite’s life cycle.
The preerythrocytic vaccines target sporozoites or schizont infected liver cells and are aimed at preventing infection by stopping the progression of hepatic stage. These vaccines are intended to induce antibody responses against sporozoites and thus prevent the entry of sporozoites into hepatocytes or to induce T cells against the antigens expressed by infected hepatocytes, so as to prevent merozoite release by killing the infected hepatocytes or by interfering with parasite development.[2,3,5,8] Such a vaccine must be 99% effective in interrupting the pre-erythrocytic stages if it has to provide sterile immunity in nonimmune individuals. Even a single sporozoite escaping vaccine-induced immunity may cause a fully pathogenic blood stage infection, as was found during the clinical trials of the latest antisporozoite RTS,S vaccine.[2,3,9]
The erythrocytic stage vaccines are aimed at reducing parasite multiplication and growth in order to protect against clinical disease, particularly severe disease.[2,5,8] These vaccines are designed to induce antibody responses against the targets on the asexual blood stage of the parasite such as merozoite surface proteins (such as MSP-1) or those contained in specialized organelles associated with invasion (such as AMA-1).[2,5,8] But the very short duration during which merozoites stay outside the red cells (about 2 minutes), the high degree of polymorphic variability, and the use of alternative invasion pathways by P. falciparum make such vaccines a difficult proposition.[2,8]
Transmission-blocking vaccines are aimed at reducing malaria transmission by interrupting the parasite life-cycle in the mosquito by inducing antibodies that prevent either fertilization of the gametes in the mosquito gut or the further development of the zygote into sporozoites. These vaccines do not protect the immunized individual but rather provide herd benefit.[2,5]
Antidisease vaccination, by preventing the parasite surface protein parasite-derived erythrocyte membrane protein (PfEMP1) from interacting with various vascular endothelial cell–surface receptors, may block the sequestration of parasite-infected erythrocytes and prevent the serious complications such as cerebral malaria or placental malaria.[2,8]
Different technology formats have been developed to produce the candidate antigens and to deliver them into the recipient. Attenuated whole parasites can deliver a vast array of antigens, much like a multiepitope vaccine, inducing effective immunity. However, it is practically not possible to produce malaria parasites on a scale required for a live or attenuated vaccine.630 Safety is also a major concern, as current technology requires that parasites are cultured in human erythrocytes, which is accompanied by the risk of serious blood-borne infections. Ensuring consistent quality and precise irradiation are the other issues. In vitro culture of P. vivax is difficult to sustain and large-scale production is not currently feasible.
Considering these difficulties, almost all efforts in malaria vaccine development today are focused on the design and delivery of subunit vaccines. Such subunit vaccines may comprise a single parasite antigen or a mix of many antigens, as in a polyvalent, multicomponent vaccine, targeting different proteins in different stages of the parasite life cycle.[2,3] Recombinant antigens or long synthetic peptides can be produced in larger scale and then delivered with appropriate adjuvants for better immunogenicity.[3,5,8] Use of DNA expression vectors such as viruses, bacteria, and plasmid DNA for delivering the parasite antigens is emerging as a promising strategy to generate the desired antibody and cell-mediated immunity by the vaccines.[2,3,5,8,10]
Priming of the immune response to chosen antigens using DNA expression vectors (plasmid or attenuated viral vectors, AVV) followed by boosting using AVV expressing the same antigens – the heterologous primeboost approach – is being employed in many of the recent vaccine candidates; different viral vectors and a range of different formulations such as liposomes, virosomes, microspheres, and nanoparticles are being tested, with the advantage of carrying multiple antigens.[2,10]
Whole Cell Sporozoite Vaccine
The possibility of using inactivated sporozoites for immunization was first demonstrated in 1910 in avian malaria.[11,12] In 1941, immunization of fowls with irradiated sporozoites was found to prevent infection and in 1967, immunizing mice with radiation-attenuated P. berghei sporozoites was reported to protect them against challenge with fully infectious sporozoites. This was followed by human studies and during the 1970s, it was established that immunizing human volunteers with the bites of irradiated mosquitoes carrying P. falciparum sporozoites in their salivary glands could protect them against challenge with fully infectious P. falciparum sporozoites. In these studies, sporozoites were collected from the mosquitoes made to feed on human volunteers with P. falciparum malaria (who were treated with chloroquine that clears only asexual forms) and the vaccine was delivered through bites of infected mosquitoes or intravenous injections (in mice). Later, mosquitoes fed on gametocytes grown in vitro cultures were used. Although many studies demonstrated the efficacy of attenuated whole parasite vaccines in generating effective and (in some cases) long-lasting protection against both sporozoites and asexual blood stages, many technical difficulties hampered the progress of this model.[1,2]
The need for precise irradiation (too little irradiation will make the sporozoite remain infectious and generate a full-blown malaria infection in “vaccinated” subjects and too much irradiation will kill the parasite, not generating effective immunity), difficulties in producing enough numbers of such attenuated sporozoites to meet global demand, and many other technical difficulties were considered as major drawbacks for further development of this vaccine.[1,2]
The postgenome era has opened up the possibility of developing genetically attenuated parasites that are not capable of causing a full-blown infection, yet immunogenic. Early studies in P. berghei with deletion of UIS3, UIS4 , and P36p  genes were reported to be successful, with both forms of the mutant parasite providing full protection against subsequent challenge with wild-type parasites.[15,16] Creation of genetically attenuated P. falciparum parasite by deletions of the sporozoite-expressed genes P52 and P36, causing parasite developmental arrest during hepatocyte infection, has now been reported. This double knockout, genetically attenuated, parasite may be an exciting candidate for whole-organism vaccine in humans. With these reported successes in the development of genetically attenuated sporozoites of P. berghei as well as P. falciparum and with other issues such as sterility, cryopreservation, and mode of administration also being on the verge of getting solved, the possibilities of an engineered sporozoite vaccine appear encouraging.[2,5]
Meanwhile, a proof-of-concept study involving inoculation to intact sporozoites to human volunteers receiving a prophylactic regimen of chloroquine has been reported to offer protection against malaria infection. Fifteen healthy volunteers – with 10 assigned to a vaccine group and 5 assigned to a control group – were exposed to the bites of mosquitoes once a month for 3 months while they were receiving a prophylactic regimen of chloroquine. The vaccine group was exposed to mosquitoes that were infected with P. falciparum, and the control group was exposed to mosquitoes that were not infected with the malaria parasite. One month after the discontinuation of chloroquine, protection was assessed by homologous challenge with five mosquitoes infected with P. falciparum. All 10 subjects in the vaccine group were protected against a malaria challenge with the infected mosquitoes while patent parasitemia developed in all five control subjects. Adverse events were mainly reported by vaccinees after the first immunization and by control subjects after the challenge; no serious adverse events occurred. Induction of parasite-specific pluripotent effector memory T cells producing interferon-γ, tumor necrosis factor-α, and interleukin-2 was observed.
Pre-erythocytic stage vaccines are based on the circum sporozoite protein (CSP) and liver-stage antigens (LSAs). These vaccines are aimed at induction of antibodies that alter the sporozoite surface or block the mobility or inhibit their invasion into hepatocytes and/or aimed at induction of cells cytolytic to the infected hepatocyte or induction of cytokines (IFN-γ) or free radicals that inhibit intrahepatic parasite development.
The CSP has been the subject of numerous trials based on a variety of peptides, recombinant proteins, modified virus vectors, plasmids, and a large diversity of adjuvants and immunization regimens.
RTS,S/AS: At present, the RTS,S/AS02 vaccine is undoubtedly the most advanced and promising vaccine candidate. The designation “RT” refers to approximately 190 amino acids from the C-terminus of the P. falciparum CSP and “S” refers to the hepatitis-B surface antigen. RTS,S virus-like particles form when the RTS malaria–hepatitis B fusion protein is coexpressed with S antigen alone in yeast cells (Saccharomyces cerevisiae). The adjuvant AS01 consists of liposomes plus MPL plus QS21. An earlier version of the RTS,S vaccine was adjuvanted with AS02 (an oil–water emulsion plus MPL plus QS21). In Phase IIa trials, RTS,S/AS02 protected 40–86% of malaria-naive individuals after artificial challenge and two proof-of-concept Phase IIb trials demonstrated a partial delay of infection, a 30% reduction in clinical episodes of malaria, and reduction in severe malaria by 58%. A phase III trial of RTS,S has been conducted in 11 countries of sub-Saharan Africa from March 2009 through January 2011, in 15460 children in two age categories (6–12 weeks of age and 5–17 months of age), with a dose of 25 µg in a three-dose schedule delivered intramuscularly at the ages of 0, 1, and 2 months. The first results have shown a reduction in the total number of episodes of clinical malaria by 55.1% and reduction in severe malaria by 47%, both in the older group. However, recalculating the trial data has shown that RTS,S protected just 35–36% after 12 months and combining the results of both age groups cut the reduction in severe malaria to 34.8%.[19,20] Serious adverse events, such as convulsions and meningitis, was significantly higher in the vaccinated group, although the data are too preliminary to draw firm conclusions.[19,20] The mortality was similar in vaccinated and control groups. Nevertheless, these initial reports have been considered as encouraging, raising the hopes of an effective malaria vaccine in the near future. [Also see]
Long Synthetic Peptide (LSP) PfCS102: Although the LSPs coding for the C-terminal 282–383 region of the CSP showed promising results in Phase Ia trials, the Phase IIa trial performed with PfCS102 showed no protection and no delay in patent parasitemia after artificial challenge and therefore the development of this candidate was stopped.
Multiple Epitope, Thrombospondin-Related Adhesion Protein-, CS-, and LSA1-Expressing Fowlpox 9 and Modified Vaccine Ankara: A heterologous prime-boost vaccine, comprising a multiepitope string containing B-cell, CD4, and CD8 T-cell epitopes derived from six sporozoite and/or LSAs, including CS, LSA-1, and LSA-3 fused to the thrombospondin-related adhesion protein (T9/96 strain) was developed at the University of Oxford. These antigens are delivered as a DNA plasmid or expressed by recombinant attenuated viruses – fowl pox strain (FP9) or modified vaccine Ankara (MVA). The prime-boost approach – DNA or FP9 then MVA – has been shown to induce high T-cell responses, but two Phase IIb studies failed to show protection against malaria.
Other FP9 and MVA polyprotein constructs including combination vaccines with CS and LSA1 antigens are being developed at the University of Oxford with the European Malaria Vaccine Initiative and Wellcome Trust support.
Adenovirus-35-CS: Priming with a vaccine candidate consisting a replication-deficient recombinant adenovirus serotype 35 expressing P. falciparum CS followed by RTS,S/AS01B boosting demonstrated high humoral and T-cell immunogenicity. This vaccine is undergoing Phase I trial.[8,21,22]
Plasmid DNA Vaccines: Several DNA-based vaccines, encoding one or several antigens, have been tested in clinical trials and have shown to actively induce T-, but not B-cell, immunity. Priming with one CS-encoding candidate, P. falciparum CS protein DNA, followed by RTS,S/AS02, demonstrated activation of both cellular and antibody responses in malaria-naive Americans.
Liver-stage antigen (LSA)-1 is a pre-erythrocytic antigen expressed during the liver stage of P. falciparum infection. In adults living in malaria-endemic areas, levels of anti-LSA-1 IgG have been found to correlate with protection against malaria. An LSA-1-based vaccine candidate is being developed as a recombinant protein antigen expressed in E. coli. In Phase I/IIa trials, the vaccine showed a good safety profile and was immunogenic, but it did not protect against infection nor delay the parasitemia following P. falciparum experimental challenge.
Liver-stage antigen-3 is expressed by sporozoites, liver schizonts, and maturing hepatic merozoites and has been found to induce partial protection against sporozoite challenge in mice and monkeys. This candidate vaccine is being developed as long synthetic peptides, a lipopeptide formulation and a recombinant protein in a Lactococcus lactis expression system. A Phase I/IIa trial of the recombinant LSA-3 candidate vaccine is ongoing.
Asexual Blood-Stage Vaccines
Vaccines against the asexual blood stage of the parasite are aimed at preventing the disease and its complications by inducing antibodies against the merozoites. The merozoite exposes at its surface several functional proteins involved in erythrocyte invasion and most blood stage vaccines are based on the use of MSP-1–3, AMA-1, and glutamate-rich protein (GLURP).[5,8]
Merozoite surface protein-1
Merozoite surface protein (MSP)-1 is expressed at the surface of blood- and liver-stage merozoites and plays an important role in initial binding and invasion of the erythrocyte. More than 20 different MSP-1 constructs are in preclinical or clinical development. The most advanced is the MSP-142 of the 3D7 clone of P. falciparum. E. coli expressed recombinant protein MSP-1 42-kd 3D7 [falciparum malaria protein-1 (FMP-1)], E. coli expressed recombinant protein MSP-1 42-kd FVO, etc., are undergoing trials. Although Phase Ia/Ib trials confirmed the safety and immunogenicity of the FMP1/AS02A formulation, Phase IIb trial in Kenyan children showed no efficacy to reduce malaria morbidity.[5,8]
Other merozoite surface proteins
An MSP-2 vaccine candidate including equal amounts of 3D7 and FC27, the two allelic variants of MSP-2, is being developed. The MSP-3 has been developed as an LSP and the Phase I trial showed good immunogenicity with high antibody responses. Phase Ib trials of MSP-3 LSP vaccine among children in Burkina Faso and Tanzania have shown promising results.[23,24] MSP-4 and -5 are also being developed as subunit vaccines against malaria.
Apical membrane antigen-1
The apical membrane antigen (AMA)-1 is another leading asexual blood-stage vaccine candidate and several variants have been tested. FMP2.1/AS02A (Phase I/IIb trial), AMA1-C1/alhydrogel (Phase Ia/Ib/IIb), and recombinant Pichia pastoris AMA-1 (25-545FVO) (Phase Ia trial) are some examples.[5,8]
The serine-repeat antigen, also known as P126 antigen, is the largest protein that accumulates in the parasitophorous vacuole of trophozoites and schizonts. A recombinant vaccine candidate including the N-terminal part of the serine repeat antigen-5 is under development and Phase I trial has been conducted.[5,8]
The Glutamate-rich protein (GLURP) is a P. falciparum protein expressed in both the pre-erythrocytic and erythrocytic stages. Antibodies to the GLURP 85-213 sequence (LR67) mediate the strongest biological effect in vitro and a Phase Ia trial showed the candidate vaccine to be safe and immunogenic, with a high level of antibodies.[5,8]
Erythrocyte-binding antigen (EBA)-175 mediates erythrocyte invasion. A Phase I trial with a recombinant PfEBA-175 region II-nonglycosylated (EBA-175 RII-NG) is ongoing and other candidates, including the N-terminal, conserved, cysteine-rich region of EBA-175 or the Duffybinding antigen, are currently being developed as recombinant vaccine candidates or as DNA vaccines in prime-boost regimens.[5,8]
PfEMP1 mediates the binding of P. falciparum–infected erythrocytes to the vascular endothelium and to uninfected erythrocytes. Its extreme variability is proving to be a major challenge for the development of an anticytoadhesion vaccine; immunization studies with a recombinant conserved CD36-binding portion of PfEMP1 failed to confer protection in Aotus monkeys.
A single PfEMP1 variant, termed VAR2CSA, which is structurally distinct from all other PfEMP1 family members, has been identified to play a key role in sequestration to the placenta by binding to chondroitin sulfate A. The increased ability of multigravidae women to control pregnancy-associated malaria has been attributed to the acquisition of anti-PfEMP1 immunity during successive pregnancies. Work is on to develop a candidate vaccine based on PfEMP1 antigens aimed at the prevention of pregnancy-associated malaria.
Multiantigen Blood-Stage Vaccines
The belief that a single malaria antigen is unlikely to induce the desired level of protection has led to the development of multicomponent vaccines that combine more than one antigen in a formulation, but it is unclear whether this approach will speed up the vaccine development process or not.
RTS,S-based combination vaccines
Several options for improvement of RTS,S-based vaccines are under investigation; sequential immunization schedules with RTS,S/AS02 combined to prior PfCSP DNA vaccination or CS-expressing adenovirus 35 or followed by CS-expressing live MVA have been investigated and a multistage, multiantigen recombinant vaccine based on RTS,S, and MSP-1 from the 3D7 strain is also under evaluation.
Recombinant hybrid GLURP plus MSP-3 (GMZ-2)
GMZ-2 is a recombinant hybrid of GLURP and MSP-3 expressed in L. lactis. A Phase Ia trial in malaria-naive volunteers has been completed and a Phase Ib trial is ongoing.[5,8]
Chimeric fusion protein MSP-1 plus AMA-1 (PfCP-2.9)
An MSP-1/AMA-1 fusion antigen vaccine, consisting of the C-terminal region of AMA-1 and the 19-kDa fragment of MSP-1 using the yeast P. pastoris is undergoing Phase Ia trials and found to be safe and immunogenic.[5,8]
PEV 301, PEV 302
A new antigen-delivery system, based on synthetic peptides displayed on the surface of reconstituted influenza virosomes, is under development. It comprises phospholipid-anchored antigenic peptides mixed with nonbounded phospholipids and nfluenza surface glycoproteins, creating virus-like particles supposed to be strong inducers of B- and T-cell immunity. This allows the inclusion of several antigenic peptides in a multiantigen, multistage vaccine. Presently, two virosome formulations using synthetic peptide mimicking a CS-like sequence (PEV 301) and an AMA-1 like sequence (PEV 302), are undergoing Phase I/Phase Ib/Phase IIa trials.[5,8]
NMRC-M3V-Ad-PfCA vaccine candidate is a mixture of two recombinant adenovectors comprising codon optimized sequences of the transgenes from the 3D7 strain of P. falciparum expressing CS or AMA1 and the replication deficient adenovector derived from adenovirus serotype 5. Phase I/IIa trials are ongoing.[5,8]
Combination B: MSP-1, MSP-2, and ring-stage infected erythrocyte surface antigen combination
Another vaccine candidate combining MSP-1, MSP-2, and the ring-stage infected erythrocyte surface antigen did not show a reduction or delay in parasitemia in a small Phase IIa challenge study, but in a subsequent Phase I/IIb trial in children, it showed a reduction in parasite density in some vaccinees compared with controls, thus becoming the first successful blood-stage vaccine to show some vaccine efficacy. Future vaccine candidates will include the opposite dimorphic form of MSP-2, FC27.
Synthetic peptide vaccine SpF66
SPf66, developed in 1987 by Dr. M.E. Patarroyo in Columbia, was the first synthetic polymeric vaccine to be tested in humans. It was based on polymeric synthetic peptides consisting of a number of epitope sequences from the blood as well as sporozoite stage proteins. Although the initial studies indicated a significant reduction in malaria morbidity, further trials of the same vaccine failed to reduce clinical disease in different locations in Latin America and Africa, resulting in the termination of its further development. SPf66 paved the way for future design and field trials of subunit malaria vaccines and highlighted the complex nature and variability in field trials of malaria vaccines.
Transmission-blocking vaccines aim at induction of neutralizing antibody responses against gametocyte and ookinete surface proteins that can block the parasite cycle in the mosquito so as to interrupt malaria transmission. The main antigens assessed as vaccine candidates are the surface antigens Pfs25, Pfs28, Pfs48/45, and Pfs230. The surface antigens of ookinetes, Pvs25 of P. vivax and its P. falciparum analog, Pfs25, expressed as a recombinant protein in S. cerevisiae, have demonstrated moderate immunogenicity but suboptimal levels of transmission blocking activity in Phase Ia trials. Further improvements are being developed.[5,8]
Malaria Vaccine: Indian Research
In India, the Malaria Group at the International Center for Genetic Engineering and Biotechnology (ICGEB), New Delhi, has undertaken efforts to develop vaccines for both P. vivax and P. falciparum malaria. The N-terminal conserved cysteine-rich region II (PvRII) has been identified as the receptor-binding domains of P. vivax Duffy-binding protein and antibodies against PvRII are expected to block the parasite invasion of erythrocytes. Methods to produce recombinant PvRII have been developed and attempts are on for the production of clinical grade recombinant PvRII for use in human clinical trials to test the safety, immunogenicity, and efficacy of a vaccine based on PvRII. The vaccine for P. falciparum malaria, being developed at ICGEB, contains a physical mixture of recombinant PfMSP-119 and PfF2, expressed in E. coli. Immunogenicity studies in small animals have demonstrated that immunization with the recombinant PfMSP-119 and PfF2 mixture elicits high titers of invasion inhibitory antibodies against both antigens and plans are afoot to study the safety, immunogenicity, and efficacy of the vaccine in a series of Phase I and II trials.
It is hoped that the vigorous pursuit of an effective and safe malaria vaccine may yield results in the near future. An ideal malaria vaccine should be safe, highly effective, and provide long-term immunity, besides being stable, easy to administer, inexpensive to manufacture, and affordable in poor malaria-endemic countries.
However, from the existing knowledge and experience, it is more likely that the first-generation malaria vaccine will be partially protective, although safe but not entirely free of small side effects and provide protective immunity for a limited period. These vaccines will be expensive to manufacture and not easily affordable by those who will need them the most, without the help of donor funds.
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