Trial and success

A five-year-old boy with a severe case of malaria, at a hospital in western Darfur, Sudan. - CRIS BOURONCLE/AFP

The success of a trial in Mozambique marks an important step forward in the scientists' prolonged endeavour to find a safe, affordable and efficacious vaccine for malaria.

ONE of the biggest challenges of medical research is to develop a safe, affordable and efficacious vaccine against malaria with a reasonable lasting effect. Malaria is endemic in 91 countries, with an estimated 500 million clinical cases every year and an annual mortality of 1-2.7 million, most of which are deaths of children under the age of five in Africa. The most virulent species of the causative parasite is Plasmodium falciparum, which is distributed globally but is common in Africa.

Despite several approaches to the goal on the basis of seemingly sound immunological rationale and decades of international effort, such a vaccine has been elusive. Rapid advances in recent times in basic biology and in biotechnology - which have, on the one hand, resulted in the sequencing of the P. falciparum genome and, on the other, led to novel approaches such as synthetic peptide vaccines and deoxyribonucleic acid (DNA) vaccines - have not really provided the much-needed breakthrough to prevent malaria.

Many factors make malaria vaccine development an enormously difficult proposition. The foremost one is the complex lifecycle of the parasite. And, because of the genetic complexity of the organism, a complete understanding of the host-parasite interaction is still lacking. Each infection presents 6,000 antigens to the human immune system. The parasite presents a different subset of these antigens at each stage in its lifecycle for the immune system to recognise and mount a response. To date, about 40 antigens have been identified as being potentially useful for vaccine development. Vaccines contain one or more of these antigens that serve as targets for the immune system to mount a response.

The parasite has evolved a series of strategies that allow it to confuse, hide and misdirect the immune system. These include, besides the large number of genes that make up the plasmodium, a high degree of genetic diversity between parasite strains and extreme genetic variations or polymorphisms within strains. During the blood stage, the parasite resides in the blood cells (erythrocytes), where the immune system's mechanism to identify foreign proteins is not effective. On the basis of single nucleotide polymorphisms (SNPs) - the most common type of genetic variation arising from a change at a single base in a DNA molecule - the time to the most recent common ancestor has been estimated to be about 100,000 to 180,000 years. Scientists point out that this coincides approximately with the start of human population and is consistent with a time-scale for it to evolve into a genetically complex organism with extreme adaptability to the human host. Thus, under the pressure of immune response, mutant variants get selected very rapidly, providing the parasite with a genetic escape route.

Currently, three approaches to malaria vaccine are being pursued: targeting the pre-erythrocytic stage (before reaching the blood cell), the blood stage (the merozoites) and the sexual stage (the transmission-blocking vaccine) of the parasite's lifecycle. Most scientists believe that the ultimate vaccine will have components from multiple stages of the parasite's lifecycle. The first type is designed to prevent hepatocytes (in the liver) from being invaded by sporozoites (the form in which malaria injects the parasite into the human host) and to destroy hepatocytes that have already been infected. The aim of the blood (or asexual) stage vaccine is to eliminate invasion of blood cells by merozoites and to prevent clinical manifestation of the disease. Transmission-blocking vaccines are designed to break the cycle of infection. These vaccines do not prevent the recipient from getting the disease but to prevent its further transmission.

According to the World Health Organisation, as many as 25 vaccines are undergoing clinical evaluation with many more at the pre-clinical stage. The efficacy of most candidate vaccines has, however, always turned out to be low and short-lived. The seemingly promising blood stage or the erythrocyte stage vaccine based on the antigenic protein known as SPf66, developed by the Columbian scientist Emmanuel Patarayo in the late 1980s, began with an efficacy of 30 per cent in trials in South America and Tanzania, which dropped to 8 per cent in children in The Gambia and offered no protection at all in trials in Thailand.

Is a malaria vaccine that confers long-term protection feasible at all? This is a moot question in view of the limited success even after decades of research. The evidence is certainly there to suggest that the human body is capable of mounting an effective immune response. It has been demonstrated since the late 1970s by Stephen Hoffman and co-workers that irradiated sporozoites, when delivered through mosquito bites, conferred protection against infection. Irradiated sporozoites amount to inactivated whole parasite vaccine that confers `sterile immunity'. But a deliverable vaccine based on irradiated sporozoites would be impractical because producing enough sporozoites for the purpose is not easy.

The second evidence is the observation of gradual onset of natural protection (after the age of 10) from severe malaria by repeated exposure to infection in endemic areas. The third is the `passive transfer of immunity'. Immune globulins purified from the blood of individuals who have been long residents of endemic areas have been used in experiments to treat severe malaria. However fear of transmitting other diseases has stalled progress with malaria immune globulins. These provide the optimism for an effective vaccine. But an understanding of how to elicit that through a vaccine is what is eluding the scientists. This is essentially because the exact nature of protective immune responses is not fully understood.

Given the current limited understanding, however, the possibility of an effective malaria vaccine is being doubted by scientists like Shobhona Sharma of the Tata Institute of Fundamental Research (TIFR). "Unless we fully understand how the parasite is able to selectively suppress the immune response in humans, awareness, bed-nets, surveillance, diagnosis and therapeutic agents (including antibodies used as drugs) is the way to go for cost-effective malaria control," she points out.

IN this somewhat bleak scenario, the encouraging results of a clinical trial in over 2,000 children in Mozambique of a candidate vaccine, developed under a collaborative venture of the Malaria Vaccine Initiative (MVI) and GlaxoSmithKline (GSK) Biologicals, may be good news. In a `proof-of-concept' study, researchers found that a recombinant sub-unit vaccine - derived not from the whole organism but from some antigenic determinants of it - conferred protection to a significant percentage of children against uncomplicated malaria, primary infection and even severe forms of the disease for at least six months. This is the largest malaria vaccine efficacy trial (Phase IIb) ever conducted in Africa. The study was led by Pedro Alonso of the Centre for International Health of the Hospital Clinic at the University of Barcelona and the Manhica Health Research Centre (CISM) in Southern Mozambique. The findings were reported in the October 16 issue of the medical journal Lancet.

The vaccine is designed to target the sporozoite form of the parasite. Designated as RTS,S/AS02A, the recombinant vaccine is a hybrid in which the circumsporozoite protein (CSP) of P. falciparum is fused to Hepatitis-B surface antigen (HBsAg) and is expressed in yeast as a particle along with unfused HBsAg. As the name suggests, the vaccine comprises RT component of the antigenic protein, where R refers to the repeat region and T for the T-cell epitope (the region that stimulates T-cell immune response), and S, the hepatitis-B surface antigen. The vaccine formulation uses a proprietary oil-in-water chemical emulsion called AS02A.

The feasibility of a vaccine based on fusion of CSP and HBsAg was demonstrated way back in 1997. It took nearly 15 years for an appropriately formulated vaccine based on this concept to be taken up for clinical trials. In 2001, a randomised trial among 306 semi-immune men in The Gambia was carried out. Overall vaccine efficacy was found to be 34 per cent. However, protection seemed to wane rapidly over nine weeks of surveillance - from 71 per cent to zero. The present MVI-GSK-sponsored study in Mozambique children, however, showed sustained efficacy of the same vaccine over a six-month period.

The MVI, a focussed vaccine development programme under the Programme for Appropriate Technology in Health (PATH), a non-governmental venture, was launched in 1999 with a $50 million grant from Bill and Melinda Gates Foundation. MVI was created, according to its web site, with the recognition that while a malaria vaccine was urgently needed, traditional market forces were insufficient to prompt substantial investment necessary to develop vaccines aggressively against a disease that primarily affected people in developing countries. In September 2003, the Foundation announced an additional grant of $100 million to the MVI. While the Foundation is the primary supporter of the MVI, it also receives funding from USAID (United States Agency for International Development).

According to the Mozambique study, which involved double-blind, randomised controlled trials in 2,022 Mozambique children aged 1-4 years, the vaccine efficacy against clinical attacks was 30 per cent, efficacy against primary infection with P. falciparum was 45 per cent and efficacy against severe disease was 58 per cent. It was also found that after six months, the prevalence of infection in the vaccinated group was 37 per cent lower than the control group, pointing to a sustained efficacy over a six-month period.

While the primary objective of the study was to estimate the vaccine efficacy against clinical onset of P. falciparum malaria, efficacy against several other end-points were studied as well. These included infection and protection against mild and uncomplicated malaria, clinical malaria and multiple episodes, prevalence of infection and anaemia, and time to first infection with P. falciparum. While the vaccine was found to be efficacious against all other end-points, interestingly, no statistically significant effect owing to the vaccine was found on malaria-related severe anaemia.

"This is indeed surprising," said Virender Chauhan, Director of the International Centre for Genetic Engineering and Biotechnology (ICGEB), the United Nations Industrial Development Organisation centre in New Delhi. Chauhan's group is engaged in the development of a different blood stage subunit vaccine, a project funded by the MVI, the European Malaria Vaccine Initiative (EMVI) - a European Union-funded international programme - and the Department of Biotechnology (DBT). "We have sought some more data from the group on this aspect," he added. The scientists themselves have attributed this finding to anaemia recorded among the children being generally low and also to better surveillance.

The performance of the vaccine has a two-fold significance. First, it has been found to be effective and well-tolerated among children in the 1-4 age group, a very important aspect from the perspective of clinical profile in Africa. The other is that, unlike the earlier trials of the same vaccine among adults in The Gambia and the trials of other candidate vaccines, the efficacy of this vaccine in children is not short-lived. This marks a significant scientific advancement and an important step forward in the sense that it affords the possibility of improvement in efficacy. It also is seen as proof of the scientific premise that antigenic protein-based recombinant vaccine is a viable concept and that technologically costlier strategies of developing synthetic peptide vaccines or DNA vaccines may not be warranted. Chauhan, in fact, envisages a possibility of combining the blood-stage ICGEB vaccine - which is about to enter the pre-clinical toxicology studies - and Alonso's pre-erythrocytic vaccine.

The research group has offered the following explanations for the apparently contradicting results between this study and the earlier Gambian study in adults as regards the efficacy sustenance. One, the vaccine was found to be much more immunogenic in the child population studied than in the adults of the earlier study and sustained immune responses might have resulted in persistent protective efficacy. Second, there was high level sporozoite exposure during the later trial that could have resulted in natural boosting of protective immune response. "I think the simple reason is that this study was carried out much better than the earlier one," points out Chauhan. "Also the adult study had the problem of a much smaller number of people," he added.

Despite the apparently successful trial, the vaccine falls well short of the goals set for it. Ripley Ballou, one of the authors of the study, had set the following desired characteristics for the RTS,S/AS02A product vaccine: protection for all age groups older than 1 year of age, compatibility with Expanded Programme of Immunisation (EPI), efficacy of more than 70 per cent against clinical malaria and more than 30 per cent against severe malaria and durable effect for 1-3 years. Moreover, an annual or biannual booster dose may pose a substantial burden on the health system if the vaccine is costly. As will be noted, the vaccine under study seems to meet two of the desired characteristics of compatibility with EPI (as it is synthsised with HBsAg, an EPI vaccine in many countries) and over 30 per cent efficacy against severe malaria. But to meet all the characteristics, clearly there is a long way to go.

"This vaccine has very clearly demonstrated that we now have a platform based on proteins to move forward. No other trial has so far shown such a long-term sustenance," says Chauhan. "All malaria vaccine trials seem to get stuck at about 30 per cent efficacy level," points out Shobhona Sharma. "This only shows that we do not understand the behaviour of the parasite fully. In fact, we know that CSP displays a great deal of polymorphisms and the vaccine will have highly variable efficacy. The important aspect of this vaccine, however, is the very high levels of sero-conversion for HBsAg (almost 95 per cent). Surprisingly, the authors do not seem to emphasise this point. I personally feel that if this vaccine can replace the Hepatitis-B vaccine in EPI, that would be good news with even the low 30 per cent efficacy for malaria," she adds.

Does the development have relevance for malaria in the Indian context? "Not really," says Sharma. The reason being that the epidemiological profile of patients (based on hospital and field data) is that the number of children reporting severe malaria and the consequent mortality is not very high as it is in Africa. Chauhan, who is about to publish data based on field survey in endemic regions of Orissa, believes that this is mainly due to much better access to health care in India. "Much maligned though it is, the government health care system in India is much better than in Africa and hence childhood deaths owing to malaria are not that high," he says. But, the importance of the vaccine lies in the proof-of-concept of protein-based recombinant vaccines, which could form the basis for the development of more efficacious vaccines, even as it marks a very significant development from the perspective of the malaria situation in Africa.