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Identification and analysis of parasite surface antigens and parasite-induced antigens on host cells
Veterinary Parasitology, 10 (1982) 181--189 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
181
IDENTIFICATION AND ANALYSIS OF PARASITE SURFACE ANTIGENS AND PARASITE-INDUCED ANTIGENS ON HOST CELLS
A.F. BARBET Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164 (U.S.A.)
ABSTRACT
Barbet, A.F., 1982. Identification and analysis of parasite surface antigens and parasiteinduced antigens on host cells. Vet. Parasitol. 10: 181--189. In this review a rational approach is presented toward the analysis of parasite surface antigens as they are relevant to problems of vaccination and strain variation. Techniques are described for the identification of surface proteins by radiochemical and immunological methods and the subsequent purification of these molecules. Examples are given where purified parasite surface antigens have been analyzed by radioimmunoassay and by other protein structural techniques to determine whether the antigens vary between different parasite isolates.
INTRODUCTION In t h e last t e n y e a r s m a n y t e c h n i q u e s have b e e n d e v e l o p e d to investigate t h e b i o c h e m i s t r y a n d i m m u n o c h e m i s t r y o f parasite surfaces. T h e s e t e c h n i q u e s e n a b l e o n e t o i d e n t i f y m o l e c u l e s e x p o s e d o n t h e surface t o a n t i b o d y , t o p u r i f y t h e m a n d t o d e t e c t a m i n o acid or o l i g o s a c c h a r i d e s e q u e n c e changes a c c o m p a n y i n g antigenic or strain variation. Such an analysis o f a parasite surface p r o v i d e s a r a t i o n a l a p p r o a c h t o vaccine p r o d u c t i o n . O n e can ask o r d e r e d q u e s t i o n s : firstly, w h i c h m o l e c u l e s are e x p o s e d o n t h e surface? S e c o n d l y , d o e s an i m m u n e r e s p o n s e against t h e s e m o l e c u l e s kill t h e parasites c a r r y i n g t h e m ? T h i r d l y , d o surface antigens v a r y in s e q u e n c e in parasites f r o m d i f f e r e n t g e o g r a p h i c a l areas? F o u r t h l y , can such s e q u e n c e v a r i a t i o n be o v e r c o m e using an i m m u n e r e s p o n s e d i r e c t e d against c o n s t a n t regions o f t h e m o l e c u l e ? A n s w e r s t o t h e s e q u e s t i o n s a l l o w o n e t o assess p o t e n t i a l p r o b l e m s in d e v e l o p m e n t o f a vaccine against a p a r t i c u l a r parasite. In t h e n e x t p a r a g r a p h s I will discuss t h e various strategies w h i c h have b e e n used t o p r o v i d e t h e s e answers. T h e m e t h o d s can generally be a p p l i e d t o p a r a s i t e surface antigens or t o t h e surface o f h o s t cells o n w h i c h parasite-specific antigens are i n d u c e d .
0304-4017/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing Company
182 IDENTIFICATION OF SURFACE ANTIGENS The first step generally employed is to radioactively label proteins or glycoproteins on the external cell surface with a reagent which does n o t penetrate through the membrane lipid and therefore does not label internal molecules. There are many reagents developed as such membrane probes which have the desired impermeant characteristics to a greater or lesser extent (for review see Hubbard and Cohn, 1976). Commonly employed are enzyme mediated labelling reactions, such as lactoperoxidase catalyzed radioiodination of exposed tyrosine residues in surface proteins: H:O: + I- + tyrosine lactoperoxidase H20 + OH- + monoiodotyrosine. The large size of an enzyme precludes its penetration through the membrane. In addition, use of a gamma emitter such as 12sI facilitates subsequent tracing of labelled proteins through purification procedures. The major disadvantage of enzyme labelling is that only the most accessible amino acids become labelled by a 30--40 nm diameter enzyme; less accessible residues or even proteins may not be labelled although they are on the outer surface of the cell membrane. An impermeant, non-enzymic, surface label which also uses 12sI as the radiotracer is diazotised [12sI]-iodosulfanilic acid. This c o m p o u n d can potentially react with amino and imidazole residues in addition to tyrosine, therefore success in labelling is less dependent on the amino acid composition of exposed proteins. If labelled proteins are to be examined b y SDS-gel electrophoresis and autoradiography one may prefer to use the ~ emitters 3sS or ~4C as the radiotracer for o p t i m u m resolution. These isotopes produce less diffuse bands than ~2sI. Diazotised [35S]-sulfanilic acid could be used or one of many different impermeant, amino group reactive reagents. An example of the latter is the amino acylating reagent [ 3sS ]-formyl-methionine-sulphone methyl phosphate. Oligosaccharides in surface glycoproteins may be labelled b y an enzyme mediated technique. Galactose oxidase oxidizes D-galactose and N-acetyl-Dgalactosamine residues to aldehydes which can then be reduced with tritiated sodium borohydride. Examples of the above surface labelling techniques follow: lactoperoxidase catalyzed iodination of erythrocytes (Phillips and Morrison, 1971), of spleen cells (Baur et al., 1971), of viruses (Stanley and Haslam, 1971), of Trypanosoma congolense (Rovis et al., 1978), of erythrocytes infected with Babesia bovis (Howard et al., 1980); labelling of Trypanosoma congolense with diazotised [35S]-sulfanilic acid (Reinwald et al., 1978); labelling with [3sS]-formyl-methionine-sulphone methyl phosphate of erythrocytes (Bretscher, 1971 ), of Trypanoso ma b rucei (Cross, 1975); labelling of erythrocytes infected with Babesia bovis with [3H] sodium borohydride and galactose oxidase {Howard et al., 1980). To identify molecules which have been surface-labelled, t w o complementary techniques are available which provide good resolution and characterize physical properties of surface proteins or glycoproteins. These are SDS-gel electrophoresis (reviewed b y Tak~cs, 1979), which characterizes the molecu-
183
lar weight of the radiolabelled molecules, and isoelectric focusing (reviewed by Braun et al., 1979) which characterizes the isoelectric pH. These techniques can be employed individually or, in combination, using a two-dimensional procedure (Anderson and Anderson, 1978a, b). There are many different ways of performing these techniques which each have advantages and disadvantages. For o p t i m u m resolution there is little d o u b t that t w o dimensional electrophoresis using polyacrylamide gel isoelectrofocusing in one dimension, and SDS-gradient slab gel electrophoresis in the second dimension, is superior in ability to resolve the largest number of labelled proteins. However, extreme care must be taken to standardize run conditions if one wishes to compare several different gels; for example, surface labelling patterns of parasites from different geographical locations. Such a comparison may be made more easily employing one dimension slab gel procedures where samples are run side by side on the same gel. The position of labelled spots or bands obtained in these techniques is most easily revealed by autoradiography (direct exposure of the dried gel to X-ray film) or fluorography. In the latter case, the gel is impregnated with scintillant before exposure to film; this technique is most often used for [3H]-labelled samples where the energy of the isotope would be inadequate to expose film directly. Therefore, the film is exposed by the light generated when ~ particles interact with scintillant in the gel (Bonnet and Laskey, 1974). ['4C]-methylated proteins prepared by reductive methylation of amino groups with [ 14CI-formaldehyde and sodium borohydride (Rice and Means, 1971) are available as standards for SDS-gel electrophoresis and autoradiography. A second approach has become available in the last ten years to identify cell surface antigens: the use of monoclonal antibodies. Myeloma proteins, which are monoclonal antibodies produced in lympho-proliferative disorders, have been used for many years to study immunoglobulin structure. However, the antigen binding specificity of such antibodies was not generally known. In 1975, Kohler and Milstein succeeded in overcoming this limitation by fusing spleen cells from a mouse immunized with sheep red blood cells with cells from a line of mouse myeloma cells. The m y e l o m a cells lacked the enzyme hypoxanthine phosphoribosyl transferase; therefore unfused cells could be eliminated from the population using a selective growth medium. The mouse spleen cells died naturally. Hybrid cells were screened for the desired antibody activity (anti-sheep red blood cell) and the antibodyproducing hybrids cloned. The technique has wide applicability in producing monoclonal antibodies of defined specificity. To identify surface antigens of parasites with this technique a mouse could be immunized with parasites or with a partially purified fraction known to contain surface antigens. Following fusion of the mouse spleen with a m y e l o m a cell line, the resulting hybrids might be screened for antibody activity by immunofluorescence or neutralization of live parasites. When " h y b r i d o m a " clones secreting antibody against the parasite surface were obtained, the antigen recognized b y that monoclonal antibody could be
184 identified in various ways. 1. Parasite antigens could be separated by SDS-gel electrophoresis then: (a) the gel incubated with radiolabelled antibody, washed and autoradiographed (Olden and Yamada, 1977) or; (b) proteins transferred from the gel and coupled to nitrocellulose paper ("western" blotting, Renart et al., 1979; Burnette, 1981). As in (a) the specific proteins recognized could be revealed b y incubation with radiolabelled antibody. Alternatively, if the monoclonal antibody b o u n d to protein A from Staphylococcus aureus (SjSquist et al., 1972), the paper containing coupled proteins could be reacted first with antibody, then with 12SI-labelled protein A. 2. Parasite proteins could be radiolabelled by.metabolic incorporation of radioactive amino acids, or by surface labelling. The proteins would then be solubilized (see later) and immunoprecipitated with monoclonal antibody and a second anti-antibody; or, again, with formalin fixed Staphylococcus aureus instead of second antibody. Monoclonal antibody techniques have already been extensively applied to studying parasite surface antigens. Such antibodies have been developed, for example, against: Plasmodium berghei sporozoites (Yoshida et al., 1979), which confer protection on mice against sporozoite-induced infection; Plasmodium falciparum merozoites (Freeman et al., 1980); Trypanosoma brucei variable surface glycoprotein (Pearson et al., 1981); Trypanosoma rhodesiense surface glycoprotein (Lyon et al., 1981); schistosome membranes (Verwaerde et al., 1979). In some studies the antibodies have been used with the analytical techniques described to identify and characterize the specific antigens recognized e.g. the use of monoclonal antibodies, two dimensional gel electrophoresis and surface labelling for studying T. brucei surface glycoproteins (Pearson et al., 1981). A.F. Barbet, W.C. Davis and T.C. McGuire (1981, unpublished data) have made 15 monoclonal antibodies to one T. brucei surface glycoprotein which we are using in combination with immunofluorescence, protein fragmentation and sequencing to map exposed and buried regions of the glycoprotein when it is in a normal conformation on the trypanosome surface. PURIFICATION OF SURFACE ANTIGENS If exposed proteins or glycoproteins on the parasite surface have been identified, one should then purify them to obtain more detailed structural and immunological information. The first problem encountered is to devise a method for solubilizing the antigens of interest. Membrane antigens can be categorized as more or less firmly integrated into the hydrophobic regions of the membrane. Some are essentially "peripheral" others "integral" components (Singer and Nicolson, 1972). As ~ result o f this different membrane interaction, the ease with which surface antigens may be solubilized is also different. In the most convenient case peripheral components can sometimes be released from the parasite surface by physical disruption, such as sonica-
185 tion or blending, and then become water soluble. This is the situation with envelope glycoproteins of certain R N A t u m o u r viruses (Strand and August, 1976) which are released simply by freeze-thawing the virus. Also, trypanosome variable surface glycoproteins are released by mechanical blending (Cross, 1975) or even, simply by incubating isolated organisms in buffer at 37°C (A.F. Barbet, 1981, unpublished observations). In other situations, however, surface antigens remain associated with parasite membranes even after physical disruption of organisms and it is necessary to resort to more drastic solubilization methods. A favourite method of immunologists is detergent solubilization. Various detergents are available. Generally, the nonionic detergents such as Nonidet P-40 and Triton X-100 are not as efficient solubilizing agents as ionic detergents such as sodium deoxycholate or sodium dodecyl sulphate, but the likelihood of retaining some native immunologic structure after solubilization is greater when using non-ionic detergents. Also, antigen--antibody binding can usually occur in the presence of nonionic detergents which facilitates subsequent immunochemical analysis of antigens solubilized in this way. To purify the desired antigens from the solubilized extract many techniques are available such as: gel filtration, ion exchange chromatography, isoelectric focusing, preparative gel electrophoresis, high pressure liquid chromatography, affinity chromatography. A detailed description of all these techniques is b e y o n d the scope of the present paper. I will, however, mention two techniques which offer substantial purification in a single step procedure, and a third technique which is useful when only small amounts of starting material are available. An affinity chromatography column can be prepared using monoclonal antibodies covalently attached to cyanogen bromide-activated Sepharose. if a mixture of antigens is passed through the column the specific antigen recognized will be b o u n d and can be eluted later after washing the column free of the other antigens. This technique has been used to achieve a 5000-fold purification of human leukocyte interferon in a single step (Secher and Burke, 1980). A method based on a similar principal is lectin--Sepharose affinity chromatography. This can be used when it is k n o w n that the surface antigen of interest is a glycoprotein, for example by employing the galactose oxidase and [3H]sodium borohydride surface labeling technique. Glycoproteins will bind to the column and will therefore separate from non-glycosylated contaminants. The glycoproteins can be eluted subsequently with the specific sugar recognized b y the lectin, e.g., a-D-methylmannoside or a-D-methylglucoside, in the case of concanavalin A-Sepharose. A micromethod we have used to isolate proteins from equine infectious anemia virus when only a small amount of starting material was available, is preparative SDS-gel electrophoresis. Coomassie Blue stained proteins were extracted from a slab gel into SDS containing buffer. The SDS and SDS-bound proteins were precipitated with potassium chloride and then dodecyl sulphate and Coomassie Blue was extracted from the proteins by the
186
ion-pair extraction method of Henderson et al. (1979). Even after contact with the protein denaturants used in this process radioiodinated viral p30 still reacted with a horse serum from a natural infection down to a radioimmunoassay titre of 1 in 10000 (A.F. Barbet and T.C. McGuire, 1981, unpublished observations). When sufficient quantity of a surface protein cannot be made by any conventional technique it is likely that the m e t h o d of choice in the future will be to make the protein in bacteria by gene cloning. ANALYSIS OF PARASITE SURFACE ANTIGENS Assuming that one has identified and purified surface antigens, there are several questions one might ask. Firstly, does antibody against that antigen kill the parasite? This question could be answered by using a specific or monoclonal antiserum against the parasite in a cytotoxic or neutralization assay. Secondly, does the antigen vary in structure in different parasites, i.e., does antigenic or strain variation occur? This question should be answered both immunochemically and biochemically. One needs to know: in which sequences of the protein or carbohydrate does structural change occur? Are these regions buried or exposed when the conformation of the antigen on the parasite surface is taken into account? Are the regions of altered sequence i m m u n o d o m i n a n t or immunosilent, i.e., is an immune response produced against them in a natural infection? Are there also constant sequences in the antigen exposed on the surface of the different parasites? Are these i m m u n o d o m i n a n t or immunosilent? The various techniques which might be used to answer these questions include: inhibition radioimmunoassay, immunfluorescence, cytotoxicity, peptide mapping on isolated and surface-labelled antigens, peptide fragmentation and purification, antibody production against different peptides, peptide sequencing and, ultimately, X-ray crystallography. The ways in which some of these techniques can be applied might be illustrated by reference to recent research on the structure of the trypanosome surface. The only molecule exposed to surface radiolabelling in Trypanosorna brucei is a glycoprotein of approximately 65000 molecular weight. Methods have been devised for purification of this molecule (Cross, 1975). Antiserum against the purified glycoprotein lysed the parasites from which the molecule was derived. Antigenic variation occurs in these parasites because of amino acid sequence changes in the glycoprotein, therefore an antiserum against a glycoprotein purified from one antigenic type was generally ineffective at neutralizing trypanosomes of a different type. Immunological cross-reactions were, however, detected by radioimmunoassay between all glycoproteins that were isolated from different antigenic types of trypanosome (Barbet and McGuire, 1978). Later, peptide fragmentation studies showed that the region of the molecule causing this cross-reaction was a small glycopeptide, and probably the carbohydrate portion of the glycopeptide, near
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to the carboxy-terminus (Barbet et al., 1979; Cross, 1979; Holder and Cross, 1981; Barbet et al., 1981). This glycopeptide region was not accessible to antibody when on the surface of the trypanosome. Other surface glycoprotein sequence homologies have been found, but were common only to a small number of different trypanosomes. These homologies were in the amino acid sequence and the regions involved were exposed at the trypanosome surface, as demonstrated by cross-neutralization tests and by peptide mapping of surface-labelled trypanosomes (Vervoort et al., 1981). To assess the possibility of producing a vaccine against trypanosomes requires that the extent of such sequence homology be known. In other parasitic diseases where antigenic variation does not occur, such an assessment should be made more readily. ACKNOWLEDGEMENTS
Support for the author was provided in part by funds provided for medical and biological research by state of Washington Initiative Measure No. 171 and by Rockefeller Foundation Grant No. GA-COH-8013.
REFERENCES Anderson, N.G. and Anderson, N.L., 1978a. Analytical techniques for cell fractions XXI. Two-dimensional analysis of serum and tissue proteins: multiple isoelectric focusing. Anal. Biochem., 85: 331--340. Anderson, N.L. and Anderson, N.G., 1978b. Analytical techniques for cell fractions, XXII. Two-dimensional analysis of serum and tissue proteins: multiple gradient--slab gel electrophoresis. Anal. Biochem., 85: 341--354. Barbet, A.F. and McGuire, T.C., 1978. Cross-reacting determinants in variant-specific surface antigens o f African trypanosomes. Proc. Natl. Acad. Sci. U.S.A., 75: 1989-1993. Barbet, A.F., McGuire, T.C., Musoke, A.J. and Hirumi, H., 1979. Cross-reacting determinants in trypanosome surface antigens. In: G. Losos and A. Chouinard (Editors), Pathogenicity of Trypanosomes. International Development Research Center, Ottawa, pp. 38--43. Barbet, A.F., Musoke, A.J., Shapiro, S.Z., Mpimbaza, G. and McGuire, T.C., 1981. Identification of the fragment containing cross-reacting antigenic determinants in the variable surface glycoprotein of Trypanosoma brucei. Parasitology, 83: 623--637. Baur, S., Vitetta, E.S., Sherr, C.J., Schenkein, I. and Uhr, J.W., 1971. Isolation of heavy and light chains of immunoglobulin from the surfaces of lymphoid cells. J. Immunol., 106: 1133--1135. Bonnet, W.M. and Laskey, R.A., 1974. A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem., 46: 83--88. Braun, D.G., Hild, K. and Ziegler, A., 1979. Resolution of immunoglobulin patterns by analytical isoelectric focusing. In: I. Lefkovits.and B. Pernis (Editors), Immunological Methods. Academic Press, New York and London, pp. 107--121. Bretscher, M.S., 1971. Human erythrocyte membranes: specific labelling of surface proteins. J. Mol. Biol., 58: 775--781.
188 Burnette, W.N., 1981. "Western Blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified cellulose and radiographic detection with antibody and radioiodinated Protein A. Anal. Biochem., 112: 195--203. Cross, G.A.M., 1975. Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitology, 71 : 393--417. Cross, G.A.M., 1979. Crossreacting determinants in the C-terminal region of trypanosome variant surface antigens. Nature, 277: 310--312. Freeman, R.R., Trejdosiewicz, A.J. and Cross, G.A.M., 1980. Protective monoclonal antibodies recognizing stage-specific merozoite antigens of a rodent malaria parasite. Nature, 284: 366--368. Henderson, L.E., Oroszlan, S. and Konigsberg, W., 1979. A micromethod for complete removal of dodecyl sulphate from proteins by ion-pair extraction. Anal. Biochem., 93: 153--157. Holder, A.A. and Cross, G.A.M., 1981. Glycopeptides from variant surface glycoproteins of Trypanosoma brucei. C-terminal location of antigenically cross-reacting carbohydrate moieties. Mol. Biochem. Parasitol., 2: 135--150. Howard, R.J., Rodwell, B.J., Smith, P.M., Callow, L.L. and Mitchell, G.E., 1980. Comparison of the surface proteins and glycoproteins on erythrocytes of calves before and during infection with Babesia bovis. J. Protozool., 27: 241--247. Hubbard, A.L. and Cohn, Z.A., 1976. Specific labels for cell surfaces. In: A.H. Maddy (Editor), Biochemical Analysis of Membranes. Chapman and Hall, London, pp. 427-501. Kohler, G. and Milstein, C., 1975. Continuous culture of fused cells secreting antibody of predefined specificity. Nature, 256: 495--497. Lyon, J.A., Pratt, J.M., Travis, R.W., Doctor, B.P. and Olenick, J.G., 1981. Use of monoclonal antibody to immunochemically characterize variant specific surface coat glycoprotein from Trypanosoma rhodesiense. J. Immunol., 126 : 134--137. Olden, K. and Yamada, K.M., 1977. Direct detection of antigens in sodium dodecyl sulphate polyacrylamide gels. Anal. Biochem., 78: 483--490. Pearson, T.W., Kar, S.K., MeGuire, T.C. and Lundin, L.B., 1981. Trypanosome variable surface antigens: studies using two-dimensional gel electrophoresis and monoclonal antibodies. J. Immunol., 126: 823--828. Phillips, D.R. and Morrison, M., 1971. Exposed protein on the intact human erothrocyte. Biochemistry, 10: 1766--1771. Reinwald, E., Risse, H. and Salker, R., 1978. Diazoniobenzenesulfonate as marker for cell surface proteins: study of the surface coat of Trypanosoma congolense. HoppeSeyler's Z. Physiol. Chem., 359: 939--944. Renart, J., Reiser, J. and Stark, G.R., 1979. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc. Natl. Acad. Sci. U.S.A., 76: 3116--3120. Rice, R.H. and Means, G.E., 1971. Radioactive labelling of proteins in vitro. J. Biol. Chem., 246: 831--832. Rovis, L., Barber, A.F. and Williams, R.O., 1978. Characterization of the surface coat of Trypanosoma congolense. Nature, 271 : 654--656. Secher, D.S. and Burke, D.C., 1980. A monoclonal antibody for large scale purification of human leukocyte interferon. Nature, 285 : 446--450. Singer, S.J. and Nicolson, G.L., 1972. The fluid mosaic model of the structure of cell membranes. Science, 175: 720--731. SjSquist, J., Meloun, B. and Helm, H., 1972. Protein A isolated from Staphylococcus aureus after digestion with lysostaphin. Eur. J. Biochem., 2 9 : 5 7 2 - - 5 7 8 . Stanley, P. and Haslam, E.A., 1971. The polypeptides of influenza virus. Virology, 46: 764--773.
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Strand, M. and August, J.T., 1976. Structural proteins of ribonucleic acid tumour viruses, J. Biol. Chem., 251: 559-~564. Tak~cs, B., 1979. Electrophoresis of proteins in polyacrylamide slab gels. In: I. Lefkovits and B. Pernis (Editors), Immunological Methods. Academic Press, New York and London, pp. 81--105. Vervoort, T., Barbet, A.F., Musoke, A.J., Magnus, E., Mpimbaza, G. and Van Meirvenne, N., 1981. Isotypic surface glycoproteins of trypanosomes. Immunology, 44: 223--232. Verwaerde, C., Graych, J.-M., Bazin, H., Capron, M. and Capron, A., 1979. Production d'anticorps monoclonaux anti Schistosoma mansoni. Etude pr~liminaire de leurs activit6s biologiques. C. R. Acad. Sci., Ser. D, 289: 725--727. Yoshida, N., Nussenzweig, R.S., Potocnjak, P., Nussenzweig, V. and Aikawa, M., 1979. Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science, 207: 71--73.
181
IDENTIFICATION AND ANALYSIS OF PARASITE SURFACE ANTIGENS AND PARASITE-INDUCED ANTIGENS ON HOST CELLS
A.F. BARBET Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164 (U.S.A.)
ABSTRACT
Barbet, A.F., 1982. Identification and analysis of parasite surface antigens and parasiteinduced antigens on host cells. Vet. Parasitol. 10: 181--189. In this review a rational approach is presented toward the analysis of parasite surface antigens as they are relevant to problems of vaccination and strain variation. Techniques are described for the identification of surface proteins by radiochemical and immunological methods and the subsequent purification of these molecules. Examples are given where purified parasite surface antigens have been analyzed by radioimmunoassay and by other protein structural techniques to determine whether the antigens vary between different parasite isolates.
INTRODUCTION In t h e last t e n y e a r s m a n y t e c h n i q u e s have b e e n d e v e l o p e d to investigate t h e b i o c h e m i s t r y a n d i m m u n o c h e m i s t r y o f parasite surfaces. T h e s e t e c h n i q u e s e n a b l e o n e t o i d e n t i f y m o l e c u l e s e x p o s e d o n t h e surface t o a n t i b o d y , t o p u r i f y t h e m a n d t o d e t e c t a m i n o acid or o l i g o s a c c h a r i d e s e q u e n c e changes a c c o m p a n y i n g antigenic or strain variation. Such an analysis o f a parasite surface p r o v i d e s a r a t i o n a l a p p r o a c h t o vaccine p r o d u c t i o n . O n e can ask o r d e r e d q u e s t i o n s : firstly, w h i c h m o l e c u l e s are e x p o s e d o n t h e surface? S e c o n d l y , d o e s an i m m u n e r e s p o n s e against t h e s e m o l e c u l e s kill t h e parasites c a r r y i n g t h e m ? T h i r d l y , d o surface antigens v a r y in s e q u e n c e in parasites f r o m d i f f e r e n t g e o g r a p h i c a l areas? F o u r t h l y , can such s e q u e n c e v a r i a t i o n be o v e r c o m e using an i m m u n e r e s p o n s e d i r e c t e d against c o n s t a n t regions o f t h e m o l e c u l e ? A n s w e r s t o t h e s e q u e s t i o n s a l l o w o n e t o assess p o t e n t i a l p r o b l e m s in d e v e l o p m e n t o f a vaccine against a p a r t i c u l a r parasite. In t h e n e x t p a r a g r a p h s I will discuss t h e various strategies w h i c h have b e e n used t o p r o v i d e t h e s e answers. T h e m e t h o d s can generally be a p p l i e d t o p a r a s i t e surface antigens or t o t h e surface o f h o s t cells o n w h i c h parasite-specific antigens are i n d u c e d .
0304-4017/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing Company
182 IDENTIFICATION OF SURFACE ANTIGENS The first step generally employed is to radioactively label proteins or glycoproteins on the external cell surface with a reagent which does n o t penetrate through the membrane lipid and therefore does not label internal molecules. There are many reagents developed as such membrane probes which have the desired impermeant characteristics to a greater or lesser extent (for review see Hubbard and Cohn, 1976). Commonly employed are enzyme mediated labelling reactions, such as lactoperoxidase catalyzed radioiodination of exposed tyrosine residues in surface proteins: H:O: + I- + tyrosine lactoperoxidase H20 + OH- + monoiodotyrosine. The large size of an enzyme precludes its penetration through the membrane. In addition, use of a gamma emitter such as 12sI facilitates subsequent tracing of labelled proteins through purification procedures. The major disadvantage of enzyme labelling is that only the most accessible amino acids become labelled by a 30--40 nm diameter enzyme; less accessible residues or even proteins may not be labelled although they are on the outer surface of the cell membrane. An impermeant, non-enzymic, surface label which also uses 12sI as the radiotracer is diazotised [12sI]-iodosulfanilic acid. This c o m p o u n d can potentially react with amino and imidazole residues in addition to tyrosine, therefore success in labelling is less dependent on the amino acid composition of exposed proteins. If labelled proteins are to be examined b y SDS-gel electrophoresis and autoradiography one may prefer to use the ~ emitters 3sS or ~4C as the radiotracer for o p t i m u m resolution. These isotopes produce less diffuse bands than ~2sI. Diazotised [35S]-sulfanilic acid could be used or one of many different impermeant, amino group reactive reagents. An example of the latter is the amino acylating reagent [ 3sS ]-formyl-methionine-sulphone methyl phosphate. Oligosaccharides in surface glycoproteins may be labelled b y an enzyme mediated technique. Galactose oxidase oxidizes D-galactose and N-acetyl-Dgalactosamine residues to aldehydes which can then be reduced with tritiated sodium borohydride. Examples of the above surface labelling techniques follow: lactoperoxidase catalyzed iodination of erythrocytes (Phillips and Morrison, 1971), of spleen cells (Baur et al., 1971), of viruses (Stanley and Haslam, 1971), of Trypanosoma congolense (Rovis et al., 1978), of erythrocytes infected with Babesia bovis (Howard et al., 1980); labelling of Trypanosoma congolense with diazotised [35S]-sulfanilic acid (Reinwald et al., 1978); labelling with [3sS]-formyl-methionine-sulphone methyl phosphate of erythrocytes (Bretscher, 1971 ), of Trypanoso ma b rucei (Cross, 1975); labelling of erythrocytes infected with Babesia bovis with [3H] sodium borohydride and galactose oxidase {Howard et al., 1980). To identify molecules which have been surface-labelled, t w o complementary techniques are available which provide good resolution and characterize physical properties of surface proteins or glycoproteins. These are SDS-gel electrophoresis (reviewed b y Tak~cs, 1979), which characterizes the molecu-
183
lar weight of the radiolabelled molecules, and isoelectric focusing (reviewed by Braun et al., 1979) which characterizes the isoelectric pH. These techniques can be employed individually or, in combination, using a two-dimensional procedure (Anderson and Anderson, 1978a, b). There are many different ways of performing these techniques which each have advantages and disadvantages. For o p t i m u m resolution there is little d o u b t that t w o dimensional electrophoresis using polyacrylamide gel isoelectrofocusing in one dimension, and SDS-gradient slab gel electrophoresis in the second dimension, is superior in ability to resolve the largest number of labelled proteins. However, extreme care must be taken to standardize run conditions if one wishes to compare several different gels; for example, surface labelling patterns of parasites from different geographical locations. Such a comparison may be made more easily employing one dimension slab gel procedures where samples are run side by side on the same gel. The position of labelled spots or bands obtained in these techniques is most easily revealed by autoradiography (direct exposure of the dried gel to X-ray film) or fluorography. In the latter case, the gel is impregnated with scintillant before exposure to film; this technique is most often used for [3H]-labelled samples where the energy of the isotope would be inadequate to expose film directly. Therefore, the film is exposed by the light generated when ~ particles interact with scintillant in the gel (Bonnet and Laskey, 1974). ['4C]-methylated proteins prepared by reductive methylation of amino groups with [ 14CI-formaldehyde and sodium borohydride (Rice and Means, 1971) are available as standards for SDS-gel electrophoresis and autoradiography. A second approach has become available in the last ten years to identify cell surface antigens: the use of monoclonal antibodies. Myeloma proteins, which are monoclonal antibodies produced in lympho-proliferative disorders, have been used for many years to study immunoglobulin structure. However, the antigen binding specificity of such antibodies was not generally known. In 1975, Kohler and Milstein succeeded in overcoming this limitation by fusing spleen cells from a mouse immunized with sheep red blood cells with cells from a line of mouse myeloma cells. The m y e l o m a cells lacked the enzyme hypoxanthine phosphoribosyl transferase; therefore unfused cells could be eliminated from the population using a selective growth medium. The mouse spleen cells died naturally. Hybrid cells were screened for the desired antibody activity (anti-sheep red blood cell) and the antibodyproducing hybrids cloned. The technique has wide applicability in producing monoclonal antibodies of defined specificity. To identify surface antigens of parasites with this technique a mouse could be immunized with parasites or with a partially purified fraction known to contain surface antigens. Following fusion of the mouse spleen with a m y e l o m a cell line, the resulting hybrids might be screened for antibody activity by immunofluorescence or neutralization of live parasites. When " h y b r i d o m a " clones secreting antibody against the parasite surface were obtained, the antigen recognized b y that monoclonal antibody could be
184 identified in various ways. 1. Parasite antigens could be separated by SDS-gel electrophoresis then: (a) the gel incubated with radiolabelled antibody, washed and autoradiographed (Olden and Yamada, 1977) or; (b) proteins transferred from the gel and coupled to nitrocellulose paper ("western" blotting, Renart et al., 1979; Burnette, 1981). As in (a) the specific proteins recognized could be revealed b y incubation with radiolabelled antibody. Alternatively, if the monoclonal antibody b o u n d to protein A from Staphylococcus aureus (SjSquist et al., 1972), the paper containing coupled proteins could be reacted first with antibody, then with 12SI-labelled protein A. 2. Parasite proteins could be radiolabelled by.metabolic incorporation of radioactive amino acids, or by surface labelling. The proteins would then be solubilized (see later) and immunoprecipitated with monoclonal antibody and a second anti-antibody; or, again, with formalin fixed Staphylococcus aureus instead of second antibody. Monoclonal antibody techniques have already been extensively applied to studying parasite surface antigens. Such antibodies have been developed, for example, against: Plasmodium berghei sporozoites (Yoshida et al., 1979), which confer protection on mice against sporozoite-induced infection; Plasmodium falciparum merozoites (Freeman et al., 1980); Trypanosoma brucei variable surface glycoprotein (Pearson et al., 1981); Trypanosoma rhodesiense surface glycoprotein (Lyon et al., 1981); schistosome membranes (Verwaerde et al., 1979). In some studies the antibodies have been used with the analytical techniques described to identify and characterize the specific antigens recognized e.g. the use of monoclonal antibodies, two dimensional gel electrophoresis and surface labelling for studying T. brucei surface glycoproteins (Pearson et al., 1981). A.F. Barbet, W.C. Davis and T.C. McGuire (1981, unpublished data) have made 15 monoclonal antibodies to one T. brucei surface glycoprotein which we are using in combination with immunofluorescence, protein fragmentation and sequencing to map exposed and buried regions of the glycoprotein when it is in a normal conformation on the trypanosome surface. PURIFICATION OF SURFACE ANTIGENS If exposed proteins or glycoproteins on the parasite surface have been identified, one should then purify them to obtain more detailed structural and immunological information. The first problem encountered is to devise a method for solubilizing the antigens of interest. Membrane antigens can be categorized as more or less firmly integrated into the hydrophobic regions of the membrane. Some are essentially "peripheral" others "integral" components (Singer and Nicolson, 1972). As ~ result o f this different membrane interaction, the ease with which surface antigens may be solubilized is also different. In the most convenient case peripheral components can sometimes be released from the parasite surface by physical disruption, such as sonica-
185 tion or blending, and then become water soluble. This is the situation with envelope glycoproteins of certain R N A t u m o u r viruses (Strand and August, 1976) which are released simply by freeze-thawing the virus. Also, trypanosome variable surface glycoproteins are released by mechanical blending (Cross, 1975) or even, simply by incubating isolated organisms in buffer at 37°C (A.F. Barbet, 1981, unpublished observations). In other situations, however, surface antigens remain associated with parasite membranes even after physical disruption of organisms and it is necessary to resort to more drastic solubilization methods. A favourite method of immunologists is detergent solubilization. Various detergents are available. Generally, the nonionic detergents such as Nonidet P-40 and Triton X-100 are not as efficient solubilizing agents as ionic detergents such as sodium deoxycholate or sodium dodecyl sulphate, but the likelihood of retaining some native immunologic structure after solubilization is greater when using non-ionic detergents. Also, antigen--antibody binding can usually occur in the presence of nonionic detergents which facilitates subsequent immunochemical analysis of antigens solubilized in this way. To purify the desired antigens from the solubilized extract many techniques are available such as: gel filtration, ion exchange chromatography, isoelectric focusing, preparative gel electrophoresis, high pressure liquid chromatography, affinity chromatography. A detailed description of all these techniques is b e y o n d the scope of the present paper. I will, however, mention two techniques which offer substantial purification in a single step procedure, and a third technique which is useful when only small amounts of starting material are available. An affinity chromatography column can be prepared using monoclonal antibodies covalently attached to cyanogen bromide-activated Sepharose. if a mixture of antigens is passed through the column the specific antigen recognized will be b o u n d and can be eluted later after washing the column free of the other antigens. This technique has been used to achieve a 5000-fold purification of human leukocyte interferon in a single step (Secher and Burke, 1980). A method based on a similar principal is lectin--Sepharose affinity chromatography. This can be used when it is k n o w n that the surface antigen of interest is a glycoprotein, for example by employing the galactose oxidase and [3H]sodium borohydride surface labeling technique. Glycoproteins will bind to the column and will therefore separate from non-glycosylated contaminants. The glycoproteins can be eluted subsequently with the specific sugar recognized b y the lectin, e.g., a-D-methylmannoside or a-D-methylglucoside, in the case of concanavalin A-Sepharose. A micromethod we have used to isolate proteins from equine infectious anemia virus when only a small amount of starting material was available, is preparative SDS-gel electrophoresis. Coomassie Blue stained proteins were extracted from a slab gel into SDS containing buffer. The SDS and SDS-bound proteins were precipitated with potassium chloride and then dodecyl sulphate and Coomassie Blue was extracted from the proteins by the
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ion-pair extraction method of Henderson et al. (1979). Even after contact with the protein denaturants used in this process radioiodinated viral p30 still reacted with a horse serum from a natural infection down to a radioimmunoassay titre of 1 in 10000 (A.F. Barbet and T.C. McGuire, 1981, unpublished observations). When sufficient quantity of a surface protein cannot be made by any conventional technique it is likely that the m e t h o d of choice in the future will be to make the protein in bacteria by gene cloning. ANALYSIS OF PARASITE SURFACE ANTIGENS Assuming that one has identified and purified surface antigens, there are several questions one might ask. Firstly, does antibody against that antigen kill the parasite? This question could be answered by using a specific or monoclonal antiserum against the parasite in a cytotoxic or neutralization assay. Secondly, does the antigen vary in structure in different parasites, i.e., does antigenic or strain variation occur? This question should be answered both immunochemically and biochemically. One needs to know: in which sequences of the protein or carbohydrate does structural change occur? Are these regions buried or exposed when the conformation of the antigen on the parasite surface is taken into account? Are the regions of altered sequence i m m u n o d o m i n a n t or immunosilent, i.e., is an immune response produced against them in a natural infection? Are there also constant sequences in the antigen exposed on the surface of the different parasites? Are these i m m u n o d o m i n a n t or immunosilent? The various techniques which might be used to answer these questions include: inhibition radioimmunoassay, immunfluorescence, cytotoxicity, peptide mapping on isolated and surface-labelled antigens, peptide fragmentation and purification, antibody production against different peptides, peptide sequencing and, ultimately, X-ray crystallography. The ways in which some of these techniques can be applied might be illustrated by reference to recent research on the structure of the trypanosome surface. The only molecule exposed to surface radiolabelling in Trypanosorna brucei is a glycoprotein of approximately 65000 molecular weight. Methods have been devised for purification of this molecule (Cross, 1975). Antiserum against the purified glycoprotein lysed the parasites from which the molecule was derived. Antigenic variation occurs in these parasites because of amino acid sequence changes in the glycoprotein, therefore an antiserum against a glycoprotein purified from one antigenic type was generally ineffective at neutralizing trypanosomes of a different type. Immunological cross-reactions were, however, detected by radioimmunoassay between all glycoproteins that were isolated from different antigenic types of trypanosome (Barbet and McGuire, 1978). Later, peptide fragmentation studies showed that the region of the molecule causing this cross-reaction was a small glycopeptide, and probably the carbohydrate portion of the glycopeptide, near
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to the carboxy-terminus (Barbet et al., 1979; Cross, 1979; Holder and Cross, 1981; Barbet et al., 1981). This glycopeptide region was not accessible to antibody when on the surface of the trypanosome. Other surface glycoprotein sequence homologies have been found, but were common only to a small number of different trypanosomes. These homologies were in the amino acid sequence and the regions involved were exposed at the trypanosome surface, as demonstrated by cross-neutralization tests and by peptide mapping of surface-labelled trypanosomes (Vervoort et al., 1981). To assess the possibility of producing a vaccine against trypanosomes requires that the extent of such sequence homology be known. In other parasitic diseases where antigenic variation does not occur, such an assessment should be made more readily. ACKNOWLEDGEMENTS
Support for the author was provided in part by funds provided for medical and biological research by state of Washington Initiative Measure No. 171 and by Rockefeller Foundation Grant No. GA-COH-8013.
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