Cloning, Characterization and Immunogenicity of Merozoite Surface Protein-1 of Malaria Parasites

Abstract

Malaria is a parasitic disease caused by the haploid unicellular Plasmodium species. It continues to be one of the most prevalent of human diseases found in tropical and subtropical regions, including parts of the America, Asia and Africa. In 2009, there were an estimated 250 million cases of malaria worldwide. The vast majority of cases (85%) were in the African Region, followed by the South-East Asia (10%) and Eastern Mediterranean Regions (4%). Malaria accounted for an estimated 860,000 deaths in 2009, of which 89% were in the African Region, followed by the Eastern Mediterranean (6%) and the South-East Asia Regions (5%). The vast majority of deaths occur in Africa among young children under 5 years of age especially in remote rural areas with poor access to health services. Every 30 seconds one child in Africa dies from malaria, pregnant women are also vulnerable (WHO, 2010). Ten of the 11 countries of the South-East Asian region are endemic for malaria. Approximately 8 of 10 people in the region live at some risk for malaria, of which 3 of 10 live at high risk (areas with a reported incidence of >1 case per 1000 population per year). In 2008, 2.4 million laboratory-confirmed malaria cases and 2408 deaths were reported. In South-East region, the countries accounted for 97% of the malaria cases in 2008 are India, 55%; Myanmar, 17%; Indonesia, 15% and Bangladesh, 10% (WHO, 2009). In India, according to figures published by the union government’s National Vector Borne Disease Control Program, there were over 1.4 million cases of malaria; more than half of them caused by Plasmodium falcipaurm, and 678 deaths have been occurred in 2010. The most vulnerable areas are the eastern and central regions which include the states of Orissa followed by Chhattisgarh, Assam, Jharkhand, West Bengal, Maharashtra, Uttar Pradesh, Bihar, Andhra Pradesh, Rajasthan, Madhya Pradesh and Gujarat (NVBDCP 2010, URL: http://www.nvbdcp.gov.in). Malaria is caused by a protozoan parasite of the genus Plasmodium (phylum Apicomplexa). In human, malaria is caused by Plasmodium falciparum, P. vivax, P. ovale and P. malariae. Recently human infection with P. knowlesi, a malaria parasite of Old World monkeys has also been identified and widely distributed in Malaysia and can be fatal (Cox-Singh et al., 2008). Parasitic Plasmodium species also infect birds, reptiles, monkeys, chimpanzees and rodents (Escalante and Ayala, 1994). There have been documented human infections with several simian species of malaria, namely P. cynomolgi, P. simiovale, P. brazilianum, P. schwetzi and P. simium; however, these are mostly of limited public health importance. The life cycle of the malaria parasite is complex (Fig. 1). Infection of the human host commences when a female anopheline mosquito injects haploid sporozoites during a blood meal. The sporozoites travel to the liver and invade hepatocytes (typically 1-10 in number) develop over a period of about one week into an exoerythroctic schizont containing approximately 10,000 to 30,000 merozoites. For two of the human species (P. vivax, P. ovale), dormant hypnozoite forms can develop leading to delayed clinical attacks months or years after, but for P. falciparum and P. malariae there are no dormant forms and merozoites are released from infected hepatocytes and then invade red blood cells (RBC). Erythrocytic invasion by merozoite is dependent on the interactions of specific receptors on the erythrocytic membrane with ligands on the surface of merozoite. The entire invasion process takes about 30 seconds. In RBC, during 48h/72h cycle depending on species of malaria parasite, the single merozoite invades and develops into a ring, trophozoite, a mature trophozoite, and finally a schizont (erythrocytic schizogony), which gives rise to approximately 8-16 new merozoites. This process occurs within a parasitophorous vacuole in RBC. Some of the merozoites differentiated into sexual forms, which are macrogametocytes (female) and microgametocytes (male). These gametocytes in the course of the events are taken up by the mosquito during blood meal, and in the mosquito gut, where the temperature is lower, male and female gametes emerges from the infected RBCs. The duration of gametocytogony is assumed to be approximately 4 to 10 days depending on the Plasmodium species. Mature macrogametocytes in midgut form macrogametes. While the microgametocytes in the midgut exflagellate and forms 8 microgametes after few minutes of post infection. The microgamete moves quickly to fertilise a macrogamete and forms a zygote. Within 18 to 24 hrs, the zygote elongates into a slowly motile ookinete which traverses the peritrophic membrane and the epithelial cell of midgut, and then transforms into an oocyst beneath the basement membrane of the midgut epithelium. Between 7 and 15 days post infection, depending on the Plasmodium species and ambient temperature, a single oocyst forms more than 10,000 sporozoites. The motile sporozoites migrate into the salivary glands and accumulate in the acinar cells of the salivary glands. When an infected mosquito bites a susceptible vertebrate host, the Plasmodium life-cycle begins again. The emergence and worldwide spread of drug resistant parasite population to chemotherapeutic agents, the increasing insecticidal resistance of the mosquitoes, decay of public health infrastructure, population movements, environmental changes, and the inability of the most affected countries to mobilize and sustain the resources required for malaria control, emphasize the need for an effective vaccine against malaria. It is also predicted that areas which are now free of malaria e.g., Himachal Pradesh, might become malaria prone under the expected changing climate conditions in the 2050s. Based on ecological and man-made environmental changes (Construction of high-rise and industrial buildings, dams, deforestation etc.), malaria is changing from rural to urban malaria, from forest to plain malaria, and from industrial to travel malaria. Recent studies suggest that the number of malaria cases may double in 20 years if new methods of control are not devised and implemented (Bhattacharya et al., 2006). The development of a malaria vaccine is, however a formidable challenge. Despite a relatively intense and systematic research effort conducted since 1960s and clinical trials of a large number of candidate vaccines, few humans have been protected (Richie and Saul, 2002). Compared to developing vaccines against viruses and bacteria, development of a vaccine against malaria is complicated by the complexity of the parasite as well as the complex host’s response to the parasite. Challenges in malaria vaccine development include, the multistage life cycle of parasite, a large 23 Mb genome encoding more than 5300 proteins, the distinct stage-specific expression of proteins, the requirement for distinct immune mechanisms targeting these different stages, the poor understanding of the protective immune mechanisms, allelic heterogeneity of parasite antigens between strains, antigenic variation within a single strain, sequence polymorphism of critical target epitopes, parasite evasion of host immune responses, and variant diseases expression based on epidemiology, genetic background and age of the host (Doolan et al., 2003). Two key observations suggest that a malaria vaccine may be achievable. First, immunization with radiation–attenuated sporozoites induces sterile protection in mice, non-human primates and human volunteers, mediated predominantly by CD8+Tcells and gamma interferon (IFN-γ) and directed against the intra hepatocytic stage of the parasite (Nussenzweig et al., 1967; Clyde et al., 1973). Second, adults in malaria endemic areas develop partial immunity, which is largely mediated by antibodies directed against blood stage antigens (Rogers et al., 1999). A vaccine may need to induce both types of responses to provide optimal protection. The subunit vaccines are derived from whole or partial genes expressed in bacterial, baculovirus or plasmid systems or synthetically and purified as protein products. A drawback of this method is lack of inflammatory cytokines induced by most protein subunit and the consequent need of adjuvant for immunogenicity. When immunogenic, this approach tends to lead to a Th2 bias with good antibody induction but limited cellular responses. Addition of strong T helper epitopes is likely to improve such immunogenicity. Such vaccines could not target the liver-stage malaria parasite.