Malaria is caused by infection of one or more of malaria protozoa (Plasmodium), including the following known 4 species: falciparum malaria (Plasmodium falciparum; hereinafter abbreviated to as “Pf”), vivax malaria (P. vivax), malariae malaria (P. malariae) and ovale malaria (P. ovale). The above-described protozoa in a form of sporozoite invade and infect a human body from the salivary gland of a female malaria-carrying mosquito (Anopheles) through the bite. An outline of the life cycle of the parasite is as follows:    [Mosquito] formation of sporozoites by sexual growth of the protozoa→<bite>→    [Human] sporozoite <invasion into the blood>→<invasion into the hepatocyte>    →[Exo-erythrocyte stage] sporozoite in the hepatocyte→schizont→formation of a merozoite and release into the blood by destruction of the hepatocytes→<invasion of the merozoite into the erythrocyte>    →[Intra-erythrocyte stage] merozoite→ring→trophozoite→asexual growth of the schizont→repetition the cycle of the formation of the merozoite and release into the blood by destruction of the erythrocytes→<crisis>; or            merozoite→differentiation into male and female gametocytes→<hematophagia>→            [Mosquito] male and female gametocytes→male and female gametes→sexual reproduction→differentiation into ookinetes→differentiation into oocytes and growth→formation of the sporozoite and moving to the salivary gland.
With respect to antigens of the above-described malaria parasite (Pf) relating to the invention, it has been reported that there are various antigens as many as 40 species in total as shown below. For example, at the above-described intra-erythrocyte stage in the life cycle, the followings are exemplified: SERA (serine-repeat antigen; another called, SERP: serine-rich protein), HRP-2 (histidine-rich protein 2), etc.; in the merozoite, MSP-1 (merozoite surface antigen-1), MSP-2 (merozoit surface antigen-2), AMA-1 (apical membrane antigen-1), etc.; in the sporozoite and also at the exo-erythrocyte stage, SSP-2 (sporozoite surface antigen-1), LSA-3 (liver-specific antigen-3), etc.; and at the sexual stage, Pfs 230, Pfs 45/48, etc. (“Topley & Wilson's Microbiology and Microbial Infections”, 9th edition, volume 5, Parasitology, p. 383, L. Collier, et al., published by Arnold Co., 1998). It has energetically been attempted to develop the vaccine using an antigen alone or as a mixture or in a form of a gene DNA, though no practically usable vaccine has been known (“The Jordan Report 2000”, pp 141-142, US National Institute of Health, published in 2000).
Moreover, the so far known techniques for producing malaria vaccines using the above-described SERA (or SERP) and a gene thereof relating to the invention have been described, for example, in European Patent EP 283,882 (the 1882-1917th bases, the 2403-2602nd bases, and the 2602-2631st bases of a 140 kd antigenic gene in SERP encode hydrophilic epitopes), U.S. Pat. No. 5,395,614 (a fusion protein of SERP epitope and HRP-2), U.S. Pat. No. 6,024,966 (a gene which can be identified by two species of probes A and B encodes SERA antigenic polypeptide), and a report (Vaccine, 14, pp. 1069-1076, 1996) relating to the expression system of SE47′ derived from the 47 kd SERA and production of the SE47′ antigen in this system. However, the antigenicity and purity of these vaccine antigens are insufficient, and the purification process is not suitable to mass production. Further, their safety, efficacy or homogeneity is not clearly assured, and therefore remarkable originality and progress are necessitated in the production process for solving these problems. Reduction to practice has not yet been achieved for these antigens, accordingly.
On the other hand, SERA (serine-repeat antigen) is a protein antigen of 115 kd in molecular weight consisting of 989 amino acids in total and expressed by Pf gene at the intra-erythrocyte stage. The structure of SERA consists of 3 domains, i.e., 47 kd-50 kd-18 kd, in order of the N-terminal to the C-terminal direction. SERA working as a precursor for these domain is expressed by 4 exons distributed on the SERA gene DNA comprising 5868 bases in total, and then processed and cleaved at the intra-erythrocyte stage during release of the merozoite to yield the above-described 3 domains. (Molecular and Biochemical Parasitology, 86, pp. 249-254, 1997; and Experimental Parasitology, 85, pp. 121-134, 1997). In this connection, the full-length data of the SERA gene DNA and which encoded the amino acid sequence are open to public and available from GenBank (Accession Number: J04000). The N-terminal region of SERA (hereinafter referred to as “47 kd domain”) consists of 382 amino acids in total. The homology search between the Pf strains relative to the sequence indicates that SERA is varied since there are in some regions deletion or addition of an amino acid or acids and about 20 variation of amino acids (non-synonymous substitution) (Molecular and Biochemical Parasitology, supra; and Experimental Parasitology, supra).
Malaria is an infectious disease occurring in many places in the world, ranking after acute lower respiratory tract infections, AIDS, and diarrhea. According to the estimation by WHO (World Health Organization), the number of patients suffering from malaria was approximately 45 million in 1999, among which approximately 1.1 million were killed (The World Health Report 2000, p. 164 and p. 170, published by WHO in 2000). Such a high rate of death is attributed to severe falciparum malaria, namely, cerebral malaria. The major cause is considered as cerebral thrombosis induced by accumulation of destructed erythrocyte debris accompanied by the Pf growth, and this results in death through sensory paralysis, delirious talk, dystrophy, convulsion, etc. It is no exaggeration to say that avoidance of such cerebral malaria is the most important problem to be solved.
As a reason of frequent occurrence of malaria, it has been proposed that drug-resistant strains or multiple drug-resistant strains of malaria parasite against anti-malaria drugs such as quinine, chloroquine, pyrimethamine-sulfadoxime, mefloquine, halofantrine, etc. have emerged and spread. Since the end of 1950s at which time occurrence of chloroquine-resistant Pf was reported in South America and Southeast Asia, such resistant parasite have spread through the almost whole area of malaria-occurring tropical or subtropical zones except a part of Central America, the Caribbean Sea, and the Middle and Near East. Therefore, the control of malaria has become currently global problems to be solved in the health administration with rapidly increased diplomatic relations so that it was inevitably recommended to spread DDT as an emergency measure (WHO Expert Committee on Malaria: Technical Report Series, No. 892, pp. 1-71, 2000, published by WHO).
Additionally, a future feared problem is the expansion of malaria-occurring areas accompanied by global warming (Science, 289, 1763-1766, 2000). Now, it is an imminent and urgent problem to provide measures against malaria for the whole humankind.
In particular, in development of a malaria vaccine, though a great deal of effort and energy has been made to develop the vaccine all over the world, no effective vaccine has been provided. The main reason is considered to be the following problems (a) to (c): (a) Since the malaria antigens are various as mentioned above, it is difficult and obscure to identify a protective antigen from such various antigens; (b) Malaria antigens are of polymorphic gene, and the antigenicity is variable depending on the strain of Pf parasite. Therefore, a single antigen derived from Pf, for example, a well-known antigen as a candidate for vaccines such as MSP-1, AMA-1, etc., has a very narrow antigenic spectrum, so that it is not necessarily effective for prevention from infection by any species of Pf strains; and (c) The antigens as a candidate for vaccines such as MSP-1, AMA-1, etc., as well known, is denatured during purification, and destructed in its steric structure or epitope to decrease or lose its antigenicity.
USPTO Backgrounds
USPTO Backgrounds