Prospect of vaccination in human malaria*

Bull. Org. mond. Sant) 1974, 50, 287-291 Bull. Wld Hlth Org. Prospects for vaccination against malaria in man* A. CORRADETTI1 Rodents have been successfully protected against a challenge of viable Plasmodium berghei by employing as vaccine: (1) the irradiated blood stages of the same parasite, or (2) the water-insoluble fraction of the blood stages, or (3) irradiated sporozoites; all these vaccines were shown to be stage-specific. A method is outlined for testing in the field, in the absence of any risks, the efficacy of analogous vaccines against falciparum malaria in man. It is emphasized that: (1) the efficacy of the vaccines can be tested in no model but only against P. falciparum in man; (2) the protection should be measured in terms of (a) prevention ofmorbidity, and (b) prevention ofmortality; and (3) research must be intensified in order to meet, as needed, the requirements of mass production of merozoites and/or sporozoites, and to find ways to increase the human immune response to a higher level ofprotection against P. falciparum. During the last decade, a series of investigations have been carried out at the Istituto Superiore di Sanit'a in Rome to establish a scientific basis for different methods of active immunization against Plasmodium berghei malaria in albino rats prior to attempting vaccinations against malaria in man (4-7). The first successful vaccine employed by us was made up of erythrocytic parasites that had been irradiated with X-rays. It was first established that inoculation into rats of parasites irradiated with 20 000 r or more did not result in infection. Rats inoculated with 30 million such irradiated parasites were also seen to be resistant to a challenge by 450000 normal parasites injected 13-23 days after the immunizing inoculation. Such resistance was characterized by (a) an absence of mortality, while 80% or more of the control rats died; (b) a much shorter period of infection; and (c) a much lower level of parasites in the blood compared with that in control rats. This resistance was found to remain unaltered for 120 days after the immunizing inocula- tion, and appeared to be specific to the strain of parasite employed for the vaccine. Wellde and Sadun found that mice vaccinated with irradiated erythro- cytic schizonts of P. berghei also showed resistance to challenge with normal blood parasites (15). Later, another method of vaccination was at- tempted by inoculating the water-insoluble fraction * Presented at the Symposium on Malaria Research, Rabat, Morocco, 1-5 April 1974. 1 Istituto Superiore di Sanita, Rome, Italy. of the blood parasites, fragments of which were obtained by submitting the plasmodia liberated from the red cells to the action of a refrigerated Ribi cell fractionator. This produced a sudden change of pressure from 140 MPa to atmospheric pressure at a temperature of 6°C. The parasites " exploded " and the low temperature permitted fragmentation with- out any protein denaturation. Our vaccination ex- periments in rats using the water-insoluble portion of parasites of P. berghei showed that the vaccine protected against a challenge with the homologous strain by reducing the intensity of the parasitaemia and the duration of the infection, and by maintaining a survival rate, in 3 experiments, of 100%, 100%, and 81.25% compared with total mortality of the control rats. Similar experiments in mice by Nussenzweig et al. employed irradiated P. berghei sporozoites of differ- ent ages. With 5 bites by 30 irradiated mosquitos, having mature sporozoites (18 days) in their salivary glands, they obtained complete protection against a challenge with normal sporozoites. No protection was evident in the immunized animals against a challenge by erythrocytic stages of the parasite. The experiments by Nussenzweig et al. have therefore demonstrated that in the same model, rodent and P. berghei, repeated inoculations of irradiated mature sporozoites prevented infection by normal sporo- zoites (8, 9, 13). The results of all the above vaccination experi- ments in rodents may have some practical implica- tions for vaccination against malaria in man. 3190 -287- 288 A. CORRADETrI It must be recognized, however, that the model P. berghei-albino rat is not only different from " human " plasmodia-man, but is peculiar in rela- tion to the immunological picture, because the rat, like other rodents, is capable of developing a " sterile immunity ", an ability that has not been discovered either in man or in monkeys (1, 2). It is perhaps owing to this difference in the quality and intensity of the immune response that Ward and Hayes failed to protect rhesus monkeys, immunized with irradiated sporozoites of P. cynomolgi bastia- nellii, against the challenge of normal sporozoites (14). Possibly for the same reason, monkeys immu- nized with irradiated erythrocytic stages of P. knowl- esi could not be protected against the challenge of normal blood forms of the same parasite (3). These experiments demonstrate that in the models P. cyno- molgi bastianeiii-monkey and P. knowlesi-monkey the immunity response system of the host is unable to protect against a challenge after vaccination with irradiated parasites. Nevertheless, it is inter- esting to note that Sadun et al. were able to demon- strate in the model P. falciparum and the owl monkey (Aotus trivirgatus) protection against the injection of viable erythrocytic parasites after vac- cination with irradiated erythrocytic forms of the parasite (10). As a consequence of the results obtained in mon- key malaria, it is evident that future investigations on vaccination against malaria in animals will continue to be of scientific interest. They cannot however demonstrate the efficacy in man of vaccines elabo- rated as described above; for testing the quality and intensity of the immune response in human malaria, one must employ malaria parasites in man. Nevertheless, the knowledge acquired from vac- cination of animals will assist in planning suitable vaccination experiments against malaria in man. For a start, there is the fact that in the experiments on P. berghei a stage specificity was demonstrated, so that vaccines made with sporozoites protected against sporozoites, while vaccines made with erythrocytic parasites protected against blood forms. For the objective of preventing malarial infection, vaccination with irradiated sporozoites appears to be preferable to the others. Sporozoites are in fact the plasmodium stage inoculated by mosquitos, and a vaccine specifically protecting against this stage is certainly the best for attacking the parasite at the very moment of its entrance into the human body. Unfortunately from long experience acquired in the epidemiology of human malaria (and chiefly falci- parum malaria, which is the most important), it is difficult to conceive that any vaccination could pre- vent the infection completely. People who have recovered from falciparum malaria are not protected against reinfection with the same parasite. Those living in endemic areas are continuously submitted, during a P. falciparum infection, to superinfections by sporozoites inoculated through the bites of in- fected mosquitos; under these conditions, they often appear, after recovery, to be unprotected against a reinfection in the following months or years. Conse- quently, one is confronted with the problem of how to increase the human immune response to a higher degree than that produced by repeated infections and superinfections, so that vaccination with irradiated sporozoites could prevent P. falciparum infections in man under natural conditions in an endemic area. In this connexion, a recent experiment on human vol- unteers by Clyde et al. appears promising (16)." In order to establish definitely whether or not complete protection against malarial infection is obtainable by vaccination with irradiated sporo- zoites one could try to vaccinate some people from nonmalarious areas who for reasons of work or pleasure must reside for some time in areas where P. falciparum is prevalent. Since the sporozoite vaccine appears to be relatively harmless when ad- ministered through mosquito bites, it could be administered on a similarly reasoned basis to that found acceptable for other vaccines of uncertain efficacy (e.g., cholera vaccine). The vaccinated sub- jects could then be followed up and the efficacy of the vaccine in preventing P. falciparum infection tested. The more limited but very important objective of preventing death from malaria by means of vaccina- tion stands a better chance of success. Owing to stage specificity, this objective can be attained only with vaccines prepared from blood parasites. The two above mentioned vaccines, (a) with irradiated blood parasites, and (b) with the water-insoluble fraction of the blood parasites, both prevented mortality at the challenge in all our experiments on P. berghei and rats. This is a good indication for similar tests on falciparum parasites in man. Bearing in mind that this vaccination should be employed against P. falciparum alone (this parasite being the only one normally capable of causing a They showed that out of three human volunteers one was protected by an irradiated sporozoite vaccine which gave cross-protection against several geographic strains; the two other volunteers were not protected at all and developed a normal falciparum infection. VACCINATION AGAINST MALARIA death), and considering the present difficulty of preparing these vaccines in large quantities, a field trial should be planned in a suitable area where the population is known to have a P. falciparum infection rate of nearly 100% every year. It is well known that in highly endemic areas death from P. falciparum is very frequent in non-immunized subjects, such as babies and children under 3 years old; in many areas it stands as the main cause of infant mortality, since the adults have become relatively resistant owing to the immunity acquired from preceding infections. Consequently, those who are to be protected are the children under 3-4 years old and non-immune people of any age who come from nonmalarious areas. The villages selected for this trial should have their infants and children vaccinated before the malaria season, so that the mortality in this age group could be compared at the end of the malaria season with that of other nonvaccinated infants and children living in villages in similar malarial conditions. Each of the two vaccines mentioned above shows advantages and disadvantages. Preparation of the vaccine made with irradiated parasites is easier, but there is the danger of the presence in the donor's blood of a virus or other microorganism, still infec- tive, despite the dose of irradiation employed for inactivating the malaria parasites. In the vaccine made with the water-insoluble part of the parasites by the action of the refrigerated Ribi cell fractionator, the presence of any living organism is eliminated, but much work and time are required to produce only small amounts of the vaccine. The above brief outline of experiments and trials for testing in man the efficacy of malarial vaccines in order to prevent the disease or to prevent death from the disease shows how complex the problems still are and how far we are from a final solution. CONCLUSIONS Future action in the field of vaccination against P. falciparum infections should be focused on the following: (1) To test in the field, in limited trials, the efficacy of the irradiated-sporozoite vaccine in preventing the disease, and the efficacy of the water-insoluble frac- tion of the blood parasites in preventing serious infections and consequent deaths from the disease. (2) For the last purpose, the vaccine with irra- diated blood parasites, which is much more easily obtainable, should also be tested, provided that a method could be found for sterilizing it from any pathogenic microorganisms that may be present. In this connexion, collateral research should be under- taken to determine the dose of X-rays that would inactivate any living organisms in the donor's blood. Such research is important not only for ensuring the safety of the malarial vaccine, but also for the discovery of new irradiated vaccines for the preven- tion of other diseases. A vaccine against rabies, for example, prepared with irradiated virus, was found to be completely protective as long ago as 1907 (11, 12). This is possibly the first record of protection against an infective disease obtained with a vaccine prepared by irradiation of the specific agent. (3) If vaccination with irradiated sporozoites is completely protective, research on the mass produc- tion of sporozoites should be intensified. If the vaccine does not protect, it should be abandoned for the moment. (4) If vaccination with the water-insoluble fraction of the blood parasite protects from death, research on the mass production of erythrocytic merozoites in vitro should be intensified. If the vaccine does not prevent death from the disease, it should be aban- doned for the moment. Similar action should be applied to the vaccine with irradiated blood para- sites. (5) A series of collateral investigations should be initiated with the objective of increasing the human immune response to P. falciparum antigens deriving from both sporozoites and blood stages of the parasite, in order to try to strengthen the action of these vaccinations, in case they should prove to be unsatisfactory at the present time. UMt PERSPECTIVES DE LA VACCINATION ANTIPALUDIQUE CHEZ L'HOMME En employant comme vaccins: a) des formes erythro- cytaires de Plasmodium berghei irradi6es, ou b) la fraction insoluble dans l'eau de ces parasites, ou c) des sporozoltes irradies, on a obtenu chez des rongeurs une resistance vis-a-vis de l'inoculation de parasites virulents de la meme souche. Les essais de vaccination qui ont et6 effectues ensuite dans les infections A plasmodes des singes et de l'homme 289 290 A. CORRADETTI ont conduit i la conclusion que chaque modele pre- sente son propre comportement. Cependant si l'on veut essayer la vaccination dans le paludisme humain le plus dangereux, il faut employer le modele P. falciparum- homme. On expose ici un projet pour evaluer sur le terrain 1'efficacite de la vaccination, sans aucun danger pour la population. La valeur des vaccins doit etre jugee en termes de pr6vention de la morbidit6 et de prevention de la mortalit6. Des recherches doivent &tre poursuivies pour obtenir la production en masse de m6rozoltes et/ou de sporozoltes, et pour elever artificiellement le degre de resistance a P. falciparum que l'organisme humain a la capacite de produire. REFERENCES 1. CORRADElTI, A. Particolari fenomeni immunitari nell'infezione da Plasmodium berghei. Riv. Parassit., 11: 201-210 (1950). 2. CORRADETIr, A. Acquired sterile immunity in experimental protozoal infections. In: Garnham, P. C. C. et al., ed., Immunity to protozoa. A Sym- posium of the British Society for Immunology. Oxford, Blackwell, 1963, pp. 69-77. 3. CORRADErrTI, A. Comments on vaccination attempts. Proceedings of the Helminthological Society of Washington, 39: 538-539 (1972). 4. CORRADETTI, A. ET AL. Resistenza a Plasmodium berghei da parte di ratti albini precedentemente immunizzati con P. berghei irradiato. Parassitologia, 8: 133-146 (1966). 5. CORRADETTI, A. ET AL. Durata della resistenza del ratto albino all'infezione con Plasmodium berghei (ceppo Istisan) dopo vaccinazione con ceppo omologo irradiato. Parassitologia, 11: 129-134 (1969). 6. CORRADETI, A. ET AL. Tecnica per l'ottenimento della frazione non idrosolubile di Plasmodium berghei in condizioni adatte per studi immunologici. Parassitologia, 11: 145-150 (1969). 7. CoRRADETrI, A. ET AL. Azione immunizzante protettiva della frazione non idrosolubile di Plasmo- dium berghei (ceppo Istisan). Parassitologia, 11: 151-160 (1969). 8. NUSSENZWEIG, R. S. ET AL. Protective immunity produced by the injection of X-irradiated sporo- zoites of Plasmodium berghei. IV. Dose response, specificity and humoral immunity. Milit. Med. 134: 1178-1182 (1969). 9. NUSSENZWEIG, R. S. ET AL. Specificity of protection immunity produced by X-irradiated Plasmodium berghei sporozoites. Nature, 222: 488-489 (1969). 10. SADUN, E. H. ET AL. Resistance produced in the owl monkeys (Aotus trivirgatus) by inoculation with irradiated Plasmodium falciparum. Milit. Med., 134: 1165-1175 (1969). 11. TizzoNI, G. & BONGIOVANNI, A. Intorno all'efficacia del virus rabido scomposto dal radio nella vaccina- zione contro la rabbia. Prime esperienze lette nell'- Accademia delle Scienze dell'Istituto di Bologna, nella seduta del 10 novembre 1907. Memorie dell'- Accademia delle Scienze dell'Istituto di Bologna, Serie VI, vol. 5: 1-20 (1908). 12. TizzoM, G. & BONGIOVANNI, A. I1 radio e la rabbia. Bologna, Zanichelli, 1908. 13. VANDERBERG, J. C. ET AL. Protective immunity produced by the bite of X-irradiated mosquitos infected with P. berghei. J. Parasitol., 56: 351 (1970). 14. WARD, R. A. & HAYS, D. E. Attempted immuniza- tion of rhesus monkeys against cynomolgi malaria with irradiated sporozoites. Proceedings of the Helminthological Society of Washington, 39: 525-529 (1972). 15. WELLDE, B. T. & SADUN, E. H. Resistance produced in rats and mice by exposure to irradiated Plasmo- dium berghei. Experimentalparasitology, 21: 310-324 (1967). 16. CLYDE, D. F. ET AL. Immunization of man against sporozoite-induced falciparum malaria. Amer. J. med. Sci., 266: 169-177 (1973). DISCUSSION BRAY: I doubt whether it is possible, in an immu- nization programme, to achieve a balance between a complete immunization of some children, leaving the nonimmune to face later challenge unprotected, and not immunizing sufficiently, so that lethal attacks are still possible. McGREGoR: This balance is possible and modified immunization represents a real goal. CLYDE: Professor Nussenzweig pointed out that the method of immunizing a host against inoculated sporozoites was conceived as long ago as 1940 by Mulligan and Russell, in India. Credit for making it practicable must be given to the New York University team of which she is a leading member. The method has been applied in man in the US Army malaria programme at the University of Maryland, but the strict ethical constraints within which we VACCINATION AGAINST MALARIA function prevent us from using some of the techniques available to Professor Nussenzweig for her work in rodents and apes. Nevertheless, taking great care to observe all the safeguards fundamental to studies in man, we are obtaining interesting results that may be summarized briefly as follows: (1) Three volunteers have been vaccinated successfully against falciparum malaria. For ethical reasons the immu- nizing X-irradiated sporozoites were inoculated by mosquito bite, not by syringe. No side-effects have occurred. (2) One of these men-a member of our scientific team-has also been immunized against vivax sporozoites. (3) The immunity is species- specific and is effective against a worldwide range of strains within the species; (4) IgM and IgG levels are not affected by the procedure, even when the circumsporozoite precipitation reaction becomes highly positive. (5) Blood ingested by a mosquito feeding on an immunized man fills mature homo- logous-species sporozoites in the salivary glands of that mosquito but does not interfere with devel- oping oocysts. This confirms the work presented by Professor Nussenzweig and has obvious epide- miological importance. (6) The duration of this type of immunity is of great practical significance. The volunteer immunized against both falciparum and vivax sporozoites is being followed up from that point of view. 291