A malaria sporozoite surface antigen distinct from the circumsporozoite protein.

A malaria sporozoite surface antigen distinct from the circumsporozoite protein R.C. Hedstrom,"2 J.R. Campbell,1'3 M.L. Leef,1 Y. Charoenvit,1 M. Carter,4 M. Sedegah,1 R.L. Beaudoin,1 & S.L. Hoffman1 Monoclonal antibody NYS4 recognizes a single 140kDa antigen on the surface of Plasmodium yoelii sporozoites, an antigen which is distinct from the extensively characterized circumsporozoite (CS) protein. To more thoroughly characterize this additional surface component, a genomic expression library was screened with NYS4 and an immunoreactive clone (M4) was obtained which expressed part of the antigen gene. The deduced amino acid sequence of the M4 peptide included two unique repetitive sequences of amino acids and a conserved sequence motif which is found in several proteins including the CS protein (region 11). The cloned DNA hybridized to a PCR (polymerase chain reaction) amplified sporozoite mRNA demonstrating the sporozoite-stage expression of this gene. A synthetic peptide of one of the repeats, (Asn- Pro-Asn-Glu-Pro-Ser), was recognized by NYS4 and mice immunized with (Asn-Pro-Asn-Glu-Pro-Ser)3 conjugated to KLH (keyhole limpet haemocyanin) produced high levels of antibodies that reacted with the surface ofsporozoites and specifically to the 140 kDa antigen. Thus, at least two different proteins are on the surface of the P. yoelii sporozoite indicating that the immunoreactive exterior of the infective stage of malaria parasites is more antigenically complex than previously thought. Introduction The sporozoite of Plasmodium is uniformly covered by a proteinaceous membrane coat which is thought to be composed entirely of the circumsporozoite (CS) protein (1, 2). Being the first parasite molecule en- countered by the host, the CS protein has been in- tensively studied and evaluated as a vaccine against malaria. Indeed, experimental immunization with ra- diation-attenuated sporozoites induces solid protec- tive immunity (3-5) coincident with the stimulation of both cellular and humoral immunological responses to the CS protein (6, 7). An emerging view is that an essential feature of the protective response induced by sporozoites is cellular immunity (4-9). If this is correct, then a multicomponent vaccine containing additional pre-erythrocytic stage antigens may be required to induce sterilizing immunity in all individuals, since those regions of the CS protein that are the immuno- dominant determinants of T-cell responses are also the most variable in amino acid sequence from para- site to parasite (10-12). The search for additional antigens by Charoenvit et al. led to the production of several monoclonal 1 Malaria Program, Infectious Diseases Department, Naval Medi- cal Research Institute, Bethesda, MD 20814-5055, USA. Corres- pondence should be sent to Dr S.L. Hoffman at this address. 2 U.S. Naval Medical Research Unit 3, Cairo, Egypt. 3 Naval Research Laboratory, Washington, DC, USA. 4Department of Immunology, Walter Reed Army Institute of Re- search, Washington, DC, USA. antibodies that recognize sporozoite determinants which are distinct from the CS protein (13). One of these antibodies, designated NYS4 (Navy Yoelii Sporozoite 4), recognizes a sporozoite-specific 140 kDa antigen. This antigen is expressed on the surface of live sporozoites and is secreted along with the CS protein in vitro (unpublished results). Wortman et al. used this antibody to isolate an antigen-ex- pressing clone from a P. yoelii genomic expression library (14). Here we describe the cloning and charac- terization of a portion of the gene encoding the 140 kDa antigen and identify a hexapeptide of this protein that is a B-cell epitope on sporozoites. Materials and methods Parasites and DNA lsolation. P. yoelii 17 X (NL) para- sites were obtained by blood passage in Balb/C mice. DNA isolation from parasite infected blood was per- formed, as described (14). Genomic expression library and immunoselectlon. A P. yoelii genomic expression library was constructed using 0.5-2.0 kb fragments generated by partial DNase I digestion and commercial (Promega, Madi- son, WI) lambda gtl 1 arms and packaging extracts (15). The library was screened for antigen expressing clones with a 1:20 dilution of NYS4 (13) hybridoma culture supernatant, as described previously (16-18). NYS4 immunoreactive plaques were detected with a commercially prepared antibody detection kit 152 Bulletin of the World Health Organization, 66 (Suppl.): 152-157 (1990) Fig. 1 (a). Nucleotide sequence and derived amino acid sequence of a portion of the P. yoeIII genomic DNA insert in lambda gMSY-4 (M4). The sequences of oligonucleotide primers (+) and (-) used for PCR amplification are indicated. (+) CCT TTT CTT ACT AAA GTT TGT CAG GAA GTA GAA AGA ATT GCT CAT TGT GGA AAA TGG GAA 60 P F L T X V C Q E V E R I A H C 0 x w N GAA TOG AGT GAA TGT TCT ACT ACT TGT GAT GAA GGA AGA AAA ATT AGA AGA AGA CAA ATA 120 H w 8 E C a T T C D z a R X I R R R Q I TTA CAT CCT OGA TGT OTT AGT GAG ATG ACT ACT CCA TOT AAG GTT COT OAT TGC CCA CAA L H P 0 c ATA CCA ATA CCT CCT I P I P P CCA GTA AAT CCA AAC P V N P N AAC CCA AAC AAC CCA N P N N P AAC AAT CCA AAT AAC N N P N N V GTC V OAT D AAT N CCA p 8 Z X T T P C K V R D C P ATC CCT AAT AAA ATT CCA OAA AAa CCA TCA AAC CCA OAA I P N x I P H x P a N P S -- ----3-HER REPZATS-----------> CCA AaC OAT CCA AAC AAC CCA AAC AaC CCA AAT AaC CCA P N D P N N P N N P N N P AAC CCA AAC AAC CCA AAC ARC CCA AAC AAC CCA AAC AAC N P N N P N N P N N P N N AAT AAC CCA AAC AAC CCA AAT AAC CCA AAT AAC CCA AAC N N P N N P N N P N N P N Q OAK AAC- N CCA p AAC N --------->3-HER REPEATS-------- CCA AAT AAC CCA AAC AAC CCA AAT AAC CCA AAT AAC CCA AaT AAC CCA AAT P N N P N N P N N P N N P N N P N CCA TCA AAC CCA AAC AAC CAC CCA AAA AGO CGA AAC CCA AAA AGG CGA P a N CCA AAA CCA P K P AAG CCA AAC X P N CCA AAC aAG P N K TTA AAC CCA L N P CCA AAC GAA P N E GAA CCA TCA z P 5 TCA AAC CCA 8 N P CCA AAC GAA P N z GAA CCA TCA E P a AAC CCA aAA N P E aAA TCG AAC * 8 N CCA GAA OAT P N N H P X R R N P X R R -------6-HER REPEAT AAC AAG CCA AAC CCA AAC AAG CCA AAC CCA AAC GAA CCA N K P N P N K P N P N E P CCA AAC GAA CCA TCA AAC CCA AAC AAG CCA AAC CCA AAC P N E P 8 N P N K P N P N CCA AAC CCA AAT GAG CCA TCA AAC CCA AAC AAG CCA AAC P N P N E P 8 N P N K P N AAC GAG CCA TCA AAT CCA AAC GAG CCA TCA AAC CCA AAT N E P S N P N E P a N P N CCA TCA AAC CCA AAT GAA CCA TCA AAC CCA AAT GAG CCA P S N P N E P a N P N E P AAC CCA AAT GAA CCA TCA AAC CCA AAA AAG CCA TCA AAC N P N E P 8 N P K K P S N AAT GAO CCA TTA AaC CCA AAT GAG CCA TCA AAC CCA AAC N E P L N P N E P 8 N P N CCA TCA AAC CCA aAA GAA CCA TCA AAC CCT AAA GAG CCA P 8 N P E E P 8 N P K E P AAC CCA aAA GAO CCA N P E E P GAO CCA ATA a P I CCC AAA GAO AaT OAa AaT N E NN P E D AAC CCA AAC N P N AAC CCA AAC N P N REGION----> TCA AAC CCA a N P GAA CCA TCA E P a CCA AAT GAG P N E GCG CCA TCA A P 8 TCA AAC CCA 8 N P CCA AAT GAG P N GAA E TCA S E CCA TCA P 8 AAC CCA N P AAC CCA GAA GAA CCA TCA AAC CCT AAA GAG CCA N P z E P S N P X E P ---------H>E-NR RZPEAT REGION----------AAaC CCA BAA aAA CTA AAC CCA AAA GAO CCa TCA N P E E L N P X z P S CCA ATA AaC CCA aAA GAa TCG AaC CCC AAA GAO P I N P z E S N P X H CCA TTO ATA ATA CAA OAT GAA CCT ATA GAA CCC P L I I Q D Z P I z P OAT TCA AAT OTA ATA CCA ATT TTA CCT ATC ATC CCA CAA D 8 N v I P I L P I I P Q (-) AAT CTA CCA BAA AAT CCA TCT aAC TCA aAA GTA aAA TAT N L P s N P 5 D S z V z Y BAA AAT TCA AAT AaT ACT ATG AAA TCA AAA AAA AAT ATA N N S N N T H K S x x N I WHO Bulletin OMS: Supplement Vol. 68 1990 AAG GOT AAT AAT ATC x B N N I CCA AGA CCA AAT GAT P R P N D CCC AAC GAG CGG P N z R AAC CCA N P CCA ATA P I AGA AAT R N CCA AGC P S AAT GGT N 0 ISo 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 OAT D AAG AAC N AAC N CCA p AAC N AAC N CCA p AAC N AAC N TCA a OAA E AAC 1140 1200 1260 1320 1380 1431 153 R.C. Hedstrom et al. (Protoblot, Promega, Madison, WI) according to the manufacturer's specifications. DNA sequencing. Phage DNA of lambda gtl 1 im- munoreactive clones was prepared from liquid lysates by standard methods (19). Plasmodial DNA insert fragments released by EcoR1 digestion were cloned into M13mpl8 (20). Overlapping clones spanning the inserts were generated using the Ml 3 RF recombinant plasmid by exonuclease III resection (21). Single- stranded templates were sequenced by using Sequen- ase (United States Biochemical Corp., Cleveland, OH). Polymerase chain reaction (PCR) amplification ofa sporo- zoite mRNA. RNA from 14-day P. yoelii infected mos- quitos was purified by the guanidine thiocyanate CsCl gradient technique (22) and reverse-transcribed (50 pg whole RNA) by using a commercially available cDNA synthesis kit (Amersham Corp., Arlington Heights, IL) which uses oligo(dT) as primer. One-tenth, one- hundredth, and one-thousandth of the resulting cDNA was subjected to 35 cycles of PCR (23) with 2.5 U Taq polymerase (GeneAmp kit, Perkin Elmer Cetus, Norwalk, CT) and 5 pmol each of the following oligonucleotides (Synthecell, Gaithersburg, MD): (+), 5'-GGGAATrCGCTCATfGTGGAAAATGG-3'; (-), 5'-GGGAATTCTAC-TICTGAGTCAGATGG-3'. Each cycle included denaturation at 94°C, rean- nealing at 51 °C, and primer extension at 72 'C. Peptide synthesis. The peptide (Asn-Pro-Asn-Glu- Pro-Ser)3 was synthesized using a solid-phase method (24) on an Applied Biosystems model 430A synthes- izer with single coupling of each amino acid. Cleavage and deblocking were performed with 10% trifluoro- methanesulfonic acid in trifluoroacetic acid, followed by precipitation in diethyl ether. The peptide was finally redissolved and lyophilized. Analytical HPLC and quantitative amino acid analyses confirmed the identity and purity of the peptide product. Production ofantisera. Six to twelve week old BALB/C mice (Jackson Laboratories, Bar Harbor, ME) were immunized at two-week intervals with 4 doses of 100 pg of (Asn-Pro-Asn-Glu-Pro-Ser)3 conjugated to keyhole limpet haemocyanin. The first dose was emul- sified in complete Freund's adjuvant, and subsequent doses in incomplete Freund's adjuvant. Sera were obtained 2 weeks after the fourth dose. immunofluorescence and Western blotting. Immuno- fluorescence and Western blotting were carried out, as previously described (13). 0~ ,,E U. Y0 :a ec i IE CL E E *0 c lb o Eu. 0= o En- 0 _ . . 1. E EL IN coL i:L i:o N b * "14 b N~~~ 14~ co U to a to to a O 04 0 0. In R 14 4 q 6 a 0 1 a PI 1 14 4 a I 1l N N 0 0U U 14 4 C4 14 14 4 X %o %w aftv N .Q u N EN U U 0 0EW PLP io N ~0 14 0 . 14 W 'I U N H oL bjU 14 - N 0 0 - P P. .41P 1 9 Mt " o " WHO Bulletin OMS: Supplement Vol. 68 1990154 Malaria sporozolte surface antigen Results Sequence analysis of the 140 kDa antigen gene. A clone designated lambda gMSY-4 (M4) was selected based on its recognition by NYS4. The primary DNA and deduced amino acid sequences of a portion of the genomic DNA insert from clone M4 are shown in Fig. l(a). Like most other plasmodial antigens, the M4 peptide contains regions of repeating amino acid sequences. One region consists of a repeating trimer Pro-Asn-Asn and the other is composed of, prin- cipally, the hexamer Asn-Pro-Asn-Glu-Pro-Ser. The first 48 amino acids are compared in Fig. l(b) to several proteins that share a remarkable sequence similarity to this region of the M4 peptide. The similarity be- tween region II of the CS protein (301-315) and thrombospondin is well known (25). In addition, mouse properdin was recently shown to contain a similar sequence (26). The M4 peptide shares this conserved motif but contains a more extensive region of sequence similarity with the recently described TRAP (thrombospondin-related anonymous protein) from P. falciparum (27). A search of the PIR protein sequence databank, conducted on 12 February 1990, revealed no additional significant similarity to pre- viously described protein sequences. mRNA for the 140 kDa antigen Is present In sporozoltes. To demonstrate sporozoite expression of the mRNA for the 140 kDa antigen we utilized the polymerase chain reaction (PCR) specifically primed with M4 clone sequence-based primers (Fig. 1(b)). An amplification product of the predicted size (the dis- tance between the two primers is 1.3 kB) was clearly visible in reaction mixtures which contained, as tem- plates, sporozoite cDNA or DNA from clone M4 (positive control) (Fig. 2, left). In Fig. 2, right, the identity of the amplified cDNA subfragment was confirmed with the M4 probe. A faint signal at 1.3 kB Fig. 2. Expression of the 140 kDa antigen mRNA In sporozoltes as determined by PCR ampiificatlon of a cDNA subfragment from reverse-transcribed RNA Isolated from Infected mosquitos. Primers used in the amplification are shown in Fig. 1. Left: agarose gel stained with ethidium bromide. Right: Southern blot of the stained agarose gel probed with 32P-labelled M4 clone DNA. Lanes 1, DNA molecular weight markers; lanes 2, M4 clone DNA (15 pg); lanes 3, M4 clone DNA (1.5 pg); lanes 4, cDNA (50 pg); lanes 5, cDNA (5 pg); lanes 6, cDNA (0.5 pg); lanes 7, RNA (50 .g); lanes 8, RNA (5 jg); lanes 9, RNA (0.5 pg). 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 12 4 6 8 12 45 78I~~~ i 1. .:. .i . .'. _ _ I_E .....~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... WHO Bulletin OMS: Supplement Vol. 68 1990 155 R.C. Hodstrom et al. was detected in control reactions of RNA that were not reverse transcribed prior to PCR. We believe this is due to amplification of the genomic sequence which resulted from DNA contamination of the RNA preparation. Antibodies to Asn-Pro-Asn-Glu-Pro-Ser recognize the sur- face of sporozoites and the 140 kDas antigen. The overall hydrophilicity of the repeating amino acid sequence Asn-Pro-Asn-Glu-Pro-Ser suggested it as a potential antigenic determinant (28). A synthetic peptide con- taining 3 copies of Asn-Pro-Asn-Glu-Pro-Ser (18- mer) was strongly recognized by NYS4 in an ELISA (data not shown) and mice immunized with the pep- tide produced antibodies that reacted with sporo- zoites in an IFAT and with the 140 kDa antigen on Western blots of sporozoite extracts (Fig. 3). These results together with those of Fig. 2 indicate that the M4 peptide sequence corresponds to at least a portion of the 140 kDa antigen and that the antigenic deter- minant of NYS4 is contained within the repeating hexamer Asn-Pro-Asn-Glu-Pro-Ser. It is notable that this repetitive sequence bears no similarity to the major repeats of the P. yoelii CS protein, in which the consensus repeating elements are Gln-Gly-Pro-Gly- Ala-Pro and Gln-Gln-Pro-Pro (29). Discussion In this paper we report the molecular characterization of a portion of the gene encoding what we now call sporozoite surface protein 2 (SSP 2). Like the CS protein, SSP 2 contains an immunogenic sequence of repeating amino acids and the conserved region II domain which is found in a number of other well-characterized proteins. These include thrombo- spondin, properdin, von Willebrand factor, beta2- glycoprotein I, collagen type IV alphal & 2 chains, glycoprotein E from human (alpha) herpesvirus 1, and antistasin, all of them proteins which are thought to play roles in adhesion (30). The sequence similarity between SSP 2 and TRAP, an erythrocytic-stage spe- cific protein of P. falciparum, extends beyond the "adhesion motif" of the CS protein in both the amino terminal and carboxyl terminal directions suggesting a more expansive functional domain in these malarial proteins. Nevertheless the conservation of this se- quence among proteins from different malarial species underscores its importance to the survival of the parasite. Perhaps sporozoite entry into host cells is facilitated by SSP 2 adherence to extracellular matrices. Immunization with irradiation-attenuated sporo- zoites confers sterile immunity against challenge with large numbers of sporozoites. It has long been thought that this protective immunity is directed Fig. 3. Antibodies to (NPNEPS)3 recognize the 140 kDa sporozolte surlae protein. Western blot of P. yoeIIi sporo- zoite extract probed with serum from mice immunized with (NPNEPS)3 conjugated to KLH (lane 1), NYS4 (lane 2), and sera from mice immunized with KLH (lane 3). ..I.l.. ..u_ll _~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.... i-.i iER i.i;- entirely against the CS protein. Our findings demon- strate the existence of another protein, SSP 2, on sporozoites that may be an additional target for protective cellular or humoral immune responses. References 1. Nussenzweig, V. & Nussenzweig, R.S. Circumsporo- zoite proteins of malaria parasites. Cell, 42: 401-403 (1985). 2. Cochrane, A.H. et al. Antibody-induced ultrastructural changes of malarial sporozoites. J. immunol., 116: 859-867 (1976). 3. Nussnzwelg, R.S. et al. Protective immunity produ- ced by the injection of X-irradiated sporozoites of Plasmodium berghei. Nature, 216: 160 (1967). 156 WHO Bulletin OMS: Supplement Vol. 68 1990 Malaria sporozolte surface antigen 4. Welss, W.R. et al. CD8+ cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites. Proc. Natl Acad. Sc., USA, 85: 573 (1988). 5. Hoffman, S.L. et al. Immune response gene regulation of immunity to Plasmodium berghei sporozoites and circumsporozoite protein vaccines: overcoming gene- tic restriction with whole organism and subunit vac- cines. J. immunol., 142: 3581-3584 (1989). 6. Sedegah, M. et al. Use of a vaccinia construct ex- pressing the circumsporozoite protein in the analysis of protective immunity to Plasmodium yoelii. In: Lasky, L., ed. Technological advances in vaccine development. New York, Alan R. Liss Inc., 1988, pp. 295-309. (UCLA Symposium on Molecular and Cellular Biology, New Series, Vol. 84). 7. Egan, J.E. et al. Efficacy of murine malaria sporozoite vaccines: implications for human vaccine develop- ment. Science, 236: 453 (1987). 8. Sadoff, J.C. et al. Oral Salmonella typhimurium vac- cine expressing circumsporozoite protein protects against malaria. Science, 240: 336-338 (1988). 9. Hoffman, S.L. et al. Sporozoite vaccine induces gen- etically restricted T-cell elimination of malaria from hepatocytes. Science, 244: 1078-1080 (1989). 10. de la Cruz, V.F. et al. Sequence variation in putative functional domains of the circumsporozoite protein of Plasmodium falciparum: implications for vaccine de- velopment. J. biol. chem., 262: 11935-11939 (1987). 11. Good, M.F. et al. Human T-cell recognition of the circumsporozoite protein of Plasmodium falciparum: immunodominant T-cell domains map to the poly- morphic regions of the molecule. Proc. NatlAcad. Sci., USA, 85: 1199-1203 (1988). 12. Lockyor, M.J. et al. Wild isolates of Plasmodium falci- parum show extensive polymorphism in T-cell epi- topes of the circumsporozoite protein. Mol. biochem. parasitol., 37: 275-280 (1989). 13. Charoenvlt, Y. et al. Characterization of Plasmodium yoelii monoclonal antibodies directed against stage- specific sporozoite antigens. Infect. immun., 55: 604-608 (1987). 14. Wortman, A. et al. Cloning of Plasmodium yoelii genes expressing three different sporozoite-specific anti- gens. Micro. path., 6: 227-231 (1989). 15. Stephens, R.S. et al. Molecular cloning and expres- sion of Chlamydia trachomatis major outer membrane protein antigens in Escherichia coli. Infect. immun., 47: 713-718 (1985). 16. Young, R.A. & Davis, R.W. Efficient isolation of genes by using antibody probes-. Proc. Natl Acad. Sci., USA, 80: 1194-1198 (1983). 17. Young, R.A. & Davis, R.W. Yeast RNA polymerase 11 genes: isolation with antibody probes. Science, 222: 778-782 (1983). 18. Johnson, D.A. et al. Improved technique utilizing non- fat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene anal. techn., 1: 3-4 (1984). 19. Maniatis, T. et al. Molecular cloning, a laboratory manual. Cold Spring Harbor, Cold Spring Harbor La- boratory, 1982. 20. Messing, J. New M13 vectors for cloning. Methods enzymol., 101: 20-78 (1983). 21. Henikoff, S. Unidirectional digestion with exonuclease IlIl in DNA sequence analysis. Methods enzymol., 155: 156-165 (1987). 22. Chirgwin, J.M. et al. Isolation of biologically active ribonucleic acid from sources enriched in ribonuc- lease. Biochemistry, 18: 5294-5299 (1979). 23. Salki, R.K. et al. Enzymatic amplification of beta- globin genomic sequences and restriction site ana- lysis for diagnosis of sickle cell anemia. Science, 230: 1350-1354 (1985). 24. Merrifleld, B. Peptide synthesis, I. The synthesis of a tetrapeptide. J. Am. Chem. Soc., 85: 2149-2154 (1963). 25. Lawler, J. & Hynes, R.O. The structure of human thrombospondin, an adhesive glycoprotein with mul- tiple calcium-binding sites and homologies with sev- eral different proteins. J. cell biol., 103: 1635-1648 (1986). 26. Goundis, D. & Reid K.B. Properdin, the terminal com- plement components, thrombospondin and the cir- cumsporozoite protein of malaria parasites contain similar sequence motifs. Nature, 335: 82-85 (1988). 27. Robson, K.J.H., et al. A highly conserved amino-acid sequence in thrombospondin, properdin and in pro- teins from sporozoites and blood stages of a human malaria parasite. Nature, 335: 79-82 (1988). 28. Hopp, T.P. & Woods, K.R. Prediction of protein anti- genic determinants from amino acid sequences. Proc. Natl Acad. Sc., USA, 78: 3824-3828 (1981). 29. Lal, A.A. et al. Structure of the gene encoding the circumsporozoite protein of Plasmodium yoelii. J. biol. chem., 262: 2937-2940 (1987). 30. Holt, G.D. et al. Antistasin, an inhibitor of coagulation and metastasis, binds to sulfatide (Gal(3-SO4)#1-lCer) and has sequence homology with other proteins that bind sulfated glycoconjugates. J. biol. chem., 264: 12138-12140 (1989). WHO Bulletin OMS: Supplement Vol. 68 1990 157