Journal qf Chromatography, 440 (1988) 461472 .

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

CHROM. 20 255

ENTAMOEBA HZSTOL YTZCA ANTIGENS AS POSSIBLE VACCINOGENS?

A SHORT REVIEW

SOHAIL AHMAD*, HARIS M. KHAN, ABBAS A. MAHDI and HEMANT KUMAR

Parasitic Immunology Laboratory, Department of Microbiology, J.N. Medical College, Aligarh Muslim University, Aligarh (India)

and

NAZOORAKHAN

Department of Pathology, J.N. Medical College, Aligarh Muslim University, Aligarh (India)

SUMMARY

A few key papers which have recently been published on the characterization of amoeba antigens are reviewed. Immunofluorescence tests and immunoelectron microscopy have demonstrated the localization of certain surface antigens on axen- ically cultured trophozoites. Most of the surface antigens have largely been shown to elicit a humoral response. The elicitation of cellular response has not been well illustrated. The localization of a large number of antigens in cytoplasmic vacuoles and plasma membrane indicates that a greater stimulus to the host would be provided by intracellular antigens than by those located on the surface of amoeba trophozoites. In a few inoculation studies, amoeba antigens, in combination with several adjuvants, have been successfully employed for inducing protective immunity in various animal model systems. These and other results clearly demonstrate that amoeba antigens are fully capable of generating humoral and as well as CM1 responses. A combination of these two effector limbs of immunity can be fully exploited through effective use of future vaccines.

The identification, isolation, purification and characterization of parasite an- tigens is currently a major research activity in the field of immunoparasitology. Puri- fication of parasite antigens is increasingly important in view of their possible role as vaccinogens. Equally important is their role in the immunodiagnosis of parasitic infections, and in the study of the host’s immune responses. Needless to say, in the absence of properly defined antigens, more exact studies of certain important im- munological parameters of the host-parasite interaction would not be possible. In view of the recent advances in the field of immunology and with the advent of new technologies in the life sciences, parasitic infections no longer enjoy the dubious distinction of being the “great neglected diseases” of the world. For example, over the past 10 years substantial progress has been made in understanding the complex nature of a large number of protozoan parasites, particularly malaria and those which

0021-9673/88/$03.50 0 1988 Elsevier Science Publishers B.V

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. S. AHMAD et al.

give rise to other blood-borne infections such as babesiosis and trypanosomiasis’. However, Entamoeba histolytica though no less important than its more cele-

brated fellow protozoans, as far as its endemicity is concerned, has not received as much attention. The parasite is the causative agent of amoebiasis, a complex symp- tom which is more maligned than understood. Amoebiasis is a serious problem in several underdeveloped countries, and its worst feature is that it strikes the people on lower incomes the hardest. Lack of sanitary toilets and poor availability of good, clean drinking water are obviously the dominant factors in its endemicity. Unlike malaria it does not always kill its victims but it is certainly a serious health problem. Amoebiasis is debilitating and causes a tremendous loss of working days and eco- nomic productivity in countries whose economies are already overburdened with other more urgent problems.

The parasite E. histolytica has a conventional life cycle. It is found both in trophozoite and cystic stages, each stage complementing the other as far as perpet- uation of the species is concerned. The infection is acquired following ingestion of hardy cysts through faecally contaminated water and food and tends to be more chronic than acute. Chemotherapy has been successfully employed though it may not be a permanent solution to the problem as reinfection is common. Treatment of asymptomatic cyst passers is not economically feasible.

The immunologic control of this parasitic infection is certainly difficult if not impossible. However, in view of the tremendous advances recently made in that up till now somewhat hazy area of parasite immunology, the development of suitable strategies for achieving an immunologic control may not be altogether impossible. The causative agent, being a complex protozoan parasite, poses certain intrinsic prob- lems to immunologists, who are generally obsessed for various reasons with the purity of antigenic isolates, their identification and characterization. By and large, the para- sitic amoebae do not easily lend themselves to such manipulations, scrutiny and analysis. However, research is continuously going on to unravel the complexities of parasite antigens, in order to recognise possibly some of those immunologically rel- evant components which may be more pertinent to our immediate goal, the identi- fication of protection antigens. Trissl’? recent review of the immunology of amoe- biasis in human and animal hosts gives a good idea of the tremendous amount of work which has already been done in this area of human endeavour. More recently, further attempts have been made to characterize the amoeba antigens on the basis of their immunogenicity in different animals. With regard to parasitic antigens, no generalizations can be made, as nearly always it is the specifics of a particular host-parasite interaction which are often more compelling. For amoebiasis, the dif- ferences in host susceptibilities and the complexities of the intestinal environment makes each model system an unique case for study.

The purpose of this communication is to review briefly’a few key papers which have recently been published on the characterization of amoeba antigens. Some early attempts toward characterization of physicochemical properties of amoebic antigens through electrophoretic analysis of purified fractions clearly demonstrated the het- erogeneity of antigenic components 3,4. About fourteen precipitin bands have been clearly demonstrated in antigen-antibody pattern by using immune sera from dif- ferent geographical regions of the world5. Later, Chang et ~1.~ by using two-dimen- sional immunoelectrophoresis were able to demonstrate 32 precipitin bands. The

ENTAMOEBA HISTOL YTICA ANTIGENS AS POSSIBLE VACCINOGENS? 469

above studies are clearly demonstrative of a large number of common antigens shared by different strains of amoebae. More likely, a major share of this antigen contingent is represented in many of those humoral responses which are so readily detectable by several immunodiagnostic tests. Numerous studies have provided some idea of the location of these antigens. For example, microsomal fractions following disrup- tion of organs give a good yield of subcellular antigens7. The view that significant antigens are located in the cytoplasm was later confirmed by McLaughlin and Mee- rovitch*. Such antigens are more likely associated with vesicles of lysosomal origin. The ribosomal, as well as lysosomal, fractions have been successfully employed to obtain purified antigen samples9. Immunofluorescence tests and immunoelectron microscopy have demonstrated the localization of certain surface antigens on axen- ically cultured trophozoiteslO. Calderon et al . l l later demonstrated a capping phe- nomenon induced by antibodies. About twelve glycoproteins have been identified in isolated fractions of plasma membrane showing a complex chemical composition l 2. Most of the surface antigens have largely been shown to elicit an humoral response. However, the elicitation of cellular response has not been too well illustrated. The localization of a large number of antigens in cytoplasmic vesicles and plasma mem- brane indicates that a greater stimulus to the host, in order to mount an immune response, would be provided by intracellular antigens than by those located on the surface of amoeba trophozoites. However, a clear delineation of these antigens is still not possible due to a rapid turnover of membranes through endocytosis and exo- cytosis. The immune response from the majority of these antigens is perhaps more of serodiagnostic value, as protective immunity to E. histolytica infection is most effectively induced by a select few, high-molecular-weight antigen fractionsi3.

In a few inoculation studies, amoeba antigens in combination with several adjuvants have been successfully employed for inducing protective immunity in var- ious animal model systems. Sharma et al. I4 have shown that al,3 polyglucose or glucan, a cell wall extract of Saccharomyces cerevisiae, can be combined with amoebic antigens for immunization of guinea pigs against E. histolytica infection. The pro- tective immune responses obtained following immunization with amoeba antigens and glucan as an adjuvant partner (immunopotentiator) demonstrated that induction of immunity can be achieved in laboratory animal model systems. Similar results were also obtained in rabbits following immunization with soluble E. histolytica an- tigens along with an aqueous suspension of trehalose-6,6’-dimycolate (TDM)’ 5. The TDM-enhanced resistance to E. histolvtica in this study was most likely related to the ability of TDM to enhance macrophage activity. In yet another study16, macro- phages have been clearly shown to contribute significantly in increasing the host resistance to hepatic amoebiasis. These and other results clearly demonstrate that amoeba antigens are fully capable of generating humoral and as well as cell mediated

l 7 immunity (CMI) responses . A combination of these two effector limbs of immunity can then be fully exploited to confer patent immunity to a host through effective use of protective vaccines. The literature on amoebiasis is replete with evidence showing the development of protective CM1 responses following amoeba antigenic stimula- tion. In human amoeba infections of the liver, low reactivity of skin responses and poor macrophage inhibitor factor production generally correlates well with an active disease statei8. Once the patient is cured such responses tend to be elevated. In one study the CM1 response was shown to be more definitely associated with the devel-

470 S. AHMAD et al.

opment of protective immunity . I9 However, the types of protective immune mech- anisms which could be directly responsible for blocking the invasion of parasites are not well defined. Similarly, it is also not clear whether the effector mechanisms that kill amoebae in vitro are equally effective in vivo. What is more clear is that E. his- tofytica has a wide range of aggressive factors which are brought into action, causing tissue damage in the hostzO. Whether the interaction with the host’s immune defence mechanism would yield to such immunologic measures which help us in devising an effective way of preventing the disease is not entirely clear either.

More recently, the unique homogeneity of monoclonal antibodies has made them an attractive tool for characterizing the antigenic composition of a large number of disease-producing agents. In amoebiasis also, hydridoma technology has been put to use in identifying a specific epitope present on the membrane of E. histoZytica*‘. The specific monoclonal antibodies were able to recognize three plasma membrane polypeptides of various molecular size. Calderon and Avilaz2 showed the presence of antibody-induced caps in E. histolytica which were composed mainly of mem- branes and contained five major [35S]methionine-labelled bands. Surface immuno- precipitates contained about twelve major [35S]methionine-labelled polypeptides, five of which had molecular weights similar to those of radiolabelled polypeptides in the cap. These results suggested that only half of the membrane surface-immunoprecip- itated antigens were enriched in the cap. The E. histolytica trophozoites have been shown in vivo to possess a 30 mm thick glycocalyx coat. The r6le of this glycocalyx in stimulating the immune response, or protecting the parasite, is unclear.

About eighteen proteins have so far been identified in purified E. histolytica membranes12. The presence of structures on the surface of the parasite that contain an high concentration of mannose residues has been demonstrated following agglu- tination with concanavalin A 23 However, in spite of these findings the antigenic . components of the parasite surface membrane, which are likely to be integral in provoking the host’s immune defence mechanisms in the prevention of a recurrent disease, need to be more clearly defined. Torian et a1.24 have attempted to define the specific surface antigenic moieties through use of monoclonal antibodies. They were able to characterize and purify a 96 OOO-dalton surface antigen from a pathogenic strain of E. histolytica. Protective immunity in invasive amoebiasis is more likely to be directed against surface constituents, some of which must be thoroughly tested for use as candidate vaccines. The identification of the 96 OOO-dalton polypeptide will probably be helpful in investigating its possible role as a vaccinogen. Joyce and Ravdinz5 through use of polyclonal sera from patients cured of amoebic liver ab- scesses have recognized in western blots four surface antigens of whole trophozoites. One of these antigens had a mass of 90 000 daltons. Since protective immunity against amoebae is logically directed against surface constituents, the surface location of a 90 000 or 96 000 dalton antigen makes it a likely target for immunologic attack. Ravdin et al.26 have shown that mouse monoclonal antibodies can be successfully used in inhibiting in vitro adherence of E. histolytica trophozoites. In an earlier study2’, mapping of immunologic components, by analyses of a whole amoeba ex- tract and its fractions and of antigen-antibody precipitates, showed a variety of active antigens in a wide range of molecular weights 150 000-9000 daltons. Autoradiog- raphy after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) analysis of r251-surface-labelled amoebae revealed more than twelve bands,

ENTAMOEBA HISTOL YTZCA ANTIGENS AS POSSIBLE VACCINOGENS? 471

of which eight corresponded to those recovered from soluble extracts. Considering the pronounced phagocytosis and rapid turnover of cell membrane in E. histolytica,

it seems more likely that the same antigenic components would be represented both on and within the amoeba at different stages. Furthermore, membrane antigens have been shown to activate complement via alternate and classical pathways leading to amoeba cell deathz7. The process demonstrated by these authors appears to play an important inflammatory role in invasive amoebiasis following deeper antigenic stimu- lation. Gitler et ~1.~~ have identified an highly immunogenic surface lipid involved in the rapid surface redistribution of immune complexes, their shedding and endo- cytosis. This finding is of particular relevance to the immunoprophylaxis of amoe- biasis.

The efforts so far made to obtain purified parasite antigens are highly com- mendable. Because of the various inherent problems mainly due to the complexity of organisms, it is by no means an easy task to isolate and purify parasite proteins, for subsequent use in inoculation experiments. The probability of developing an ef- fective vaccine against a complex, adaptable and a genetically diverse parasite is clearly minimal if we have a limited array of antigens at our disposal. A larger choice of defined parasite antigens will certainly make it easier to develop strategies for immunologic control of naturally infected hosts, which vary in susceptibility to in- fection from chronic asymptomatic cyst passers to acutely ill symptomatic patients. The prospects for identifying host-protective antigens were never brighter than now, especially in view of the tremendous advances recently made in the field of immu- nology and biotechnology. Undoubtedly, there are better chances of developing a parasite vaccine today than ever before.

As the field of immunoparasitology develops further, it can be anticipated that the molecular immunoparasitologists will not only be using the advances made in other fields, but will themselves be devising novel approaches for the isolation and analysis of immunologically relevant antigens from complex mixture of molecules, the parasites.

ACKNOWLEDGEMENTS

The authors feel greatly privileged in having had the opportunity of collabor- ating in amoeba inoculation studies with Professor Edger Lederer, in recognition of whose outstanding contribution this article is specially written.

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