Malaria vaccines in pre-clinical and clinical trials
Currently, more than 90 malaria vaccines are in preclinical and clinical trials, and over 30 of these are being tested, mostly in the early phases of clinical trials (WHO, 2009). Pre-clinical studies are conducted before a malaria vaccine can be tested in humans, and which support and assist in setting up limitations for testing and safe use of the vaccine in subsequent Phase I clinical trials. Phase I clinical trials involve assessment of safety and immunogenicity of the vaccine in less than 100 human volunteers. Phase Ib are performed in non-endemic countries and Phase Ib are performed in malaria-endemic countries Phase II clinical trials involve assessment of safety, immune response and efficacy. In Phase IIa trials, malaria-naive volunteers in non-endemic countries are vaccinated and later exposed to malaria-carrying mosquitoes to observe how long it takes them to become infected. Phase IIa trials give initial indication of a vaccine's efficacy before the vaccine moves to Phase IIb endemic-country trials in up to a few thousand volunteers. Finally, in Phase III trials the vaccine safety, immunogenicity and efficacy are monitored in larger trial size. The regulatory approval for distribution is obtained (licensure) and the use of vaccine is initiated (introduction).
Potential vaccines candidates for each stage of the malaria's complex life cycle are at present in clinical development: the pre-erythrocytic, erythrocytic, and transmission-blocking stages. Here we will be discussing the most advanced agents being developed for each target of the life cycle.
4.1. Pre-erythrocytic vaccines
As discussed before, pre-erythrocytic vaccines can target either the sporozoite, or infected liver stage. Many pre-erythcroytic vaccines candidates have focused largely on the P. falciparum sporozoite's primary surface antigen, circumsporozoite protein (CSP) or epitopes from it because the host's immune response usually targets CSP when attacking the parasite. Among all malaria vaccine candidates, RTS,S is the most promising and advanced. This vaccine targets CSP and two additional non-CSP based vaccines are being developed against the liver stage, which have undergone Phase IIb trials. These are a FP9/MVA ME-TRAP vaccine, which is a fowlpox strain FP9 and modified vaccinia virus Ankar (MVA) vector that expresses the pre-erthyrocyte antigen TRAP (thrombospondin-related adhesion protein) fused to a multi-epitope (ME) string; and the DNA-MVA prime boost ME+TRAP vaccine. Combination of these vaccine with the RTS,S vaccine have been performed or are ongoing. These vaccines will be later discussed and compared with other vaccines that have been studied in advanced clinical trials.
Other antigens that are the targets for pre-erythrocytic vaccine development include: sporozoite threonine and asparagine rich protein (STARP); sporozoite and liver stage antigen (SALSA) and liver stage antigens 1 and 3 (LSA1 and 3).
4.2. Blood-stage vaccines
The targets for erythrocytic vaccines are variable; the three main targets are the merozoite, malaria toxins, which are released following rupturing of the schizont, and the antigens that stimulate the binding between parasite and vascular endothelium. The major target of current blood-stage vaccines are the merozoite surface antigens which are expressed on the surface of merozoites and are accessible to circulating human antibodies. Most progressive blood-stage vaccines are based on the use of the following antigens: apical membrane antigen 1 (AMA1), which is a type 1 integral membrane protein, merozoite surface protein 1 (MSP1), a part of a complex involved in the invasion of erythrocytes, MSP2, MSP3, and the glutamate-rich protein (GLURP).
There are currently two blood-stage vaccines that have reached the advanced clinical trial phase IIb: one is AMA1 based FMP2.1/AS02A vaccine and the other is MSP1 based FMP1/AS02A vaccine. The malaria vaccine FMP2.1/AS02A a recombinant protein (Falciparum malaria protein 2.1, FMP2.1) based on AMA1 from the 3D7 clone of P. falciparum, formulated in the Adjuvant System AS02A. The FMP1 antigen is the 42-kDa C-terminal fragment of MSP1(42) of the 3D7 clone of P. falciparum. This is formulated with AS02A to form the FMP1/AS02A candidate vaccine. These two vaccines are currently undergoing Phase IIb clinical trial.
Combination B is a blood-stage vaccine consisting of MSP1, MSP2 and ring-infected erythrocyte surface antigen (RESA), and these vaccines have completed a Phase IIb study. (Genton et al., 2002).
A MSP3 based vaccine has been developed as a long synthetic peptide (LSP) as well as a recombinant protein, MSP3 LSP. A recently completed Phase I trial of this vaccine has been tested in children in both Burkina Faso and Tanzania, where the vaccine showed increased levels of safety. Phase IIb trial of the MSP3 LSP vaccine candidate is currently underway in Mali. These four vaccines will be discussed later in detail along with other vaccines candidates that have been evaluated in advanced clinical trials.
Anti-disease vaccines: Once the schizont bursts, several malarial toxins, such as GPIs, are released, resulting in disease pathogenesis. The use of GPI as an anti-toxin vaccine is being studied in preclinical trials. A synthetic GPI as a vaccine (comprising a GPI oligosaccharide coupled to a carrier protein) in rodent models of malaria demonstrated that the vaccine had good immunogenicity and protected the animals from malaria pathology, such as cerebral malaria and death. A study showed that GPIs can stimulate the release of TNF-alpha from macrophages. In addition, individuals who were resistant to clinical malaria had high levels of persistent anti-GPI antibodies, while vulnerable children contained no or had low levels of short-lived antibody response. Individuals who were not exposed to the malaria parasite completely lack anti-GPI antibodies. The absence of a persistent anti-GPI antibody response resulted in malaria-specific anaemia and fever. This indicates that anti-GPI antibodies may provide protection against clinical malaria (Naik et al., 2000). Another study in Gambia there were increased levels of anti-GPI antibodies in infected children who did not develop clinical malaria compared to those who did. However children under the age of 2 were seronegative for anti-GPI antibodies, even when they were infected with malaria. This study indicates that anti-GPI antibodies may induce partial immunity to malaria. Consequently, the difference was not statistically significant and this study did not demonstrate that anti-GPI antibodies can provide protection against severe malaria (de Souza et al., 2002). Clinical trials in Malian children also demonstrated that increased levels of antibodies to GPI in young children were associated with disease severity and were short-lived (Cissoko et al., 2006). Since not much is known about the protective role of anti-GPI antibodies against malarial pathogenesis, further clinical trials in this field are essential (Naik et al., 2000).
In severe malaria, PfEMP1 is thought to be the primary molecule involved in the binding of infected erythrocytes to the vascular endothelium, this is known as cytoadhesion. This antigen can be targeted by anti-disease vaccines. Duffy binding-like domain 1α (DBL1α) is the most conserved domain experessed on the extracellular region PfEMP1. In preclinical trials, rats who were vaccinated with PfEMP1-DBL1α demonstated reduction in the malaria parasite sequestration from both strains. Additionaly this vaccine has shown to prevent P.falciparum sequestration significantly in monkeys (Moll et al., 2007).
Several vaccines for placental malaria target the var gene family encoding the highly variable PfEMP1 protein family- almost 60 different members. In placental malaria a single PfEMP1 variant, known as VAR2CSA, stimulates parasite sequestration by interacting with placental chondroitin sulfate A (CSA). During repeated pregnancies the acquirement of anti-PfEMP1 immunity is associated with the enhanced ability of multigravidae women to regulate pregnancy-associated malaria. Preclinical studies on the development of a candidate vaccine based on PfEMP1 antigens intended to prevent pregnancy-associated malaria are being performed. Consequently, the huge variability of PfEMP1 makes it difficult for its development. The results from these studies are promising but further clinical trials are critical for determining their clinical effectiveness.
4.4. Transmission-blocking Vaccines
Clinical studies have shown these vaccines to induce production of antibodies against gametocyte and ookinete parasite antigens. P. vivax is less deadly than P.falciparum, but it is a major cause of malaria in South America and Asia. These vaccines contain either the P. falciparum ookinete surface protein antigens Pfs25 and Pfs28 or their P. vivax homologues, Pvs25 and Pvs28. In a clinical trial the recombinant protein Pvs25 was used as vaccine, where immunized individuals produced antisera which were present in the mosquito's blood meal. The mosquito that ingested this blood meal prevented the parasite from being transmitted to any other victim. Initial human phase I trials of Pfs25 and Pvs25 formulated in both ISA51 adjuvant and alum have been performed, and no serious adverse effects were reported.
Other transmission blocking stage antigens that are being developed as vaccines are Pfs48/45 and Pfs230, which are expressed on the surface of gametocytes. These vaccines have not yet advanced to clinical trails. Pfs48/45 vaccine was found to be highly dynamic, stable and safe in mice, however, it has not yet advanced for clinical studies in humans. Pre-clinical trials of Pfs230 vaccine have demonstrated that monoclonal antibodies to Pfs230 completely block transmission in membrane feeds, indicating a clear mechanism of antiparasite activity (compliment mediated). However, further research is needed to strengthen the promising evidence obtained from these studies.
4.3. Combination multi-stage vaccines
Researchers are also focusing on the development of multi-antigen vaccines which can inhibit the parasite at different stages of its life cycle. For example, the combination of pre-erythrcytic stage CSP and blood stage Duffy Binding Protein (DBP)-based vaccines are being developed, where synergy can be expected, and will be dependent on the outcome of challenge studies for the two CSP and DBP antigens.
The SPf66 vaccine (also known as Patorraya vaccine) is the first malaria vaccine developed in 1987 and involves a combination of antigens from the pre-erythrocytic sporozoite (CS repeats) and erythrocytic merozoite parasites. This will be analysed in more details below, when comparing it with other clinically advanced vaccines in Phase IIb/III trials.
PEV3A is another combination vaccine which is being tested in phase IIa trials. In Oxford, UK, a phase I/IIa clinical trial of two vaccines, FP9/MVA ME-TRAP and PEV3A, was performed on healthy local volunteers, which are active against parasites in pre-erythrocytic liver and blood stages. PEV3A includes peptides from both the pre-eythrocyte CSP and the blood stage antigen AMA1, delivered with influenza virosome. These are virus-like particles that have similar membrane fusion and cell binding features of the native virus, but lack the viral genetic material. PEV3A and AMA1 were chemically coupled to the virosome's surface to increase their immune response. The results showed that a very strong immune response was induced in human subjects, with a long-lasting antibody response. One of the problems faced by drug developers is antigenic variation, where specific antigens can interfere with the immune response of other antigens. Consequently, the phase I studies of PEV3A demonstrated that subjects vaccinated with the two antigens did not demonstrate any interference with immunogenicity of either antigens. Although this strategy shows potential, so far studies based on multi-antigen combination vaccines have not demonstrated any synergistic or increasing advantages.
VACCINE (Vaccine type)
RTS,S/AS02A (Recombinant protein)
DNA MVA prime-boost ME string + TRAP (DNA/recombinant viral vector)
FP9 MVA prime-boost ME-TRAP (Recombinant viral vector)
MSP1 42 3D7 (FMP-1/AS02A) E. coli expressed (Recombinant protein)
AMA1 3D7 (FMP2.1)/AS02A E. coli expressed (Recombinant protein)
Combination blood stage antigens:
Combination B: RESA, MSP1, MSP2 (Recombinant protein)
Other blood-stage antigens:
MSP3-LSP (Recombinant protein/long synthetic peptide)
SPf66 (synthetic peptide)
Table: Malaria vaccine candidates that were previously or are currently being assessed in advances phases of clinical trials