Malaria vaccine

Malaria vaccines are an area of intensive research, however, no effective vaccine has yet been introduced into clinical practice.

Justification for malaria vaccine research
The need for a vaccine against this disease is intense, as mortality and morbidity associated with malaria are increasing. Despite the disease's long history and the existence of a multitude of treatments, around 2 to 3 million lives are still lost every year because of the disease with approximately 90% of these in sub-Saharan Africa. An estimated 2 billion people live in areas where malaria is transmitted, with between 300 and 500 million new infections occurring in this group every year. Individuals who have not developed immunity to the infection are most at risk from the disease. These include young children, travellers, pregnant women and people from endemic areas who are no longer regularly exposed to infection.

The global burden of malaria is increasing due to the development of drug-resistant parasites and insecticide-resistant mosquitoes; this is illustrated by re-emergence of the disease in areas that were previously safe. We are becoming less well equipped to deal with malaria and the present morbidity and mortality trends are set to continue unless new methods of control are developed. The implications of an increasing burden on the public health infrastructure and economic stability of the countries most badly affected is cause for concern, therefore making alternatives to the currently available treatment options and prevention strategies research priorities.

Vaccines are one of the most effective modes of treatment available. They are cost-effective in most instances and easily administered. They have historically presented a significant reduction in the spread and burden of infectious diseases. Yet no effective vaccine for malaria has so far been developed. Despite this, the efforts remain optimistic. This is rightly justified for several reasons. The first of these being that individuals who are frequently exposed to the parasite for long periods of time gain an acquired immunity against developing any clinical manifestation, even when the parasitic infection is evident by blood film analysis. More evidence supporting the research is that if immunoglobulin is taken from immune adults, purified and then given to individuals that have no protective immunity, some protection can be gained. In addition to this clinical and animal studies have shown that experimental vaccination has some degree of success when using attenuated sporozoites.

The process of vaccine development
The task of developing a vaccine that is of therapeutic and potentially preventative benefit for malaria is a complex process. Many different approaches have been adopted in the past regarding the principles that require consideration, however the basic rational remains the same. This can be divided into several steps. These will be discussed individually.

The evolutionary selection pressures of P.falciparum
P.falciparum has demonstrated the capability, through the development for multiple drug-resistance parasites, of evolutionary change. The plasmodium species has a very high rate of replication, much higher than that actually needed to ensure transmission in the parasite’s life cycle. This enables pharmaceutical treatments that are effective in reducing the reproduction rate but not halting it to exert a high selection pressure, thus favouring the development of resistance. The process of evolutionary change is one of the key considerations necessary when considering potential vaccine candidates. The development of resistance could cause a significant reduction in efficacy of any potential vaccine thus rendering useless a carefully developed and effective treatment.

Considering the immune responses to be induced
There are two main types of immune response than could be elicited by the parasite. These are Anti-parasitic Immunity and Anti-toxic Immunity.

A) Anti-Parasitic Immunity consists of an antibody response (humoral immunity) and a cell-mediated immune response. Ideally a vaccine would enable the development of anti-plasmodial antibodies in addition to generating an elevated cell-mediated response. Potential Antibodies against which a vaccine could be targeted will be discussed in greater depth later. Antibodies are part of the specific immune response. They exert their effect by activating the complement cascade, stimulating phagocytic cells into endocytosis through adhesion to an external surface of the antigenic substances, thus ‘marking’ it as offensive. Humoral or Cell-Mediated Immunity consists of many interlinking mechanisms that essentially aim to prevent infection entering the body (through external barriers or hostile internal environments) then killing any micro-organisms or foreign particles that succeed in penetration. The cell-mediated component consists of many white blood cells that target foreign bodies by a variety of different mechanisms, these include: monocytes, neutrophils, macrophages, lymphocytes, basophils, mast cells, natural killer cells, eosinophils etc… In the case of Malaria both systems would be targeted to attempt to increase the potential response generated thus ensuring the maximum chance of preventing disease.

B)Antitoxic Immunity refers to the immune response associated with the production of factors that either induce symptoms or reduce the effect that any toxic by-products (of micro-organism presence) have on the development of disease. For example it has been shown that Tumour Necrosis Factor-a has a central role in generating the symptoms experienced in severe P.falciparum malaria. Thus a therapeutic vaccine could target the production of TNF-a preventing the respiratory distress and cerebral symptoms experienced. This approach has serious limitations as it would have no effect on reducing the parasitic load, rather just reducing the associated pathology and therefore would present substantial difficulties when evaluating in human trials.

Taking this information into consideration an ideal vaccine candidate would attempt to generate a more substantial cell-mediated and antibody response on parasite presentation. This would have the benefit of increasing the rate of parasite clearance, thus reducing the experienced symptoms and providing a level of consistent future immunity against the parasite.

Considering the potential plasmodial antigenic targets
By their very nature, parasites are more complex organisms than bacteria and viruses, with more complicated structures and life-cycles. This presents problems in vaccine development but also increases the number of potential targets that a vaccine could be directed towards. These have been summarised into the life-cycle stage and the antibodies that could potentially elicit an immune response:

Sporozoite
- Abs that block hepatocyte invasion - Abs that kill the sporozoite via complement fixation or opsonization

Infected hepatocyte
- CTL mediated lysis - CD4+ help for the activation and differentiation of CTL - Localized cytokine release by T cells or APCs - ADCC or C' mediated lysis

Asexual erythrocytic
- Localized cytokine release that directly kills infected erythrocyte or intracellular parasite - Abs that agglutinate the merozoites before schizont rupture - Abs that block merozoite invasion of RBCs - Abs that kill iRBC via opsonization or phagocytotic mechanisms - Abs engulfed with the merozoite at time of invasion which kill intraerythrocytic parasite - Abs which agglutinate iRBCs and prevent cytoadherence by blocking receptor-ligand interactions (CD-36 is such a receptor) - Abs which neutralize harmful soluble parasite toxins

Sexual erythrocytic
- Cytokines which kill gametocytes within the iRBC - Abs that kill gametocytes within iRBC via C' - Abs that interfere with fertilization - Abs that inhibit transformation of the zygote into the ookinete - Abs that block the egress of the ookinete from the mosquito midgut (Doolan and Hoffman)

When selecting the most suitable vaccine target the following considerations are made:

a) How accessible is the antigen to the immune system? b) How susceptible is the antigen to evolutionary change? c) How critical is the antigen to parasitic biological functions? d) How likely is a protective response in animal models? e) Does the antigen contain epitopes that are recognisable by HLA allele superfamilies? f) How compatible is the antigen with other potential antigens?

Considering the antigenic components necessary
Increasing the potential immunity generated against the plasmodia can be achieved by attempting to target multiple phases in the life-cycle. This is additionally beneficial in reducing the possibility of resistant parasites developing. The use of multiple parasite antigens can therefore have a synergistic or additive effect. The most successful vaccine candidates currently in clinical trials consist of recombinant antigenic proteins to the circumsporozoite protein (This is discussed in more detail below.).

Considering an appropriate vaccine delivery system.
The use of adjuvants and delivery systems is discussed in greater detail below. The selection of an appropriate system is fundamental in all vaccine development but especially so in the case of malaria. A vaccine targeting several antigens may require delivery to different areas and by different means in order to elicit an effective response. Some adjuvants can direct the vaccine to the specifically targeted cell type e.g. the use or Hepatitis B virus in the RTS,S vaccine to target infected hepatocytes, but in other cases, particularly when using combined antigenic vaccines this approach is very complex. Some methods that have been attempted include the use of two vaccines, for example, one directed at generating a blood response and the other a liver-stage response. These two vaccines could then be injected into two different sites thus enabling the use of a more specific and potentially efficacious delivery system.

Adjuvants and delivery systems
To increase, accelerate or modify the development of an immune response to a vaccine candidate it is often necessary to combine the antigenic substance to be delivered with an adjuvant or specialised delivery system. These terms are often used interchangeably in relation to vaccine development; however in most cases a distinction can be made. An adjuvant is typically thought of as a substance used in combination with the antigen to produce as more substantial and robust immune response than that elicited to the antigen alone. This is achieved through three mechanisms: by effecting the antigen delivery and presentation, by inducing the production of immunomodulatory cytokines and by effecting the antigen presenting cells (APC). They can consist of many different materials from cell microparticles to other particulated delivery systems e.g. liposomes. Whereas delivery systems are substances designed purely to deliver antigens into the target cells. Adjuvants are crucial in effecting the specificity and isotype of the necessary antibodies. They are thought to be able to potentiate the link between the innate and adaptive immune responses. Due to the diverse nature of substances that can potentially have this effect on the immune system it is difficult to classify adjuvants into specific groups. In most circumstances they consist of easily identifiable components of micro-organisms that are recognised by the innate immune system cells. The role of delivery systems is primarily to direct the chosen adjuvant and antigen into target cells to attempt to increase the efficacy of the vaccine further therefore acting synergistically with the adjuvant. There is increasing concern that the use of very potent adjuvants could precipitate autoimmune responses, making it imperative that the vaccine is focused on the target cells only. Specific delivery systems can reduce this risk by limiting the potential toxicity and systemic distribution of newly developed adjuvants. Studies into the efficacy of malaria vaccines developed to date have illustrated that the presence of an adjuvant is key in determining any protection gained against malaria. A large number of natural and synthetic adjuvants have been identified throughout the history of vaccine development. Options identified thus far for use combined with a malaria vaccine include mycobacterial cell walls, liposomes, monophosphoryl lipid A and squalene.

Vaccine targets
The life cycle of the malaria parasite is particularly complex, presenting initial developmental problems. It should be noted that despite the huge number of vaccines available at the current time, there are none that target parasitic infections. Despite this, the distinct developmental stages involved in the life cycle present numerous opportunities for targeting antigens, thus potentially eliciting an immune response. Theoretically, each developmental stage could have a vaccine developed specifically to target the parasite. The initial stage in the life cycle, following inoculation is relatively short _pre-erythrocytic or hepatic phase_. A vaccine at this stage must have the ability to protect against sporozoites invading and possibly inhibiting the development of parasites in the hepatocytes (through inducing cytotoxic T-lymphocytes that can destroy the infected liver cells). However, if any sporozoites evaded the immune system they would then have the potential to be symptomatic and cause the clinical disease. The second phase of the life cycle is the _erythrocytic_ or blood phase. A vaccine here could prevent merozoite multiplication or the invasion or red blood cells. This approach is complicated by the lack of MHC molecule expression on the surface of erythrocytes. Instead malarial antigens are expressed and it is this towards which the antibodies could potentially be directed. Another approach would be to attempt to block the process of erythrocyte adherence to blood vessel walls. It is thought that this process is accountable for much of the clinical syndrome associated with malarial infection. Therefore a vaccine given to during this stage would be therapeutic and hence administered during clinical episodes to prevent further deterioration. The last phase of the life cycle that has the potential to be targeted by a vaccine is the _sexual stage_. This would give any protective benefits to the individual inoculated but would prevent further transmission of the parasite by preventing the gametocytes from producing multiple sporozoites in the gut wall of the mosquito. It therefore would be used as part of a policy directed at eliminating the parasite from areas of low prevalence or to prevent the development and spread of vaccine-resistant parasites. This type of transmission-blocking vaccine is potentially very important. The evolution of resistance in the malaria parasite occurs very quickly therefore potentially making any vaccine developed redundant within a few generations. This approach to the prevention of spread is therefore essential. Any vaccine produced would ideally have the ability to be of therapeutic value as well as preventing further transmission and is likely to consist of a combination of antigens from different phases of the parasite’s development.

Vaccines developed thus far
The epidemiology of Malaria varies enormously across the globe, and has led to the belief that it may be necessary to adopt very different vaccine development strategies to target the different populations. A Type 1 vaccine is suggested for those exposed mostly to P.falciparum malaria in sub-Saharan Africa, with the primary objective to reduce the number of severe malaria cases and deaths in infants and children exposed to high transmission rates. The Type 2 vaccine could be thought of as a ‘traveller’s vaccine’, aiming to prevent all cases of clinical symptoms in individuals with no previous exposure. This is another major public health problem, with malaria presenting as one of the most substantial threats to travellers’ health. Problems with the current available pharmaceutical therapies include costs, availability, adverse effects and contraindications, inconvenience and compliance many of which would be reduced or eliminated entirely if an effective (greater than 85-90%) vaccine was developed.

There are many antigens present throughout the parasite life cycle that potentially could act as targets for the vaccine. More than 30 of these are currently being researched by teams all over the world in the hope of identifying a combination that can illicit immunity in the inoculated individual. Some of the approaches involve surface expression of the antigen, inhibitory effects of specific antibodies on the life cycle and the protective effects through immunization or passive transfer of antibodies between an immune and a non-immune host. The majority of research into malarial vaccines has focused on the Plasmodium falciparum strain due to the high mortality caused by the parasite and the ease of a carrying out in vitro/in vivo studies. The earliest vaccines attempted to use the parasitic circumsporozoite (CS) protein. This is the most dominant surface antigen of the initial pre-erythrocytic phase. However, problems were encountered due to low efficacy, reactogenicity and low immunogenicity.

The first vaccine developed that has undergone field trials, is the SPf66, developed by Manuel Elkin Patarroyo in 1987. It presents a combination of antigens from the sporozoite (using CS repeats) and merozoite parasites. During phase I trials a 75% efficacy rate was demonstrated and the vaccine appeared to be well tolerated by subjects and immunogenic. The phase IIb and III trials were less promising, with the efficacy falling to between 38.8% and 60.2%. A trial was carried out in Tanzania in 1993 demonstrating the efficacy to be 31% after a years follow up, however the most recent (though controversial) study in the Gambia did not show any effect. Despite the relatively long trial periods and the number of studies carried out, it is still not known how the SPf66 vaccine confers immunity; it therefore remains an unlikely solution to malaria. The CSP was the next vaccine developed that initially appeared promising enough to undergo trials. It is also based on the circumsporoziote protein, but additionally has the recombinant (Asn-Ala-Pro15Asn-Val-Asp-Pro)2-Leu-Arg(R32LR) protein covalently bound to a purified Pseudomonas aeruginosa toxin (A9). However at an early stage a complete lack of protective immunity was demonstrated in those inoculated. The study group used in Kenya had an 82% incidence of parasitaemia whilst the control group only had an 89% incidence. The vaccine intended to cause an increased T-lymphocyte response in those exposed, this was also not observed.

The NYVAC-Pf7 multistage vaccine attempted to use different technology, incorporating seven P.falciparum antigenic genes. These came from a variety of stages during the life cycle. CSP and sporozoite surface protein 2 (called PfSSP2) were derived from the sporozoite phase. The liver stage antigen 1 (LSA1), three from the erythrocytic stage (merozoite surface protein 1, serine repeat antigen and AMA-1) and one sexual stage antigen (the 25-kDa Pfs25) were included. This was first investigated using Rhesus monkeys and produced encouraging results: 4 out of the 7 antigens produced specific antibody responses (CSP, PfSSP2, MSP1 and PFs25). Later trials in humans, despite demonstrating cellular immune responses in over 90% of the subjects had very poor antibody responses. Despite this following administration of the vaccine some candidates had complete protection when challenged with P.falciparum. This result has warranted ongoing trials.

In 1995 a field trial involving [NANP]19-5.1 proved to be very successful. Out of 194 children vaccinated none developed symptomatic malaria in the 12 week follow up period and only 8 failed to have higher levels of antibody present. The vaccine consists of the schizont export protein (5.1) and 19 repeats of the sporozoite surface protein [NANP]. Limitations of the technology exist as it contains only 20% peptide and has low levels of immunogenicity. It also does not contain any immunodominant T-cell epitopes.

RTS,S is the most recently developed recombinant vaccine. It consists of the P.falciparum cirumsporozoite protein from the pre-erythrocytic stage. The CSP antigen causes the production of antibodies capable of preventing the invasion of hepatocytes and additionally elicits a cellular response enabling the destruction of infected hepatocytes. The CSP vaccine presented problems in trials due to its poor immunogenicity. The RTS,S attempted to avoid these by fusing the protein with a surface antigen from Hepatitis B, hence creating a more potent and immunogenic vaccine. When tested in trials an emulsion of oil in water and the added adjuvants of monophosphoryl A and QS21 (SBAS2), the vaccine gave 7 out of 8 volunteers challenged with P.falciparum protective immunity.

The future of malarial vaccine therapy: Vaccine development strategies
The development of a vaccine of therapeutic and protective benefit against the malaria parasite requires a novel approach as to date there are no vaccines available that effectively target a parasitic infection. The focus so far has been predominately on the use of sub-unit vaccines. The use of live, inactivated or attenuated whole parasites is not feasible and therefore antigenic particles, or subunits, from the parasite are isolated and tested for immunogenicity i.e. the ability to elicit an immune response. The majority of subunits tested have been discussed above and are frequently combined with adjuvants and specialised delivery systems to increase the very variable level of immune response. The most recent advances in the field of sub-unit vaccine development include the use of DNA vaccines. This approach involves removing sections of DNA from the parasitic genome and inserting the sequences into a vector, examples including plasmid genomes, attenuated DNA viral genomes, liposomes or proteoliposes, and other carrier complex molecules. When inoculated the plasmid or attenuated virus is endocytosed into a host cell, the DNA sequence is then incorporated into the host DNA and replicated by protein synthesis. The proteins then produced are expressed on the cell surface membrane of the ‘infected’ cell. These bind to the HLA molecules, priming T cells and therefore creating a population of memory T cells specific to the inoculated DNA sub-unit. This technique has been shown to produce a high rate of T cell response but poor level of antibody production. The efficacy of DNA vaccines can be assessed using an ELISPOT assay. The development of this method of testing for immune responses is extremely beneficial when examining the potential efficacy of a vaccine candidate and is hoped to enable critical analysis of the mechanisms that provide ‘partial’ protection, thus facilitating a greater understanding of vaccine technology. This approach of potentially allowing the modification of vaccine candidates to improve development techniques and further scientific understanding is known as ‘iterative development’. The advantage of DNA vaccines over classical attenuated vaccines are numerous and include being able to mimic MHC class 1 CD8+ T cell specific responses that potentially could reduce some of the safety concerns associated with vaccine therapy and additionally provide a substantial reduction in production cost and due to the nature of DNA vaccines, increased ease of storage.

The most successful candidate developed to date is the RTS,S recombinant vaccine. The RTS,S/AS02A, one of the key vaccines produced using this technique, has been used in field trials in The Gambia. Three repeat doses were administered in the 6 months leading up to the period of highest malaria transmission. The vaccine efficacy was reported at approximately 71% (with 95% confidence intervals spanning from 46 to 85%) during the first 2 months of follow-up, but falling to 0% in the last 6 weeks in 250 male volunteers. Explanations for this are likely to be complex. On further analysis it was noticed that the majority of control subjects had become infected towards the end of the follow up period thus only the remaining (and therefore potentially more immune) subjects were included in the comparison against inoculated individuals, therefore as the efficacy of the vaccine decreased it was being tested against an increasingly immune cohort of controls, potentially explaining the massive decrease in protective immunity seen. Another reason to explain this is that an increase in the rate of malaria transmission during the final follow-up period occurred; this is particularly plausible and would coincide additionally with the suspected decrease in protection given by the last vaccine booster.