Studies of immunity in typhoid fever

Bull. Org. mond. Sante 1 1971, 44, 667-672 Bull. Wid Hith Org. Studies of Immunity in Typhoid Fever Protection Induced by Killed Oral Antigens or by Primary Infection * H. L. DUPONT,' R. B. HORNICK, M. J. SNYDER, A. T. DAWKINS, G. G. HEINER & T. E. WOODWARD Adult male volunteers were orally vaccinated with two " killed " antityphoidpreparations. The recommended doses of both vaccines resulted in serum antibody development in only afew ofthe subjects. When the dose ofthe monovalent preparation (Taboral) was doubled, serological responses occurred more frequently, with a rise in 0 agglutinins in nearly one-fifth of the subjects, in H agglutinins in approximately one-fourth, and in Vi anti- bodies in nearly half. When vaccinated volunteers were fed virulent typhoid organisms, disease occurred less frequently among those men vaccinated with Taboral at twice the recommended dose (38 %) than among those not vaccinated (54 %). Thispreparation did not conferprotection at the recommended dose. Volunteers who had previously recovered from an induced typhoid infection received a further challenge with virulent organisms. These persons developed typhoid fever less frequently (23 %) than individuals without prior typhoid exposure (30 %). Immunity in typhoid fever is not clearly defined. Not only does the disease occur among well- vaccinated individuals (Benenson, 1964) but recur- rences of clinical typhoid fever have been well documented (Marmion et al., 1953). Two forms of cell suspensions of killed bacteria have been widely employed as antityphoid vaccines. The vaccines, an acetone-inactivated (K) and a heat-inactivated phenol-preserved (L) suspension of Salmonella typhosa (strain Ty2), are administered in the form of two or three subcutaneous or intra- muscular injections. Cvjetanovi6 & Uemura (1965) have summarized the results of various typhoid vaccine field trials conducted between 1954 and 1964. In a more recent field trial comparing the two vaccines, protection rates after 7 years were 88% for the acetone-killed and 65% for the heat- * From the University of Maryland School of Medicine, Baltimore, Md. 21201, USA. This study was supported by the US Army Research and Development Command Con- tract DA-49-193-MD-2867 and in part by the Clinical Study Center Contract RR-33. The results of this study were briefly discussed in a review article on typhoid fever published in the New England Journal of Medicine, 1970, 283, 686-691, 739-746. 1 Assistant Director, Division of Infectious Diseases, University of Maryland School of Medicine. phenolized typhoid vaccine (Ashcroft et al., 1967). In the same study, the degree of protection showed little diminution until the fifth year. Efficacy of these parenterally administered vaccine preparations has been quantified in adult male volunteers (Homick et al., 1967). They were effec- tive when the number of live virulent S. typhosa administered to vaccinated volunteers did not exceed an ID25 for the unvaccinated control group. The protection afforded by vaccines K and L in these volunteers following this low challenge dose was similar to that seen in various field trials. As a result of these studies, a better method of vaccination or else an improved vaccine seemed desirable. Oral vaccination was one logical alternative. The oral route would probably minimize the local and systemic post-vaccinal reactions found with paren- teral vaccines and might offer the additional ad- vantage of direct stimulation of local intestinal immune mechanisms. The first line of defence against systemic invasion by typhoid bacilli must be the gastrointestinal tract. In many individuals the pathogen never multiplies after being swallowed. Also, as seen with typhoid carriers, intra-lumenal 2681 667- 668 H. L. DUPONT AND OTHERS growth may occur without the development of disease: these carriers may excrete as many as 1000 million organisms per gram of faeces. Whether the ability of the gastrointestinal tract ta prevent the development of systemic infection is a result of intestinal immune proteins (coproantibodies), inhibition by indigenous microflora, or other factors remains to be elucidated. However, it appears that local factors almost certainly play an important role in conferring protection against progressive disease. Through direct stimulation of intestinal immune factors, oral vaccination has been shown to be the most effective means of immunization against a variety of enteric patho- gens (Sabin, 1957; Couch et al., 1963; Mel et al., 1968; Cvjetanovic et al., 1970). Preliminary work with an oral " killed " typhoid vaccine in children was encouraging in that humoral antibodies were produced and there were no post- vaccinal complications (Vladoianu et al., 1965). The present report includes studies of the immuno- genicity of two oral " killed " antityphoid pre- parations in man. Also reported is a study of the efficacy of one of the vaccines in which the frequency of induced clinical typhoid fever is compared among vaccinated and unvaccinated adult male volunteers. The protection afforded by oral vaccine is compared with that following a similar challenge study of men who had recovered from a previously induced typhoid infection. MATERIALS AND METHODS The first vaccine examined, Typhoral,' was an enteric-coated tablet containing 33 x 109 organisms of S. typhosa (strain Ty58) with equal numbers of paratyphoid A and B. During manufacture the organisms were harvested in a Sharples centrifuge and were inactivated in acetone. The suspension containing all three organisms was frozen and thawed 5 times, and then lyophilized, incorporated into tablets, and enteric-coated. The second European oral vaccine, Taboral,2 was a monovalent preparation each tablet of which contained 100 x 109 acetone-killed S. typhosa (strain Ty2) organisms and 20% sorbitol. The tablets were coated with Keratine and were manufactured by the Swiss Serum Vaccine Institute. 1 Manufactured and kindly furnished by Behringwerke, Marburg-Lahn, Federal Republic of Germany. I Manufactured and kindly furnished by Swiss Serum Vaccine Institute, Berne, Switzerland. The two vaccines were administered to healthy, informed, adult male, volunteer inmates of the Maryland House of Correction.3 Three Typhoral tablets were administered on 3 successive days (total 9 tablets per man) to 94 volunteers. A total of 43 men received 2 Taboral tablets a day for 3 days (total 6 tablets), while 29 additional volunteers received the monovalent Taboral preparation in a dose of 4 tablets per day for 3 days (total 12 tablets). Reactions to the vaccine were assessed by recording subjective complaints. Serum agglutinins were determined by standard techniques on weekly blood specimens obtained over 6 months. Challenge studies were performed only with those volunteers who ingested the more concen- trated, monovalent oral vaccine (Taboral). The challenge with 100 000 virulent S. typhosa (Quailes strain) was given, in a manner previously described (Hornick et al., 1967), 8-10 weeks after vaccination, to 35 volunteers who had previously received the lower Taboral dose (6 tablets) and to 21 men who had received the higher dose (12 tablets). Reactions were compared with those seen among 52 unvaccinated volunteers who received the same virulent challenge. Twenty-two volunteers who had previously re- covered from induced typhoid fever with positive blood and stool cultures and rising serum agglutinins received a second typhoid challenge. Within 12 months of the initial infection, they were fed 100 000 S. typhosa (Quailes strain) and their reactions were compared with those in 34 control volunteers who received the same oral inoculum In each of the studies, once induced typhoid fever had developed, the volunteers were admitted to a special research ward maintained by the Divi- sion of Infectious Diseases, University of Maryland School of Medicine, appropriate antimicrobial therapy was begun, and the patient's clinical course was carefully monitored by a staff of nurses and physicians. Criteria for specific therapy included fever of 103°F (for over 36 hours) and clinical signs and symptoms of typhoid fever, including abdominal pain, constipation, and presence of rose spots. The diagnosis was confirmed by recovery and culture of S. typhosa from blood and stools and by rising titres of typhoid agglutinins. In this ' Each volunteer agreed to participate after the nature of the study had been explained. No coercion was used and each man was free to withdraw at any time. The conditions relating to volunteer studies outlined in the Declaration of Helsinki were adhered to in these studies. See also the Postscript on p. 672. IMMUNITY IN TYPHOID FEVER 669 study, only those individuals who received anti- Table 2. Clinical and bacteriological findings among biotic treatment were considered to have typhoid vaccinated volunteers and unvaccinated controls disease and only the data from these individuals following ingestion of 100 000 virulent S. typhosa were included in the analysis of vaccine- or disease- No.with No.with No.with induced resistance. Group No. of positive clinical relapse volunteers stool typhoid following isolation fever therapy RESULTS Vaccinated with 6 Taboral tablets Table 1 shows the serological response of volun- Vaccinees 35 21 (60 %) 14 (40 %) 3 (21 %) teers vaccinated with Typhoral and of those vacci- nated with the 2 dosage levels of Taboral. In general, ons 28 14 (50%) 12 (43%) 3 (25%) the serum agglutinin titres did not show a 4-fold rise consistently when the vaccines were administered Vaccinated with 12 Taboral tablets in the recommended dosage schedules. Only 3 Vaccinees 21 7 (33 %) 8 (38 %) 0 of 94 men (3 %) experienced a significant rise of Controls 24 15 (63 %) 13 (54 %) 2 (15 %) flagellar or H-agglutinin following vaccination with Typhoral, while 12% and 17%, respectively, showed 4-fold rises of the 0 and Vi agglutinins. With twice the recommended dose of Taboral (12 tablets) immunity following oral vaccination. The fre- serological response occurred more frequently quency of development of typhoid agglutinins (Table 1). following the virulent challenge did not differ between either vaccine group or its corresponding control group. Table 1. Serological response following oral typhoid The data presented in Table 3 show that a 4-fold vaccine rise in serum agglutinins following oral vaccination No. with 4-fold rise in serum could not be correlated with subsequent protection No. of No. of aguiiVaccine tablets volun- agglutinin teers 0 H v Table 3. Correlation of 4-fold serum agglutinin rise following vaccination with Taboral and typhoid fever Typhoral 9 94 11 (12 %) 3 (3 %) 16 (17 %) development following virulent S. typhosa challenge Taboral 6 43 2 (5 %) 3 (7 %) 3 (7 %) Serum No. No. with clinical Taboral 12 29 5 (17 %) 7 (24%) 14 (48%) agglutinin _ ' typhoid fever 4-fold rise 0 7 3 (43%) In these studies, no intestinal or systemic reactions H 5 2 (40%) were found among the volunteers following oral vaccination with either vaccine or dosage schedule. vi 11 4 (36%) Table 2 shows the frequency of development of 0, H, and Vi 2 1 (50%) typhoid fever following a virulent challenge among volunteers vaccinated with two dosage levels of No agglutinin rise 34 13 (38%) Taboral as compared with that among unvaccinated _ controls. No evidence of protection against disease was seen when only 6 Taboral tablets were adminis- tered. Fewer stool isolations were positive for against virulent challenge. Note that the develop- S. typhosa from adult males given 12 tablets and ment of clinical typhoid fever could not be related clinical typhoid fever was less frequent in this to the presence or absence of prior humoral anti- group than in the unvaccinated controls, although body development. the difference in frequency of fever was not statis- The results of the second challenge in volunteers tically significant. It may be that absence of patho- who had fully recovered from clinical typhoid gen multiplication is a measurable index of intestinal fever are given in Table 4. Following the ingestion 670 H. L. DUPONT AND OTHERS Table 4. Clinical findings among volunteers with prior of subjects. The absence of H antibodies following induced typhoid fever and non-typhoid-exposed Typhoral vaccination was probably due to the controls following ietyhos sensitivity of the flagellar antigens to the action ofS._______ pho a __ __________ digestive enzymes (Vladoianu et al., 1965) and to _-No. with T No. with the elimination or destruction of H antigen during Group volunteers clinical folaowing centrifugation. That a higher percentage of indi-typhoid fever therapy viduals developed humoral antibody when the dose __________ - -of Taboral was increased may have been due to Prior disease 22 5 (23%) 0 increased absorption of typhoid antigen from thegastrointestinal tract.Controls 34 1 1 (30%) 2 (18%) The 12-tablet dose of Taboral (12 x 1Oll killed organisms) may have conferred a limited degree of antityphoid immunity (Table 2) but this appeared to be unrelated to agglutinin development follow- of 100 000 virulent S. typhosa the attack rate among ing vaccine administration. This is not surprising, 22 volunteers who had the disease between 2 months however, as antibody titre cannot be correlated and year previously was 23%, compared with a with immunity in typhoid fever and relapse is rate of 30% among 34 controls with no prior known to occur when the antibody level is at its typhoid exposure. peak (Hornick et al., 1967). With the small num- bers of men employed in this study and the dose of virulent S. typhosa used, the incidence of typhoid fever among controls and vaccinees was not signi- The presence of typhoid organisms in the gastro- ficantly different. Similar results have recently intestinal tract does not ensure disease progression. been obtained using Taboral in a field trial in These bacteria must enter the body from the intesti- India (Chuttani, 1971). nal lumen probably via the intestinal lymphoid In the evaluation of typhoid vaccines, it is useful system. In chimpanzees orally infected with S. to compare their efficacy with the degree of immunity typhosa there was early invasion of the intestinal following recovery from the disease. It would be epithelial lining but this site was cleared of bacilli difficult to understand how an inactivated vaccine within a short time; multiplication then occurred against typhoid fever could offer more protection in the intestinal lymph follicles and draining mesen- than the disease itself. The present studies indi- teric lymph nodes (Gaines, Sprinz et al., 1968). cate that the immunity conferred by an attack of The gastrointestinal tract appears to act as a barrier typhoid fever is not complete even when the infecting to infection and is probably not essential to disease dose is approximately ID25.30 (Table 4). It is interest- development. Gaines, Tully & Tigertt (1968) have ing to note that the passage of virulent organisms shown that disease can be successfully established through the intestinal barrier did not necessarily by intravenous or intramesenteric lymph node immunize the individual. Perhaps in typhoid fever challenge in chimpanzees. In chimpanzees that had the organisms that reach the intestinal lymphoid recovered from typhoid illness, after a second system and the systemic circulation quickly do not challenge bacterial multiplication and invasion in stimulate locally produced antibodies, or perhaps the intestine was suppressed and there was a less the early administration of chloramphenicol in such intense lymphoidal reaction (Gaines, Sprinz et al., patients interferes with the synthesis of protective 1968). antibodies (Ambrose & Coons, 1963). In the present Because the major barrier to progressive disease study clinical typhoid fever did not appear to be appears to be the gastrointestinal tract, we have as protective as parenterally administered commer- turned our attention to this organ and to local cial vaccine (Hornick et al., 1967). It is probable immune factors in relation to the development that host factors are of prime importance in sus- and evaluation of typhoid vaccines. ceptibility to typhoid bacilli and that those who When two killed preparations were orally ad- develop disease following a low-dose inoculum ministered in the recommended doses to adult represent the more susceptible individuals. Even males in the present investigation, humoral antibody following clinical infection, such a population may development resulted in only a small proportion be more susceptible than most immunized persons IMMUNITY IN TYPHOID FEVER 671 without prior experience of the disease. Also in a study of naturally occurring typhoid fever, only a moderate degree of protection was found when patients with confirmed typhoid fever were exposed a second time (Marmion et al., 1953). As the use of various parenteral killed vaccines has given protection in field trials against naturally occurring typhoid fever, while only low immunity to a challenge dose was found among prison volun- teers vaccinated with the same vaccines (Homick et al., 1967), it may be that most outbreaks of naturally occurring disease result from the ingestion of low numbers of organisms. The disparity of vaccine efficacy in natural and experimental condi- tions could also be explained by the possibility of a low degree of immunity among populations who may have been exposed to typhoid antigens in the past. Vaccination of such individuals may thus represent a " booster" to this low degree of existing immunity. RtSUMt tTUDE DE L'IMMUNITE ANTITYPHOIDIQUE: PROTECTION CONFtREE PAR L'ADMINISTRATION ORALE D'ANTIGENES TUES OU PAR UNE INFECTON PRIMAIRE On a administre par la voie orale a des volontaires deux vaccins antityphoidiques tu6s. Dans le s6rum des sujets qui avaient requ les doses recommandees on a observe une faible frequence d'agglutinines antityphoi- diques, mais des anticorps humoraux sont apparus plus regulierement lorsque l'une des preparations avait ete administree a dose supdrieure. Parmi les volontaires qui avaient requ du vaccin monovalent tue a base de S. ty- phosa (souche Ty2) et auxquels on avait ensuite fait ingerer 105 germes typhoidiques virulents, la frequence d'apparition de la maladie a ete moindre que chez les sujets non vaccines. Ces donnees donnent a penser que la vaccination orale par un antigene typhoidique tue confere un certain degre d'immunite mais, compte tenu de la dose de contre-epreuve utilisee et du nombre des volontaires etudies, la protection obtenue n'6tait pas sta- tistiquement superieure a celle qui a et observ6e chez les sujets non vaccines. Parmi les volontaires remis d'une fievre typhoide clini- quement induite, et qui ont et soumis a une contre- 6preuve virulente a base de S. typhosa, la maladie n'a pas et6 beaucoup moins frequente que chez les sujets indemnes de toute exposition anterieure a la typhoide. L'immunit6 conferee par l'infection typholdique dans ce groupe experimental est apparue inf6rieure ai celle qui, d'apres les 6tudes precedentes, resulte de la vaccination par des sus- pensions de cellules bacteriennes tuees. ACKNOWLEDGEMENTS The authors acknowledge the help they received from the officials of the Maryland Prison System in the conduct of the study and thank the volunteer participants for their willing co-operation. REFERENCES Ambrose, C. T. & Coons, A. H. (1963) J. Exp. Med., 117, 1075-1088 Ashcroft, M. T., Singh, B., Nicholson, C. C., Ritchie, J. M., Sobryan, E. & Williams, F. (1967) Lancet, 2, 1056-1059 Benenson, A. S. (1964) Bull. Wld Hlth Org., 30, 653- 662 Chuttani, C. S. (1971) Bull. Wid Hith Org., (in press) Couch, R. B., Chanock, R. M., Cate, T. B., Long, D. L., Knight, V., Huebner, R. J. (1963) Amer. Rev. resp. Dis., 88, 394-403 Cvjetanovic, B., Mel, D. & Felsenfeld, 0. Bull. Wld Hith Org., 42, 499-507 Cvjetanovic, B. & Uemura, K. (1965) Bull. Wld Hith Org., 32, 29-36 Gaines, S., Sprinz, H., Tully, J. G. & Tigertt, W. D- (1968) J. infect. Dis., 118, 293-306 Gaines, S., Tully, J. G. & Tigertt, W. D. (1968) J. infect. Dis., 118, 393-401 Hornick, R. B., Woodward, T. E., McCrumb, F. R., Snyder, M. J., Dawkins, A. T., Bulkeley, J. T., De La Macorra, F. & Carozza, F. A. (1967) Med. Clin. N. Amer., 51, 617-623 Marmion, D. E., Naylor, G. R. E. & Stewart, I. 0. (1953) J. Hyg. (Camb.), 51, 260-267 Mel, D. M., Arsic, B. L., Nikolic, B. D. & Radovanic, M. L. (1968) Bull. Wld Hlth Org., 39, 375-380 Sabin, A. B. (1957) J. Amer. med. Ass., 164, 1216-1223 VlAdoianu, I. R., Dimache, G., Antohi, S., Vladoianu, C. & Zarma, 0. (1965) Bull. Wid Hlth Org., 32, 37-45 672 H. L. DUPONT AND OTHERS POSTSCRIPT All individuals who developed typhoid fever were complete antibiotic schedule had been given. This study promptly hospitalized and given chloramphenicol was part of a long-term investigation into the patho- therapy. Close clinical and laboratory control was main- genesis and prophylaxis of typhoid fever and during the tained on all volunteers, and all stools were collected and 10 years that these studies have been in progress no serious examined to ensure that no carrier states were induced. complications have occurred and no carriers have been Patients were discharged from hospital only after the revealed.