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BSI symposium on parasite antigens and vaccination
288
Parasitology Today, vol. 4, no. I0, 1988
BSI Symposium on Parasite Antigens and Vaccination K.C. Carter and J.E. Smith British Society for Immunology July 1988, Glasgow Vaccination is now an accepted strategy for control of parasitic protozoan diseases. Speakers at the British Society for Immunology (BSI) summer meeting at Strathclyde University reviewed the current status of research on vaccines for malaria, Toxoplasma, Theileria, Babesia, Leishmanio and trypanosomes. With malaria trials for the first of the 'newgeneration' vaccines underway we are beginning to see a new realism in approaches to selection of target antigens and expectations of control. There was a uniform opinion that the main aim of immunization is to minimize clinical episodes of disease and morbidity rather than to create sterile immunity. Target antigens had been selected and characterized in molecular terms but there was a paucity of knowledge on how best to present these antigens to elicit cellmediated immunity in addition to humoral responses.
Malaria Three main stages of the malaria life cycle are open to immunological attackthe infective sporozoites, the asexual bloodstages, and the stages present within the mosquito vector. Elizabeth Nardin (NYU, New York) reported that when conjugated to tetanus toxoid the (NANP)3 peptide, derived from the circumsporozoite (CS) protein, produced a level of immunity similar to that obtained with whole irradiated sporozoites. In man this antigen awarded some protection against sporozoite challenge but a more effective adjuvant was needed to reduce the risk of serum sickness. A more significant problem however, was that the peptide contained no T-cell epitopes and would therefore not induce memory. Trials with recombinant CS protein resulted in effective protection and memory in mice, whereas similar studies in squirrel monkeys using adjuvants showed that protection was not solely mediated by antibody. In the supporting programme, J.R.L. Pink (Basle, Switzer-
land) and M.J. Lockyer (Wellcome Foundation, London) confirmed the existence of T-cell epitopes on the CS protein but cautioned that strain-specific polymorphism of these regions would abrogate memory produced by vaccination against one strain. The pathology of malaria is associated with asexual parasitaemia. Among the symptoms, cerebral malaria caused by cytoadherence of parasites in the brain, is the most severe. At present, no adequate model exists to identify the immunological mechanisms that limit the pathology in infected individuals. Strains that display cytoadherence lose this ability when cultured in vitro, and in animal models such as Plasmodium chabaudi, parasites are sequestered in the liver rather than the brain (C. Newbold, John Radcliffe Hospital, Oxford). Many mechanisms, including antigenic variation of proteins expressed at the surface of the red cell and antigenic overlap between discrete parasite proteins, impair the development of effective immunity. However, the ability to survive a malaria attack does increase with successive infections. Until these important in viva mechanisms that limit pathology are identified, the performance of vaccines in reducing morbidity rather than producing sterile immunity cannot be evaluated. An alternative approach to vaccine development is the production of transmission-blocking vaccines, to be given in conjunction with a sporozoite or asexual vaccine (R.E. Sinden, Imperial College, London). These would kill vector stages of the life cycle and prevent malaria transmission. Priming of mice with a 21 kDa antigen, expressed on the surface of the P. berghei macrogamete and zygote, induced a high antibody titre. At concentrations above 100 gg/ml, this antibody completely blocked transmission of the parasite to mosquitoes, but as the antibody level fell below 40 gg/ml transmission was actually enhanced by up to 250%. A further caution was added with regard to the parallel molecule in P. falciparurn. Gene sequence data revealed the existence of four redundant repeats homologous to the human low density lipoprotein (LDL) receptor. This raises concern that vaccination may generate autoimmunity.
Molecular Mimicry D. Russell (NYU, New York) presented some exciting data on the promastigate surface protease - GP63 - the prime vaccine candidate against cutaneous leishmaniasis. In vitro studies had shown that a monoclonal antibody raised against this antigen prevented the penetration of macrophages by promastigotes. By monitoring the uptake of purified GP63/HPLC bead conjugate in macrophages depleted of specific receptors, it was shown that the receptor for GP63 was the complement receptor CR3. Further DNA sequence data revealed a region encoding for an RGD tripeptide (arginine-glycineaspartic acid) homologous to the binding region of complement C3bi. Thus the promastigote may employ molecular mimicry to facilitate entry of the host cell. Alternatively, if the RGD region is common to related protozoa, we may have an example of evolutionary conservation. A second example of molecular mimicry may exist in the parasite Toxoplasma gondii. Immune sera from patients and infected animals recognise four major antigens on the surface of the tachyzoite (20, 30, 35 and 42 kDa). Passive transfer experiments with a monoclonal antibody raised against the 30 kDa antigen (p30) significantly increased survival in mice challenged with RH-strain parasites. The monoclonal antibody was found to have invasion-blocking activity in vitro. Administration of the antibody to pregnant mice, in an attempt to protect against congenital infection, gave surprising results since abortion or foetal resorption occurred. Minute doses of antibody caused abortion even in uninfected mice suggesting that there may be homology between p30 and the trophoblast membrane.
Invasion Blocking Antibodies Surface antigens, implicated in the process of invasion, have been selected as candidate vaccines for two major cattle diseases, babesiosis and theileriosis. A monoclonal antibody against Theileria annulata sporozoites inhibited lymphoid) 1988,ElsevierPublications,Cambridge O1694758/88/$02.00
ParasitologyToday,vol. 4, no. I O, 1988 cyte invasion by 80% and schizont development by 30% in vitro (R.H. Hall, Glasgow University).This antibody was used to screen an expression library, and two recombinant proteins of 147 kDa and 135kDa (inserts 30kDa and 20 kDa) were cloned. Antisera raised in rabbits immunized with the 135 kDa protein recognized the native antigen and could cause 100% inhibition of sporozoite penetration in vitro. In Babesia divergens a 24 kDa antigen on the merozoite surface is involved in invasion (C. Winger, Imperial College, London). Purified antigen injected with Freund's adjuvant induced a strong antibody response and conferred some resistance against lethal infection in gerbils. Acetone precipitation of antigen, saponin and liposomes was used in an attempt to improve immunogenicity. Enhancement was obtained only with saponin which elevated antibody levels tenfold over controls and conferred total protection against lethal challenge.
289 A repeat experiment failed, possibly because of the variation in virulence of different batches of parasites - a problem which seems to dominate work with this parasite.
Trypanosomes There are major problems with the development of vaccines against trypanosomes. In Chagas disease there is extensive cross-reaction between human and parasite antigens resulting in chronic autoimmune disease. This means that candidate molecules for vaccination must be selected with extreme caution. G. Takle (St Georges Hospital Medical School, London) described a molecular approach to identify stage-specific protective antigens on the trypomastigote of Trypanosomo cruzi. Two trypomastigote-specific DNA clones have been selected and expressed as fusion
proteins in Escherichia coil and are currently being characterized. In African trypanosomiasis, bloodform trypomastigotes escape immune attack by means of antigenic variation. Metacyclics have fewer variable antigen types (27 are known at present) but current research shows that the repertoire is not stable and new types arise spontaneously (C.M.R. Turner, Glasgow University). A transmission-blocking vaccine which would act against the naked procyclic form now seems unlikely as procyclics develop in the rear of the tsetse midgut where antibodies are inactivated. When asked to give his opinion on the prospects of obtaining a vaccine against African trypanosomiasis Dr Turner replied 'there are none'.
Katharine Carter is with the Immunology Division~Pharmacy Department, Strathclyde University, GlasgowG4 ONR,UK.]udith Smithis at the Department of Pure and Applied Biology, Universityof Leeds,LS29IT, UK.
Viruses of Parasitic Protozoa M.A. Miles Associations are increasingly recognized between parasitic protozoa and smaller organisms such as bacteria (see Parasitology Today I, 143), and viruses. Their revelation, originally by ultrastructural studies, may now be indicated by observations of'rogue bands' on gel electrophoresis of parasite nucleic acids. The origins and roles of parasite 'parasites' are still uncertain, but, as Michael Miles suggests here, they may prove of particular importance as agents to introduce genes for important immunogens into non-pathogenic protozoa for vaccine delivery. Early reports of viruses in parasitic protozoa were almost entirely based on ultrastructural observations. Virus-like particles (VLPs) have been described in Entamoeba, Naegleria, Leishmania, Endotrypanum, Trypanosoma, Plasmodium, and others I - 5 (see Fig. I ). Such tantalizing glimpses need cautious interpretation v and left many virologists and molecular biologists sceptical - were they real, transmissible infectious agents? Only for E. histolytica, where filamentous, polyhedral and beaded particle morphologies had been described, was there evidence that particles were transmissible and lytic to susceptible amoebal strains ~. Doubts that protozoan viral infections exist have finally been dispelled by the purification and characterization of an RNA virus from Giardia 7-9. In addition, sensitive techniques of electrophoresis are revealing small nucleic acids suggestive of further RNA or DNA viruses. Protozoan viruses are not simply of academic interest but offer exciting new ways for the dissection and manipu~ ) 1988, Elsevier Publications, Cambridge 0169~t758/88/$02.00
tation of the parasite genome, and for the production of parasite antigens. The consistent presence of a minor 7 kb band after agarose gel electrophoresis of nucleic acids provided the first evidence of the Giardia intestinalis (lamblia) virus (GLV). Experiments suggested the band consisted of RNA - it was absent from samples pre-treated with alkali or boiled ribonuclease A, but was unaffected by enzymes that degrade DNA 7'8. Electron microscopy revealed spherical (37 nm) VLPs in the nuclei of. sectioned trophozoites, in cell fractions and also free in the culture media. A single major viral protein had an estimated molecular mass of 100kDa (although in earlier experiments a presumed contaminant was seen at 66kDa) 9. Prior to treatment with sodium dodecyl sulphate or proteinase K, the RNA was protected against the action of ribonuclease A - perhaps by the I00 kDa protein. Electron microscopy of the purified RNA preparations showed linear double-stranded struc-
tures of approximate length 1.5 I~m. The radiolabelled RNA (3' end-labelled using T4 RNA ligase) failed to hybridize to Southern blots of G. intestinalis DNA, and this was considered to indicate absence of any sequence homology 9. In two similar studies, GLV was sought among 38 Giardia strains from different hosts (man, cat, guinea-pig, beaver, llama, sheep)from several countries 8't°. In one survey 18 of 38 strains were found to be infected 8, in the second ten of 38 strains I°. Interestingly, one Portland strain (Portland-I-CCW) from which the original discovery was made, carried GLV whereas another (Portland-I-ATCC30888) did not. GLV could be transmitted by filter sterilized medium (in which it did not replicate); some Giardia strains were highly susceptible but others were totally resistant even at l0 6 viral particles per trophozoite. In susceptible strains, GLV reached 5 x l 0 S particles per cell and although the trophozoites did not tyse with these high infections they failed to adhere, and growth ceased. GLV could not be transmitted to Trichomonas vaginalis, Tritrichomonas foetus, Trypano. soma brucei or Leishmanla major . Antlsera raised in mice to the GLV I O0 kDa protein could both block (I in 104 dilutions) and eliminate (I in I0 3 dilutions) GLV infections, although the ,
I O
'
Parasitology Today, vol. 4, no. I0, 1988
BSI Symposium on Parasite Antigens and Vaccination K.C. Carter and J.E. Smith British Society for Immunology July 1988, Glasgow Vaccination is now an accepted strategy for control of parasitic protozoan diseases. Speakers at the British Society for Immunology (BSI) summer meeting at Strathclyde University reviewed the current status of research on vaccines for malaria, Toxoplasma, Theileria, Babesia, Leishmanio and trypanosomes. With malaria trials for the first of the 'newgeneration' vaccines underway we are beginning to see a new realism in approaches to selection of target antigens and expectations of control. There was a uniform opinion that the main aim of immunization is to minimize clinical episodes of disease and morbidity rather than to create sterile immunity. Target antigens had been selected and characterized in molecular terms but there was a paucity of knowledge on how best to present these antigens to elicit cellmediated immunity in addition to humoral responses.
Malaria Three main stages of the malaria life cycle are open to immunological attackthe infective sporozoites, the asexual bloodstages, and the stages present within the mosquito vector. Elizabeth Nardin (NYU, New York) reported that when conjugated to tetanus toxoid the (NANP)3 peptide, derived from the circumsporozoite (CS) protein, produced a level of immunity similar to that obtained with whole irradiated sporozoites. In man this antigen awarded some protection against sporozoite challenge but a more effective adjuvant was needed to reduce the risk of serum sickness. A more significant problem however, was that the peptide contained no T-cell epitopes and would therefore not induce memory. Trials with recombinant CS protein resulted in effective protection and memory in mice, whereas similar studies in squirrel monkeys using adjuvants showed that protection was not solely mediated by antibody. In the supporting programme, J.R.L. Pink (Basle, Switzer-
land) and M.J. Lockyer (Wellcome Foundation, London) confirmed the existence of T-cell epitopes on the CS protein but cautioned that strain-specific polymorphism of these regions would abrogate memory produced by vaccination against one strain. The pathology of malaria is associated with asexual parasitaemia. Among the symptoms, cerebral malaria caused by cytoadherence of parasites in the brain, is the most severe. At present, no adequate model exists to identify the immunological mechanisms that limit the pathology in infected individuals. Strains that display cytoadherence lose this ability when cultured in vitro, and in animal models such as Plasmodium chabaudi, parasites are sequestered in the liver rather than the brain (C. Newbold, John Radcliffe Hospital, Oxford). Many mechanisms, including antigenic variation of proteins expressed at the surface of the red cell and antigenic overlap between discrete parasite proteins, impair the development of effective immunity. However, the ability to survive a malaria attack does increase with successive infections. Until these important in viva mechanisms that limit pathology are identified, the performance of vaccines in reducing morbidity rather than producing sterile immunity cannot be evaluated. An alternative approach to vaccine development is the production of transmission-blocking vaccines, to be given in conjunction with a sporozoite or asexual vaccine (R.E. Sinden, Imperial College, London). These would kill vector stages of the life cycle and prevent malaria transmission. Priming of mice with a 21 kDa antigen, expressed on the surface of the P. berghei macrogamete and zygote, induced a high antibody titre. At concentrations above 100 gg/ml, this antibody completely blocked transmission of the parasite to mosquitoes, but as the antibody level fell below 40 gg/ml transmission was actually enhanced by up to 250%. A further caution was added with regard to the parallel molecule in P. falciparurn. Gene sequence data revealed the existence of four redundant repeats homologous to the human low density lipoprotein (LDL) receptor. This raises concern that vaccination may generate autoimmunity.
Molecular Mimicry D. Russell (NYU, New York) presented some exciting data on the promastigate surface protease - GP63 - the prime vaccine candidate against cutaneous leishmaniasis. In vitro studies had shown that a monoclonal antibody raised against this antigen prevented the penetration of macrophages by promastigotes. By monitoring the uptake of purified GP63/HPLC bead conjugate in macrophages depleted of specific receptors, it was shown that the receptor for GP63 was the complement receptor CR3. Further DNA sequence data revealed a region encoding for an RGD tripeptide (arginine-glycineaspartic acid) homologous to the binding region of complement C3bi. Thus the promastigote may employ molecular mimicry to facilitate entry of the host cell. Alternatively, if the RGD region is common to related protozoa, we may have an example of evolutionary conservation. A second example of molecular mimicry may exist in the parasite Toxoplasma gondii. Immune sera from patients and infected animals recognise four major antigens on the surface of the tachyzoite (20, 30, 35 and 42 kDa). Passive transfer experiments with a monoclonal antibody raised against the 30 kDa antigen (p30) significantly increased survival in mice challenged with RH-strain parasites. The monoclonal antibody was found to have invasion-blocking activity in vitro. Administration of the antibody to pregnant mice, in an attempt to protect against congenital infection, gave surprising results since abortion or foetal resorption occurred. Minute doses of antibody caused abortion even in uninfected mice suggesting that there may be homology between p30 and the trophoblast membrane.
Invasion Blocking Antibodies Surface antigens, implicated in the process of invasion, have been selected as candidate vaccines for two major cattle diseases, babesiosis and theileriosis. A monoclonal antibody against Theileria annulata sporozoites inhibited lymphoid) 1988,ElsevierPublications,Cambridge O1694758/88/$02.00
ParasitologyToday,vol. 4, no. I O, 1988 cyte invasion by 80% and schizont development by 30% in vitro (R.H. Hall, Glasgow University).This antibody was used to screen an expression library, and two recombinant proteins of 147 kDa and 135kDa (inserts 30kDa and 20 kDa) were cloned. Antisera raised in rabbits immunized with the 135 kDa protein recognized the native antigen and could cause 100% inhibition of sporozoite penetration in vitro. In Babesia divergens a 24 kDa antigen on the merozoite surface is involved in invasion (C. Winger, Imperial College, London). Purified antigen injected with Freund's adjuvant induced a strong antibody response and conferred some resistance against lethal infection in gerbils. Acetone precipitation of antigen, saponin and liposomes was used in an attempt to improve immunogenicity. Enhancement was obtained only with saponin which elevated antibody levels tenfold over controls and conferred total protection against lethal challenge.
289 A repeat experiment failed, possibly because of the variation in virulence of different batches of parasites - a problem which seems to dominate work with this parasite.
Trypanosomes There are major problems with the development of vaccines against trypanosomes. In Chagas disease there is extensive cross-reaction between human and parasite antigens resulting in chronic autoimmune disease. This means that candidate molecules for vaccination must be selected with extreme caution. G. Takle (St Georges Hospital Medical School, London) described a molecular approach to identify stage-specific protective antigens on the trypomastigote of Trypanosomo cruzi. Two trypomastigote-specific DNA clones have been selected and expressed as fusion
proteins in Escherichia coil and are currently being characterized. In African trypanosomiasis, bloodform trypomastigotes escape immune attack by means of antigenic variation. Metacyclics have fewer variable antigen types (27 are known at present) but current research shows that the repertoire is not stable and new types arise spontaneously (C.M.R. Turner, Glasgow University). A transmission-blocking vaccine which would act against the naked procyclic form now seems unlikely as procyclics develop in the rear of the tsetse midgut where antibodies are inactivated. When asked to give his opinion on the prospects of obtaining a vaccine against African trypanosomiasis Dr Turner replied 'there are none'.
Katharine Carter is with the Immunology Division~Pharmacy Department, Strathclyde University, GlasgowG4 ONR,UK.]udith Smithis at the Department of Pure and Applied Biology, Universityof Leeds,LS29IT, UK.
Viruses of Parasitic Protozoa M.A. Miles Associations are increasingly recognized between parasitic protozoa and smaller organisms such as bacteria (see Parasitology Today I, 143), and viruses. Their revelation, originally by ultrastructural studies, may now be indicated by observations of'rogue bands' on gel electrophoresis of parasite nucleic acids. The origins and roles of parasite 'parasites' are still uncertain, but, as Michael Miles suggests here, they may prove of particular importance as agents to introduce genes for important immunogens into non-pathogenic protozoa for vaccine delivery. Early reports of viruses in parasitic protozoa were almost entirely based on ultrastructural observations. Virus-like particles (VLPs) have been described in Entamoeba, Naegleria, Leishmania, Endotrypanum, Trypanosoma, Plasmodium, and others I - 5 (see Fig. I ). Such tantalizing glimpses need cautious interpretation v and left many virologists and molecular biologists sceptical - were they real, transmissible infectious agents? Only for E. histolytica, where filamentous, polyhedral and beaded particle morphologies had been described, was there evidence that particles were transmissible and lytic to susceptible amoebal strains ~. Doubts that protozoan viral infections exist have finally been dispelled by the purification and characterization of an RNA virus from Giardia 7-9. In addition, sensitive techniques of electrophoresis are revealing small nucleic acids suggestive of further RNA or DNA viruses. Protozoan viruses are not simply of academic interest but offer exciting new ways for the dissection and manipu~ ) 1988, Elsevier Publications, Cambridge 0169~t758/88/$02.00
tation of the parasite genome, and for the production of parasite antigens. The consistent presence of a minor 7 kb band after agarose gel electrophoresis of nucleic acids provided the first evidence of the Giardia intestinalis (lamblia) virus (GLV). Experiments suggested the band consisted of RNA - it was absent from samples pre-treated with alkali or boiled ribonuclease A, but was unaffected by enzymes that degrade DNA 7'8. Electron microscopy revealed spherical (37 nm) VLPs in the nuclei of. sectioned trophozoites, in cell fractions and also free in the culture media. A single major viral protein had an estimated molecular mass of 100kDa (although in earlier experiments a presumed contaminant was seen at 66kDa) 9. Prior to treatment with sodium dodecyl sulphate or proteinase K, the RNA was protected against the action of ribonuclease A - perhaps by the I00 kDa protein. Electron microscopy of the purified RNA preparations showed linear double-stranded struc-
tures of approximate length 1.5 I~m. The radiolabelled RNA (3' end-labelled using T4 RNA ligase) failed to hybridize to Southern blots of G. intestinalis DNA, and this was considered to indicate absence of any sequence homology 9. In two similar studies, GLV was sought among 38 Giardia strains from different hosts (man, cat, guinea-pig, beaver, llama, sheep)from several countries 8't°. In one survey 18 of 38 strains were found to be infected 8, in the second ten of 38 strains I°. Interestingly, one Portland strain (Portland-I-CCW) from which the original discovery was made, carried GLV whereas another (Portland-I-ATCC30888) did not. GLV could be transmitted by filter sterilized medium (in which it did not replicate); some Giardia strains were highly susceptible but others were totally resistant even at l0 6 viral particles per trophozoite. In susceptible strains, GLV reached 5 x l 0 S particles per cell and although the trophozoites did not tyse with these high infections they failed to adhere, and growth ceased. GLV could not be transmitted to Trichomonas vaginalis, Tritrichomonas foetus, Trypano. soma brucei or Leishmanla major . Antlsera raised in mice to the GLV I O0 kDa protein could both block (I in 104 dilutions) and eliminate (I in I0 3 dilutions) GLV infections, although the ,
I O
'