Studies on immunization against Brugia malayi infection in the rhesus monkey*

Bull. Org. mond. Sante 1969, 40, 493-501Bull. Wid Hlth Org. Studies on Immunization against Brugia malayi Infection in the Rhesus Monkey* M. M. WONG,' H. J. FREDERICKS2 & C. P. RAMACHANDRAN3 Recent studies on immunity to helminthic infection have shown that some degree of protective immunity may be stimulated by inoculations of attenuated living worms or their metabolites. The present experiments were designed to observe the effects of atiempted immunization in the rhesus monkey by the use of attenuated infective larvae of Brugia malayi. Some effect was observed in animals inoculated with larval incubate, in which micro- filaraemia did develop, but at low levels andfor short durations. However, the most strik- ing finding was that persistent immunity to challenge infections (expressed as failure to cause microfilaraemia) was obtained in animals vaccinated with large numbers (200) of infective larvae attenuated by X-irradiation at 20 000 R. Experiments with different numbers oflarvae attenuated by different doses of irradiation suggest that there is an optimum combined effect of these two factors in elicitingfunctional antibody in a quantity sufficient to prevent patent infection in Malayan filariasis. Excellent reviews on the subject of helminth immunity have been presented by Soulsby (1960), Stoll (1961) and Urquhart et al. (1962). Although vaccinatiop against helminthic infections is far from being a common practice, recently reported studies in this area have contributed much to the knowledge that will probably bring us nearer to making active immunization against worms a reality. The classical method of producing immunity with vaccines made from dead whole-worm material or from extracts of such material has been generally disappointing (Urquhart et al., 1962; Thorson, 1963). Attempts to provide protective immunity against filarial infection by inoculation with either ground adult worms or infective larvae have not been * These studies were carried out at the Institute of Medical Research, Kuala Lumpur, Malaysia, with financial support from the World Health Organization. 1 Formerly Lecturer in Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur. Present address: National Center for Primate Biology, University of Cali- fornia, Davis, Calif., USA. ' Formerly Senior Officer, Malaria and Filariasis Re- search Division, Institute of Medical Research, Kuala Lum- pur; at present, Senior Officer, Malaria Research Division, Institute of Medical Research, Kuala Lumpur. ' Formerly Lecturer in Parasitology, Faculty of Medicine, University of Malaya; at present, Senior Officer, Filariasis Research Division, Institute of Medical Research, Kuala Lumpur. successful (McFadzean, 1953; Krishnaswami & Pattanayak, 1959). Some degree of protective immunity has been shown to be stimulated by metabolites of living worms. The metabolites have been introduced by the following procedures: (1) implantation of non- wandering forms into an abnormal site (Stoll, 1958); (2) inoculation with living worms attenuated by artificial means, e.g., X-irradiation (Jarrett et al., 1957; Dow et al,. 1959; Poynter et al., 1960; Hsu et al., 1962; Radke & Sadun, 1963; Miller, 1965); (3) inoculation of a strain of the parasite which would not become patent in the host (Hsu & Hsu, 1961; Hunter et al., 1961; Sadun et al., 1961); and (4) utilization of the metabolites (exoantigens) themselves, obtained from culture media or incu- bates of the living worms (Thorson, 1953; Campbell, 1955; Soulsby et al., 1959). The present studies were designed in an attempt to elicit protective immunity in a vertebrate host against filarial infection by inoculations of various vaccines made of or from infective-stage larvae. MATERIALS AND METHODS Parasite The subperiodic strain of Brugia malayi, the major human filarial parasite in West Malaysia and 2316 -493 M. M. WONG, H. J. FREDERICKS & C. P. RAMACHANDRAN a natural zoonotic infection in several local animal species, was used in the preparation of vaccines and for the challenge inoculations. Experimental animals Rhesus monkeys (Macaca mulatta) from India, 1-11/2 years old, were used as experimental hosts. This species of monkey has been found to be quite susceptible to this strain of parasite experimentally. All the animals were screened for microfilaraemia at the beginning of the experiment by examination of 2 night blood samples and were found to be negative. Vector Infective larvae were obtained mainly from laboratory-bred Aedes togoi, supplemented occa- sionally by wild Mansonia uniformis when sufficient numbers of Ae. togoi were not available. The mosquitos were fed on 2 cats which had been experimentally infected with Brugia malayi (sub- periodic) originally isolated from a human infection. Microfilarial counts in the cats at various times of feeding averaged 432 microfilariae per 60 ml of blood. Only third-stage larvae (L3) were selected. The average recovery rates were 7.6 L3/mosquito from Ae. togoi and 12.7 L3/mosquito from M. uniformis; and the infectivity rates after feeding were approximately 70% and 89% respectively. Preparation of vaccines Three types of vaccine were prepared: (1) " SE vaccine", presumably containing secretory and excretory products (exoantigens) of infective larvae in saline incubate; (2) " IL vaccine ", consisting of infective larvae treated by incubation in immune serum; (3) "XL vaccine ", consisting of infective larvae attenuated by X-irradiation. As these were initial studies of filarial vaccines, the methods employed in these preparations were some- what exploratory in nature. SE vaccine was pre- pared by incubation of 100, 200, or 400 active L3 in 1.25 ml of sterile Krebs-Ringer solution for 1 hour at 37°C. After incubation, 1.0 ml of each incubate was removed and injected subcutaneously as 100, 200, or 400 SE vaccine respectively. It was assumed that the incubate would contain larval metabolic products in quantities directly proportional to the number of larvae in the incubate. The above larvae were then placed in tubes con- taining 1.0 ml of immune serum, also in groups of 100, 200, and 400. After incubation for 1 hour at 37°C, they were removed for subcutaneous injection as 100, 200, or 400 IL vaccine respectively. The immune serum was obtained from an older rhesus monkey which had never developed patent infection in spite of repeated infective inoculations of infective larvae of subperiodic Brugia malayi (Wong, in preparation). It was thought that specific antibodies to the filarial worm might be present in the serum and that infective larvae incubated in these antibodies might be attenuated. Immediately after dissection from the mosquitos, infective larvae to be X-irradiated were placed in groups of 100, 200, or 400 in flat-bottomed glass dishes (1.5 cm diameter) containing Krebs-Ringer solution. The depth of the solution was then ad- justed to 5.0 mm. To reduce exposure time to a minimum, a short X-ray cone giving a target specimen distance of 10 cm and a field area of 50 cm2 was used. At this distance the dishes were completely covered during X-irradiation. X-irradiation was delivered by a Dermopan unit with an exposure rate of 670 roentgen/min at the level of the larvae. Radiation factors were 50 KVp, 25 mA with 1.0 mm aluminium filtration. The half- value layer for the beam was 0.75mm aluminium. The distance from the target to the surface of the suspen- sion was 9.0 cm. Total irradiation doses in the region of 10 000 R, 20 000 R, or 40 000R were given to produce 9 different XL vaccines-3 each of 100, 200, or 400 infective larvae. These vaccines are designated 100 XL/100000R, 200 XL/10000R ... 200 XL/40 000 R, 400 XL/40 000 R. All larvae were as actively motile after irradiation as before. Inoculation into the monkeys was carried out within half-an-hour after irradiation was com- pleted. Vaccination and challenge The different types of vaccines were tested in essentially the same manner. Experimental monkeys were inoculated subcutaneously twice, generally at 2- or 4- week intervals, and challenged one month after the last vaccinating inoculum with 100 normal infective larvae. At the same time, a control monkey was inoculated with each vaccinating or challenge inoculum to determine its viability. Assessment of the host's reactions was made by examination of fortnightly samples of night blood for microfilarae- mia by the concentration technique of Knott (1939), leucocyte and differential counts, measurement of body temperature, palpation of lymph nodes, and serological tests. In addition, some animals were later sacrificed for recovery of worms, using methods previously described by Buckley & Edeson (1956) 494 IMMUNIZATION AGAINST BRUGIA MALAY! INFECTION IN RHESUS MONKEYS and Ash & Little (1964). This paper reports only the data from parasitological examinations. RESULTS Experiment 1 As seen in Table 1, only 1 of the 9 animals vaccinated with SE vaccine failed to develop a patent infection; however, its microfilarial counts were considerably lower than those of the control animal. In 3 animals the counts were never more than 3 microfilariae/0.1 ml of blood and on several occasions no microfilaria was found even by Knott's concentration method. Patency in these animals lasted 9 months or less, in comparison with the 10 or more months of patency seen in the other 5. The prepatent period, which is counted from the day of the challenge inoculum, is normally found to be about 3 months. On the basis of the range of from 81 days to 131 days found among all the challenge controls (in all experiments), no alteration in prepatency among the test monkeys could be discerned. Experiment 2 A total of 9 monkeys was inoculated with the 3 different IL vaccines. Three animals were addi- tionally injected with 100 IL vaccine to serve as vaccine controls, i.e., to determine whether or not the so-called attenuated larvae were themselves infective. Table 2 shows that both experimental and control animals became infected. Four animals, representing each of the IL vaccines, developed a low microfilaraemia of short duration (3 months or less). Patent infections of more than 12 months' duration were seen in 5 of the remaining 7 animals. The short prepatent periods observed in 6 of the monkeys in this group are considered to have been derived from the vaccinating inocula. Experiment 3 As shown in Table 3, microfilaraemia occurred in only 4 of the 9 test animals. None of the animals receiving 400 L3, regardless of the amount of irradia- tion received, became patent. When the number of attenuated larvae in the inoculum was 100 or 200, only those receiving approximately 20 000 R did not cause patent infection upon challenge inocula- tion. Low microfilaraemia of short duration (11/2 and 4 months) was seen in the animals vaccinat- ed with 200 XL/10 000R or 200 XL/40 000 R vaccine respectively. Microfilaraemia in the aninmals inocu- lated with 100 XL/10000R or 100 XL/40000R vaccine persisted for more than 12 months. None of the 6 vaccine control monkeys developed micro- filaraemia. Since these results were observed in single animals, this experiment was repeated using a total of 11 monkeys and testing only 2 of the 9 previous XL vaccines. Four monkeys were each vaccinated with 100 Ls exposed to 20000 R and 3 were vaccinated with 200 L3 also exposed to 20000 R. Again the interval between the 2 vaccine doses given to each monkey was 2 weeks, with the challenge inoculation with 100 infective larvae carried out a month later. Two monkeys, used as vaccine controls, were given only irradiated larvae, and 2 monkeys were injected only with the challenge inoculum to serve as challenge controls. Table 4 shows that 5 of the 7 test animals remained negative up to 6 months after challenge (observations are continuing at the time of the writing of this report). The 2 monkeys in which the infection became patent had microfilarial counts of 12 and 45 microfilariae/0.1 ml respectively. Both vaccine controls remained negative while the challenge controls showed good levels of microfilaraemia after a normal prepatent period. Subsequent experiments Rechallenge of immune monkeys. Three XL vaccinated animals (A124, A141, and A102 of Experiment 3), which had remained negative for microfilariae for 10 months after the challenge inoculation, were each given a second challenge of 100 L3. Blood films examined regularly for the following 6 months remained negative for micro- filariae in all 3 animals. Delayed challenge of X-irradiated vaccine controls. Three (A55, A103, and A122) of the 6 vaccine controls, which had not developed microfilaraemia during the 12-month period following their inocula- tions, were challenged with 100 L3 each. Two developed no microfilaraemia during the following 6 months while the third became positive after a prepatent period of 143 days. The microfilarial count was very low and reverted to negative after 2 months. Recovery of adult worms Two immune monkeys (i.e., those in which micro- filaraemia did not occur) and 1 vaccine control (Experiment 3) plus 2 challenge controls were 495 M. M. WONG, H. J. FREDERICKS & C. P. RAMACHANDRAN TABLE I RESULTS OF ATTEMPTED IMMUNIZATION OF RHESUS MONKEYS WITH SE VACCINE (EXPERIMENT 1) Vaccination dose Monkey Chall No. Interval doNo. lst inoc. (weeks) 2nd inoc. A120 100 SE 2 100 SE 100 A104 100 SE 2 100 SE 100 A71 100 SE 4 100 SE 100 A138 200 SE 2 200 SE 100 A107 200 SE 2 200 SE 100 A146 200 SE 4 200 SE 100 A129 400 SE 2 400 SE 100 A136 400 SE 2 400 SE 100 A116 400 SE 4 400 SE 100 Highest Prepatency microfilarial (days) count per 0.1 ml 74 88 88 130 74 130 74 74 70 2 225 3 225 292 95 Challenge control A130C - - - 100 L3 88 1265 >12 TABLE 2 RESULTS OF ATTEMPTED IMMUNIZATION OF RHESUS MONKEYS WITH IL VACCINE (EXPERIMENT 2) Vaccination dose Highest Duration of Monkey Challenge Prepatency microfilarial microfilaraemiaNo. lst inoc. Interval 2nd inoc. dose (days) count per (months)1stnoc. (weeks) 0.1 ml (ots A137 100 IL 2 100 IL 100 L3 88 122 >12 A147 100 IL 2 100 IL 100 L3 47 376 >12 A131 100 IL 4 100 IL 100 Ls 87 30 8 A115 200 IL 2 200 IL 100 L3 74 6 3 A125 200 IL 2 200 IL 100 L3 74 29 7 A140 200 IL 4 200 IL 100 L3 60 3 Died A105 400 IL 2 400 IL 100 Ls 74 2 1 A119 400 IL 2 400 IL 100 L3 59 197 >12 A112 400 IL 5 400 IL 100 L3 33 85 >12 Vaccine controls A113C 100 IL 4 400 IL _ 63 510 >12 A117C 100 IL - - _ 174 2 1 A126C 100 IL 63 127 5 Challenge control A133C - 100 L3 104 74 >12 496 Duration of microfilaraemia(months) >12 7 >12 9 >12 4 >12 10 - 497IMMUNIZATION AGAINST BRUGIA MALA YI INFECTION IN RHESUS MONKEYS TABLE 3 RESULTS OF ATTEMPTED IMMUNIZATION OF RHESUS MONKEYS WITH XL VACCINE (EXPERIMENT 3) Vaccination dose a Highest Duration Monkey Challenge Prepatency microfilarial of micro- No. Itstainoc.Interval 2nd inoc. dose (days) count per filaraemia1stinoc. (weeks) IndInoc.0.1 ml (months) A127 100 XL/10 000 R 2 100 XL/10 000 R 100 L3 102 95 >12 A66 200 XL/10 000 R 3 200 XL/10 00 R 100 L3 102 5 4 A124 400 XL/10 000 R 2 400 XL/I0 000 R 100 Ls - 0 0 A109 100 XL120 000 R 2 100 XL/20 000 R 100 Ls - 0 0 A128 200 XL/20 000 R 4 200 XL/20 000 R 100 L3 - 0 0 A141 400 XL/20 000 R 2 400 XL/20 000 R 100 L3 - 0 0 AII I00 XL/40 000 R 2 100 XL/40 000 R 100 L3 88 188 >12 A132 200 XL/40 000 R 4 200 XL/40 000 R 100 L3 103 3 1.5 A102 400 XL/40 000 R 2 400 XL/40 000 R 100 L3 - 0 0 Vaccine controls A55C 100 XL/10 000 R _ - - - 0 0 A61C 100 XL/20000 R _ - - - 0 0 A103C 100 XL/40 000 R _ - - - 0 0 A121C - 100 XLIIO 000 R - - 0 0 A122C - - 100 XL20 000R - - 0 0 A46C - - 100 XL/40 000 R - - 0 0 Challenge controls A145C - _ - 0Ls 130 63 11 A68C - - | 100 Ls 88 400 >12 a XL/10 000 R, XL/20 000 R and XL/40 000 R indicate vaccines incorporating larvae irradiated at total doses of 10 000 R, 20000 R and 40000 R, respectively. TABLE 4 RESULTS OF ATTEMPTED IMMUNIZATION OF RHESUS MONKEYS WITH XLI20000 R VACCINE (EXPERIMENT 3) Vaccination dose Highest Duration Monkey __________ - _________ - Challenge Prepatency microfilarial of micro- No. n Istinoc nterval 2nd inoc. dose (days) count per fllaraemiaNo. 1st inoc. ~~~(weeks) dio.0.1 ml (months) R358 100 XL/20 000 R 2 100 XL/20 000 R 100 La 98 12 >3 A51 100 XL/20 000 R 2 100 XLI20 000 R 100 Ls 98 45 >3 R141 I00 XL/20 000 R 2 100 XL/20 000 R 100 L3 - 0 0 A93 I00 XL/20 000 R 2 100 XL/20 000 R 100 L3 - 0 0 A94 200 XL/20 000 R 2 200 XL/20 000 R 100 L3 - 0 0 A391 200 XL/20 000 R 2 200 XL/20 000 R 100 L3 - 0 0 A419 200 XL/20000 R 2 200 XL/20900 R 100 Ls - 0 0 Vaccine controls A58C 00 XL/20 000 R 2 100 XL/20 000 R - - 0 0 A79C - - 200 XL/20000 R - - 0 0 Challenge controls A69C - _ _00 L3 81 435 >3 A143C - _ _00 LJ 98 84 >3 a The data reported represent observations made during the first 6 months after challenge inoculation. M. M. WONG, H. J. FREDERICKS & C. P. RAMACHANDRAN TABLE 5 RECOVERY OF ADULT WORMS FROM 5 EXPERIMENTAL MONKEYS Monkey Status Vaccination dose Challenge Micro- Adult wormsNo. dose Ifllaraemia recovered A94 A102 A58 A143 A130 Immune monkey Immune monkey Vaccine control Challenge control Challenge control 200 XLI20 000 R (twice) 400 XL/40 000 R (twice) I100 XL/20000 R (twice) sacrificed. Attempts were made to recover adult worms to gauge the fate of infective challenge larvae in the immune animal, the viability of X-irradiated larvae after inoculation, and to compare the worm recovery rate of infected animals (challenge controls). As shown in Table 5, a total of 15 adult worms was recovered from the infected challenge control animals. Two worms, a male and a gravid female with fully developed microfilariae in utero, were found in 1 of the 2 immune monkeys. No worm was recovered from the vaccine control. DISCUSSION Since absolute resistance to helminthic infection is rarely achieved, one is left to contend with relative or partial immunity. This immunity is usually indicated by significant decreases in rate of migra- tion, degree of development, reproductive capacity and longevity of adults, as well as the final worm burden. In filarial infections, however, the assess- ment of degrees of immunity is often difficult and problematic. For lack of better parameters, these same indices were considered in the present experi- ments in comparing the vaccinated and the control monkeys. The rate and degree of development were interpreted from the length of prepatent period; difficulties existed with those which never became patent, no attempt having been made to determine either the migration rate of the larvae or their degree of development. Whether the reproductive capacity and longevity of adult worms could possibly be expressed in the appearance, level and duration of microfilaraemia is questionable-it is not known how many microfilariae are produced by each B. malayi, nor whether there is any correlation between the number of microfilariae and the number of adults present. Moreover, the possibility exists that, as with Dirofilaria immitis, no microfilaria may be 100 L3 100 L3 100 L3 100 L3 + 2 (- 1 ;) 4 (1 S3 $) 11 (4S7 Y) found in the circulating blood although fully mature worms may be found in the heart (Thrasher, 1965). Finally, the assessment of the total worm load depends on the actual recovery of all worms in each host, an infallible technique for which is still lacking. The use of metabolic products (SE antigens or exoantigens) as vaccines has been shown to elicit some degree of immunity in certain experimental infections: Trichinella spiralis (Campbell, 1955; Chute, 1956; Chipmann, 1957), Nippostrongylus muria (Thorson, 1953), and Ascaris lumbricoides (Soulsby, 1957). Difficulties exist in the collection, concentration, and preservation of such products. It is probable that in the present experiment (Experiment 1) these same difficulties accounted for the result obtained. Moreover, the exsheathing fluid rather than the metabolites may, as pointed out by Soulsby (1960) in the case of Ascaris, serve as the main source of functional antigen. Whether this may also be the case in filarial infection has not been determined. The exact nature of the so-called "functional antigen" is still not clearly defined. Techniques under development in recent years for harvesting, concentrating, characterizing, and standardizing antigens produced during various stages of in vitro cultivation (Mills & Kent, 1965; Silverman et al., 1966; Fife et al., 1967; Sadun & Gore, 1967) will undoubtedly aid in achieving this goal. The deleterious effect of immune serum on worms has been considered in several experiments (Mauss, 1940; Oliver-Gonzalez, 1941; Schwabe, 1957; Wong, 1964). In this experiment (Experiment 2), the immune serum was obtained from a monkey in another experiment (Wong, in preparation) which had been exposed to 20 doses of 20 infective larvae each and had remained negative for microfilaraemia all through the year of the experiment. It was as- 498 IMMUNIZATION AGAINST BRUGIA MALAYI INFECTION IN RHESUS MONKEYS sumed that antibodies which in some way interfered with the larval development had been formed. Un- fortunately, no attempt was made to characterize the nature of the immunity until later in the experiment, when a fluorescent antibody technique was designed to test for antibodies against the infective stage as well as the microfilarial stage (Wong & Guest, unpublished data). It was found that antimicro- filarial, but not antilarval, antibodies were de- monstrable in the serum. This finding may explain the failure to attenuate the infective larvae used in the vaccinating doses. The question of the existence of stage-specific antibodies becomes of considerable importance in this respect. Perhaps the most rewarding results were obtained with X-irradiated larvae (Experiment 3). These data lead to speculation on the importance of the suffi- ciency of functional antigen necessarily present before protective immunity is elicited. Apparently both the degree of attenuation and the number of larvae in the vaccine are contributing factors. The degree of attenuation of the worm should be directly proportional to the dose of radiation to which it was exposed; at any rate, a patent infection was not seen in any of the vaccine controls. Neither did micro- filaraemia develop in any of the monkeys which received 400 larvae; however, 2 of the 3 monkeys which received 100 larvae produced patent infections upon challenge. It is possible that the larvae exposed to the highest dose of irradiation (40 000 R) were over-irradiated and probably did not survive long enough to have produced a sufficient amount of functional antigen except when the highest number of larvae (400) was present. The degree of attenuation which seemed most promising was that accomplished by exposure to a total dose of 20 000 R. However, as shown in Table 4, 2 of the 4 monkeys vaccinated with 100 larvae produced patent infections while none of the 3 monkeys which received 200 larvae exhibited microfilaraemia. These findings again suggest that not only the degree of attenuation, represented by the total irradiation dose, but also the number of larvae used in each vaccine might be contributing to the total effect. The duration of immunity for more than 10 months, as shown when vaccinated, challenged monkeys were subjected to rechallenge (see under " Subsequent experiments "), is of considerable interest. In addition, the effect of the vaccine seems to persist for a long period. This was demonstrated when vaccine controls were challenged with an infective dose a year later. Assessment of immunity by recovery of adult worms in Malayan filariasis in the monkey is not an easy task. The techniques developed for their recovery are, to say the least, extremely time- consuming. Moreover, to determine the fate of the attenuated worms used in vaccination would require even more careful and frequent examination. Although the number of monkeys examined in this experiment barely represents each immune effect seen, the fact that a pair of fully developed adult worms was recovered in a monkey which had consistently shown no microfilaraemia deserves fresh attention and consideration. One wonders if the " immunity " developed was an immune response which either prevented the circulation of micro- filariae (Wong, 1964) or inhibited the release of the progeny from the female worm (Taliaferro, 1948). In either case, the possibility of an occult infection cannot be dismissed. Occult filariasis in the form of " eosinophilic lung " (Danaraj, 1958) has been reported in man in Singapore and Malaysia. Micro- filariae are undoubtedly produced but are trapped in the lung and other tissues (Webb et al., 1960; Lie, 1962; Danaraj et al., 1966). The presence of filarial antibodies (Danaraj et al., 1959), and speci- fically microfilarial antibodies (Wong & Guest, 1969), is associated with the syndromes of occult filariasis. It is hoped that the analysis of the various data collected in the present studies, especially the serological studies, may reveal the roles played by the various immune responses in the etiology of filarial diseases. Although at least one helminthic vaccine has been shown to be of economic value (Urquhart et al., 1962), the results of the present studies do not indicate the practicability of vaccine development and use for Malayan filariasis in the near future. Although some degrees of immunity may be in- terpreted as having been brought about by vaccina- tion, the problems of assessment of these in Malayan filariasis seem to be compounded by the occurrence of occult infections. Undoubtedly, a better under- standing of the basic biology of the filarial parasite would be most helpful towards solving some of these problems. 499 500 M. M. WONG, H. J. FREDERICKS & C. P. RAMACHANDRAN ACKNOWLEDGEMENTS Grateful thanks are due to the late Professor Dato Ungku Omar-Ahmad, Director, Institute for Medical Research, Kuala Lumpur, for his encouragement and for providing facilities to carry out this work. The authors also wish to express their gratitude to members of the Entomology Division of the Institute (Senior Officer: Mr W. H. Cheong) for supplying the large numbers of infective larvae required for the experi- ments, and to the Radiotherapy Department, General Hospital, Kuala Lumpur, for the use of their machine for the X-irradiation of larvae. The technical assistance of Mr S. Sivanandan through- out these experiments was indispensable and is deeply appreciated. Grateful acknowledgement is also due to Dr M. F. Guest for valuable assistance in the preparation of the manuscript. RESUME ETUDES SUR L'IMMUNISATION CONTRE L'INFECTION A BRUGIA MALA YI CHEZ LE SINGE RHtSUS Le singe rhesus (Macaca mulatta) est riceptif a l'infec- tion experimentale par Brugia malayi, agent de la fila- riose humaine subperiodique. On a recherche chez cet animal le pouvoir immunisant de trois preparations vacci- nales: a) un vaccin SE, obtenu par incubation de larves infectantes, et suppose contenir des produits du metabo- lisme du parasite (exoantigenes); b) un vaccin IL, consis- tant en des doses variables de larves infectantes traitees par contact avec un immunserum; c) un vaccin XL, larves infectantes traitees par des doses plus ou moins elevees de rayons X. Quatre semaines apres l'administration de l'un ou l'autre vaccin, les singes ont requ une injection d'epreuve de 100 larves infectantes de B. malayi. La r6ponse immunitaire a ete evaluee sur la base de I'appari- tion ou de la non-apparition d'une microfilaremie, sur son taux et sa duree; on a egalement compare la charge en parasites adultes des singes vaccines et des animaux temoins. L'administration des vaccins SE et IL n'a pas empechE l'infection de se developper, mais 1'intensit6 et la dur6e de la microfilaremie ont e plus faibles chez les animaux vaccines que chez les singes temoins. Le resultat le plus marquant a e obtenu par l'emploi d'un vaccin XL contenant 200 larves infectantes attenuees par applica- tion d'une dose totale de 20 000 R: chez aucun des singes ainsi traites, on n'a pu deceler d'infection apres l'inocula- tion d'epreuve. II semble que le nombre et le degre d'attenuation des larves contenues dans le vaccin jouent tous deux un role dans le d&clenchement de la reponse immunitaire. 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