Measuring the efficacy of vaccination in affording protection against plague*

Bulletin of the World Health Organization, 57 (2): 309-313 (1979) Measuring the efficacy of vaccination in affording protection against plague* JAMES E. WILLLAMS1 & DAN C. CAVANAUGH 2 The relationship of F1 antibody titre to protection against plague was investigated by subjecting seropositive laboratory rats to virulent challenge and observing for survival. The passive haemagglutination (PHA) test in microtitre was employed for serology. Rats vaccinated with live vaccine EV76(51f), killed U.S.P. vaccine, or Fl antigen and challenged by subcutaneous inoculation of 1 x 10 3 to 5 x 10 5 Yersinia pestis survived at similar rates that, overall, equalled 6% at titres less than 1:16, 46% at titres of 1:32- 1 :64, 90% at titres of 1:128-1:256, and 96% at titres of 1:512-1:1024. Rats vaccinated with Fl antigen and rats that had been infectedpreviously were challenged intranasally with 8.9 x 10 4 Y. pestis and subsequently demonstrated similar rates of survival that was zero at titres less than 1:128, 86% at titres of1:128-1:256, and 100% at titres of 1:512- 1:1024. The significance of titre of Fl antibody as a measure of seroimmunity against acute bubonic or pneumonic plague is discussed for rats, monkeys, and man. The plague bacillus ( Yersinia pestis) charac- teristically possesses the ability to elaborate a specific envelope or capsular antigen known as fraction 1 (Fl) that functions as an important determinant of virulence (1-3). The Fl content of a vaccine has a direct effect on the degree ofimmunity to acute plague infection produced in laboratory animals (4, 5). Exposure to Fl, either through vaccination or infection, stimulates the production of antibodies to the antigen that can be measured quantitatively (6). At the present time, the passive haemagglutination (PHA) and haemagglutination- inhibition techniques (7) are most widely employed for this purpose because these procedures are convenient and exhibit great sensitivity and speci- ficity (8). Although the occurrence of antibody to Fl in man or animals suggests that some degree of protection against reinfection has been acquired (9-12), the relationship between the serological titre of Fl antibody and immunity to plague has not been clearly defined. The work reported here investigated the correlation between titre and * From the Department of Hazardous Microorganisms, Division of Communicable Disease and Immunology, Walter Reed Army Institute of Research, Washington, DC 20012, USA. 1 Chief, Plague Section, Department of Hazardous Microorganisms. 'Chief, Department of Hazardous Microorganisms. protection with reference to the PHA procedure for measuring Fl antibody. MATERIALS AND METHODS The WR strain of laboratory rat (Rattus norve- gicus), derived from the Wistar strain, was employed in the experiments,a as unvaccinated individuals characteristically exhibit a high susceptibility to plague infection (LD5O <20 virulent organisms) regardless of age, sex, or season of the year. Challenge infections were done by subcutaneous inoculation or nasal instillation of virulent Y. pestis of the 195/P strain. Plague bacilli to be inoculated subcutaneously were cultured at 25°C to obtain phagocytosis-sensitive Y. pestis similar to those transmitted by fleas, whereas intranasal challenge employed encapsulated phagocytosis-resistant plague bacilli grown in vivo to simulate organisms implicated in interhuman transmission of pneumonic plague (13). Intranasal challenge employed Y. pestis 195/P taken from the peritoneum of a moribund WR rat by the technique of Cavanaugh & Randall (13) and administered by the method of Meyer et al. (14). a In conducting the research described in this report, the investigators adhered to the Guide for the care and use of laboratory animals. US Department of Health, Education and Welfare. DHEW Publication No. (NIH) 73-23, 1972. 3802 -309 310 J. E. WILLIAMS & D. C. CAVANAUGH Table 1. Experiments with laboratory rats Method ofchallenge Treatment before challenge Fl antibody titre Percentage survival Average day Method of ~~~~~~~~~~~~~~~atchallenge a (survivors/total) of death Subcutaneous inoculation Vaccination with None b 0 (0/23) 2.8 of 1 x 103 to live avirulent 61 :16 0 (0/24) 4.2 5 x 105 Y. pestis Y. pest/s strain 1 :32-1 :64 46 (6/13) 5.4 195/PC EV76(51f) 1 :128-1 :256 91 (10/11) 6 1: 512-1 :1024 100 (6/6) - Vaccination with None b 0 (0/6) 3.2 killed whole- <1 :16 9 (6/70) 4.3 organism plague 1 :32-1 : 64 50 (3/6) 13.3 vaccine U.S.P. 1:128-1 :256 89 (8/9) 10 1 :512-1 :1024 80 (4/5) 13 Vaccination with None b 0 (0/5) 4.2 the Fl antigen 1: 32-1 :64 40 (2/5) 9.0 of Y. pestis 1:512-1:1024 97 (34/35) 11 Nasal instillation of 8.9 x 104 Noneb 0 (0/7) 3.0 Y. pestis 195/p d Vaccination with 1:32-1 :64 0 (0/4) 9.2 the Fl antigen 1 :128-1 :256 50 (1/2) 13 of Y. pestis 1: 512-1 :1024 100 (13/13) - Infection with <1 :16 0 (0/3) 3.7 virulent Y. pestis 1: 32-1 :64 0 (0/4) 6.2 195/P and antiserum 1:128-1 :256 100 (5/5) - injected 2 days later 1: 512-1:1024 100 (4/4) a Final serum dilutions in the PHA test. b Unvaccinated controls. c Simulated flea challenge with phagocytosis-sensitive Y. pestis. d Simulated pneumonic challenge with phagocytosis-resistant Y. pestis. Three groups of seropositive rats, with appro- priate controls, were challenged subcutaneously. Rats in the first group had been vaccinated with 103-107 Y. pestis of the live avirulent strain EV76(51f) and were challenged either 26 days later with 5 x 105 Y. pestis 195/P or 10 weeks later with 2 x 105 Y. pestis 195/P. The second group consisted of rats that had received killed whole-organism plague vaccine U.S.P.,a with injections of either 1 ml on days 1, 32, and 49 followed by 0.2 ml on day 83 or 0.5 ml on days 1, 32, and 49 followed by 0.1 ml on day 83. These rats were challenged 100 days after vaccination with 108, 104, or 105 Y. pestis 195/P. The third group contained rats vaccinated with Fl antigen (Walter Reed lot 4, July 1971) in Freund's complete adjuvant. The rats received an initial subcutaneous injection of 500 ,ug of Fl followed by booster injections of 200 ,ug of Fl at 7 and 14 days. These rats were challenged 6 weeks later with 3.5 x 103 Y. pestis 195/P. Two groups of rats were challenged concurrently by nasal instillation of 8.9 x 104 phagocytosis- a Cutter Laboratories, Berkeley, CA 94710, USA. resistant Y. pestis 195/P. One group consisted of rats vaccinated with Fl antigen, as outlined above, 6 weeks earlier. The other group contained rats that had been inoculated subcutaneously with 48 virulent Y. pestis 195/P and, 2 days later, given an intra- peritoneal injection of antiplague serum-globulin b equivalent to 15 ml of rabbit serum. Rats surviving subcutaneous infection without discernable buboes were challenged intranasally after 6 months when it could be reasonably assumed, from previous studies (15), that the antiserum administered had been eliminated and any titre present was derived from active immunization. Serum titres of specific Fl antibody were determined before and after every challenge using the PHA microtitration test (15). To provide continuity with earlier studies (9-12), the titres reported are the highest final dilutions of serum giving clear-cut 4+ haemagglutination. As a further precaution to provide the best data possible, new microtitration loops were employed and these were recalibrated before use to confirm accuracy. b Lederle Laboratories, Pearl River, NY 10965, USA. VACCINATION AGAINST PLAGUE RESULTS Whereas unvaccinated rats always died after challenge infection, many vaccinated rats survived subcutaneous challenge with phagocytosis-sensitive Y. pestis or intranasal challenge with phagocytosis- resistant Y. pestis. Survival among vaccinated rats was in direct proportion to the titre of Fl antibody present at the time of challenge (Table 1). In vaccinated rats that died, death was delayed and the average time before death was directly correlated with the Fl antibody titre at the time of challenge. Patterns of survival following subcutaneous challenge were similar in groups of rats vaccinated with avirulent live vaccine, whole-organism killed vaccine, or Fl subunit vaccine. Overall survival in rats challenged subcutaneously was 6% (6/94) at Fl antibody titres < 1:16, 46% (11/24) at titres of 1:32-1:64, 90% (18/20) at titres of 1:128-1:256, and 96% (44/46) at titres of 1:512-1:1024. Patterns of survival after intranasal challenge were similar in groups of rats that had derived Fl anti- body titres from vaccination with Fl antigen or from a previous infection with virulent Y. pestis. However, unlike those challenged subcutaneously, rats with titres < 1:64 never survived intranasal challenge, reflecting the high virulence of phago- cytosis-resistant Y. pestis. An increased mortality was not evident in rats possessing higher levels of antibody at the time of challenge. Overall survival from intranasal challenge was 86% (6/7) in rats with Fl antibody titres of 1:128-1:256 and 100% (17/17) at titres of 1:512-1:1024. DISCUSSION The results of our studies with laboratory Norway rats suggest that the PHA titre of Fl antibody is a significant indicator of protection from acute bubonic plague in this species. In fact, retrospective analyses of published data for bubonic plague in wild-caught Norway rats, and also in laboratory monkeys, showed strikingly similar results (Table 2): in all cases, greater numbers of animals survived experimental infections where the levels of Fl anti- body present at the time of challenge were higher. In Norway rats and monkeys, the majority of animals possessing final Fl antibody titres > 1:128 (e.g., initial serum dilutions > 1:64 in the PHA test) survived severe challenges with plague bacilli of classical virulence. Some protection was evident even in vaccinated animals that succumbed to experi- mental plague, including individuals that had no measurable titre at challenge, as death was delayed. Possibly very small amounts of antibody to the Fl antigen were present that were not detected by the serological method used, or perhaps other immuno- logical mechanisms had functioned for a time to delay death. Alternatively, some protection against virulent challenge may have been derived from exposure to other antigens of the plague bacillus such as antigen T (toxin) (16, 17) or antigen L (18). Table 2. Experiments with wild rats and laboratory monkeys Species of animal and treatment Mehod f haln Fl antibody titre Percentage survival Average day Source before challenge etho o chalenge at challenge a (survivors/total) of death of data Wild Norway rats Subcutaneous .1:16 14 (1/7) Not Chen &(R. norvegicus) from Oahu, inoculation 1 : 32-1 64 50 (1/2) reported Meyer (12) Hawaii infected with 1.2 of 7.7 x 107 1:128-1 :256 80 (4/5) x 104 Y. pestis 195/P and Y. pestis 195/P 1: 512-1: 2048 100 (10/10) survived spontaneously Hanuman langurs Subcutaneous None b 0 (0/11) 6 Meyer (9);(Presbytus entellus) inoculation .1 :16 0 (0/2) 12 Chen et al. vaccinated with killed of 6.4 x 104 1 : 32-1 : 64 17 (2/12) 11 (11) whole-organism plague to 2.3 x 105 1 :128-1 :256 67 (6/9) 19 vaccine U.S.P. or living Y. pestis 195/P 1:512-1:2048 100 (5/5) - Y. pestls strain EV Saigon African green vervets Subcutaneous None b 0 (0/2) 7 Hallett(Cercopithecus aethiops) inoculation 1 : 32-1 : 64 0 (0/2) 9 et al. (10) vaccinated with living of 8.8 x 1 03 1 128-1: 256 86 (6/7) 12 Y. pestis strain Y. pestis EV 51f strain F357 a Final serum dilution in the PHA test. b Unvaccinated controls. 311 312 J. E. WILLIAMS & D. C. CAVANAUGH Similarly, a good correlation between Fl anti- body titre and survival was found from our experi- ments with intranasal challenge of laboratory rats. Meyer (14) estimated that only 10% of the bacilli administered intranasally reach deeper respiratory passages (e.g., 8900 Y. pestis in the present study), and infection of the larynx, tonsils, or other parts of the upper respiratory tract may also result. Although intranasal challenge is not as reliable as other methods (e.g., Henderson apparatus) for inducing a relatively uncomplicated plague pneu- monia, our data indicated intranasal challenge to be much more severe than subcutaneous challenge, with no survivors among intranasally challenged rats possessing Fl antibody titres lower than 1:128. The experimental data suggest that Fl antibody titre may be a good measure of immunity to pneu- monic as well as to bubonic plague. Although anti-Fl PHA titre in Norway rats is an indicator of protection against acute plague, there is good evidence that this relationship does not extend to protection from chronic plague in these rats (19). Indeed, Fl antibody may have some influence on the development of chronic disease (2). The vaccines employed in our study with rats gave essentially identical results in terms of the correla- tion between Fl antibody titre and protection. However, this relationship may be altered with some experimental vaccines or animal species. Chen et al. (11) found that the administration of a combined plague-cholera-typhus vaccine to Hanuman langurs and silver leaf monkeys (Presbytus cristatus) considerably enhanced survival in animals with very low titres of specific Fl antibody. Fournier et al. (20) observed that two baboons with titres of 1:32 and 1:64 after exposure to the living vaccine strain EV40 succumbed to plague, while a baboon vac- cinated with EV76(51f) that did not produce a detectable titre of Fl antibody survived virulent challenge. The mouse protective antibody index has been employed to measure the immune response of man to vaccination against plague (21). A strong correla- tion between the PHA titre of Fl antibody in human serum and the protection afforded mice injected with serum just prior to virulent challenge has recently been reported (22-24). These results suggest that the relationship between immunity from plague and the titre of antibody to Fl found in rats and monkeys may also apply to man. RtSUMt AVALUATION DE LA PROTECTION CONFiREE PAR LA VACCINATION ANTIPESTEUSE La relation entre le titre d'anticorps Fl et la protection contre la peste a ete etudi6e en exposant des rats de laboratoire s6ropositifs i des formes virulentes du bacille et en observant les taux de survie. Pour les examens s6rologiques, on a employ6 le microtitrage par h6magglu- tination passive. Des rats ayant requ le vaccin vivant EV76(51f), le vaccin tue U.S.P. ou l'antigene Fl et infectes ensuite par inoculation sous-cutan6e de Yersinia pestis A raison de 1 x 103 a 5 x 105 organismes ont pr6sent6 un taux de survie uniforme pour des valeurs d6terminees du titre d'anticorps Fl, soit 6% pour des titres <1:16, 46% pour des titres compris entre 1:32 et 1:64, 90% pour des titres compris entre 1:128 et 1:256 et 96% pour des titres compris entre 1:512 et 1:1024. Des rats vaccin6s avec F'antigene Fl et des rats qui avaient 6t6 infectes ant6rieurement ont requ des aerosols de Y. pestis a raison de 8,9 x 104 organismes, et ils ont 6galement pr6sente des taux de survie unifor- mes - mais tres inferieurs a ceux constat6s chez les rats infectes par voie sous-cutan6e - soit 0% pour des titres d'anticorps <1:64, 86% pour des titres compris entre 1:128 et 1:256 et 100% pour des titres se situant entre 1:512 et 1:1024. La valeur du titrage des anticorps Fl en tant que moyen de mesurer l'immunite s6rologique contre la peste bubonique aigue ou la peste pulmonaire est examin&e ici chez les rats et les singes, mais il a sans doute une signification analogue chez 1'homme. REFERENCES 1. ENGLESBERG, E. ET AL. Virulence in Pasteurella pestis. Science, 119: 413-414 (1954). 2. BURROWS, T. W. Virulence of Pasteurella pestis and immunity to plague. Ergebnisse der Mikrobiologie, Immunitdtsforschung und experimentellen Therapie, 37: 59-113 (1963). 3. CHEN, T. H. 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