SOME CHARACTERISTICS OF THE IMMUNE RESPONSE TO SPOROZOITES OF SIMIAN AND HUMAN MALARIA

SOME CHARACTERISTICS OF THE IMMUNE RESPONSE TO SPOROZOITES OF SIMIAN AND HUMAN MALARIA Ruh S. Nussenzweig, M.D., Ph.D.;2 and David Chen, M.A., Ph.D.3 The ques for a vaccine agains malaria has focused considerable aenion on sporozoie-induced immuniy in recen years. This aricle reviews our presen knowledge of his subjec and summarizes he resuls of curren research, concenraing especially on he degree o which sporozoie anisera are specific for paricular human and simian malaria srains and species. Inroducion The purpose of his aricle is o review he sae of he ar of immunizaion agains malaria using he organism s sporozoie sage, and o summarize some recen progress in his area, mainly by defining he specificiy of he immune response o sporozoies of human and simian malaria. I also repors preliminary resuls obained by immunizing Rhesus monkeys wih irradiaed sporozoies of Plasmodium cynomozgi and aemps o correlae hese findings wih he resuls obained in preliminary human rials (Clyde, e al., 1973). Finally, i seeks o poin ou a number of he gaps in our knowledge of sporozoie-induced immuni y. To summarize he sae of he ar on immunizaion wih sporozoies is no a difficul ask, since i has been an unorhodox approach followed by few invesigaors. In fac, mos immunizaion aemps have used blood Paper presened a he Thireenh Meeing of he PAHO Advisory Commiee on Medical Research held in Washingon, D.C., on 24-27 June 1974. Also appearing in Spanish in Bolein de la Oficina Saniaria Panamericana, Volume LXXVII, 1974. Porions of his repor were previously presened a he Symposium on Malaria Research held a Raba, Morocco, in April 1974 and will be ublished in a special issue of he Bullein of he Word P Healh Organizaion. All he sudies on simian and human malaria are he subjec of he Ph.D. hesis of David Chen. 2Professor of Prevenive Medicine, Deparmen of Prevenive Medicine, New York Universiy School of Medicine. New York Civ. 3Graduae suden,. Deparmen of Prevenive Medicine, New York Universiy School of Medicine, New York Ciy. sages of he parasie, parly because of dificulies involved in working wih infeced mosquioes, bu also because of he quie deeply rooed, erroneous idea ha sporozoies are no immunogenic. On he oher hand, immunizaion wih sporozoies is no really a new approach, since i was shown quie convincingly in he 1940 s ha avian malaria sporozoies which have been exposed o ulraviole radiaion induce anisporozoie bodies and a parial, alhough very significan, proecion in immunized birds (Mulligan, e al., 1941). Immunizaion of Rodens The work on immunizaion wih sporozoies of roden malaria began abou seven years ago, when we observed ha a single inravenous injecion of irradiaed sporozoies oally pro- eced a variable proporion of injeced mice agains an oherwise invariably lehal P. berghei infecion (Nussenzweig, e al., 1967). The percenage of mice proeced agains sporozoie challenge afer his single immunizing dose varied from 15 o 100 per cen in differen experimens. I should be poined ou ha wo of he basic quesions raised by hese experimens, namely, (1) he reasons for his considerable variaion in he immunogeniciy of differen sporozoie preparaions, and (2) he relaed maer of how o increase his proecion so as o obain resisance in 100 per cen of he animals, sill remain o be answered. The closes 198

Nussenzweig and Chen IMMUNE RESPONSE TO MALARIA SPORO Z&ES 199 approach o he laer goal was achieved by reaing he mice wih Corynebacerium parvum before immunizaion. C. parvum. a poen simulaor of he reiculoendohelial sysem (RES), can subsanially increase nonspecific resisance o sporozoie-induced infecions (Nussenzweig, 1967). When adminisraion of his RES simulan was combined wih injecion of a single dose of X-irradiaed sporozoies, we observed a considerable increase in he percenage of proeced animals. Wheher his was simply an addiive effec of non-specific plus specific resisance, or wheher C. pawum aced as an adjuvan, amplifying he proecive response induced by X-irradiaed sporozoies, is sill unclear. Meanwhile, he effecs of C. parvum on he simian malaria sysem are being explored in order o see if i also enhances he immune response o P. cynomolgi sporozoies. If one increases he number of immunizing sporozoie injecions per mouse o four or five, he resuls become very reproducible, close o 100 per cen proecion being observed in mos of our experimens (Nussenzweig, e al., 1969a). This experimenal model has permied us o deermine a number of characerisics of sporozoie-induced immuniy in roden malaria, characerisics which were reviewed in a repor o he Iner-American Malaria Research Symposium organized by he Pan American Healh Organizaion in 1972 (Nussenzweig, e al., 1972). Recen Work on Human and Simian Malaria Sporozoies This iniial work is wha led us ino our more recen invesigaions of immune responses ha can be produced agains sporozoies of various human and simian malaria species. Vaccinaion of Monkeys Aemps o vaccinae simian hoss by using irradiaion-aenuaed sporozoies are sill in a somewha preliminary phase. Findings repored by ohers (Collins and Conacos, 1972), as well as our own unpublished observaions, provide some evidence of parial immuniy resuling from several of hese aemps. However, opimal condiions for simian immunizaion sill remain o be esablished. Apar from he need o find he bes dosage, roue, and schedule for immunizaion, he success achieved by hese vaccinaion aemps will probably depend heavily on selecion of an appropriae anigen preparaion. The daa repored on he pages ha follow provide informaion concerning he immunogeniciy of differen sporozoie preparaions ha relaes direcly o his poin. The subjec has been invesigaed by examining he anibody response (a) o sporozoies of differen srains and species of simian and human malaria, and (b) o differen developmenal sages of sporozoies. In addiion, we have obained iniial daa on he infeciviy of some of hese developmenal sages. Inducion of Ani-Sporozoie Anibodies in Ras Invesigaion of he immune response o simian and human malaria sporozoies was faciliaed by using ras as anisporozoie anibody producers. This was made possible by he fading ha injecion of simian malaria sporozoies ino his unnaural hos causes parasie developmen o sop and induces a very rapid and consisen circumsporozoie (CSP) anibody response (Nussenzweig, e az., 1973). These anibodies can be deeced by incubaing he sporozoies wih immune serum, which causes a hread-like precipiae o form, usually a one end of he sporozoie. The reacion can be observed easily under a phase conras microscope and is essenially similar o he roden malaria CSP reacion previously described (Vanderberg, e al., 1969). We have recenly found CSP anibody formaion o resul from injecion of ras wih a oal dose of l-o-2.0 x lo5 irradiaed or non-irradiaed sporozoies adminisered in one or wo inravenous injecions. The anibody is deecable less han wo weeks afer he iniial immunizaion.

200 PAHO BULLETIN - vol. VIII, No. 3, I974 This paern of anibody response has been found consisenly in all our aemps o immunize ras wih sporozoies of various simian and human malarias. Furhermore, inravenous immunizaion of Rhesus monkeys wih irradiaed sporozoies of Plasmodium cynomolgi has shown hese animals o produce a similar bu delayed CSP anibody response. Deerminaion of Anigenic Mauriy of P. cynomolgi Sporozoies The developmen of cerain anigens in sporozoies of simian malaria has been invesigaed by esing he immunogeniciy and infeciviy of he various sages of P. cynomolgi (srain B) during sporogony. For his purpose infeced Anopheles sephensi mosquioes were secioned, separaing he horax from he abdomen, and sporozoies were colleced from hese abdominal and horacic regions as well as from disseced salivary glands and midgus. This was done under condiions designed o minimize reciprocal conaminaion of he various sporozoie populaions. Ras were hen immunized wih he sporozoies from hese differen locaions, he sporozoies having been obained a differen ime inervals following he mosquio s infecive blood meal-i.e., from day seven up o day 25 afer infecion. The purpose of hese experimens was o deermine wheher sporozoie mauraion a any given locaion was ime-dependen or, alernaively, wheher parasie populaions from he midgus ever became infecive and anigenically maure before moving o he hemocele4 and laer o he salivary glands. All he sporozoie populaions were analyzed in erms of he following characerisics: (1) heir infeciviy for Rhesus monkeys, (2) heir capaciy o induce he formaion of CSP anibodies, and (3) heir capaciy o serve as anigenic maerial in he CSP reacion. I was iniially observed ha he immunogeniciy of he sporozoie populaions varied considerably, depending on heir locaion in he 4The general body caviy of he mosquio. mosquio. I was furher observed ha a prolonged period of mosquio infecion failed o promp addiional sporozoie mauraion, unless migraion of he sporozoies oward he salivary glands had also occurred. Midgu sporozoies only induced minimal amouns of CSP anibodies-and only in excepional cases. Moreover, hey consisenly failed o reac wih known posiive anisporozoie anisera. The basic anigenic characerisics of sporozoies from he hemocele of he abdominal region were raher similar. They induced only minimal amouns of anibodies, and very few produced a ail-like precipiae characerisic of he CSP reacion. Midgu sporozoies so far have been non- infecive o monkeys. Sporozoies from he hemocele of he abdominal regions were also non-infecive in several insances. In oher experimens hese hemocele sporozoies pro- duced paency afer a significanly prolonged incubaion period, indicaing a considerable loss of infeciviy. Quie differen resuls were obained wih sporozoies from he horacic hemocele. These parasies were infecive, hough apparenly less so han salivary gland sporozoies. They induced a considerable CSP anibody response, bu relaively few individual sporozoies (no more han 1 per cen) yielded a posiive CSP reacion. Finally, all hree characerisics were fully presen in salivary gland sporozoies. Their infeciviy was also considerable, alhough i varied a grea deal in differen baches of mosquioes. This variabiliy made i difficul o compare he infeciviy of sporozoie populaions from differen regions. Comparisons became meaningful only when parallel sudies were conduced using he same baches of mosquioes. The ime elapsed beween infecion of he mosquio and is dissecion did no seem o play a major role in deermining he degree of sporozoie infeciviy. Thus he earlies salivary gland sporozoies (appearing 10 o 11 days afer infecion) were on some occasions jus as infecive as sporozoies obained 25 days afer infecion.

Nussenzweigand Chen * IMMUNE RESPONSE TO MALARIA SPOROZOITES 201 However, we did noice a progressive degree of anigenic mauraion in salivary gland sporozoies obained a differing lenghs of ime afer infecion. Early salivary gland sporozoies (colleced 10 days afer he mosquioes blood meal) reaced poorly upon incubaion wih immune sera. Maximum reaciviy was obained from parasies colleced 17 and 21 days afer infecion. Furher experimens on he comparaive infeciviy and immunogeniciy of hese differen sporozoie populaions are currenly underway. The Species Specificiy of Anibodies Produced agains Sporozoies of Simian Malaria Anisera produced agains salivary gland sporozoies of a number of differen simian malaria species have been esed for CSP reaciviy. In each insance he sera were firs screened, using homologous sporozoies as anigen, o confirm he presence of anisporozoie anibodies. When posiive, hese sera were esed wih sporozoies of oher human and simian malaria species and srains o deec any possible cross-reacions. The resuls obained (see Table 1) indicae ha posiive reacions occurred only beween homologous sporozoies and anisera. Even when he simian malaria species were believed o be raher closely relaed, as in he case of he wo ovale-ype parasies P. simiovale and P. fieldi, no cross-reacions were observed. Nor did anisera produced agains sporozoies of simian malaria cross-reac wih sporozoies of human malaria. Thus, anisera prepared agains he vivax-ype parasie P. cynomolgi did no cross-reac wih eiher he Rio Mea or he Sal II srain of P. vivax. The anisera produced agains sporozoies of oher simian malarias also failed o reac wih sporozoies of eiher P. falciparum or P. +vax. Addiional daa of a more deailed naure, including informaion abou he srain-specificiy of hese anibodies, are presenly being obained. The Species Specificiy and Srain Cross- Reaciviy of Anibodies Produced agains Sporozoies of Human Malaria We have also invesigaed he specificiy of anisera produced by inravenous immunizaion of ras wih sporozoies of various ypes of human malaria. So far we have obained anibodies agains he Thau srain of P. falciparum and he Sal II srain of P. vivax. These anisera have produced posiive CSP reacions only when incubaed wih sporozoies of he same species. No cross-reacions have been observed beween sporozoies of P. vivax, P. falcipancm, TABLE 1-Circumsporozoie (CSP) reaciviy observed in sera of ras immunized wih various simian malaria sporozoies. Sporozoie anigen Vivaxype Ovale- We Oher ypes P. cynomolgi P. gonderi P. fieldi P. simiovale Anisera produced wih sporozoies of: P. cynomolgia P. fieldib P. simiovaleb P. knowlesic P. knowlesi athis aniserum was also esed agains sporozoies of he Sal II and Rio Mea srains of P. vivax and he Thau srain of P. falciparum, bu did no cross-reac wih any of hese. bno posiive reacions occurred when hese anisera were esed agains sporozoies of P. vivax (Sal II) and R falciparum (Thau srain). cno posiive cross-reacions were observed when his aniserum was esed agains P. falciparum (Thau srain).

202 PAHO BULLETIN Vol. VIII,ivo. 3, 1974 TABLE 2-Species-specificiy and srain cross-reaciviy of CSP anibodies induced in ras by inravenous injecion of P. falciparum and P. vivax sporozoies. Sporozoie anigen Species and Srain P. falciparum (Burma) P, falciparum (Mark) P. coaneyi P. vivax (Sal II) P. cynomolgi (B) P. know Iesi (H) Anisera produced wih sporozoies of: P. fakipanrm (Thau) PO?.. P. vivax (Sal II) -* - - - * - -No done. c or any of several simian malaria species (see Table 2). Sporozoie srains from differen geographic regions have reaced as srongly wih heerologous as wih homologous anisera. These serologic findings have recenly been confirmed in human voluneers immunized by he bie of X-irradiaed P. falciparum-infeced mosquioes (Clyde, e al., 1973a). One of hese voluneers is repored o have developed deecable ani-sporozoie anibodies and resisance o repeaed sporozoie challenge. Afer a long period of repeaed immunizaion his serum yielded a posiive CSP reacion wih sporozoies of he homologous Thau srain as well as wih sporozoies of hree oher P. fakipamm srains. No cross-reacion wih P. vzimx sporozoies was observed (Clyde, e al., 1973b). The proecive immuniy acquired by his voluneer paralleled hese serological resuls. He was shown o be oally resisan o challenge wih sporozoies of he hree oher P. falciparuwl srains, bu was fully suscepible o sporozoies of P. vivax. Conclusions These resuls agree closely wih our earlier daa (Nussenzweig, e al., 1969b, and Vanderberg, e al, 1969) on he correlaion beween cross-reaciviy of CSP anibodies and cross-proecion in sporozoieinduced roden malaria. Furher findings on he cross-reaciviy of differen simian and human malaria sporozoies migh herefore help o predic he range of cross-proecion fo be obained from hese anigen preparaions. The finding ha sporozoies are subjec o a process of anigenic mauraion during he sporogonic cycle of P. cynomolgi is cerainly noeworhy. This mauraion process shows some similariy o wha had previously been observed in roden malaria (Vanderberg, e al., 1972). Anigenic mauraion, herefore, seems o follow a similar paern and o represen a common feaure of sporogonic developmen in all mammalian malaria parasies. This sill leaves open he basic quesion of wha facors promp he appearance and/or expression of cerain sporozoie anigens. To he exen ha heir expression parallels he capaciy of sporozoies o induce proecive immuniy, characerizaion of hese anigens becomes a problem of fundamenal imporance. In his regard here is hope ha comparaive anigenic analysis of differen sporozoie popu- laions migh lead o characerizaion of he proecive anigens involved. I was recenly demonsraed ha sporozoies concenraed and purified by gradien cenrifugaion reain heir immunogeniciy and infeciviy (Kreli, e az., 1973). Thus characerizaion of proecive anigens migh be achieved using his approach. In addiion, he considerable yield of parasies colleced by his mehod makes i easier o obain large sporozoie doses for purposes of vaccinaion. From he poin of view of immunizaion i is of paramoun imporance o use immunolog- P. Y 4. *

Nussenzweig and Chen IMMUNE RESPONSE TO MALARIA SPOROZOITES 203 ically maure sporozoies ha are equipped imum mauraion and migraion o he hemowih he proecive anigens. For vaccinaion cele and salivary glands have occurred; his ime aemps hese parasies should herefore be does no necessarily coincide wih he ime of harvesed a he sage of infecion when max- maximum parasie yield.?9 4 SUMMARY 4 c cl r Previous work on roden malaria has demonsraed ha Inravenous vaccinaion wih X-irradiaed sporozoies frequenly resuls in oal proecion agains an oherwise lehal sporozoie inoculum. Similar vaccinaion aemps are being pursued in simian hoss and more recenly in human voluneers. The purpose of he experimens repored here was o esablish (1) some of he anigenic characerisics of various simian and human malaria sporozoies, (2) he sage specificiy of heir anigen or anigens, and (3) he exen of parasie infeciviy of he differen developmenal sages. Ras were immunized wih a number of preparaions, providing anisera specific for various human and simian malaria species and srains. Rhesus monkeys were injeced wih differen developmenal sages of sporozoies o deermine heir infeciviy. Only sporozoies colleced from he horacic region (hemocele and salivary glands) of mos- quioes induced significan anibody formaion and were consisenly infecive. An addiional degree of mauraion ook place in he salivary glands. Sporozoies obained from he laer sie were he mos infecive and he bes inducers of circumsporozoie (CSP) anibody formaion. These sporozoie populaions also conained a higher percenage of parasies which produced CSP precipiaes deecable upon incubaion wih immune serum. I was furher esablished ha CSP (anisporozoie) anibodies o he simian and human malaria species involved are sricly speciesspecific, in ha we observed no cross-reacion, even beween malarial species believed o be closely relaed. However, inense reacions were obained wih anisera from differen srains of he same species and from differen geographic isolaes. This knowledge should be valuable in choosing he ype of sporozoie preparaion o be used in developing a malaria vaccine. ACKNOWLEDGMENTS The auhors wish o hank Dr. W. Collins of from he Pan American Healh Organizaion 1 he Primae Research Uni, Naional Insiues and World Healh Organizaion, by a Naional of Healh, for his collaboraion in he sudies Insiues of Healh Training Gran, and by 3 on cross-reaciviy among simian and human Conrac No. DADA 17-71-C-1109 from he 4 malaria sporozoies. Unied Saes Army Medical Research and This research has been suppored by grans Developmen Command. Y 3..s.+ + of monkeys agains P. cynomolgi by X-irra- * diaed svorozoies. Naure [New Bioll 236: 176-177: 1972. (4) Kreli, A., e al. Immunogeniciy and infeciviy of sporozoies of mammalian malaria isolaed REFERENCES (1) Clyde, D. F., ei al. Immunizaion of man agains by densiy gradien cenrifugaion. J Prosporozoie-induced fakiparum malaria. Am J ozool20: 662-665, 1973. Med Sci 266: 169-177. 1973a. (5) Mulligan, e al. Acive immunizaion of fowls (2) Clyde, D. F., el al. Spe&ficiy of proecion of agains Plasmodium gallinaceum by injecions man immunized agains sporozoie-induced of killed homologous sporozoies. J MaZ Zns falciparum malaria. Am J Med Sci 266: Indin. 4: 25-34, 1941. 398403,1973b. (6) Nussenzweig, R. S. Increased non-specific resis- (3) Collins, W. E., and P. G. Conacos. Immunizaion ane o malaria produced by adminisraion of killed Corynebakerium pa&m. Exp Parasiol 21: 224-231, 1967. (7) Nussenzweig, g. S., and D. Chen. Characerisics of he anibody response o sporozoies of simian and human malaria: Is sage and

PAHO BULLETIN. Vol. VIII, No. 3,1974 species specificiy and srain cross-reaciviy. Bull WHO. (In press). (8) Nussenzweig, R. S., e al. Proecive immuniy produced by he injecion of X-irradiaed sporozoies of Plasmodium berghei. Naure 216: 160-162,1967. (9) Nussenzweig, R. S., e al. Proecive immuniy produced by he injecion of X-irradiaed sporozoies of Plasmodium berghei: IV. Dose response, specificiy, and humoral immuniy. MiZi Med 134 (Suonl): 1176-1182. 1969a. (IO) Nussenzweig, R. &-i al. Specificiy of proecive immuniy produced by X-irradiaed Plasmedium berghei sporozoies. Naure 222: 488489,1969b. (II) Nussenzweig, R. S., e al. Sporozoie-induced immuniy in malaria: A review. Am J Trap Med Hyg 21: 722-728, 1972. (12) Nussenzweig, R. S., e al. Anibodies agains sporozoies of human and simian malaria produced in ras. J Zmmunol 110: 600-601,, 1973. (13) Nussenzweig, R. S., e al. Anicuerpos conra esporozoios de malaria humana y simica producidos en raas. Bol Of Sani Panam 75: 114-118, 1973. (14) Vanderberg, J., ef al. Proecive immuniy pro- duced by he injecion of X-irradiaed sporo- i zoies of Plasmodium berghei: V. In viro effecs of immune serum on sporozoies. Mili Med 134 (Suppl): 1183-l 190,196V. (15) Vanderberg, J., e al. Sage specificiy of anisporozoie anibodies in roden malaria and is relaionship o proecive immuniy. Proc Helm So Wash 39: 514-525, 1972. 4