Pre-erythrocytic stage malaria parasites: non-circumsporozoite protein antigens.

Pre-erythrocytic stage malaria parasites: non-circumsporozoite protein antigens* M.R. Hollingdale, M. Aikawa, Guoxian Chen, J.F.G.M. Meis, K. Sakhuja, B. Sina, & Jingdong Zhu A series of non-circumsporozoite proteins found in pre-erythrocytic parasites are being developed as putative vaccine candidates. It is anticipated that these will be useful in addition to, rather than instead of, the CS (circumsporozoite) vaccines. It is likely that a greater understanding of the basic biology of malaria parasite-host relationships will lead to development of improved malarial vaccines. Introduction The circumsporozoite (CS) proteins of malarial sporo- zoites have been the focus of intense research for development of anti-malarial vaccines. Studies on human volunteers have demonstrated that CS re- combinant or synthetic peptide subunit vaccines, though poorly immunogenic in man, can elicit pro- tection in some volunteers to sporozoite challenge. The identification of T-cell epitopes, and the use of improved carriers, may be expected to increase im- munogenicity, and thus protection. The rationale for such vaccines is the induction of protective anti- sporozoite antibodies that neutralize sporozoite in- fectivity for hepatocytes, and immune T-cell effector mechanisms that destroy exoerythrocytic (EE) para- sites within hepatocytes, either by secretion of soluble lymphokines or by direct cytotoxicity. However, in- vestigation of the basic biology of sporozoite-hepato- cyte interactions and EE development has also led to the identification of additional antigens that are now receiving consideration as alternative vaccine candidates (1). CSP-2 The immunodominance of the repeat region of CS proteins has made identification of other sporozoite antigens, particularly those on the surface, difficult using antisera from immunized animals or man. How- ever, alternative strategies have revealed novel sporo- zoite antigens. The capacity of irradiated or live sporo- zoites to elicit protection to sporozoite challenge has * From the Biomedical Research Institute, Rockville, MD 20852, USA; Case Western Reserve University, Cleveland, OH 44106, USA; and the University of Nijmegen Medical School, Nijmegen, Netherlands. Correspondence should be sent to Dr M.R. Hollingdale, Biomedical Research Institute, 12111, Parklawn Drive, Rockville, MD 20852, USA. been considered species-specific (1, 2). Sera from sporo- zoite-immunized animals or man strongly react with CS proteins on Western blots (3, 4). Recently, mice immunized with Plasmodium falciparum sporozoites were found to be immune to challenge with P. berghei sporozoites (5). Protection was specific as immunized mice were not immune to challenge with P. yoelii sporozoites. Cross-protection was unlikely to be mediated by P. falciparum CS proteins, since mice immunized with R32tet32 vaccine were completely susceptible to P. berghei sporozoite challenge. This represented the first demonstration that a non-CS antigen could elicit protective anti-sporozoite immun- ity. Proteins (CSP-2) of almost similar molecular mass (42 and 54 kDa) have now been identified on P. falciparum and P. berghei sporozoites that share cross-reactive epitopes. These proteins are being cloned and expressed for direct immunogenicity studies. Since the antigen is common to both P.falciparum and P. berghei, passive protection studies performed in mice with a monoclonal antibody (MAb) to CSP-2 characterize the efficacy of this novel P. falciparum antigen by demonstrating protection to P. berghei sporozoites. Thus, the protective capacity of this P. falciparum antigen can be studied in a model system without the need to directly conduct human trials, as has been the case for CS vaccines. E12 A second non-CS protein antigen has been identified using an MAb from mice immunized with P. yoelii blood-stage parasites (Taylor et al., in preparation). This MAb, E12, reacted by IFA (immunofluorescence antibody) with the apical region of P. yoelii and P. falciparum blood-stage merozoites, P. falciparum sporozoites, P. berghei EE merozoites, as well as Toxoplasma and Eimeria. In inhibition of sporozoite invasion (ISI) assays, MAb E12 reduced P.falciparum sporozoite invasion of HepG2-A16 cells. By Western blots, MAb E12 recognized a conserved protein in 178 Bulletin of the World Health Organization, 68 (Suppl.): 178-180 (1990) Plasmodlum sporozoite non-CS antigens Fig. 1: (A) Agarose gel electrophoress of Eco RI digestd clone C2GI DNA of P. b.h showing the Insert size of 6.8 Kb. (B) Agarose gel eiectrophoresis of Eco RI digedsd clone SK47 DNA of P. b.rhel showing Insert size of 6.0 Kb. (C) Western blot analysis of expressed protein of SK47 clone under f-galactosidaso promotion. (D) Southern blot of P. taklparum genomic DNA against 32P-labelled oligonuclotide probe spanning the 17 amino acid rpeat of LSA. (E) Western blot analysis of P. haklparum blood stages with MAb E12. D 1 0v 6kl E i,6 57kd - I§5, 48kd 7kb "; ;. several Plasmodium parasites, and Eimeria. Further studies have shown that MAb E12 reacted on West- ern blots P. falciparum blood-stage merozoites with two proteins of 48 and 57 kDa (Fig. 1E). This protein complex has been partially purified from P.falciparum lysates, and retains serological reactivity. Thus far, screening of Plasmodium genomic libraries has been unsuccessful. Alternative strategies of screening Plas- modium cDNA libraries, or purification and N-ter- minal sequencing for construction of oligonucleotide probes are being pursued. LSA-1 A P. falciparum liver-stage-specific antigen (LSA-1) has been identified (6), and a 196 bp fragment of the gene cloned and sequenced, revealing three and a half repeats of the amino acid sequence E(G)QQSDLEQERLAKEKLQ (6). Rabbit anti- bodies to this peptide recognized P.falciparum EE par- asites, and using immunoelectron microscopy the re- activity in EE parasites was localized in the flocculent material found in the parasitophorous vacuole (1), and similar flocculent material of P. berghei EE para- sites has previously been shown to be the target of cellular infiltration on rats (7). Therefore, LSA ex- pression, as well as delivery in systems that elicit cellular immune mechanisms (8), is being pursued. Mice have been immunized with either EQQSDLE- QERLAKEKLQ (EQQ peptide) or LEQERLAK- EKLQEQQSD (LEQ peptide) peptide constructs conjugated to tetanus toxoid at either terminus. Sera from these immunized mice were reacted with the flocculent material and were used to identify reactive epitopes within the full length peptide sequence by the method of Geysen (8). Epitope specificity depended on the sequence of the peptide and orientation of the carrier tetanus toxoid. Anti-EQQ peptide sera recog- nized different epitopes from anti-LEQ peptide sera. A gene fragment representing about 1.6 bp has been cloned (Fig. 1D), sequenced and expressed in E. coli, and contains at least 20 repeats of the EQQ peptide sequence interspersed with three variant repeats. Further cloning has determined the complete se- quence, and the N- and C-terminal sequence (1). WHO Bulletin OMS: Supplement Vol. 68 1990 A B C 200 - kd I i 4-6kt 1*4kt 5kb 1*Bkb 4.f.i. 179 M.R. Holllngdale et al. Heat shock proteins Heat shock proteins (hsp7O) are localized in the nucleus and 78-kDa glucose regulated proteins (grp78) have signal sequences and remain in the endo- plasmic reticulum (ER) of eukaryotic cells. Clones expressing P. falciparum hsp70 and a protein similar to grp78 have been described (9, 10). Pfgrp78 contains a C-terminal sequence homologous with P.falciparum CS protein NI region hepatocyte binding region (11). By immunoblots and immunoelectron microscopy, Pfhsp7O was not found in sporozoites, but was detec- ted in newly invaded sporozoites, and the nuclei of P. berghei EE parasites in hepatoma cells, whereas Pfgrp78 was found in the ER of P. berghei EE para- sites. Since antibodies to heat-shock proteins are found in sera from malaria-endemic areas (10), the relationship of anti-sporozoite or anti-EE parasite immunity is being further studied. Antigens common to EE and blood-stage parasites EE and erythrocytic stage parasites share many com- mon epitopes (12,13). Since the major erythrocytic merozoite surface coat precursor protein has been shown to completely protect Aotus monkeys (14), MAb C2G1 has been developed that reacts with a 250 kDa antigen common to P. berghei erythrocytic and EE merozoites (13). A fragment of this gene has been cloned (Fig. IA), and is being sequenced. A second clone (Fig. IB), SK47, was isolated using mouse polyclonal anti-P. berghei erythrocytic-stage antisera, and expressed as a ,B-galactosidase fusion protein in E. coli, encoding a protein of about 86 kDa (Fig. IC). This protein is being used to raise antisera for further immunochemical and immunological studies. References 1. Hollingdale, M.R. et al. Non-CS pre-erythrocytic pro- tective antigens. Immunol. lefters, 25: 71-76 (1990). 2. Clyde, D.F. et al. Specificity of man immunized against sporozoite-induced falciparum malaria. Am. j. med. sci., 266: 398-403 (1973). 3. Yoshida, N. et al. Hybridoma produces protective antibody against the sporozoite stage of malaria para- site. Science, 207: 71-73 (1980). 4. Nardin, E.H. et al. Circumsporozoite proteins of human malaria parasites Plasmodium falciparum and P. vivax. J. exp. med., 156: 20-30 (1982). 5. do Remrlo, V.E. et al. Plasmodium falciparum sporo- zoites protect mice to challenge with P. berghei sporozoites. Proc. Ann. Mtg Am. Soc. Trop. Med. Hyg., Honolulu, 1988, Abstract 381. 6. Guerln-Marchand, C. et al. A liver stage-specific anti- gen of Plasmodium falciparum characterized by gene cloning. Nature, 32: 164-167 (1987). 7. Mlol, J.F.G.M. et al. Cellular response against exo- erythrocytic forms of Plasmodium berghei in rats. Am. j. trop. med. hyg., 37: 506-510 (1987). 8. Geysen, H.M. et al. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl Acad. Sci., 81: 3998-4002 (1984). 9. Kumar, N. et al. Plasmodium falciparum gene en- coding a protein similar to the 78-kDa rat glucose- regulated stress protein. Proc. Nati Acad. Sci., 85: 6277-6281 (1988). 10. Poteren, M.G. et al. A second antigenic heat shock protein of Plasmodium falciparum. DNA, 7: 71-78 (1988). 11. Aley, S.8. et al. Synthetic peptides from the circum- sporozoite proteins of Plasmodium falciparum and P. knowlesi recognize specific hepatic receptors. J. exp. med., 164: 1915-1922 (1986). 12. Holllngdale, M.R. et al. Serological reactivity in vivo cultured exoerythrocytic stages of P. berghei in indi- rect immunofluorescent or immunoperoxidase anti- body tests. Am. j. trop. med. hyg., 32: 24-30 (1983). 13. Szarfman, A. et al. Mature liver stages of cloned Plasmodium falciparum share epitopes with proteins from sporozoites and asexual stages. Parasite im- munol., 10: 339-351 (1988). 14. SIddiqul, W.A. et al. Merozoite surface coat precursor protein completely protects Aotus monkeys against Plasmodium falciparum malaria. Proc. Natl Ac*d. Sci., 84: 3014-3018 (1987). iso WHO Bulletin OMS: Supplement Vol. 68 1990