Recent developments in the assessment of the immune response to malaria, especially as related to vaccination: Malaria vaccination with irradiated sporozoites: serological evaluation of the antigen and antibody responses*

Bulletin of the World Health Orgnazaitor., S5 (Suppi. 1). 205-209 (1979) Malaria vaccination with irradiated sporozoites: serological evaluation of the antigen and antibody responses* M. P. BAWDEN,I T. T. PALMER,2 M. F. LEEF,3 & R. L. BEAUDOIN4 Vaccir?ation against Plasmodium falciparum with atrenuaredsporozoites is the goal of the US Nayyskfalaria Vaccine Program. One requirement in the developmnent of this vac- cine is an imnntinological lest ro study the sporozoite andgen and irmtune responses it in- duces. Using an indirect fluorescent antibody test (IFA T) anzd P. berghei in the mouse or rat as a model, we have mnade sign ificantprogress toward this goal. Four antigens were detected in vaccine preparations: sporozoite-specific antigens, mnosquito antigens, antigens on the sporozoite that are common to erythrocytic stages, and bovine serum albumin, an anttigenic element of the isolarion mediumi2 no longer employed. The IFA T was a reliable monitor of vaccination in a mouise and rat model in conjunction wsith protection? to challenge. The test was a sensitive mionitor of vaccine quality. Anamnestic responses ro bites of infected mos- quitos were derected in mice previously immunized wirh wrradiated sporozoites. The primary objective of the US Navy's Malaria Vaccine Program is to develop a vaccine effective against Plasmiodiumfalciparurn. At present, this vac- cine is based upon the attenuated sporozoite as the immunizing antigen. Such an agent will be used to prevent infection in relatively small groups of non- immunes who enter endemic areas and remain for de- fined periods of time, and will be especially beneficial where chloroquine resistance is present. Immuniz- ation with irradiated sporozoites of P.falciparuin tentatively meets this objective (1, 2, 3 ). but the prep- arations require refinement before they can be practi- * This work was supported by the Naval LMedical Rcsearch and Development Command, Work Unit Nos. ZF51.524 009.0069 and M0095-PNO02.5050. The opinions or assertions contained herein are the private ones of the aulbors and are nol to beconstrued as official or reflecting the views of the US Navy or the naval service at large. The experiments reported herein werc conducted according to the principles set forth m the Guide for the care and ase of laboratorv animals, Bethesda, MD, Institute of LaboraLory Resources, National Research Council, Department of Health, Education. and Welfare, 1978 (PubiLcation No. (NIH) 78-23). Head, Sestologl Branch of ,he Malaria Division Immuno- parasitology DepartmenL, Nasal Miedicai Research Institute. Bethesda, MD 20014, USA. ! AssistanL to lhe Officer-in-Charge. US Naval Nledica: Reserrch and Training Unit, CaalaZorne, Panama, and Adjunct AAssociate of the Gorgas Memonal Laboratory, Balboa Heights. Canal Zone ' Head Technician, Serology Branch of the 'Malaria. Diision, Immunoparasitology Departmenl. Naval Medical Research Institu;e. Bethesda, MD 20014, UISA. Head, Malaria Division, Jmmunoparasitology Department. Naval Medical Research InstiLute, Belltesda, NMD 20014, USA. cally adninistered to any sizeable number of people. A requisite tool for development of this vaccine is an immunological test that can be used to study the anti- gen and immune responses it elicits. This paper will briefly review immunological tests that have been used to study the immune response to sporozoite vaccines and will describe an indirect fluorescent antibody test (IFAT) developed in our laboratorv for this purpose (4). Emphasis will be placed upon information we have gathered since 1974 from the 1FAT about the irradiated sporozoite as the immunizing antigen and the antibody responses that it induces. REV[EW OF {NIMUNOLOGICAL METHODS FOR THE STUDY OF SPOROZOITE ANTIGEN Protection against challenge Historically, the first immunological method used to study sporozoite 'mmunity was protection against challenge with infective sporozoites (5). This method is the ultimate standard with which all others must be compared, since iL is a direct measure of vaccine- induced immunity. In the laboratory. it can be applied by injecting infective sporozoites (6) or by allowing infected mosquntos ro inoculate the sporozoites (2, 3). In addition, one can test the species, strain and stage specificity of the immunity by selecting the challenge inoculum from erythrocytic stages or sporozoites of different strains and species. 3389 -205- M P BAWDEN ET AL. Agglutination This method, which has theoretical potential because one can observe the reaction and discern its specificity, was successfully applied by Mulligan et al. (7). They demonstrated a correlation between high titres of agglutinin and the quality of immunity in- duced by vaccination of birds with ultraviolet-atten- uated sporozoites ofP. gaiinaceum. Of equal import- ance was the observation that immunization of birds by blood-stage infection (which includes only eryth- rocytic and exoerythrocytic stages) also produced ag- glutinin reactive with freshly isolated, viable sporo- zoites (8). This is, in our opinion, a clear demon- stration of stage-common antigen on viable sporo- zoites freshly dissected from mosquitos in contrast with the results presented by Nardin & Nussenzweig (9). We tried the agglutination technique for studving the immune response in mice vaccinated with irradi- ated sporozoites of P. berghei (Jarvinen & Bawden, unpublished results). In our hands, this approach proved impractical for technical reasons; the main ob- jection was the need for excessive numbers of sporo- zoites to titrate the agglutinin in each serum. We have therefore abandoned further study of this serological reaction. Passive transfer and sporozoite-neutralizing activity (SNA) Passive transfer of immune serum is closely allied, both historically and in practice, with protection against challenge. However, it is practical only in animal models. Nussenzweig et al. (10) made an ex- tensive study of this technique and the related phenomenon of sporozoite neutralization by immune serum from mice vaccinated with irradiated sporo- zoites of P. berghei. They demonstrated neutralizing antibody by reducing infectivity of sporozoites, in vitro, with immune serum. In addition, they showed increased blood clearance of sporozoites from mice pretreated with immune serum. However, these tech- niques have limited application in studying malarial immunity in man. Circurnsporozoite precipitin reaction (CSP) Vanderberg et al. (11) found that the serum of mice vaccinated with sporozoites of P. berghei contained a specific antibody that precipitated around viable sporozoites in vitro, hence the name circumsporozoite precipitin reaction. This test was used extensively to evaluate the immune response of vaccinated mice and rats (12, 13). It was also used to study the antigenic character of rodent, primate, and human malarial sporozoites (14, 15). From the small amount of data available, it appears that the CSP reaction has limited usefulness in moni- toring experimental immunization, since the appear- ance of CSP antibody does not consistently correlate with the onset of protective immunity in man (16, 17). However, further experimnents should be done to de- termine the appearance of CSP antibody in vacci- nated persons. In animal models, CSP antibody pro- duction did not correlate with protection (12, 18, 19). Its most effective and extensive use was to study anti- genic relationships between sporozoites of different strains and species. Hypersensitivity From a practical point of view, a simple skin test would be desirable for assaying sporozoite immunity in man. With this in mind, we examined hypersen- sitivity reactions by footpad swelling in vaccinated mice. After extensive study (Smrkovski & Wood, un- published data), we concluded that if there was a sporozoite specific response it was masked by a strong response to antigens of mosquito origin, which con- taninate all vaccine preparations. When purer sporo- zoite preparations are available, these studies should be repeated to find out whether sporozoite-specific hypersensitivity responses can be used as monitors of immunization. Fluorescent antibody tests Serological tests that employ fluorescent stains at- tached to specific antibody have added significantly to our understanding of malarial immunity (20). This type of assay has a distinct advantage; the specificity of the antigen-antibody reaction cani be visualized. Also, the test requires relatively small quantities of re- agents and can be used to study the irmmune response of man and animals. Employing fluorescent antibody techniques, various investigators demonstrated that sporozoites are antigenic (21, 22) and that they pos- sess antigens that are commnon to the erythrocytic stages of the same species (23, 24, 25). Conmmon anti- gens were also dcmonstrated between exoerythrocytic stages and erythrocytic stages of the same species (23). It is this feature of the IFAT, that is, its potential to differentiate reactions with all three major stages of the malarial parasite, that prompted us to choose it to study sporozoite antigens and imimnune responses. In our opinion, the IFAT offers the best means of inves- tigating these complex specificities because it can be modified to meet the needs of the system and the operator can actually localize the reaction at its sitC of action. This feature is invaluable, and in this paper we shall summarize the advances we have made in under- standing sporozoite immunology. We shall not, how- ever, indcluce all details of methods or results; these 206 SPOROZOITE VACCINE: ANTIGEN AND ANTIBODY RESPONSES will follow in later publications. The IFAT we use is briefly outlined by Palmer (26) and Bawden et al. (4), and has been discussed in detail at several recent scientific meetings. All of this work was done with the ANKA strain of P. berghet main- tained in Anopheles stephensi and the NMRI mouse (6). Antigen employed in the IFAT was dried and frozen before use. The conjugated antisera used were reactive with IgG, IgM and IgA. REVIEW OF ADVANCES MADE AT THE N-AV4L MEDICAL RESEARCH INSTITUTE WITH AN [FA TEST Antigens in sporozoite preparations We have clearly demonstrated 4 distinct antigens in the irradiated sporozoite preparations used for vac- cination (4, 27). The first group comprises sporozoite- specific antigens. The second group comprises mos- quito antigens that contaminate all preparations of sporozoites produced by current methods (28, 29, 30). The third group comprises antigens on the sporo- zoite that are common to the erythrocytic (and probably exoerythrocytic) stages of the malaria para- site. Last is material employed in the isolation of sporozoites from infected mosquitos. For example, bovine serum albumin was used extetisively for den- sity gradient isolation of sporozoites (31, 32). This protein suppressed the immune response to sporo- zoite vaccination and predisposed the recipients to anaphylaxis upon booster or challenge injection (33). This type of antigen can be avoided by substituting homologous serum (28) as a component of the iso- lation medium. Sporozoire-speciJfic and stage-commnon antigens. Our criterion for defining sporozoite-specific anti- gens is that they induce antibody that reacts only witlh sporozoites in the IFAT. Thus, these antisera do not induce antibody that is detectably reactive with eryth- rocytic stages. (Exoerythrocytic stages must also be included as having potentially common antigens but we have not yet tested any of our sera for this reaction. This will be done when these stages are readily avail- able.) The occurrence of sporozoite-specific antibody in immunized mice, rats, or rabbits was dependent upon 3 factors. The first was the route of inoculation. When 2 rabbits were immunized with about 16 million viable sporozoites in 5 intravenous doses. both de- veloped antibody that reacted with erythrocytic stage- common antigens. They also produz-d antibody reac- tive with sporozoites. When 6 rabbits were immumized by allowing infected mosquitos to inoculate the sporozoites by bite, none ever produced antibody Lo common antigens. They all produced high-ditre anti- body (about 1:2000 average) that was striCtly sporo- zoite-specific. The route of inoculation accounted for this difference in response. The second factor that affected the specificity of the immune response to sporozoite vaccination was the species of animal used. Of 350 mice injected intra- venously with 1-3 doses of 30 000 iiradiated sporo- zoites, only 10 (about 3 %o) had detectable antibody to common antigen. The time of appearance of this anti- body was irregular. In contrast, 40 (93%7) of 43 tats injected intravenously with 7 weekly doses of 25 000 irradiated sporozoites, and challenged witlh viable sporozoites, produced antibody reactive with com- mon antigens. The third factor that appeared to affect the production of antibody to common antigen was the number of doses administered. In rats, this anti- body was detected only aftei the 7th vaccLnation, but in mice it occurred in an irregular way after one or more doses. Mosquito-specijic anrigen. The current methods of isolating sporozoites from infected mosquitos do not remove all the antigenic debris tbat is produced when mosquitos are disrupted to release sporozoites (29, 30). Also. one could postulate that sporozoites would have mosquito antigens attached to their surface since they are in constant contact with mosquito tissues. We were unable to detect thelatter by using mosquito- specific antiserum or by absorbing antisporozolte sera witb mosquito antigen. However, imosquiLo anLigens were present in vaccine inocula as discrete antigens. In mice vaccinated with one or more doses of irradiated sporozoites, the antibody responsc to this debris was equal to the response to the sporozoites (27). In the rat, the response to the spotozoite was stronger than the response to mosquito debris, even though the doses were the same as those used in the mouse. Im- proved isolation techniques reduced the quantity of this debris (29), but mosquito-specific antibody res- ponses were still detectable. IFA T as a monitor of vaccination ln conjunction Witb protection-against-challenge, the IFAT was a successful monitor of vaccination. For example, in the mouse a single dose of 30 000 ir- radiated sporozoites induCed immunity in 10007o of the vaccinated individluals (34). The maximnum plo- tection was demonstrated about 10 days after the vac- cination. Prior to this, all imnmune animals had a sporozoite-specitic antibody responise that became dctectable on the third day, reached its peak on the fourth and fiffh days, and thlereafter declincd i apidly. (These results arc an extension of those previously reported (4)). Thus, all immune mice had a sporo- zoite-specific response prior to the onset of solid im- munitw. The relationship in N accinated rats was some- what different. To induce 90-1007o immunity in a 207 2M P BAWDEN Er AL group of rats, it required 7 weekly doses of 25 000 ir- radiated sporozoites. Sporozoite-specific antibody was not detected in all rats until after the second vac- cinating dose when the average titre was 1:128. Sub- sequent doses caused a slight increase in antisporo- zoite antibody, so that the average titre was 1:512. This high level of antibody was sustained throughout the immunization schedule. All immune rats had an intense, sustained, sporozoite-specific immune res- ponse prior to the onset of protection. IFA Tr a monitor of vaccination In conjunction with protection-against-challenge, the IFAT was a successful monitor of vaccination. For example, in the mouse a single dose of 30 000 ir- radiated sporozoites induced immunity in 100% of the vaccinated individuals (34). The maximum pro- tection was demonstrated about 10 days after the vac- cination. Prior to this, all imnmune animals had a sporozoite-specific antibody response that became detectable on the third day, reached its peak on the fourth and fifth day, and thereafter declined rapidly. (These results are an extension of those previously reported (4)). Thus, all immune mice had a sporo- zoite-specific response prior to the onset of solid im- munity. The relationship in vaccinated rats was some- what different. To induce 90-100%o immunity in a group of rats, it required 7 weekly doses of 25 000 ir- radiated sporozoites. Sporozoite-specific antibody was not detected in all rats until after the second vac- cinating dose when the average titre was 1:128. Sub- sequent doses caused a slight increase in antisporo- zoite antibody, so that the average titre was 1:512. This high level of antibody was sustained throughout the immunization schedule. All immune rats had an intense, sustained, sporozoite-specific immune res- ponse prior to the onset of protection. IFAT as a monitor of vaccine quality The IFAT is a powerful tool when applied as a monitor of vaccine quality. There are two types of quality that can be considered. The first is the purity of the vaccine, i.e., its freedom from unwanted antigens. This is illustrated in the paper by Wood et al. (29). Particulate debrs in a sporozoite vaccine prepared by filtration was reduced by an average of 97/o, but 2 immunizing doses of this cleaner antigen caused a substantial antibody response to mosquito contaminants in 8 of 10 vaccinated niice. The IFAT can also be used to study the antigenic quality of vaccine preparations or variations pro- duced by an alternate route of immunization. In this case, comparison is always made to the antibody res- ponse observed with intravenous doses of 30 000 irradiated sporozoites in mice. Using this approach, we found that other methods of attenuation such as formalin treatment, freezing, and lyophylization are less effective in producing immunity and the anti- genicity of the product is correspondingly poorer. The efficacy of these preparations as booster doses is also under study. Quality of natural booster by infected mosquitos We have employed the IFAT in recent expeliments to study the cffectiveness of bites by infected mos- quitos as booster inoculations in immune mice. These studies are not yet complete but the preliminary re- sults indicate that the bite of 1-4 infected mosquitos caused a significant increase in the average titre of sporozoite-specific antibody in mice vaccinated 5-7 weeks before being bitten. CONCLUSION The lFA test is an important research tool. It has made possible significant advances in understanding the antigenic properties of sporozoite vaccine prep- arations and the various immune responses they in- duce. Methods previously applied to the study of sporozoite vaccines have drawbacks. Themost import- ant disadvantage is that these methods are too restric- tive. They focus only on responses to the sporozoite itself and cannot be used to characterize the unwanted antigens present in candidate sporozoite vaccine prep- arations. With the IFAT, we have characterized the major classes of antigens of vaccine preparations. We are confident that this approach will also be useful in evaluating the effectiveness of P.falciparum sporo- zoite vaccines in malaria-free humans. On the other hand, the ability to detect exposure to sporozoites in an endemic area would have great sero- epidemiological significance as well as importance in evaluating the immune status of a resident population in which immunization trials are to be conducted. Be- cause people in these areas may have antibody cross- reactive with common antigens, a different approach is required for detecting their sporozoiLe experience. In this case, a sporozoite-specific test is needed and we are currently developing such a method (27). 208 SPOROZOlTE VACCiNE; ANTIGEN AND ANTIBODY RESPONSES 209 RESUNM VACCINATION ANTIPALUDIQUE AU MOYEN DE SPOROZOITES IRRADESS: tVALUA1 ION StROLOGIQUE DE LANTIGkNE ET DES RkPONSES IMMUNITAlRES Les travaux du programme de la MIarine des Etats-Unis d'Am&ique pour la mise au point d'un vaccin anEipailudique portent sur la vaccinaLion contre la maladie caus6e per Plasmodium fa1ciparum en utilisant des SporozoLtes attW- nu&s. Des epreuves immunologLques sont requises pour htudier V'antigEne lie au sporozoite et La reponse immunitaire suscit6e par celui-ci. Des progres appr&iables ont kt fats A cet fgard au moyen des epreu'ves d'immunofJuorescenice indirecte (IF) et en se servant d'un modIec constEtu& par la souris ou le rat infect6s par P. berghei. Quatre antigenes ont 6tt identfiMs dans les pr6parations vaccinales: des antigenes sp6cifiques du spotozoite, des antigenes du moustique. deb antigEnes portEs par le spot ozoite mais communs aux divers stades eryrhrocytaires du parasite. enfini de la s6rumalbu- mnine bovine, element antig6nique du milieu prtc6demment employe pour lPisolement. mais auquel on peut substituer tin serum homologue. Les 6preuves IF ont permis de contr6ler d'une manifre efficace la prolecoon contiree paz la vaccination lors d'une infection exp6rimentale chez la sourLs ou le rat et d'&valuer la qualht6 du vaccin dans les condLtlons de fiabiliib requises. Des reponses anamnnsiques i des piqfires de moustiques infect6s ont &6 constat6es chez des souris ant6rieurement vaccinees avec des spoiozoltes irradies REFERENCES 1. CLYDE, D. F. ET AL. Americanl jourrnal oJ tJe nedical sctences, 266: 169-177 (1973). 2. RiECKNMAN.N, K.IH. ET AL. Transacmions of the Royal Society of Trop,cal Mfedicine and Hygiene, 68: 258-259 (1974). 3. RJECKMANN. K. H. ET AL. Bulletin ofthe WorldHealth Organization, 57 (Suppl. 1): 261-265 (1979). 4. BAWDEN, M. P. ET AL. In: Abstracts o,f the Fifth Inter- national Congress of Protozoology, New York City, 26 June-2 July 1977, Society of Protozoologists, 1977. 5. SERGENT. E. & SERGENT, E. Cotnpie' rendus de lAca- demie des Sciences, 151: 407-409 (1910). 6. BEAUDOIN, R. L. ET AL Experrmental parasitology, 39. 438-443 (1976) 7. MULLIGAN, H. W. Er AL Journal of the Malaria IJsti- rute of India, 4: 25-34 (1941). 8. RUSSELL, P. F. Er AL Journal ol the Aalaria Insttute of India, 4: 15-24 (1941). 9. NARDIN, E. 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