Malaria continues to pose a tremendous public health burden for people living in the tropical region, particularly in Asian and Africa countries. Globally, 300 to 500 million people infected annually, which is more than 40% of the global population (>2.1 billion people) and it causes more than 2 million deaths each year.
Malaria is caused by single-celled protozoan parasites of the genus Plasmodium and transmitted by mosquitoes. Four species infect humans by entering the bloodstream: Plasmodium falciparum, which is the main cause of severe clinical malaria and death; Plasmodium vivax; Plasmodium ovale; and Plasmodium malariae. Inoculation of parasite sporozoites occurs via the bite of infected blood-feeding female mosquitoes of the genus Anopheles. In humans, the parasites multiply exponentially in the liver, releasing merozoites that develop and multiply in infected red blood cells. With a blood meal, mosquitoes ingest Plasmodium gametocytes, which undergo another reproductive phase inside the mosquito before being transferred to another human host. History of vaccine development
Malaria parasites have complex life cycles and distinct developmental stages, each of which has multiple antigens that could serve as targets of an immune response. A pre-erythrocytic vaccine would protect against the infectious form injected by a mosquito (sporozoite) and/or inhibit parasite development in the liver. In a previously unexposed individual if a few parasites were to escape the immune defenses induced by a pre-erythrocytic vaccine they could eventually multiply and result in full-blown disease. An erythrocytic or blood stage vaccine would inhibit parasite multiplication in the red cells, thus preventing (or diminishing) severe disease during the blood infection. An optimal vaccine would have the ability to elicit protective immunity that blocks infection as well as prevents pathology and interrupts transmission of parasites.
Human populations residing in malaria endemic areas naturally acquire protective immunity against disease, although the patterns of immunity vary with malaria transmission patterns. Several studies showed that immunoglobulin purified from the blood of immune adults from endemic regions can passively transfer protection against parasite falciparum. Clinical studies carried out since the 1970's demonstrated that experimental vaccination with attenuated sporozoites can effectively immunize patients against a subsequent malaria infection. Animal models of malaria clearly substantiate the potential for induction of protective immunity with defined vaccines.
To date, most of the effort on vaccine development has focused on P. falciparum for the following reasons:
*High mortality from infection
*Capability for experimental challenge infection
*Relative ease of in vitro studies
*Availability of animal models for in vivo studies.
While P. vivax has a wider geographic distribution than P. falciparum, including in emerging economies such as Southeast Asia, India and Brazil, work on vaccine development has been impeded by several technical obstacles, such as the difficulty of culturing the parasite in vitro.
Recent developments
More than 30 distinct antigens identified in various life cycle stages of the malaria parasite have been proposed, at some level, as potential vaccine candidates based on observations such as: surface expression of the antigen on one or more life cycle stages; in vitro inhibitory (e.g. invasion-blocking) effects of specific antibodies; or, in vivo experiments showing protective effects of either direct immunization or passive transfer of antibody in animal models.
Several early vaccine candidates, many based on the circumsporozoite (CS) protein, the dominant surface antigen of the sporozoite stage, progressed into
Phase I/II clinical trials but were halted by problems of low immunogenicity and efficacy. Only one candidate vaccine, SPf66, based on antigens from both merozoite and sporozoite stages, has undergone extensive field trials. Efficacy was reported in several early clinical trials in South America, and one in Africa, but results from subsequent trials in Southeast Asia and Africa were not as promising. Recent clinical studies of a vaccine composed of CS antigen and hepatitis B surface antigen (RTS, S) demonstrated that adjuvant plays a critical role in successful immunization; these studies employed the same antigenic construct with 3 different adjuvant formulations, only one of which (SBAS2, an oil-in-water emulsion also containing 3-deacy-lated monophosphoryl lipid A and QS21) induced significant protective immunity.
Recombinant vaccines
The Walter Reed Army Institute of Research (WRAIR) and SmithKline Beecham Biological (SBBio), through a partnership extending uninterruptedly over the past 17 years, are developing a multi-antigen, multi-stage malaria vaccine based upon recombinant protein antigens3. This collaboration has led to development of the CS-based RTS, S vaccine, which in combination with SBAS2 adjuvant repeatedly induced protection of volunteers in a Phase IIa trial. Subsequent re-challenge of volunteers revealed that protection waned substantially by 6 months after the last immunization. In partnership with SBBio, USAID, the Naval Medical Research Center (NMRC) and others, WRAIR is conducting preclinical, clinical, and field trials to:
*Optimize RTS,S/SBAS2 vaccine regimens;
*Evaluate RTS,S with improved adjuvant;
*Develop the blood-stage antigen MSP-1 as a potential component of a multi-stage,multi-antigen vaccine
*Explore prime/boost strategies.
Other WRAIR initiatives include development of: the pre-erythrocytic Liver Stage Antigen-1 (LSA-1); blood-stage antigens Merozoite Surface Protein (FVO strain and 3D7 mutant) and Apical Membrane Antigen-1 (AMA-1); and, attenuated Venezuelan Equine Encephalitis Virus as a platform for antigen delivery.
The NIAID Malaria Vaccine Development Unit (MVDU) is focusing on recombinant proteins derived from blood stages and sexual stages of parasite development. The MVDU has facilities for protein expression in a variety of recombinant systems as well as subsequent process development. Once produced and purified, blood-stage antigens are being tested in Aotus monkeys to identify the most promising candidates. A region of variant parasite antigen that mediates binding of the infected cell to D36 on vascular endothelial cells has shown promising results in an Aotus trial. The MVDU is also conducting research on other blood-stage candidates, including MSP3, MSP4, MSP5, and AMA1, in most cases as a collaborative effort with other investigators. Transmission-blocking vaccines under development at the MVDU include Pfs25 and Pvs25, sexual stage antigens expressed by P. falciparum and P. vivax, respectively. Clinical grade Pfs25 has been produced and a plan is underway for evaluating these antigens for safety and immunogenicity in clinical trials later this year. Clinical grade Pvs25 is also being prepared for Phase I testing. Investigators at New York University are investigating the use of CS-based multiple antigenic peptides (MAP) for induction of anti-sporozoite immunity. A synthetic MAP vaccine containing minimal T and B cell epitomes from the repeat region of the P. falciparum CS protein with lump and QS21 elicited high levels of parasite-specific antibodies in a recent clinical trial, but immunogenicity was HLA-restricted. Newer methods for MAP synthesis and inclusion of universal epitomes are currently being explored.
The multivalent, multistage malaria vaccine development strategy, which is aimed at inducing "multiple layers" of long-lasting, effective immunity, takes into consideration the immunogenicity and genetic diversity of antigenic fragments contained in stage-specific proteins. Two P. falciparum candidate vaccines are under investigation. One, an 41 kd protein called FALVAC-1, contains 21 B-and T-cell epitopes from a variety of pre-erythrocytic, erythrocytic and sexual stages: CS, LSA1, MSP1, SSP2, MSP2, AMA1, RAP1, EBA-175, and Pfg27. FALVAC-1 has been expressed in a baculo-virus expression system in collaboration with National Institute of Immunology, New Delhi, India, and Protein Sciences Corporation, Connecticut. Mouse, rabbit, and monkey immunization studies of FALVAC-1 with various adjuvant demonstrated induction of immune responses that recognizes different stages of the parasite. These observations provide proof of the principal that recombinant antigen containing antigenic fragments from different stage-specific antigens can induce responses against different stages of parasites. A second candidate, FALVAC-2, containing the 19 kd fragment of MSP1, the third epidermal growth factor domain of Pfs25, Region II of EBA-175, as well as 30 B-cell epitopes and 25 T cell epitopes from a total of 13 stage-specific antigens, is under development. Similar approaches are underway for the development of multivalent, multistage P. vivax vaccines. CDC has entered into a Collaborative Research and Development Agreement (CRADA) with the Bharat Biotech International Limited (BBIL), Hyderabad, India, for production of GMP-grade candidate vaccine antigens. The goal for the next 5 years is to test multivalent, multistage P. falciparum and P. vivax recombinant vaccines in non-immune persons and individuals living in malaria endemic areas. The Australian malaria vaccine program is developing prototype asexual stage vaccines based on the 190L fragment of MSP1, MSP2, the Ring Associated Surface Antigen RESA, AMA1, and the Rhoptry Associated Proteins RAP1 and RAP2. Five human vaccine trials have been conducted with combinations of recombinant MSP1, MSP2 and RESA in the Montanide ISA 720 adjuvant (SEPPIC), with the most recent being a Phase IIb trial in children in a highly endemic area of Papua New Guinea.
Further extensive trials are planned to extend these promising results to younger children and further optimize this formulation. In test animals, including monkeys, AMA1 and RAP2 have given very encouraging protection. The first Phase I human trial has been conducted with AMA1, and further Phase-I trials with AMA1 and RAP2 are planned for later this year.
Indian development
India is likely to begin phase one trial of a malaria vaccine in 2007 after conducting animal safety trials next year even as the malaria drug policy is being reviewed to guard against the parasite's drug resistance. The sites for vaccine trials are being prepared in Orissa and Madhya Pradesh. A recombinant, protein-based malaria vaccine is being developed by the Delhi-based International Centre for Genetic Engineering and Biotechnology (ICGEB) in collaboration with the Bharat Biotech Ltd. In about one-and-half year, the first phase clinical trials would begin after its toxicity was tested in animals.
Military scientists from the Shanghai Medical College No 2 of the Chinese People's Liberation Army (PLA) have developed PfCP2.9, an indigenous malaria vaccine that will soon undergo human trials in Shanghai. The trial is being funded by Partnership for Appropriate Technology in Health (PATH) and the Malaria Vaccine Initiative (MVI). In addition to the $2 million funding for the project, PATH will also provide technical support and data analysis. On the Chinese front, Shanghai-based Wanxing Biological Pharmacy Co Ltd is also engaged in the research and development of the vaccine.
Malaria remains a major problem in Asian and African countries. Important achievements have been made in the past few years, which will contribute significantly to malaria control. They include exploitation of the P. falciparum, A. gambiae and human genomes, development of new drugs and new vaccine candidates, development and implementation of combination therapy, intermittent preventive treatment and home management of malaria.
The main challenges include overcoming the spread of drug-resistant P. falciparum through the use of combination therapy and appropriate early detection and monitoring of drug resistance, ensuring that the implementation of IPT on a large scale does not interfere with the expanded programme of immunization (EPI) and developing appropriate mechanisms for sustainable and effective implementation of the home management strategy over time and on a much larger scale.
These challenges will only be alleviated, and the control strategies achieve maximum impact, if additional resources are deployed to strengthen malaria research and control communities in countries where the new tools will be used. Continued and sustained efforts are needed to develop control tools through research and development
in partnership.
E-mail: pm_mandal@yahoo.co.in