Parasite antigens*

MEMORANDA Parasite antigens* The currently available preparations used as antigen in the serological investigation of parasitic diseases are ill-defined heterogeneous mixtures, and there is an evident need for better characterized reagents. Antigens of different parasite species (schistosomes, filariae, trypanosomes, and plasmodia) are discussed and parasite sources enumerated. Modern methods for the preparation of antigenic extracts and their fractionation are described, together with certain guidelines as to their biochemical characterization and their immuno- logical activity. In order to implement this endeavour and to make better use of serological techniques in parasitic diseases, proposals are made concerning collaborative research and field application among a number of laboratories on schistosome, onchocercal, trypanosome. and plasmodial antigens. The serology of parasitic diseases suffers from a lack of specific and characterized antigens. The various techniques widely used for indirect diagnosis or for seroepidemiological purposes have not received adequate consideration. Most, if not all, of the currently available prepara- tions used as antigen are ill-defined heterogeneous mixtures. Cross-reactions with unrelated parasitic conditions are the rule rather than the exception in the serology of parasitic diseases, and particularly in helminthiasis. To make full and better use of sero- logy, more and better-defined antigens are necessary. With the development of modern biochemical and immunochemical techniques there is a good chance of producing specific antigens from a variety of parasitic organisms. It is felt that a concerted effort should be made to sort out the problems and to establish a coordinated programme for evaluating and defining the best techniques for making available reliable and repro- ducible antigens. With this aim in mind, this Memo- randum limits its considerations to antigens of schistosomes, filarial parasites, American trypano- somes, and malaria parasites. ANTIGENS In this Memorandum crude extracts of the para- site material are referred to as " antigen mixtures " * This Memorandum was drafted by the signatories listed on page 247. Reprints can be obtained from: Epidemiological Methodology and Clinical Pathology, Division of Malaria and Other Parasitic Diseases, World Health Organization, 1211 Geneva 27, Switzerland. A French translation of the Memorandum will be published in a future issue of the Bulletin. and the isolated materials thought to be single entities are referred to as " pure antigens " or " puri- fied antigens ". It is, however, recognized that a " pure antigen " could have more than one antigenic determinant and a " purified antigen " might consist of more than one molecular species. Antigens can be subdivided into those that are soluble and those that are particulate (i.e., difficult to dissolve). They can also be subdivided into somatic antigens (i.e., parasite body material) or exoantigens (i.e., those that are secreted or excreted). Attention is drawn to the special importance of antigens at the surface of parasites. In serology the main requirement is that the antigens should be able to detect and differentiate antibodies induced by specific parasitic infections. Schistosomiasis Immunodiagnostic tests are required to supple- ment parasitological information. In particular they are needed to: (a) detect light infections; (b) determine the efficacy of chemotherapy; (c) assess the importance of immune-complexes in pathogenesis; (d) detect single sex infections; (e) conduct seroepidemiological investigations. A wide variety of immunological tests ranging from skin tests to serological methods have been used to detect schistosomal antibody. Antigens can be whole parasites (e.g., cercariae or sections of worms in immunofluorescent antibody tests) or extracts (e.g., in skin tests, passive haemagglutina- 3345 - 237 BULL. WORLD HEALTH ORGAN., Vol. 52, 1975 MEMORANDA tion tests, etc.). Usually extracts are crude; an exam- ple of obtaining a purified entity is given under Proteolytic enzyme below. This purified proteolytic enzyme gives rise to immediate type hypersensitivity in patients infected with schistosomes. It appears to be slightly less reactive, but perhaps more specific than the crude antigenic preparations. Most of the schistosome antigens used are saline extracts of different life-cycle stages of Schistosoma mansoni and represent antigen mixtures. Antigen extracts have been prepared from adult worms, eggs, miracidia, and cercariae. The hatching fluid of schistosome eggs also constitutes an antigen mixture. Considerable efforts have been made to isolate purified schistosome antigens. Techniques used for this purpose include column chromatography, iso- electric focusing, immunoadsorption, etc. However, only a limited number of actually purified antigens are available: (a) proteolytic enzyme; (b) circulating polysaccharide antigen (high mole- cular weight); (c) circulating polysaccharide antigen (low mole- cular weight); (d) acetylcholinesterase. Proteolytic enzyme. The isolation of a proteolytic enzyme from adult worms has been performed by separation of an adult worm antigen extract in an acid solution (pH 4.0). The extract is concentrated on an Amicon P-10 membrane and fractionated on a Sephadex G-75 column. The enzyme-containing fractions are passed through a column filled with phenylalanine-coupled Sepharose, after which the enzyme can be eluted by lowering the pH to 2.5. This antigen is available from: Division of Biological and Medical Sciences, Brown University, Providence, RI, USA; and the Laboratory of Parasitology, Univer- sity of Leiden, Leiden, Netherlands. Circulating polysaccharide antigen (high molecular weight). A schistosome derived antigen, circulating in the serum of heavily infected hamsters, has been isolated both from adult worms and from the infected-hamster serum. Isolation procedures for recovery of the antigen from adult worms include heating (100°C, 30 min), trichloroacetic acid (TCA) treatment and desalting of the TCA-soluble fraction on a Sephadex G-25 column. The same antigen could be isolated from serum from S. mansoni- infected hamsters by means of TCA treatment of the serum, desalting on a Sephadex G-25 column, and gel filtration on a Sephadex G-200 column. The antigen is heat-stable, TCA-soluble, and strongly negatively charged at pH 8.2. It has a molecular weight of over 200000. This antigen is available from the Laboratory of Parasitology, University of Leiden, Leiden, Netherlands. Circulating polysaccharide antigen (low molecular weight). A schistosome derived antigen has been isolated from the urine of a hamster heavily infected with S. mansoni. Isolation procedures include TCA treatment of the urine and desalting of the soluble fraction on a Sephadex G-25 column. The antigen is heat-stable, TCA-soluble, positively charged at pH 8.2, and has a molecular weight lower than 30 000. This antigen is available from the Labora- tory of Parasitology, University of Leiden, Leiden, Netherlands. Acetykcholinesterase (ACHE). Isolation of ACHE from S. mansoni has been reported from the Labora- toire de Parasitologie, Lille, France. Warm ACHE has been eluted after binding to a specific inhibitor on an affinity column. Binding studies of various drugs against parasite ACHE are in progress at the Division of Biological and Medical Sciences, Brown University, Providence, RI, USA, and at the School of Hygiene and Public Health, Johns Hopkins Uni- versity, Baltimore, MD, USA. Filarial infections Immunological tests are needed to: (a) identify specific infective larvae in the vectors (i.e., to differentiate these from animal filariae); (b) identify antibodies to infective stage larvae; (c) identify antigens and antibodies to micro- filariae; (d) identify antigens and antibodies to adult worms; (e) aid the seroepidemiology of onchocerciasis; (f) aid the evaluation of control programmes; (g) study the pathogenesis of Onchocerca volvulus. Apparently, living microfilariae are not recognized by the host; once killed they are recognized as " non-self ". It is known that in filarial helminthiasis the imme- diate type hypersensitivity reactions are common and cross-reactions are frequent. A more specific skin-test antigen is needed, hence the need for obtaining purified antigens. 238 PARASITE ANTIGENS At present the following serological tests are applied to filarial infections: The immunofluorescent antibody (IFA) test has been employed in infections with Onchocerca, Wuchereria, Brugia, etc., using frozen sections of adult worms but not necessarily of the same species as that present in a patient. The passive haemagglutination (PHA) test, using as antigen fractions of Dirofilaria immitis (for which a purified preparation is available) or of other filarial species, has been employed for the detection of antibodies in most filarial infections. Immunodiffusion, immunoelectrophoresis, and enzyme-linked immunosorbent assay (ELISA) have been used to detect antibodies using parasite extracts derived from filarial species and even other nema- todes (e.g., Ascaris lumbricoides). Antigen-antibody complexes have also been demonstrated in onchocer- ciasis, employing gel filtration and isoelectric focus- ing. The skin test with D. immitis antigen has found a wide application, particularly for seroepidemiologi- cal studies of filarial infections. However, skin test- ing has produced conflicting results. The complement fixation test has also been used but to a limited extent. Owing to the wide range of non-specific reactions and the cross-reactivity of the preparations used as antigen, attempts are being made to prepare better-defined and more purified antigen preparations (e.g., from 0. volvulus). For investigative purposes it has been found useful to label 0. volvulus extracts with radioactive iodine of high specific activity, since this permits the study of micro-quantities of antigenic material. On the other hand, the use of the detergent Triton X-100 during the extraction resulted in a better solubiliza- tion of some antigens, the molecular weights of which were reduced. A further advantage is that in subsequent double immunodiffusion some precipita- tion lines became sharper. Isoelectric focusing, immunoelectrophoresis, and gel filtration of the radioactive 0. volvulus extracts indicated the pres- ence of several antigens. Sera from donors in a non- endemic area (Finland) produced no precipitation lines when diffused against the antigen extracts, whereas sera from infected Africans produced vari- ous precipitation line patterns. Extracts from hook- worm, Litomosoides carinii, and 0. gutturosa pro- duced lines with the sera of 0. volvulus patients. Similar cross-reactions were observed with the ELISA. Attempts are in progress to isolate 0. volvulus antigens by absorption to and elution from cyanogen bromide activated Sepharose columns to which immunoglobulins from 0. volvulus patients have been covalently bound. American trypanosomiasis (Chagas' disease) Immunodiagnosis is currently the only practical way of identifying chronic infections and ofdetermin- ing whether some pathological conditions (e.g., megacolon, megaoesophagus) are due to Trypano- soma cruzi infection. It is important that any " im- proved antigen" should be able to differentiate between Chagas' disease, leishmaniasis, and T. ran- geli infections in the Americas. The ready availabil- ity of large amounts of in vitro cultured parasites is recognized as a valuable asset in the preparation of T. cruzi antigens. The many variations in antigen preparation and diagnostic methods used in different laboratories make valid comparisons difficult. Common prepara- tive forms are: (a) aqueous extracts; (b) delipidized aqueous extracts; (c) aqueous extracts lyophilized, then delipidized (e.g., benzene and chloroform or methanol); (d) somatic protein antigens from epimastigote culture forms; (e) epimastigote exoantigens; (f) amastigote exoantigens from tissue culture. Most commercially available reagents are a crude antigen preparation derived from in vitro culture of epimastigote forms; in some cases antisera or com- plete diagnostic kits are sold: (a) latex agglutination test kit; (b) complement fixation antigens and test kits; (c) PHA antigens and test kits; (d) antigens for immunofluorescence; (e) antigen for counter-immunoelectrophoresis. African trypanosomiasis Immunodiagnosis is important in the epidemio- logical assessment of the disease. It can be done by non-specific means, i.e., estimation of IgM of which high levels, particularly in cerebrospinal fluid, are 239 MEMORANDA indicative of trypanosomiasis. It is more satisfactory, however, to use specific immunological tests such as immunofluorescence. The fact that African trypanosomes can readily vary their surface antigens can pose problems for immunodiagnosis. Because of the complexity of this antigenic variation it is not dealt with in this Memo- randum. Malaria In epidemiological surveys of malaria, immuno- diagnosis can give prevalance data that can usefully supplement the parasitological information. Al- though less valuable in individual infections, sero- logy can be used to exclude malaria in infected blood donors and to exclude or confirm malaria as the causative agent in certain pathological conditions The antigens used are derived from blood forms of the parasites. They can be soluble (e.g., in PHA, gel- diffusion, and ELISA) or whole parasites (e.g., in IFA). Antibodies induced by blood stages are usual- ly detected and are " malaria-specific " but rarely species-specific. Malaria parasites can readily undergo antigenic variation and they have a complex mosaic of anti- gens. At present, malarial antigens are available in the form of blood films from humans or primates containing Plasmodium falciparum, P. vivax, P. malariae, P. brasilianum, P. cynomolgi and P. fieldi. All these materials can be used in IFA tests and can detect antibody induced by malarial infec- tion. The homologous antigens give the highest titres. These antigen preparations are normally kept at -70°C but can be distributed in suitably adapted containers in which they retain reactivity at room temperature for some weeks. Lyophilized soluble malarial antigens suitable for use in gel-diffusion, PHA, or ELISA and originating from human infections or from experimentally infected monkeys are also available (P. falciparum, P. knowlesi, and P. fieldi). Lyophilized erythrocytes sensitized with malarial antigen and suitable for PHA can be obtained, and polystyrene plates already sensitized with malarial antigen for ELISA could be made available. SOURCES OF PARASITES FOR ANTIGEN PREPARATION Schistosomiasis Generally speaking, S. mansoni has been used for various physiological, biochemical, and immunologi- cal studies. This is because S. mansoni was the first species to be found easy to cultivate in mice and a few other rodents. Recently it became possible to maintain S. haematobium and S. japonicum in vari- ous small mammals. It can be expected, therefore, that all three human species will become available for both biochemical studies and antigen prepara- tion. Besides these three common human forms, species such as S. intercalatum and S. bovis can also be maintained in the laboratory. It is technically feasible to obtain various life-cycle stages of schistosomes. These would include eggs, sporocysts, miracidia, cercariae, schistosomula, developing lung-stage worms, and fully mature adults. Antigens could be extracted from any one of these stages. It is currently believed that the two most promising sources of antigens are the adult worms and eggs. Within the limits of present know- ledge, S. mansoni will probably be the most useful source of both of these stages. There are many useful laboratory animal models for the recovery of various schistosome stages. Some of these are indicated in Table 1, which shows the available sources of raw material for antigen prepa- ration. Recovery techniques are described in the Annex. Filarial infections The causative agents of filarial infection in man are: Wuchereria bancrofti, Brugia malayi, Oncho- cerca volvulus, Dipetalonema streptocercum, D. per- stans, Loa loa, and Mansonella ozzardi. Of these only W. bancrofti, B. malayi, and 0. volvulus are con- sidered to be of major public health importance. Table 2 shows the available laboratory models that are most used. Complete life cycles can be maintained in the laboratory, including the vectors. They are important as future sources of raw material (e.g., adult worms and microfilariae) for antigen preparation, and for the study of immunological phenomena (e.g., host-parasite relationship). The complete life cycles of only two of the filarial species causing infection in man are being main- tained in the laboratory: B. malayi and L. boa. 0. volvulus infections can be maintained in the chimpanzee but suitable insect vectors for experi- mental transmission have not yet been found. For this reason, only infected persons in endemic areas can serve as sources of homologous antigen for the other filarial species. Sources of adult filarial worms. Sources of W. ban- crofti are extremely scarce and are limited to mate- rial obtained from the few medical centres in ende- 240 241PARASITE ANTIGENS Table 1. Sources of schistosomes Parasite species Laboratory animal Useful for Remarks or intermediate host recovering: S. mansoni Biomphalaria glabrata sporocysts, use albino strain cercariae B. pfeifferi sporocysts, difficult to maintain cercariae B. alexandrina sporocysts, easy to maintain cercariae white mouse adult worms, will yield 25-50 (Mus musculis) eggs, miracidia worms/animal golden hamster adult worms, will yield about (Mesocricetus auratus) eggs, miracidia 100-300 worms/animal Nile rat (Arvicanthus adult worms, currently available only in niloticus) eggs, miracidia Egypt; will yield 1000 worms/ animal S. haematobium Bulinus truncatus & sporocysts, moderately difficult to rear B. obtusispirus cercariae in laboratory golden hamster adult worms will yield 40-100 worms/animal; few eggs Nile rat & multimammate adult worms, rat (Mastomys natalensis) eggs man eggs specially handled urine speci- mens (washing in saline to remove ammonia & other toxic products S. japonicum Oncomelania spp. cercariae difficult to cultivate; relatively(e.g., hupensis) few cercariae/snail multimammate rat, golden worms, eggs worm yield: multimammate hamster, & mouse rat (20-30 worms/rat) > mouse > golden hamster (eggs, but few worms) Table 2. Laboratory models in filariasis Parasite species Dirofilaria immitis Litomosoides carinii Dipetalonema witeae Brugia pahangi & B. malayi Onchocerca gutturosa Onchocerca cervicalis Loa loa Laboratory animal dog cotton rat (Sigmodon hispidus) Mastomys natalensis Meriones spp., golden hamster (Mesocricetus auratus) & Gerbillus hirtipes cat cattle horse Macaca spp. Useful for recovering: adult worms, micro- filariae adult worms microf ilariae adult worms, micro- filariae adult worms, micro- filariae MEMORANDA mic areas where surgical removal of elephantoid tissue and restoration of collateral lymphatic circula- tion is done on a regular basis. Small quantities of adult antigen of B. malayi can be obtained from experimentally infected monkeys and cats, in addition to the extremely limited mate- rial from patients undergoing surgery for elephan- tiasis. The closely related species, B. pahangi, has been maintained in cats in various laboratories of academic institutions and pharmaceutical com- panies. Because of the high cost of maintaining these experimental infections in the laboratory, mass pro- duction of worms does not seem feasible at present. The subcutaneous nodules (onchocercomata) of patients with onchocerciasis harbouring the adult 0. volvulus worms are the only source of adult antigen in this species. Following nodulectomy, the worms can be either extracted or removed by diges- tion of the surrounding tissue. Nodulectomy is widely practised in certain countries where oncho- cerciasis is endemic. Recovery techniques for Oncho- cerca are shown in the Annex. Sources of microfilariae. W. bancrofti and B. malayi can be obtained from blood samples of patients with a heavy microfilaraemia by means of Millipore concentration techniques. Small quantities of microfilariae of 0. volvulus can be harvested from skin snips or skin removed by nodulectomy. Many of the microfilariae in the tissue will emerge when the skin biopsy material is kept in saline solution or distilled water. Because of the pronounced cross-reactions to anti- gens prepared from different species of the Fila- rioideae, most immunodiagnostic tests have em- ployed antigens prepared from Dirofilaria immitis, the heartworm of dogs. American trypanosomiasis (Chagas' disease) Trypanosoma cruzi can be cultured easily and is, therefore, readily available as a source of antigen. The culture medium may even be of plant origin, thus avoiding all mammalian materials that might be troublesome contaminants of antigens. The tissue forms of T. cruzi can be grown in large amounts in tissue culture. There are many laboratories that either have the crude antigen mixture available or can provide it at short notice. Malaria The supply of erythrocytic antigen material ori- ginating from human malarial parasites is limited. Available sources of supply are indicated below. Plasmodium falciparum. The only sources of sup- ply are: (a) placentae of naturally infected women; (b) blood from infected people, cultured to produce mature parasites; and (c) blood from experimentally infected Aotus trivirgatus (these douroucoulis mon- keys are in very short supply). Plasmodium vivax. Available sources are: (a) blood of naturally infected people; and (b) blood of experimentally infected A. trivirgatus. Since P. vi- vax usually gives low parasite densities, it is difficult to produce a large mass of parasite material. For many purposes, however, it may be possible to use the simian analogue, P. cynomolgi, which infects rhesus monkeys; in this way, large amounts of infected blood can be obtained. Plasmodium malariae. Available sources are: (a) blood of naturally infected people; and (b) blood of experimentally infected A. trivirgatus. Since P. malariae causes low parasitaemias it is difficult to produce large amounts of parasite material. P. brasi- lianum, an analogous parasite of South American monkeys, is virtually identical to P. malariae and may also be used as a source of antigen. A limited number of laboratories could supply all these parasites in the form of blood films containing washed infected erythrocytes (suitable for immuno- fluorescence). Material for the provision of antigen mixtures for analysis, extraction, etc., is not gener- ally available. Other malarial parasites not of human origin can be used as a source of antigen for some purposes. P. knowlesi, for example, produces massive infec- tions in rhesus monkeys; P. fieldi, P. inui, and P. cynomolgi also infect rhesus monkeys and yield reasonable amounts of material. Sporozoite antigen material is produced only with great difficulty and is not generally available. The same applies to purified merozoites. PREPARATION OF ANTIGEN EXTRACTS FROM PARASITES Although fresh material is preferable, parasites could be stored and transported: (a) in the frozen state; (b) lyophilized; (c) in saturated ammonium sulfate solution; or (d) in glycerol. In many instances, before other manipulations, it may be preferable to wash the parasite and store, incubate, or grow it in a suitable medium (care being taken to avoid immunogenic substances in the me- dium) to obtain antigens that are secreted, excreted, or easily diffused from the parasite and that presum- _24-2 PARASITE ANTIGENS ably cause an early antibody response in the host. During or after this, it may be possible to extract further material by manipulating the composition of the medium (e.g., the ionic environment) without actually disrupting the organism. In principle, disruption of the organism may be achieved by means of any one of the methods currently used for tissue homogenization. Methods widely used include physical procedures such as grinding (with or without glass beads), pressing, treatment with ultrasound, freezing-thawing, osmo- tic shock, and pressing-decompressing. Disruption may also be achieved chemically, e.g., by means of enzymes, lipid solvents, or detergents. Most protozoan parasites, including T. cruzi, have been disrupted by treatment with ultrasound, by freezing-thawing or by hypotonic breakage. Hel- minths can be disrupted by grinding with a Pot- ter-Elvehjem or other type of homogenizer. Some steps may be necessary before homogenization. Lyophilized material is usually rehydrated. Ammo- nium sulfate or glycerol used to preserve the material can be removed by dialysis. Bacteriostatic agents such as streptomycin, sodium azide, and thiomersal may also have to be removed. Protein antigens Frequently, delipidization seems to be an impor- tant step in the treatment of the parasite material, either before or after homogenization. This has been shown for trypanosomes as well as for schistosomes and other helminths (e.g., Echinococcus) where removal of lipids results in less non-specific reac- tivity. Delipidization has been achieved with lipid solvents such as benzene and acetone (lyophilized T. cruzi) and cold ethanol (- 15°C) followed by anhydrous diethyl ether (e.g., for S. manzoni, Ascaris and other nematodes). Detergents constitute a valu- able alternative, since they may replace the lipid moiety of lipoprotein complexes and may also help to solubilize proteins. A widely used neutral deter- gent is Triton X-100. It is used especially for Schisto- soma, Onchocerca, and Plasmodium. Besides lipids, there are other substances that should be removed because, even though they are not usually antigenic, they may cause complications. Glycogen, which is a significant component of most helminths, can easily be eliminated by enzyme treat- ment. A suitable procedure consists essentially in a preliminary treatment of the crude parasitic extract with f-amylase (3.2.1.2) for 30 min at room tempera- ture. A second similar treatment may be applied after the crude material has been submitted to digestion procedures with sodium hydroxide or pro- teolytic enzymes. Normally, small molecules must be removed to obtain an optimal antigen preparation. The methods most commonly used for this are dialysis, ultrafiltra- tion, and desalting on Sephadex G-25 columns. Fractionation. For purification, a whole range of established procedures can be used to isolate the proteins. One approach is to produce characteristic patterns by such techniques as electrophoresis, double diffusion, and immunoelectrophoresis. These techniques, however, have drawbacks for parasitic antigen analysis in that: (a) it is difficult to repro- duce such patterns reliably; and (b) a wide variety of variables (e.g., composition of antigen mixtures, differences in host response, presence of other infec- tious agents) can affect the patterns. However, for some parasite populations, such as Plasmodium and Leishmania, isoenzyme patterns can represent a highly specific and reliable indicator. Another ap- proach is to isolate and define single or limited numbers of antigens. Before resorting to sophis- ticated procedures, simple ones such as salt frac- tionation and isolectric precipitation should be tried. Further isolation can be tackled in two ways: (a) by chemical fractionation followed by immuno- logical identification or fractionation (e.g., ion- exchange chromatography and/or gel filtration) and then by identification of antigens through double diffusion or immunoelectrophoresis; and (b) by immunological isolation followed by further charac- terization. Columns containing matrices covalently coupled to immunoglobulins from a pool of sera from patients with proven specific infection can be used to combine antigens from parasite extracts. Such com- plexes can then be dissociated to yield the specific antigens. Generally, dissociation can be achieved by altering ionic composition (pH), or by using protein unfolding agents. Affinity chromatography can be used to purify enzymes. The substrate or a substrate-like com- pound is coupled to the matrix, thus permitting the specific absorption of the enzyme. The complex can be dissociated to release the enzyme by a variety of means, such as elution with high substrate concen- trations and the use of protein unfolding agents (e.g., urea, guanidine). Lectins, such as concanavalin A, may also be coupled to a matrix, which may then react with specific proteins. 243 MEMORANDA It is advisable to carry out preliminary purification before applying samples to the fractionation column, because the crude extracts can cause clogging or other undesirable effects. The antigen composition of preparations eluted from immunosorbents will, of course, depend on the antisera coupled to the matrices. Purified immuno- globulins from a pool of sera are usually preferable to a single serum if a comprehensive range of antigens is to be isolated. Monospecific antisera have been produced in animals by cutting out a single precipitation line produced by large-scale immuno- electrophoresis and using this for immunization. Other antigens Antigens other than pure polypeptide proteins, such as polysaccharides, glycoproteins, lipoproteins, and nucleic acids, may be of importance for immu- nological studies. Polysaccharide antigens have been demonstrated in various parasites, e.g., Trypano- soma, Echinococcus, and Schistosoma. Lipoproteins have been isolated from Fasciola and Echinococcus. Commonly used isolation methods for carbo- hydrate antigens are: heating, TCA-precipitation, pepsin digestion, phenol extraction, alkali extrac- tion, and affinity chromatography (based on lectins). Extraction methods for lipoproteins are normally based on the use of organic solvents. It should be noted that it has proved very difficult to obtain antibodies against pure carbohydrates. Host antigens. The possible presence of material of host origin should be considered when dealing with extracts of parasites. Intact organisms, sub-cellular fractions, and resi- dues. Particulate or intact whole organisms are widely employed as antigens, especially in immuno- fluorescence tests. Subcellular fractions are often used for the same or similar purposes. Residues from single extraction procedures should not be discarded until all extraction methods have been employed (e.g., buffer followed by detergent and enzyme digestion). The final residues may still have antigenic activity detectable by some immuno- logical techniques, especially complement fixation and immunofluorescence. CHARACTERIZATION OF ANTIGENS Isolated material may differ from the raw mate- rial. Consequently both immunological and a certain amount of biochemical characterization is necessary. Biochemical characterization The usual methods (e.g., isoelectric point, mole- cular size, and biological activity in the case of enzymes) can be used for protein characterization. The protein content of antigen preparations should be measured and should be expressed in terms similar to those used for some generally available proteins, e.g., albumin or standardized serum. In addition, the prosthetic groups should be described and any enzyme activity indicated by means of generally understood terms and units. Characterization of carbohydrate antigens is car- ried out by measuring molecular size (gel filtration), sugar analysis, etc. Immunological characterization The specificity and reactivity of the final product must then be determined. This could be done by carrying out immunological tests with a defined range of reference human homologous positive sera, sera from heterologous infections, and negative sera. Such sera should, in general, be described in terms of age, sex, duration of infection, status of chemo- therapy, etc. Immunological activity The immunological activity should be expressed in terms of reactivity with reference serum prepara- tions. It is essential that sera from specific parasito- logically proven cases be collected, possibly pooled, and dispensed in small aliquots so as to be made available for such purposes (see General proposals, section 3, p. 245). PROVISION OF ANTIGENS In general, the antigens should be provided in a form in which they can easily be used by the worker in the field. Lyophilization is a generally accepted procedure and there is considerable commercial expertise available in this field. In some instances it may be preferable to provide the antigens in liquid form, in which case glycerol may be useful as a preservative. Alternatively, antigens may be made available for specific tests in the form of materials bound to a solid support such as polystyrene, aga- rose, or stabilized erythrocytes. Control antigens should also be made available. For parasites grown in vivo, tissue of an uninfected host could be used (e.g., blood or skin); for parasites grown in vitro, the initial culture medium could be used. 244 PARASITE ANTIGENS RECOMMENDATIONS General pr-oposals 1. It is suggested that small-scale workshops and the exchange of laboratory personnel between the various institutions engaged in specific programmes be arranged. For example, in the case of the isola- tion of parasite antigens, meetings could be arranged between those who produce the parasite material, those who subsequently extract and fractionate it, and those who test the purified antigens under laboratory and field conditions. The establishment of such a close contact between the developing areas where the parasitic diseases occur and the developed areas where the qualified laboratory research is performed, will result in the diffusion of specialized knowledge to the developing areas where it would then become possible to initiate qualified laboratory research. In particular, workers from the developing countries should be encouraged to visit the special- ized laboratories. 2. Current collaborative projects in the field of parasitology should be summarized periodically. Such reports might provide information on the institutions and investigators involved in these pro- jects, as well as indicate the progress and findings of their work. In this way a link could be established with other laboratories that are involved in similar investigations and wish to participate in such col- laborative projects. Information ought to appear on a regular basis and should be directly distributed to investigators. Interested workers should also receive regularly information from computerized biblio- graphies, such as the one now existing on filariasis. Means should be investigated of using available abstracting services to provide information about literature on specific parasitic diseases. 3. Certain reference preparations of human anti- bodies to parasites and of parasite antigens may be of value in the standardization of immunological tests for parasitic infections in the various areas throughout the world. It is felt that WHO should encourage all those engaged in antigen production to describe fully the preparative and storage steps used in obtaining a product. Efforts should be undertaken to use rigorously defined physical harvesting methods, many of which are mentioned in this Memorandum. Standardization and purity checks of the preparations are recommended. WHO could also encourage cooperative studies and the exchange of experimentally produced anti- gens. A start has already been made in this direction by the establishment of the International Collabora- tive Study of Assay of Malarial Antibodies. In this programme reference antisera from persons with proved infection have been distributed to different laboratories for comparative serological testing. It is thought that antibody/antigen preparations relevant to schistosomiasis, Chagas' disease, and filariases should now receive priority. The aims of such a programme are: (a) to standardize the antigens that will be made available; (b) to assess the suitability of the reference pre- parations for the relevant immunological tests; (c) to assess, in a wide variety of situations, the precision, specificity, and sensitivity of the relevant immunological tests currently used. Strictly defined sera and antigens would be pre- pared in a form suitable for distribution. The prepa- rations would then be coded and distributed to the interested investigator or institution, together with a detailed description of specific methods to be em- ployed for the assessment of the preparations. The participating laboratories would also be requested to include their own antigens and sera in the tests for comparative purposes. The results would be re- turned to a central coordinating centre where they would be analysed. It is possible that this proposal will in due course lead to the establishment of international standard reference materials. It is essential that the coopera- tion of a unit dealing with biological standardization be sought for the implementation of these pro- grammes. 4. It would be very profitable if biochemists and serologists could see for themselves the progress being made in the actual field application of antigen and immunological tests developed in the labora- tory. By being able to see first-hand the extent of the problem to be solved and the practical possibilities of realization in the field, their work would become still more efficient and directly applicable. Specific proposals A few specific proposals are made here, all of which could be implemented within a short time and all of which are highly relevant to current practical problems in the field. These specific proposals con- cern projects of limited duration from which early results could be expected. 245 246 MEMORANDA Schistosomiasis. A collaborative project is pro- posed in which two laboratories will participate to: (a) produce skin-test antigen; (b) characterize further the kinetics and physical-chemical properties of this antigen; and (c) extend observations to determine its use in other immunological procedures (develop- ment of assays for the demonstration of circulating antigen or of enzymatic activity in blood or urine). The schistosome proteolytic enzyme being at pre- sent the only clearly defined antigen, it is necessary to obtain a better index of its sensitivity, reliability and stability. This will require comparative tests of these parameters as well as large-scale field trials in the order of 2000-3000 tests at first and later 10 000 tests). A preliminary plan for a cooperative project between Brown University, USA (Division of Bio- logical and Medical Sciences) and the University of Leiden, Netherlands (Laboratory of Parasitology) is outlined below: Brown University University ofLeiden 1. Produceenzymeantigens Produce enzyme antigens from fresh or lyophilized from fresh or lyophilized worms worms 2. Test substrate specificity Develop coupling proce- on proteins and on an dures on solid supports artificial substrate (e.g., DASS a or ELISA) 3. Initial trials to develop a Assay of enzyme in urine rapid dose skin test oper- and blood by agarose- ative for field use (skin antibody affinity reactions test gun) 4. Characterize enzyme or Fluorescent or other solid protein contents toge- support reaction develop- ther with amino acid ment analysis 5. Prepare 5000-10 000 Test ELISA with this standardized skin-test antigen in the field doses for trials (e.g., in Puerto Rice with the co- operation of the Com- municable Disease Cen- ter, Atlanta, USA; later in South America or in Egypt) 6. Extension of collaboration with other laboratories (e.g., Laboratory of Immunochemistry, Istituto Supe- riore di Sanita, Rome; London School of Hygiene and Tropical Medicine; National Bacteriological Laboratory, Stockholm) is proposed. Short training courses in the application of specialized techniques (e.g., ELISA) would be arranged. a Defined antigen substrate spheres (DASS) system. Further characterization and testing of the two polysaccharide schistosome circulating antigens is to be considered. A collaborative effort on (a) the production of these antigens and the development of relevant assays and (b) their characterization by sugar-analysis is proposed. The University of Leiden (Laboratory of Parasitology), Netherlands, and the Istituto Superiore di SanitA (Laboratory of Immuno- chemistry), Rome, would participate in this project. The isolation of different purified schistosome antigens and the development of sensitive assays using these antigens should be encouraged. This research project would require the collaboration of a number of laboratories. Studies would include, for instance, the recovery and purification of the larger proteins involved in purine salvage pathways-e.g., adenosine kinase (2.7.1.20), adenosine deaminase (3.5.4.4), and purine nucleoside phosphorylase (2.4.2.1)-and these would be considered from the standpoint of their possible use as immunogens. Onchocerciasis. At present the following institutes are involved in a project on antigens of Onchocerca: Minerva Institute for Medical Research, Helsinki; Service de Parasitologie, Universite de Grenoble, France; Institut d'Ophtalmologie tropicale de l'Afrique (IOTA), Bamako; Nuffield Institute of Comparative Medicine, London; Istituto Superiore di Sanita, Rome; and the Swiss Tropical Institute, Basle. These laboratories represent a network in which raw parasitic material (skin nodules) is sent from Mali to the laboratory in Grenoble where the parasites are dissected out and then sent on to the institutes in Helsinki and London for the isolation of antigens. The raw material will also be sent to Rome for isolation of polysaccharide antigens. The isolated antigens will then be tested immunologically in the laboratories where their isolation was achieved, as well as at the Swiss Tropical Institute in Basle, the Service de Parasitologie in Grenoble, and other specialized laboratories. The same network of labo- ratories will also collaborate in the provision and testing of corresponding reference and defined sera. The Minerva Institute for Medical Research in Helsinki is already actively engaged in the isolation and identification of onchocercal antigens as de- scribed in Filarial infections, p. 238. However, owing to the lack of this parasite and of the corresponding sera in Finland, this laboratory is dependent on the functioning of the network described and the same will apply to the Istituto Superiore di SanitA, where polysaccharide antigens are to be isolated. This collaborative programme is to be coordi- PARASITE ANTIGENS 247 nated by WHO (Division of Malaria and Other Parasitic Diseases), and the antigens thus isolated and tested will then be sent to the endemic areas for testing under field conditions by laboratories such as IOTA in Bamako and possibly the Centre Muraz in Bobo-Dioulasso, as well as other laboratories. The WHO Regional Office for Africa should be kept informed of this activity, as this will also facilitate the eventual field evaluation of the antigens. In connexion with the work oti Onchocerca, small quantities of other filarial species, together with the corresponding antisera, are already being distributed through this network of laboratories for compara- tive studies. The same network is prepared to undertake work on other filariae, such as W. bancrofti, provided the raw material is made available. The South-Western Pacific, particularly Western Samoa, would appear to be the most suitable source of both W. bancrofti and the corresponding sera. American trypanosomiasis (Chagas' disease). It is proposed that a collaborative programme be estab- lished to evaluate the ELISA and to compare this with currently available serological tests for Chagas' disease as well as with the DASS system. In parti- cular, it is envisaged that a field trial of ELISA using both commercial and other available antigen pre- parations be designed. Such a study could be carried out in the near future in Brazil (at the Instituto de Medicina Tropical, Sao Paulo) or combined with current seroepidemiological field studies in several South American countries. The aim would be to determine whether the ELISA is a practical field test. In this context it is necessary that one person experienced in the T. cruzi ELISA technique should visit the Instituto de Medi- cina Tropical for an initial period of about one month. The further development of the programme should be decided only after the completion of this preliminary evaluation. The following institutions have agreed to colla- borate in this programme: Laboratory of Parasito- logy, University of Leiden, Netherlands; Behring- werke AG, Marburg/Lahn, Federal Republic of Germany; and Nuffield Institute of Comparative Medicine, London, England. Malaria. The following recommendations, al- ready made by a WHO Scientific Group,a which a WHO Technical Report Series, No. 579, 1975 (Develop- ments in malaria immunology: report of a WHO Scientific Group). met in Geneva from 23 April to 2 May 1975, are endorsed: 1. Studies should be made on the purification and serological properties of antigens that are derived from the specific stages of malaria parasite popula- tions. 2. Studies on the nature and diversity of malarial antigens require an adequate source of supply of plasmodial material. So far as the malarias of man are concerned, the need for new animal hosts and for an improved method of cultivation are clearly indi- cated. 3. New and more sensitive tests such as radio- immunoassay and ELISA should be further devel- oped and evaluated, particularly when specific anti- gens become available. * * * A. A. Buck, Division of Malaria and Other Parasitic Diseases, World Health Organization, Geneva, Switzerland. A. Davis, Division of Malaria and Other Parasitic Diseases, World Health Organization, Geneva, Switzer- land. A. M. Deelder, Parasitology Laboratory, University of Leiden, Leiden, Netherlands. B. Enders, Behringwerke AG, Marburg/Lahn, Federal Republic of Germany. M. Faure, Chef du Service de Biochimie des Antiganes, Institut Pasteur, Paris, France. R. Grasbeck, Director, Minerva Institute for Medical Research, Helsinki, Finland. J. Haworth, Division of Malaria and Other Parasitic Diseases, World Health Organization, Geneva, Switzer- land. N. H. Kent, Division of Malaria and Other Parasitic Diseases, World Health Organization, Geneva, Switzer- land. G. Marcoullis, Minerva Institute for Medical Research, Helsinki, Finland. A. W. Senft, Division of Biological and Medical Sciences, Brown University, Providence, RI, USA. G. Vicari, Chief, Immunochemistry Laboratory, Istituto Superiore di Sanita, Rome, Italy. A. Voller, Head, WHO Collaborating Centre for Malaria Reference and Research on the Development of Sero- logical Techniques, Nuffield Institute of Comparative Medicine, London, England. N. Weiss, Swiss Tropical Institute, Basle, Switzerland. MEMORANDA Annex RECOVERY OF SCHISTOSOMA AND ONCHOCERCA FOR ANTIGEN PREPARATION RECOVERY OF SCHISTOSOMA At present there appear to be no significant differences among the techniques used for the recovery of the various Schistosoma species from small rodents. Typical recovery procedures for adult worms or eggs are therefore de- scribed in this Annex. Adult worms Recovery of adult worms from the golden hamster is described below as an example of the procedures used. 1. Recovery of worms is generally initiated about two weeks after the onset of egg production in any species. However, certain developmental or time variations should be noted to ensure optimal recovery. Time of recovery in a golden hamster is about 45 days after exposure to infection for S. mansoni and 65 days for S. haematobium. 2. Perhaps the most common perfusion technique consists of sodium pentobarbital anaesthesia adminis- tered intraperitoneally, followed by opening of the portal vein, perfusion via the dorsal aorta with citrated saline, and removal of the worms from both the portal system and the liver. 3. It is felt that the above technique should be modified to include physiological perfusion fluids composed of balanced salts, amino acids, and glucose in order that the worms recovered may be kept in optimum physiological condition. Perfusion at 37°C may be preferable to collec- tion at ambient temperature, particularly when recovery of exoantigens or secreted antigens is desired. 4. Most previous recovery procedures have empha- sized thorough washing of worms in order to remove host antigenic materials. However, excessive rinsing may be undesirable if recovery of secreted or regurgitated anti- gens is the object. In this case, the wash fluid could be usefully added to a lyophilized preparation. 5. While it is recognized that axenic techniques are difficult to perform, the use of special sterile recovery techniques will nevertheless have to be considered for long-term maintenance, e.g., in excess of 4 hours. Mul- tiple rinsing of the parasites in sterile fluids and the use of a laminar flow hood will usually suffice. In special situations antibiotics at a low concentration may have to be added. Storage. For the preservation of all known enzymatic and antigenic activity, storage at low temperature (-20 to -78°C) appears adequate. Lyophilization, however, may present the risk of possible protein denaturation or loss of enzyme activity; suggested guidelines are therefore given, as follows: (a) The initial freezing procedure should be extremely rapid. A new technique that appears to be helpful is the Leybold-Heraeus liquid N2-jet apparatus. (b) Care should be taken to achieve extremely low water and salt content, and to avoid bacterial contamina- tion of the product. (c) Until the long-term survival characteristics of the product are fully known, storage in carefully sealed ampoules at -20°C or lower would be advisable. (d) Special protein or enzyme stabilization techniques may have to be considered, such as storage in glycerol or sodium glutamate. (e) As regards short-term transportation between labo- ratories, it is believed that despatch at ambient tempera- ture does not significantly alter potency. (f) At the present time it is suggested that lyophiliza- tion and storage in glassware may be preferable to the use of plastics, in which chemical reactions or adsorption problems may be encountered. (g) Complete information regarding recovery proce- dures should always be indicated on the label of the preserved material. Eggs There are several well-known methods for recovering cleaned eggs from the liver and/or gut of infected animals, the most common being as follows: 1. Flotation recovery of eggs by means of detergents and separation by saline density gradient (Fig. 1). The following procedure is used: (a) Homogenization of tissues at room temperature in 17-g/litre saline solution with a small amount (8-10 drops) of Triton X-100. (b) Passage of the homogenate through graded sieves. (c) Transfer to sidearm flask. (d) Sublayering with higher density (20 g/litre) saline solution. 2. Homogenization and trypsinization of egg-contain- ing tissues followed by sieving or centrifugation. The following procedure is used: (a) Homogenization of livers in Dulbecco's balanced salt solution. (b) Passage of the homogenate through graded sieves. (c) Incubation of the mixture for 1 h at 37°C in Dulbecco's solution containing trypsin, with a magnetic stirrer. Penicillin and streptomycin may be added. (d) Either passage of the mixture through a sieve with a mesh that retains the eggs, or centrifugation. 248 PARASITE ANTIGENS Homogenate of liver and intestine with Triton X-100 in 17 g/l line solution Egg Fig. 1. Flotation recovery of Schistosoma eggs. ,Glass plate Nylon sieve or net for / n snails (netting catches the snail fecal material) 1°°0| / Siliconized glassware \50 1/ l///Cercariae WHO 75950 Fig. 2. Recovery of Schistosoma cercariae from the snail intermediate host. After recovery, the eggs are generally washed free with saline solution and then stored according to the proce- dures followed for adult worms. To prevent hatching, the eggs are stored in 1 5-g/litre saline solution and main- tained at a moderately low temperature (4°C). Miracidia A number of procedures have been described for hatching eggs from aqueous faecal preparations, from homogenized livers, or from clean egg concentrates. The use of egg concentrates is recommended. Eggs can be hatched in either sterile aerated tap water, or possibly filter-sterilized aquarium water. Special techniques for the illumination and handling of recovered miracidia have been well described in the literature. The hatching fluid itself, because of its non-constituent antigens, may be an important factor. Sporocysts Only a few laboratories are currently studying sporo- cysts and their value as a source of antigen seems limited. Cercariae About 10 snails, 5-6 weeks after exposure to miracidia, are placed for the shedding of cercariae in a vessel having a limited volume (100-300 ml) (Fig. 2). Cercariae are not centrifuged but are sedimented in a refrigerator at 40C, after which the excess fluid may be removed. Exoantigens There is increasing interest in special metabolic anti- gens, notably enzymes recovered in vitro from schisto- somes. Among these are membrane antigens (e.g., those recovered by saponization of worms) or regurgitant materials (e.g., haemoglobin-proteolytic enzyme). The use of special worm handling techniques (e.g., temperature shock) to increase regurgitation of intestinal contents may be valuable. In addition, the serum and urine of the infected host may be a useful source of such antigens. RECOVERY OF ONCHOCERCA Onchocercal nodules are recovered from patients from endemic areas undergoing nodulectomy. The nodules, after being washed, are immediately placed in plastic tubes and frozen. They are then shipped in liquid nitrogen to a central parasitology laboratory. The nodules are dissected after thawing, and the adult worms separated, washed, and immediately lyophilized. The lyophilized material is used for further extractions. Other suggested methods for the preparation of raw material are: (a) nodules are placed directly in plastic tubes containing a saturated solution of ammonium sulfate or glycerol and shipped; and (b) the worms recovered from the nodules are similarly placed in a saturated solution of ammonium sulfate or glycerol. For these two methods no cold storage or cryogenic container is required. 2490