A New Leptospiral Vaccine for Use in Man. n, Clinical and Serologic Evaluation of a Field Trial with Volunteers

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1 THE JOURNAL OF INFECTIOUS DISEASES VOL. 128, No.5 NOVEMBER by the University of Chicago. All rights reserved. A New Leptospiral for Use in Man. n, Clinical and Serologic Evaluation of a Field Trial with Volunteers M. Torten, Esther Shenberg, C. B. Gerichter, P. Neuman, and M. A. Klingberg From the WHO/FAD and National Leptospirosis Reference Laboratory, Israel Institute for Biological Research, Ness-Ziona; Government Central Laboratories, Ministry of Health, Jerusalem; Sick Fund, Kibbutz Neot Mordechai; and Department of Epidemiology, Israel Institute for Biological Research, Ness-Ziona, Israel A double-blind field trial for clinical and serologic evaluation of a new leptospiral vaccine was performed. The new vaccine, consisting of killed Leptospira, the only one grown in a chemically defined medium, was innocuous, and led to seroconversion in 57% of all volunteers who received two doses of the vaccine. The vaccine had a booster effect in 100% of volunteers with previous records of clinical leptospirosis. The new vaccine, named Shen-Tor, was approved by the Israeli Ministry of Health for vaccination of 700 irrigation workers in a trial designed to measure the potency of the vaccine to protect against leptospirosis in a natural setting. The development of a new vaccine for leptospirosis has been described [1]. The Leptospira for the new vaccine were grown on a chemically defined medium to avoid any possible contamination by foreign proteins, which are present in all commercially available leptospiral vaccines. The vaccine was tested for potency and safety in laboratory animals and was approved by the Israeli Ministry of Health for a field trial on volunteers. The trial was conducted with persons in two rural settlements in the Hula Valley in the Upper Galilee, Israel. The Hula Valley was chosen because Received for publication February 20, 1973, and in revised form June 6, This study was supported in part by a WHO grant to the Ness-Ziona WHO/FAO Leptospirosis Reference Laboratory, and by the Israeli Ministry of Health. We thank Ms. R. Sapiro-Hirsh of the Central Sick Fund Laboratory in Haifa and Mr. J. Lindenbaum of the Central Ministry of Health Laboratory in Abu Kabir for their help in checking some of our serological data, and Dr. M. Robotnik, Northern District Medical Officer and Head of the Government Hospital in Safad and Mr. S. Levy, Health Educator of the Hula Valley for their advice in planning the field trial. The technical assistance of Ms. H. Porath and Ms. S. Meir was appreciated as well as the cooperation of all the nurses in the settlements of Neot-Mordechai and Mahanaim. Please address requests for reprints to the WHO / FAG and National Leptospirosis Reference Laboratory, P.O. Box 19, Ness-Ziona, Israel. it is an endemic area for the serotypes grippotyphosa and szwajizak, from which the vaccine was prepared [2, 3]. The finding of volunteers was no problem, since the people in the area are well aware of the damage in human suffering and economic losses that leptospirosis has caused them. The results obtained in a double-blind study are presented in this report. Materials and Methods Study group. Volunteers were chosen from two kibbutzim in the Hilla Valley. As it had been already established that secondary manifestations of leptospirosis could be caused by immunologic hypersensitivity, we wanted to be able to identify such reactions if they appeared in vaccinated persons with previous contact with leptospiral antigens. In order to check this possibility we chose one farm located in an area where human cases of leptospirosis had been previously recorded, and the other in an area where no leptospirosis cases had been diagnosed. in use. The vaccine administered was the newly developed Shen-Tor vaccine previously described [1]. Equal numbers of identically labeled vials of vaccine and placebo in saline were given random numbers, which were recorded as a code by a third party not involved in the vaccination. The 647

2 648 Torten et al. vials were mixed and then handed to the vaccinating team as one batch. The code was broken only after all the serologic and clinical examinations were completed and recorded. Each volunteer received 1 ml sc injections in his arm. Injections, containing 2 X 10 8 formalinkilled Leptospira, were given 21 days apart. All persons included in the study were inoculated with the vaccine, irrespective of the status of their immunity to leptospirosis before vaccination. Intradermal testing for possible allergic manifestations was accomplished by injection of 0.1 ml of the vial's content into the ventral side of the forearm of each volunteer. In every case the material for the intradermal injection was taken from the vial to be used for the vaccination of the same person. Each of the volunteers was examined 45 min after intradermal injection and then given a sc injection. Serology. Two samples of blood for serologic tests were drawn immediately before each of the two vaccinations, while a third sample was taken 21 days after the second vaccination. Sera were separated within 24 hr and stored at - 20 C until the serologic tests were performed. Agglutination titers of the sera were determined by the microscopic agglutination test with 29 serotypes [3]. The control serum obtained before initial vaccination and the two sera obtained after each of the vaccinations from each subject were tested on the same day with the same batch of antigen by the same person to avoid any discrepancies due to human error and antigen variability. Clinical follow-up. Each of the vaccinated volunteers was seen on the three days after vaccinations,and then at one-week intervals. Data on each of the volunteers were recorded on a special form designed for the trial by the medical staff, which included a physician and nurses. Results Clinical observations. Intradermal tests. Table 1 presents the reactions observed after intradermal injection of 0.1 ml of the vaccine or placebo. Whenever local redness and/or swelling appeared within 45 min of the skin test they were recorded as positive. With the exception of two cases, all positive reactions were very mild. Only two of the six volunteers who had a positive skin test the second time had a positive skin Table 1. Intradermal reactions in volunteers receiving 0.1 ml of leptospiral vaccine or placebo. Time of testing Before first vaccination Before second vaccination No. intradermal reactions/total tested 6/51 14/51 0/45 0/45 test before the first vaccination; the other twelve had not reacted the first time. Of the six volunteers reacting for the first time only one person had a record of previous infection with Leptospira. Of the positive volunteers, only two subjects developed a wheal in addition to redness of skin. One of the two had a minimal redness after the first skin test and a more pronounced reaction the second time. The second time this volunteer had a wheal of about 15 mm in diameter, with a zone of erythema 8 mm in diameter. This subject had no adverse local or systemic effects after the vaccination on either occasion. The second volunteer who developed a wheal (15 mid in diameter in a zone of erythema of 9 mm) the first time reacted also after the second skin test. However, a local reaction (redness and swelling) appeared only after the first vaccination. This subject had no other adverse effects. Fever. Seven volunteers showed a very slight rise in oral temperature after the first vaccination; no rises were observed after the second. Table 2 presents the data on oral temperature. There was no association between vaccination and fever; three of 51 vaccine showed a slight rise Table 2. Data on the seven volunteers who had rises in oral temperatures after vaccination with killed Leptospira. Oral Days Treatment temper- after and sub- ature vacciject no. (OC) nation Symptoms None Local reaction Pharyngitis Conjunctivitis, pharyngitis Nausea, dizziness, general aching Fatigue None

3 Human Leptospiral II 649 Table 3. Local reactions after primary sc vaccination in 51 volunteers who received leptospiral vaccine and 45 who received placebo. Reaction Swelling alone Redness alone Swelling and redness Redness and induration Swelling, redness, and induration No. of reactions in temperature, but four of 45 of placebo also showed rises. Local reactions. Mild local reactions to sc administration of the vaccine were observed in 22 volunteers after the first vaccination. The reactions after the second injection were almost undetectable. Table 3 presents the data on local reactions. Other symptoms. Table 4 presents the data on general symptoms observed after the first sc administration of either placebo or vaccine. No general symptoms were observed after the second vaccination. Follow-up of subjects with previously recorded clinical leptospirosis. Table 5 presents the data accumulated for those subjects who could possibly exhibit allergic manifestations due to previous contact with leptospiral antigens. Table 4. General symptoms observed in 51 volunteers who received leptospiral vaccine and 45 who received placebo. Subject no. No. with symptoms* Vac- Pla- Symptoms cine cebo Fatigue 69 2,32,78,11 4 Headache on first day None 50, Headache after first day 25, Dizziness Muscular pains in limbs None Back pain 25 None 1 0 Eye pain 25, Abdominal pain None 26 0 * As some of the showed more than one symptom, it should be realized that all of the general symptoms appeared only in three persons receiving the vaccine and in seven of placebo. It is assumed that a number of the reactions, even in the vaccinated persons, may have been due to concurrent illness or to psychologic factors involved In a trial of a new vaccine. Table 5. Symptoms after administration of vaccine or placebo to volunteers who had a previous record of clinical leptospirosis. Skin test reaction Subject Previous lepto- After 1st After 2nd Other reactions* no. spirosis (year) vaccination vaccination Local Systemic Mild, None Redness Headache on positive days 12-13, pains in eye and back None None Redness, None swelling None None None None Positive None None None None None Redness None None None None Fatigue None None None None None None None Nausea on days 1-3 * Reactions after first vaccination, since none were observed after second vaccination.

4 650 Torten et al, Table 6. Serologic reactions in volunteers with records of previous leptospirosis who received two injections of either Shen-Tor leptospiral vaccine or placebo. Titer 21 days Subject Previous lepto- Before 1st Before 2nd after 2nd no. spirosis (year) Serotypes injection injection injection Hebdomadis 1:20 1:80 1:80 Swaiizak 0* 1:160 1:160 Grippotyphosa 0 1:160 1: Swajizak 0 1:20 1:40 Grippotyphosa 0 0 1: Swajizak 0 1:160 1:160 Hebdomadis 0 1:40 1:40 Grippotyphosa 0 1:80 1: Swajizak 0 NDt 1:80 Hebdomadis 0 ND 1: Swajizak 0 1:160 1: 160 Grippotyphosa 0 1:40 1: Hebdomadis 1:80 1:80 1:80 Swajizak 1:40 1:40 1:40 Grippotyphosa 1:20 1:20 1:20 Swajizak 1:40 1:40 1:40 Hebdomadis 1:320 1:320 1:320 Swajizak 1:80 1:80 1:80 NOTE. Although a titer <20 was observed before vaccination, the increase in titer of vaccine was considered as a booster effect, because data recorded in the diagnosis of past infection showed presence of positive agglutinin titers. * 0 =less than 1:20. t ND =not done. Results of serologic tests. In most cases, the titers before vaccination were < 1:20, which was the initial dilution of the sera to be tested. Positive seroconversion was recorded in cases of titer increase of at least two twofold dilutions with any of the antigens tested. In most cases when an increase in titer was observed, it was < 1: 160. This is not surprising in view of our previous findings with experimental animals that showed good protection with low titers [1]. Table 6 describes the serologic data obtained from the subjects known to have had previous clinical leptospirosis, while table 7 presents the data on seroconversion of all volunteers participating in the trial. Discussion A double-blind field trial has shown that the newly developed leptospiral vaccine does not produce any adverse systemic reactions (including fever). Local reactions due to sc injection of the vaccine were equivalent to what would be expected from any serum-free vaccine and were unnoticeable after the second vaccination. Intradermal skin tests proved that there is little or no danger of systemic allergic response. Positive skin tests were attributed to either circulating antibodies or an irritation reaction caused by the vaccine itself or by formalin, which was used to inactivate the Leptospira. The vaccine produced a booster effect in Table 7. Seroconversion in all volunteers who received two injections of either Shen-Tor leptospiral vaccine or placebo. No. of seroconversions/total injected Injection (% ) * 29/51 (57) 1/45 (2) * Seroconversion was defined as an increase in titer of at least two twofold dilutions with anyone of the leptospiral antigens tested.

5 Human Leptospiral II % of the volunteers who had a history of clinical leptospirosis and also led to seroconversion in 57% of the volunteers with no history of leptospirosis. An additional 8% of the volunteers had a serologic response that could be considered as possible seroconversion (from negative, i.e., < 1:20 to 1:20). The agglutination titers 21 days after clinical leptospirosis are usually quite high in comparison with the low titers after vaccination (~ 1: 160). This was not surprising, since in previous trials with experimental animals we observed low titers with good protection, as measured by challenge [1]. At that time we postulated that we may be dealing with a cellular response due to the adjuvantlike effect of the fatty acids in the chemically defined medium. We also stated that in leptospirosis titers of agglutinins and protection do not necessarily correlate. The ability of the vaccine to protect humans from clinical leptospirosis would be evaluated by epidemiologic surveillance on vaccinated and nonvaccinated persons in the endemic area of the Upper Galilee. Because of the variability in the number of cases from year to year it is felt that at least a five-year period of continued vaccination of irrigation workers will be needed before a final conclusion can be drawn as to potency in the field. The results obtained thus far with the Shen-Tor vaccine warrant such a trial, and the Ministry of Health has approved the vaccination of 700 irrigation workers in the Hula Valley. All 700 irrigation workers have already been vaccinated without any adverse clinical effects. The evaluation of the epidemiologic and serologic data on these and other persons will be concluded in several years after a follow-up. References 1. Shenberg, E., Torten, M. A new leptospiral vaccine. I. Development of a vaccine from leptospira grown in a chemically defined medium. J. Infect. Dis. 128: , Hoeden van der, J., Shenberg, E. Infections with leptospira mini szwaiizak in man and animals in Israel. Zoonoses Research 1: , Torten, M., Birnbaum, S., Klingberg, M. A., Shenberg, E. Epidemiologic investigation of an outbreak of leptospirosis in the Upper Galilee, Israel. Am. J. Epidemiol. 91:52-58, 1970.