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Conclusions and recommendations

The workshop focused initially on the current status of technologies to identify and characterize potentially protective parasite antigens. Substantial progress has been made in the use of polyclonal and monoclonal antisera to identify and characterize antigens of Plasmodium, Theileria, Anaplasma and Trypanosoma. The advantages and disadvantages of the polyclonal and monoclonal antibody approaches to the identification of potential immunogens were described. Generally, where protective immunity in the host is mediated by antibodies, polyvalent sera from immune animals should be used to identify potentially protective antigens from the various stages of parasites with complex lifecycles. Where a specific stage of the parasite lifecycle, e.g. the sporozoite, has been chosen as a target for a vaccine, the monoclonal antibody approach can be used successfully, as is being shown with malaria and East Coast fever. Where individual antigens have been identified by polyvalent sera, monoclonal antibodies made to the isolated antigens may be used to identify relevant epitopes on the antigen for further structural analysis. While monoclonal antibodies can clearly be used in the identification and characterization of certain parasite antigens, it should be remembered that differences exist in the antigens of a given parasite species recognized by different host species. Monoclonal antibodies produced in the mouse may not always recognize the important immunogenic determinants of the parasite in other host species.

While antigen identification by products of the humoral immune system is now a powerful tool in the development of potential vaccines against certain parasitic diseases, identification of parasite antigens which directly induce protective cell-mediated responses in the host is an important field which still requires further study. This problem is particularly acute in the analysis of cell-mediated responses and the antigens which induce them in outbred populations such as man and domestic animals, as genetic similarity between the inducer and responder populations is required for the induction and expression of cell-mediated immune responses. In such populations the current development of techniques such as histocompatibility antigen typing, cloning and maintenance of immune cells in vitro, transfection of cells in vitro with defined genetic materials and, in domestic animals, the ability to obtain genetically identical siblings by embryo splitting and transfer should in future allow both the definition of parasite antigens relevant to cell-mediated immune protection and the assessment of their ability to induce protective cell-mediated immune responses. It is important that the technologies for identification of parasite antigens responsible for protective cell-mediated responses continue to be developed and refined, as such responses are most likely fundamental to immunity against many parasitic diseases, including East Coast fever.

Following the identification and characterization of potentially protective parasite antigens, the next step in vaccine development involves production of sufficient material for immunization trials. In many instances, due to parasite size and availability, it is difficult if not impossible to isolate sufficient native antigen for this purpose directly from the parasites. The workshop considered two approaches to this problem as applied to antigens which are basically protein in nature. The first involves production of antigens using recombinant DNA technology and the second the in vitro biochemical synthesis of peptides with the relevant antigenic specificity.

The first step in recombinant DNA technology is to identify and isolate the genetic information coding for the relevant antigen or epitope. This can be done in two ways. The first and most direct is to identify and isolate the mRNA coding for the antigen in question and construct cDNA libraries. The second is to digest nuclear DNA with restriction enzymes and construct a genomic library of these fragments, usually in an expression vector such as lambda gt11. The workshop discussed the recent advances in both technologies, especially the improved efficiency in cDNA library construction and the ability of mung bean nuclease to cleave genomic DNA into gene-sized fragments. Both procedures have advantages and disadvantages and the choice between them will be largely determined by factors such as the types of material available for DNA or RNA isolation and present knowledge of the structure of the antigen and its epitopes.

The identification of relevant mRNA or DNA sequences has been greatly facilitated by the use of polyvalent antisera specific for a particular antigen, either by immunoprecipitation of polysomes or screening the products of a genomic library in an expression vector. Polyvalent antisera, because of their ability to recognize multiple epitopes on an antigen molecule, are much more efficient in precipitation and screening assays than monoclonal antibodies.

It can be concluded that recombinant DNA technology provides a practical means of identifying and isolating parasite genetic material coding for potentially protective antigens. In combination with immunoselection, these techniques have already led to the successful identification of relevant genetic material for antigens of Plasmodium, Theileria and Anaplasma.

When suitable genetic sequences have been identified, isolated and characterized, sufficient amounts of the antigen coded for by the genetic material must be obtained in a protective immunogenic form for immunization trials. The selection of suitable expression systems is a complex issue. Factors to be considered include: quantity and purity of the desired antigen, requirements for post-transcriptional and/or post-translational molecular modification and epitope structure. Expression systems are now available using bacteria, yeasts or mammalian cells. Each system has advantages and disadvantages, but the complexity increases when the mRNA or its protein product must be processed to produce an immunogenic product. Suitable expression systems have been successfully developed for large-scale production of viral antigens as demonstrated by the hepatitis B vaccine. It is unlikely, therefore, that the development of suitable systems for parasite antigens will prove to be a major constraint to future vaccine production.

Synthesis of defined peptides is a conventional biochemical technique when a short amino-acid sequence has already been determined. New technologies in micro-sequencing now make it possible to obtain accurate amino-acid sequences from minute quantities of purified parasite protein antigens. In both malaria and cholera, it is now possible to synthesize the short peptides of an immunogenic epitope whose sequence has already been determined. When appropriate immunization protocols have been established, such peptides can induce protective antibody responses. One obstacle to the widespread use of synthetic peptides as immunogens has been the feet that not all epitopes are made up of linear stretches of contiguous amino acids. In certain eases the epitope comprises amino acids which come together in a spatial relationship due to folding of the protein chain, and the amino acids which form the epitope are therefore not linearly associated. The development of a new technology for synthesizing peptides was described which is cheap, efficient and suitable for large numbers of simultaneous synthetic steps. This system has the potential for producing synthetic peptides which specifically combine with the combining sites of monoclonal antibodies and recognize epitopes of protein antigens, for example, of the foot-and-mouth disease virus. The use of such synthetic systems to form peptides which immunologically mimic protective epitopes on parasite antigens should be explored.

At present there do not appear to be any substantial obstacles to the identification and characterization of relevant parasite protein antigens and the genetic material coding for such antigens or their significant epitopes. Nor are there major obstacles to the in vitro synthesis of antigenic materials in quantities sufficient to assess their potential as vaccines in vivo. Indeed, rapid advances are being made in these technologies which make them more widely applicable.

The final step, and ultimately the most important one, is the development of appropriate immunization schedules using in vitro produced materials to produce vaccines against important diseases of man and livestock. The most commonly used adjuvant in experimental models is complete Freund's adjuvant (CFA). This is undoubtedly a powerful adjuvant and in most eases induces good antibody responses even to short peptides. However, since it contains mycobacteria, CFA causes unacceptable reactions at the injection site and sensitizes the host to tuberculosis, so it cannot be used in vaccines applied to man or to domestic livestock where tuberculosis eradication schemes are in place. Alternative strategies include the use of the active moeity of the tuberculin molecule of the mycobacteria which does not have significant side effects, and the use of alternative adjuvanting substances such as alum.

The major problem in the induction of humoral responses using peptide antigens seems to be that such peptides are generally poorly immunogenic by themselves or when combined with possible adjuvants. One approach to this problem has been to link the peptide to an unrelated carrier protein to obtain a modified hapten-carrier effect. In some eases antigens of other important pathogens have been used as carriers to explore the combined vaccine approach. The possibility should not be overlooked that the best carrier for a peptide epitope is some portion of the original parasite protein molecule from which the epitope was identified. It is clear that enhancing peptide immunogenicity with carriers and/or adjuvants is of substantial importance in the development of new and successful vaccines against major parasitic diseases. A better understanding of the epidemiology of these diseases and the nature of the protective humoral responses induced in natural acquired immunity would considerably aid the development of effective vaccines. A great deal of research still needs to be done.

While the development of effective antibody-mediated vaccines can be visualized in the near future, the problems of inducing protective cell-mediated immune responses remain. Only when relevant parasite antigens have been identified and the immunological reactions they induce are defined, can the question of how to induce such responses by immunization be approached systematically.

The workshop closed after considering the usefulness of monoclonal antibodies, DNA probes and DNA analysis for parasite characterization and diagnosis. Considerable progress has been made in these areas, especially in the mayor protozoan diseases such as malaria, trypanosomiasis, anaplasmosis and theileriosis.

It is anticipated that many new reagents and techniques will be developed in the future, but the question remains of adopting the technologies developed in sophisticated laboratories to conditions in the regions where these diseases are significant problems. Encouraging results have been obtained in the substitution of radioactive isotopes by enzyme-linked bases in the preparation of DNA probes. However, there are still problems of the specificity and sensitivity of enzyme-labeled probes which must be resolved, or alternative labeling methods must be devised, before the benefits of these new technologies can be made widely available to the human and animal populations exposed to these diseases.


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