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Immunity induced by live attenuated salmonella vaccines
Res. MicrobioL 1990, 141, 757-764
~) INSTITUTPASTEUR/ELsEVIER Paris 1990
IMMUNITY
INDUCED
BY L I V E
ATTENUATED
SALMONELLA
VACCINES
C.E. Hormaeche(t) ('), H.S. Joysey(l), L. Desilva(l), M. I z h a r ( l ) a n d B. A , D . Stocker(2)
(I)Microbiology and Parasitology Division, Department o f Pathology, University o f Cambridge, Tennis Court Road, Cambridge CB2 IQP, (UK) " and (2)Department o f Microbiology and Immunology, Stanford University School o f Medicine, Stanford, C 94305 (USA)
Summary. Studies on the degree and specificity of protection conferred by immunization with aroA salmonella live vaccines in BALB/c mice are described. Animals were immunized i.v. and challenged orally 3 months later to ensure that the vaccine had been cleared from the tissues. Vaccination with Salmonella typhimurium aroA SL3261 conferred very good protection against virulent S. typhimurium C5 (over 10,000 × LDs0). The specificity of cross protection was studied using S. typhimuriurn, Salmonella enteritidis and Salmonella dublin for vaccination and challenge, including challenge with variants of S. typhimurium and S. enteritidis of similar virulence which differed in the main LPS (lipopolysaccharide) antigen (0-4 or 0-9). S. typhimurium SL3261 gave very good protection against S. typhimurium C5 (0-4), but no protection against S. enteritidis Se795 (0-9). However, challenge with strains differing in the main 0 antigens showed that, although protection was generally better to strains expressing the same LPS type as the vaccine, specificity of protection was determined more by the background (S. typhimurium or S. enteritidis) of the parent strain used for the challenge than by 0 factors 4 or 9, suggesting that other factors could be involved. The nature of the antigen(s) responsible for protection in this model is unclear, but it would not appear to be the main 0.specific antigen. An S. enteritidis Se795 aroA vaccine was far less effective than S. typhimurium SL3261 ; it conferred good protection against the homologous wild type at 2 weeks post-vaccination, but far less at three months (approx 10-200 x LDs0). This was unexpected, as the persistence of the S. enteritidis vaccine in the liver and spleen was similar to that of S. typhimurium SL3261, and the S. enteritidis and S. typhimurium challenge strains were of similar virulence. An S. dublin aroA vaccine conferred similar protection against wild types S. dublin (approx 300 × LDs0). KEY-WORDS"Salmonella, Vaccine, Mutant aroA, Lipopolysaccharide; Protection.
(') Correspondingauthor.
758
C.E. H O R . M A E C H E E T A L .
Introduction.
The mechanisms of immunity to salmonellosis are still unclear. In experimental models, live vaccines, which induce both humoral and cellular immunity, confer much better protection than killed vaccines, which do not elicit cellular immunity (Collins, 1974; Eisenstein and Sultzer, 1983). The encouraging results obtained with live attenuated vaccines suggest that cellular immunity is important in human typhoid (Murphy et al., 1989). Salmonellae wi:h mutations in genes of the aromatic pathway are effective as vaccines and as carriers of recombinant antigens from other parasites (Dougan et al., 1989). However, some live salmonella vaccines confer very poor protection (Collins, 1974; Hormaeche et al., 1981 ; Smith et aL, 1984). Effective vaccines can persist for a limited period in the tissues after which non-specific immunity disappears, with protection requiring the specific recall of immunity (Collins, 1974). The specificity of the protective response is still unclear and may be complex, involving lipopolysaccharide (LPS) and other antigens (Acharya et al., 1987; Eisenstein et al., 1984; Fierer et al., 1988; Killar and Eisenstein, 1985; Kuusi et al., 1981 ; Lyman et aL, 1979; Muotiala et ai., 1989; Nnalue and Stocker, 1987; Robertson et al., 1982a,b; Smith et al., 1984; Udhayakumar and Muthukkaruppan, 1987). We have studied the specificity of protection conferred by aroA salmonellae in mice challenged three months after immunization, In particular, the role of the LPS O-specific antigen was investigated using challenge strains of S. typhimurium and S. enteritidis expressing the main LPS 0 antigen of S. typhimurium (0-factor 4) or S. enteritidis (0-factor 9). The virulent S. typhimurium SL1344 (04,5,12) and C5 (01,4,5,12 (Hormaeche, 1979)) and the aroA S. typhimurium SL3261 (Hoiseth and Stocker, 1981) have been described. SL5559 and SL5560 are derivatives of C5 expressing 0 antigens, respectively, 1,4,12 and 1,9,12, referred to below as C5-04 and C5-09. The virulent S. enteritidis strains were Se795 (01,9,12) (Collins, 1968a) and two transductionai derivatives, SL7137 (04,12) and SL7138 (09,12) which are referred to as Se795-04 and Se795-09. CU58, an aroA derivative of Se795, is referred to as Se795 aroA. The aroA S. dublin (BRD061) and its virulent parent (BRD060) were received from G. Dougan, Wellcome Biotech (UK). Virulence and growth kinetics of vaccine and challenge strains. S. typhimurium C5 and S. enteritidis Se795 were of similar virulence in BALB/c mice, as shown by their i.e. and oral LDs0 (tables I-V) and their growth patterns in liver and spleen (figure 1). The vaccine strains S. typhimurium SL3261 and S. enteritidis Se795 aroA also behaved similarly in vivo (fig. 2); the bacterial count in vivo never exceeded the inoculum dose and 100 organisms or less were present at 1 month. No organisms were detected two months after vaccination. Protection conferred by $. typhimurium aroA.
a) i.e. challenge. - Mice challenged 3 months after i.e. immunization were well protected against S. typhimurium, as assessed by LDs0 determinations (table I); the LDs0 were > 104 in immune mice vs. < 102 in controls: Cross-protection experiments (table I) showed that the mice were much better protected against S. typhimurium than against S. enteritidis. Surprisingly, protection was almost as good against S. typhimurium C5-09, i.e. expressing the S. enteritidis LPS (0-factor 9), as against C5-04 expressing the homologous LPS (0-factor 4), suggesting that the LPS 0 antigen is not the main antigen involved in the specific recall of the protective immune response in this system. However, the dose-response pattern was often uneven, with scattered deaths over a wide dose range, suggesting that protection against i.e. challenge in this system was incomplete.
LIVE SALMONELLA
VACCINE AND IMMUNITY
759
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FIG. 1. - -
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.
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Growth o f S. t y p h i m u r i u m C5 (solid symbols) and S. enteritidis Se795 (open symbols) in livers (circles) and spleens (triangles) of BALB mice.
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FIG. 2. - -
1'0
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Growth o f S. t y p h i m u r i u m SL3261 (solid symbols) and S. enteritidis Se795aroA in livers (circles) and spleens (triangles) o f BALB/c mice.
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C.E. H O R M A E C H E E T A L .
b) Oral challenge. - IV vaccinated mice were better protected against oral challenge than against IV challenge (table II); the irregular pattern of deaths seen with IV challenge was not observed. Protection was somewhat better against C5-04 (4.8 log) than against C5-09 (3.2 log). By contrast, there was no protection against S. enteritidis Se795 or Se795-09, and very little protection against Se795-04 (1.4 log). As with the i.v. challenge system, the main determinant of immunity appeared to be the parent background, S. O,phimurium or S. enteritidis, of the challenge, rather than its 0-factor antigen. Protection conferred by S. enterMdis Se795 aroA. Strain Se795 aroA was not as effective as SL3261 (table III); 3 months after vaccination, protection against S. enteritidis was at best 200 x the LDs0 in controls. Protection was better against S. typhimurium variants expressing the homologous antigen, i.e. C5-09, but overall protection was very poor when compared with that afforded by S. typhimurium SL3261 (cf. tables II and III). This lower protection with the S. enteritidis live vaccine was unexpected, given the similarity in the in vivo growth kinetics of the two vaccine strains, and the similar virulence of the challenge strains. In contrast, there was extensive cross-protection between S. enteritidis and S. typhimurium when mice were challenged orally two weeks after i.v. vaccination (table IV). Both S. typhimurium SL3261 and S. enteritidis Se795 aroA conferred equally high protection ( > 10,000 x LDs0) against either S. typhimurium or S. enteritidis, indicating that the poor protection against S. enteritidis at three months post vaccination is not due to an inherent incapacity of BALB/c mice to control S. enteritidis.
TABLE I.
-
-
Vaccination with S. typhimurium SL3261 aroA: IV challenge.
Challenge strain
S. S. S. S. S.
typhimurium SLI344(a) typhimurium C5 typhimurium C5-04(b) typhimurium C5-09(b) enteritidis Se795
LPS 1,4,5,12 1,4,5,12 1,4,5,12 1,9,12 1,9,12
log 10 LDs0 Vacc. Cont. >4.9 4.38 4.0 3.83 <2.3
<2.1 < 1.9 <2.3 2.2 <2.3
Protect. yes yes yes yes no
BALB/c mice vaccinated i.v. with l06 organisms, challenged i.v. at three months. (i) Parent of SL3261. (b) 0 antigen variants of S. typhimuriumC5.
TABLE II. - - Vaccination with S. typhimurium 8L3261 aroA: oral challenge. Challenge strain
S. $. S. S. S.
typhimurium C5-04(a) typhimurium C5-09(a) enteritidisSe795 enterMdis Se795-04(b) enteritidis Se795-09(b)
LPS 1,4,5,12 1,9,12 1,9,12 1,4,12 1,9,12
log 10 LDs0 Vacc. Controls 10.59 9.17 < 6.17 8.14 6.63
5.76 5.94 < 6.37 6.72 7.04
log 10 protection 4.83 3.23 ? 1.42 ?
BALB/c mice vaccinated i.v. with 106 organism~,oral cha)le,ge at 3 months. Sister transductants expressingdifferent LPS 0 antigens on an S. typhimuriumCSp) or S. enteritidis 8e795(b)background.
LIVE SALMONELLA VACCINE AND IMMUNITY
761
Proteetien conferred by S. dublin aroA. The protection conferred by S. dublin aroA against its virulent parent was a hundredfold lower than with the S. typhimurium system (table V); there were scattered deaths over a wide dose range. Protection was nearly as good against S. typhimurium C5-09, but there was no immunity to C5-04.
TABLE I I I .
--
Vaccination with S. typhimurium Se795 areA: oral challenge.
Challenge strain
S. S. S. S. S.
LPS
enteritidis Se795 enteritidis Se795-04(b) enteritidis Se795-09(b) typhimurium C5-04(a) typhimurium C5-09(a)
1,9,12 1,4,12 1,9,12 1,4,5,12 1,9,12
log 10 LDs0 Vacc. Controls 8.35 7.8 8.04 6.74 7.84
log 10 protection
5.9 6.58 6.99 6.04 5.50
2.36 1.22 1.05 0.7 2.34
BALB/c mice vaccinated i.v. with 106 orgav.isms, oral challenge at 3 months. Sister transductants expressing different LPS 0 antigens on an S. typhimuriumC5(~)or S. enteriti-
dis Se795(b)background.
TABLE IV. - - Vaccination with S. typhimurium SL3261 and S. enteritidis Se795 aroA: oral challenge at 14 days. Vaccine
Challenge strain
S. typhimurium SL3261
S. S. S. enteritidis Se795 aroA S. S. None S. S.
typhimurium C5-04 enteritidis Se795 typhimurium C5-04 enteritidis Se795 typhimurium C5-04 enteritidis Se795
log 10 LDs0
log 10 protection
> 10.8 10.39 9.7 > 10.9 5.8 5.67
>5 4.72 3.9 > 5.23
BALB/c mice were vaccinated i.v. with approx. 106organisms and challenged orally 14 days later.
TABLE V . Challenge strain
--
Vaccination with S. dublin aroA: oral challenge. LPS
S. dublin 1,9,12 S. typhimurium C5-04(a) 1,4,5,12 S. typhimurium C5-09(a) 1,9,12
Vacc.
log 10 LDso Controls
log 10 protection
8.74 6.25 g.80
<6.31 6.93 6.98
>2.43 none 1.81
BALB/c mice were vaccinatedi.v. with approx. 10~organismsand challengedorally 3 months later. p) Sister transductants expressing different LPS 0 antigens on an S. typhimurium C5 background.
762
C.E. H O R M A E C H E E T A L .
Discussion and conclusions.
The present results suggest that thc specificity of protection in this system is determined more by the species of the challenge strain than by the LPS 0 antigen. However, further studies with a wider variety of strains are desirable to see whether this constitutes a general case, or if it applies only to the strains tested here. The results also show that aroA vaccines can differ significantly in their immuidzing capacity for mice. There is evidence obtained with different experimental models for both LPS and non-LPS antigens conferring protection in salmonellosis. LPS antigens are important in protection against intraperitoneal ch~lenge (Lyman et al., 1979; Svenson and Lindberg, 1981). Protection in mice vaccinated with 0-4,12 and 0-6,7 derivatives of an aroA S. typhimurium and challenged with S. typhimurium and S. cholerae-suis was 0-specific with strains of moderate virulence, but serotype-specific for a more virulent challenge (Nnalue and Stocker, 1987). Mice immunized with S. dublin (group D) cured of its virulence plasmid were protected from i.p. challenge with salmonellae of groups B (0-antigens (1),4,(5), 12) and D (0-(0,9,12) but not group C (0-6,7) (Fierer et al., 1988). Several authors have presented evidence suggesting that LPS antigens contribute to immunity from challenge by other routes. Cross-protection to i.e. challenge between S. typhimurium and S. enteritidis, which decreased as the vaccine was cleared, has been described (Collins, 1968b). Calves challenged orally three weeks after immunization with aroA salmonellae showed cross protection between S. typhimurium and S. dublin (Rankin et al., 1967; Sm'~th et al., 1984, ~/ray et al., 1977). Calves vaccinated with aroA salmonellae showed delayed hypersensitivity and in vitr, proliferative responses to LPS antigens, suggesting that cell-mediated immunity to LPS may be involved in protection (Robertsson et al., 19g2a,b). In vitro proliferative responses to S. typhi LPS antigens have been reported in humans (Murphy et al., 1989). However, there is also evidence for a role of non-LPS antigens in immunity. Live "rough" salmonellae can protect against smooth organisms (Muotiala et ai., 1989). LP~-unresponsive C3H/I-Iel mice acquire long-lasting protection following vaccination with aroA S. typhimurium (Eisenstein eta/., 1984; Killar and Eisenstein, 1985). The present results show that vaccination with aroA salmonellae confers shortlived non-speciflc protection which is followed by solid but species-specific immunity to oral challenge; the mechanisms remain unclear. It is not known whether the T-cell/macrophage interactions operating in immunity to Listeria and mycobacteria (Kaufmann, 1988) also occur in salmonellosis. It is also unknown whether adaptation tO the host environment (Finlay and Falkow, 1989) causes the expression in vivo of other protective antigens. The role of heat-shock proteins in immunity is being investigated (Young et ai., 1988); mice recognize a putative heat-shock salmonella protein during infection (Brown and Hormacche, 1989). The failure of S. typhimurium to confer protection against S. enteritidis could be due to unknown virulence factors in S. e,:teritidis different from those of S. typMmurium; however, BALB/c mice can control S. enteritidis Se795, as shown by the protection observed two weeks after vaccination with either organism. These results are not easily explainable in terms o f LPS-specific protection. It is not clear why the S. enteritidis and S. dublin vaccines gave much less protection than the S. typhimurium vaccine, especially as the patterns of persistence in the liver and spleen of SL3261 and Se795 were so similar. We have obtained similar preliminary results in BI0.M mice. Persistence in the reticuloendothelial system is believed to be important in determining vaccine efficacy (Collins, 1968a; O'Callaghan et al., 1988; Sigwart et al., 1989). The present results suggest that factors other than persistence of the vaccine and virulence of the challenge as measured by in vivo net growth rates or LDs0 determine vaccine efficacy in this model. Further work on
LIVE SALMONELLA
VACCINE AND IM3~UNITY
763
the specificity o f protection and the immune response induced by effective and noneffective vaccines could yield useh:l informe*~<,o on the ~ e c h a n i s m s o f immunity to salmonellae and the requ~remems for an eti¢ctive vaccine strain. Acknowledgements.
This work was supported by a grant from the Medical Research Council (UK).
References.
ACHARYA,I.L., Low~, C.U., THAPA, R., GURUBACHARYA,V.L., SHRESTHA,M.•., CADOZ,M., Sctlucz, D., AV.M~, D., BRYLA,D.A., TROU~VO~,B., C ~ O N , T., SCU~eP.SON,R. & RonmNs, J.B. (1987), Prevention of typhoid fever in Nepal with the Vi capsular polysaceharide of SMmone//a typM. New Eng/. J. Meal., 317, i101-1104. BROWN,A. & HOP~AECHE,C.E. (1989), The antibody response to salmonellae in mice and humans studied by immunoblots and ELISA. Microbial Pathogenesis, 6, 445-454. COLLINS,F.M. (1968a), Cross-protection against Salmonella enteritidis infection in mice. J. Bact., 95, 1343-1349. COLUNS,F.M. (1968b), Recall of immunity in mice vaccinated with Salmonella enteritidis and Salmonella t.vphimurium. J. aact., 95, 2014-2021. COLLINS, F.M. (1974), Vaccines and cell-mediated immunity. Bact. Rev., 38, 371-402. D o u o ~ , G., SMm~, L. & H~w~o~, F. (1989), Live bacteria[ vaccines anti their application as carriers for foreign ann~ens. Advanc. Vet. Sci. Comp. Meal., 33, 271-300. EISENSTEIN, T.K. & SULTZBR, B.M. (1983), Immunity to salmonella infections. Advanc. exp. Med. Biol., 261-296. EISENSTem,T.K., KmLAR,L.M., SXOCKEn,~.A.D. & SuLxzen, B.M. (1984), Cellular immunity induced by avirulent salmonella in LPS.defective C3H/HeJ mice. ,L. ImmunoL, 133, 958-961. FmR~R, 1., C m ~ l , G., HAL~N, L., H~RNAN, E.J. & GullY, D. (1988), Active immunization w/th LD842, a plasmid-cured strain of S¢lmonella dublin, protects mice against group B and grout3 D salmonella infection. J. infect. D/s., 158, 460-463. Fm~.~,z, B.B. & FALKOW,S. (1989), Common themes in microbial pathogenicity. Microbiol. Rev., 53, 210-230. HOIS~TH, S.K. & STOCK,R, B.A.D. (1981), Aromatic.dependent Salmonella tyohimurium are non-virulent and effective as live vaccines. Nature (Lond.), 291, 238-239. HORMA~C~e,C.E. (1979), Natural resistance to Salmonella t,vphimurium in mice. Immunology, 37, 311-318. Ho~M~cue, C.E., FAU~SK~OG,M.C., Pe~svon, R.A. & B~ocK, J. (1981), Acquireo immunity to Salmonella typhimurium and delayed (footpad) hypersensitivity in BALB/c mice. Immunology, 43, 547-554. KAUFMA~, S.H.E. (1988), Immunit~ against intracellular bacteria: biological effector functions and antigen specificity of T lymphocytes. Curr. Top. MicrobioL ImmunoL, 138, 141-176. KtCUAR, L.M. & Etse~sxem, T.K. 0985), Immunity to Salmonella typhimurium infection in C3H/HeJ and C3H/HeNCrlBR mice: studies with an aromatic-dependent live S. typhimurium strain as a vaccine. ,:,~fect. Immun., 47, 605-612. Kuus~, N., Nu~N~4, M., SAxon, H. & M ~ , P.H. (1981), Immunization with ma~or outer membrane protein (porin) pregara~ion in experimental salmonenosis: effect of lipopolysaccharide. Infect. Immun., ~ , 328-332. LVMA~,M.B., STOCKS, B.A.D. & R o ~ r p ~ , R. L (1979), Evaluation of the immune response directed against the salmonella antigenic factors 04,5 and 09. Infect. Immun., 26, 956-965. MUOTIALA,A., Hovl, M. & MAKELA,P.H. (1989), Protective immunity in mouse salmonellosis: comparison of smooth and rough live and killed salmonella vaccines. Microbial Pathogenesis, 6, 51-60. MURPHY, J.R., WASSERMAN,S.S., BAQAR,S., SCHLESINGER,L., FERRECCIO,C., LINI)BER¢3,A.A. & L~vme, MoM. (1989), Immunity to Salmonella typhi: considerations relevant to measurements of cellular imunity in typhoid-endemic regions. Clin. exp. Immunol., 75, 228-233.
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NNALUE,N.A. & STOCKER,B.A.D. (1987), Tests of the virulence and live vaccine efficacy of auxotrophic and galE derivatives of Salmonella choleraesuis. Infect. Immun., 5S, 955-962. O'CALLAGHAN,D., MaSKELL,D.J., LIEW,F.Y., EASMON,C.S.F. & DOUOAN,G. (1988), Characterisation of aromatic and purine dependent Salmonella typhimurium: attenuation, persistence and ability to induce protective immunity in BALB/c mice. Infect. Immun., 56, 419-423. RANKJN,J.D., TAYLOR,R.J. & NEWMAN,G. (1967), The protection of calves against infection with Salmonella typhimurium by means of a vaccine prepared from Salmonella dublin (strain 51). Vet. Record., 80, 720-726. ROB~RTSSON,J.A., FOSSUM,C., SVENSON,S.B. & LINDnERG,A.A. (1982a), Salmonella typhimurium infection in calves: specific imune reactivity against 0-antigenic polysaccharide detectable in in vitro assays. Infect. Immun., 37, 728-737. ROBER~SSON,J.A., SVENSON,S.B. & LINDB~RG,A.A. (1982b), Salmonella typhimurium infection in calves: delayed specific skin reactions directed against the 0-antigenic polysaccharide chain. Infect. Immun., 37, 738-748. SIGWART,D.F., STOCKER,B.A.D. & CLEMENTS,D. (1989), Effect of a purA mutation on efficacy of Salmonella typhimurium live vaccine vectors. Infect. lmmun., 57,1858-1861. SMITH,B.P., REINA-GUERRA,M., HOISETH,S.K., STOCKER,B.A.D., HABASHA,F., JOHNSON,E. & MERRITT,F. (1984), Aromatic-dependent Salmonella typhimurium as modified live vaccines for calves. Amer. 3. vet. Res., 45, 59-66. SVENSON,S.B. & LINDBERG,A.A. (1981), Artificial salmonella vaccines: Salmonella typhimurium 0-antigen specific oligosaccharide-protein conjugates elicit protective antibodies in rabbits and mice. Infect. Immun., 32, 490-496. UDHAYAKUMAR,V. & MUTm'KKARUPPAN,V.R. (1987), Protective immunity induced by outer membrane proteins of Salmonella typhimurium in mice. Infect. Immun., 55, 816-821. WRAY,C., SJOKA,W.J., MORRIS,J.A. & BRINLEYMORGAN,W.J. (1977), The immunization of mice and calves with gale mutants of Salmonella typhimurium. J. Hyg. (Camb.), 79, 17-24. YOUNG,D., LATHIGRA,R., HENDRIX,R., SWEETSTER,D. & YOUNG,R.A. (1988), Stress proteins are immune targets in leprosy and tuberculosis. Proc. nat. Acad. Sci. (Wash.), 85, 4267-4270.
~) INSTITUTPASTEUR/ELsEVIER Paris 1990
IMMUNITY
INDUCED
BY L I V E
ATTENUATED
SALMONELLA
VACCINES
C.E. Hormaeche(t) ('), H.S. Joysey(l), L. Desilva(l), M. I z h a r ( l ) a n d B. A , D . Stocker(2)
(I)Microbiology and Parasitology Division, Department o f Pathology, University o f Cambridge, Tennis Court Road, Cambridge CB2 IQP, (UK) " and (2)Department o f Microbiology and Immunology, Stanford University School o f Medicine, Stanford, C 94305 (USA)
Summary. Studies on the degree and specificity of protection conferred by immunization with aroA salmonella live vaccines in BALB/c mice are described. Animals were immunized i.v. and challenged orally 3 months later to ensure that the vaccine had been cleared from the tissues. Vaccination with Salmonella typhimurium aroA SL3261 conferred very good protection against virulent S. typhimurium C5 (over 10,000 × LDs0). The specificity of cross protection was studied using S. typhimuriurn, Salmonella enteritidis and Salmonella dublin for vaccination and challenge, including challenge with variants of S. typhimurium and S. enteritidis of similar virulence which differed in the main LPS (lipopolysaccharide) antigen (0-4 or 0-9). S. typhimurium SL3261 gave very good protection against S. typhimurium C5 (0-4), but no protection against S. enteritidis Se795 (0-9). However, challenge with strains differing in the main 0 antigens showed that, although protection was generally better to strains expressing the same LPS type as the vaccine, specificity of protection was determined more by the background (S. typhimurium or S. enteritidis) of the parent strain used for the challenge than by 0 factors 4 or 9, suggesting that other factors could be involved. The nature of the antigen(s) responsible for protection in this model is unclear, but it would not appear to be the main 0.specific antigen. An S. enteritidis Se795 aroA vaccine was far less effective than S. typhimurium SL3261 ; it conferred good protection against the homologous wild type at 2 weeks post-vaccination, but far less at three months (approx 10-200 x LDs0). This was unexpected, as the persistence of the S. enteritidis vaccine in the liver and spleen was similar to that of S. typhimurium SL3261, and the S. enteritidis and S. typhimurium challenge strains were of similar virulence. An S. dublin aroA vaccine conferred similar protection against wild types S. dublin (approx 300 × LDs0). KEY-WORDS"Salmonella, Vaccine, Mutant aroA, Lipopolysaccharide; Protection.
(') Correspondingauthor.
758
C.E. H O R . M A E C H E E T A L .
Introduction.
The mechanisms of immunity to salmonellosis are still unclear. In experimental models, live vaccines, which induce both humoral and cellular immunity, confer much better protection than killed vaccines, which do not elicit cellular immunity (Collins, 1974; Eisenstein and Sultzer, 1983). The encouraging results obtained with live attenuated vaccines suggest that cellular immunity is important in human typhoid (Murphy et al., 1989). Salmonellae wi:h mutations in genes of the aromatic pathway are effective as vaccines and as carriers of recombinant antigens from other parasites (Dougan et al., 1989). However, some live salmonella vaccines confer very poor protection (Collins, 1974; Hormaeche et al., 1981 ; Smith et aL, 1984). Effective vaccines can persist for a limited period in the tissues after which non-specific immunity disappears, with protection requiring the specific recall of immunity (Collins, 1974). The specificity of the protective response is still unclear and may be complex, involving lipopolysaccharide (LPS) and other antigens (Acharya et al., 1987; Eisenstein et al., 1984; Fierer et al., 1988; Killar and Eisenstein, 1985; Kuusi et al., 1981 ; Lyman et aL, 1979; Muotiala et ai., 1989; Nnalue and Stocker, 1987; Robertson et al., 1982a,b; Smith et al., 1984; Udhayakumar and Muthukkaruppan, 1987). We have studied the specificity of protection conferred by aroA salmonellae in mice challenged three months after immunization, In particular, the role of the LPS O-specific antigen was investigated using challenge strains of S. typhimurium and S. enteritidis expressing the main LPS 0 antigen of S. typhimurium (0-factor 4) or S. enteritidis (0-factor 9). The virulent S. typhimurium SL1344 (04,5,12) and C5 (01,4,5,12 (Hormaeche, 1979)) and the aroA S. typhimurium SL3261 (Hoiseth and Stocker, 1981) have been described. SL5559 and SL5560 are derivatives of C5 expressing 0 antigens, respectively, 1,4,12 and 1,9,12, referred to below as C5-04 and C5-09. The virulent S. enteritidis strains were Se795 (01,9,12) (Collins, 1968a) and two transductionai derivatives, SL7137 (04,12) and SL7138 (09,12) which are referred to as Se795-04 and Se795-09. CU58, an aroA derivative of Se795, is referred to as Se795 aroA. The aroA S. dublin (BRD061) and its virulent parent (BRD060) were received from G. Dougan, Wellcome Biotech (UK). Virulence and growth kinetics of vaccine and challenge strains. S. typhimurium C5 and S. enteritidis Se795 were of similar virulence in BALB/c mice, as shown by their i.e. and oral LDs0 (tables I-V) and their growth patterns in liver and spleen (figure 1). The vaccine strains S. typhimurium SL3261 and S. enteritidis Se795 aroA also behaved similarly in vivo (fig. 2); the bacterial count in vivo never exceeded the inoculum dose and 100 organisms or less were present at 1 month. No organisms were detected two months after vaccination. Protection conferred by $. typhimurium aroA.
a) i.e. challenge. - Mice challenged 3 months after i.e. immunization were well protected against S. typhimurium, as assessed by LDs0 determinations (table I); the LDs0 were > 104 in immune mice vs. < 102 in controls: Cross-protection experiments (table I) showed that the mice were much better protected against S. typhimurium than against S. enteritidis. Surprisingly, protection was almost as good against S. typhimurium C5-09, i.e. expressing the S. enteritidis LPS (0-factor 9), as against C5-04 expressing the homologous LPS (0-factor 4), suggesting that the LPS 0 antigen is not the main antigen involved in the specific recall of the protective immune response in this system. However, the dose-response pattern was often uneven, with scattered deaths over a wide dose range, suggesting that protection against i.e. challenge in this system was incomplete.
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10
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FIG. 1. - -
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Growth o f S. t y p h i m u r i u m C5 (solid symbols) and S. enteritidis Se795 (open symbols) in livers (circles) and spleens (triangles) of BALB mice.
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FIG. 2. - -
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Growth o f S. t y p h i m u r i u m SL3261 (solid symbols) and S. enteritidis Se795aroA in livers (circles) and spleens (triangles) o f BALB/c mice.
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C.E. H O R M A E C H E E T A L .
b) Oral challenge. - IV vaccinated mice were better protected against oral challenge than against IV challenge (table II); the irregular pattern of deaths seen with IV challenge was not observed. Protection was somewhat better against C5-04 (4.8 log) than against C5-09 (3.2 log). By contrast, there was no protection against S. enteritidis Se795 or Se795-09, and very little protection against Se795-04 (1.4 log). As with the i.v. challenge system, the main determinant of immunity appeared to be the parent background, S. O,phimurium or S. enteritidis, of the challenge, rather than its 0-factor antigen. Protection conferred by S. enterMdis Se795 aroA. Strain Se795 aroA was not as effective as SL3261 (table III); 3 months after vaccination, protection against S. enteritidis was at best 200 x the LDs0 in controls. Protection was better against S. typhimurium variants expressing the homologous antigen, i.e. C5-09, but overall protection was very poor when compared with that afforded by S. typhimurium SL3261 (cf. tables II and III). This lower protection with the S. enteritidis live vaccine was unexpected, given the similarity in the in vivo growth kinetics of the two vaccine strains, and the similar virulence of the challenge strains. In contrast, there was extensive cross-protection between S. enteritidis and S. typhimurium when mice were challenged orally two weeks after i.v. vaccination (table IV). Both S. typhimurium SL3261 and S. enteritidis Se795 aroA conferred equally high protection ( > 10,000 x LDs0) against either S. typhimurium or S. enteritidis, indicating that the poor protection against S. enteritidis at three months post vaccination is not due to an inherent incapacity of BALB/c mice to control S. enteritidis.
TABLE I.
-
-
Vaccination with S. typhimurium SL3261 aroA: IV challenge.
Challenge strain
S. S. S. S. S.
typhimurium SLI344(a) typhimurium C5 typhimurium C5-04(b) typhimurium C5-09(b) enteritidis Se795
LPS 1,4,5,12 1,4,5,12 1,4,5,12 1,9,12 1,9,12
log 10 LDs0 Vacc. Cont. >4.9 4.38 4.0 3.83 <2.3
<2.1 < 1.9 <2.3 2.2 <2.3
Protect. yes yes yes yes no
BALB/c mice vaccinated i.v. with l06 organisms, challenged i.v. at three months. (i) Parent of SL3261. (b) 0 antigen variants of S. typhimuriumC5.
TABLE II. - - Vaccination with S. typhimurium 8L3261 aroA: oral challenge. Challenge strain
S. $. S. S. S.
typhimurium C5-04(a) typhimurium C5-09(a) enteritidisSe795 enterMdis Se795-04(b) enteritidis Se795-09(b)
LPS 1,4,5,12 1,9,12 1,9,12 1,4,12 1,9,12
log 10 LDs0 Vacc. Controls 10.59 9.17 < 6.17 8.14 6.63
5.76 5.94 < 6.37 6.72 7.04
log 10 protection 4.83 3.23 ? 1.42 ?
BALB/c mice vaccinated i.v. with 106 organism~,oral cha)le,ge at 3 months. Sister transductants expressingdifferent LPS 0 antigens on an S. typhimuriumCSp) or S. enteritidis 8e795(b)background.
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Proteetien conferred by S. dublin aroA. The protection conferred by S. dublin aroA against its virulent parent was a hundredfold lower than with the S. typhimurium system (table V); there were scattered deaths over a wide dose range. Protection was nearly as good against S. typhimurium C5-09, but there was no immunity to C5-04.
TABLE I I I .
--
Vaccination with S. typhimurium Se795 areA: oral challenge.
Challenge strain
S. S. S. S. S.
LPS
enteritidis Se795 enteritidis Se795-04(b) enteritidis Se795-09(b) typhimurium C5-04(a) typhimurium C5-09(a)
1,9,12 1,4,12 1,9,12 1,4,5,12 1,9,12
log 10 LDs0 Vacc. Controls 8.35 7.8 8.04 6.74 7.84
log 10 protection
5.9 6.58 6.99 6.04 5.50
2.36 1.22 1.05 0.7 2.34
BALB/c mice vaccinated i.v. with 106 orgav.isms, oral challenge at 3 months. Sister transductants expressing different LPS 0 antigens on an S. typhimuriumC5(~)or S. enteriti-
dis Se795(b)background.
TABLE IV. - - Vaccination with S. typhimurium SL3261 and S. enteritidis Se795 aroA: oral challenge at 14 days. Vaccine
Challenge strain
S. typhimurium SL3261
S. S. S. enteritidis Se795 aroA S. S. None S. S.
typhimurium C5-04 enteritidis Se795 typhimurium C5-04 enteritidis Se795 typhimurium C5-04 enteritidis Se795
log 10 LDs0
log 10 protection
> 10.8 10.39 9.7 > 10.9 5.8 5.67
>5 4.72 3.9 > 5.23
BALB/c mice were vaccinated i.v. with approx. 106organisms and challenged orally 14 days later.
TABLE V . Challenge strain
--
Vaccination with S. dublin aroA: oral challenge. LPS
S. dublin 1,9,12 S. typhimurium C5-04(a) 1,4,5,12 S. typhimurium C5-09(a) 1,9,12
Vacc.
log 10 LDso Controls
log 10 protection
8.74 6.25 g.80
<6.31 6.93 6.98
>2.43 none 1.81
BALB/c mice were vaccinatedi.v. with approx. 10~organismsand challengedorally 3 months later. p) Sister transductants expressing different LPS 0 antigens on an S. typhimurium C5 background.
762
C.E. H O R M A E C H E E T A L .
Discussion and conclusions.
The present results suggest that thc specificity of protection in this system is determined more by the species of the challenge strain than by the LPS 0 antigen. However, further studies with a wider variety of strains are desirable to see whether this constitutes a general case, or if it applies only to the strains tested here. The results also show that aroA vaccines can differ significantly in their immuidzing capacity for mice. There is evidence obtained with different experimental models for both LPS and non-LPS antigens conferring protection in salmonellosis. LPS antigens are important in protection against intraperitoneal ch~lenge (Lyman et al., 1979; Svenson and Lindberg, 1981). Protection in mice vaccinated with 0-4,12 and 0-6,7 derivatives of an aroA S. typhimurium and challenged with S. typhimurium and S. cholerae-suis was 0-specific with strains of moderate virulence, but serotype-specific for a more virulent challenge (Nnalue and Stocker, 1987). Mice immunized with S. dublin (group D) cured of its virulence plasmid were protected from i.p. challenge with salmonellae of groups B (0-antigens (1),4,(5), 12) and D (0-(0,9,12) but not group C (0-6,7) (Fierer et al., 1988). Several authors have presented evidence suggesting that LPS antigens contribute to immunity from challenge by other routes. Cross-protection to i.e. challenge between S. typhimurium and S. enteritidis, which decreased as the vaccine was cleared, has been described (Collins, 1968b). Calves challenged orally three weeks after immunization with aroA salmonellae showed cross protection between S. typhimurium and S. dublin (Rankin et al., 1967; Sm'~th et al., 1984, ~/ray et al., 1977). Calves vaccinated with aroA salmonellae showed delayed hypersensitivity and in vitr, proliferative responses to LPS antigens, suggesting that cell-mediated immunity to LPS may be involved in protection (Robertsson et al., 19g2a,b). In vitro proliferative responses to S. typhi LPS antigens have been reported in humans (Murphy et al., 1989). However, there is also evidence for a role of non-LPS antigens in immunity. Live "rough" salmonellae can protect against smooth organisms (Muotiala et ai., 1989). LP~-unresponsive C3H/I-Iel mice acquire long-lasting protection following vaccination with aroA S. typhimurium (Eisenstein eta/., 1984; Killar and Eisenstein, 1985). The present results show that vaccination with aroA salmonellae confers shortlived non-speciflc protection which is followed by solid but species-specific immunity to oral challenge; the mechanisms remain unclear. It is not known whether the T-cell/macrophage interactions operating in immunity to Listeria and mycobacteria (Kaufmann, 1988) also occur in salmonellosis. It is also unknown whether adaptation tO the host environment (Finlay and Falkow, 1989) causes the expression in vivo of other protective antigens. The role of heat-shock proteins in immunity is being investigated (Young et ai., 1988); mice recognize a putative heat-shock salmonella protein during infection (Brown and Hormacche, 1989). The failure of S. typhimurium to confer protection against S. enteritidis could be due to unknown virulence factors in S. e,:teritidis different from those of S. typMmurium; however, BALB/c mice can control S. enteritidis Se795, as shown by the protection observed two weeks after vaccination with either organism. These results are not easily explainable in terms o f LPS-specific protection. It is not clear why the S. enteritidis and S. dublin vaccines gave much less protection than the S. typhimurium vaccine, especially as the patterns of persistence in the liver and spleen of SL3261 and Se795 were so similar. We have obtained similar preliminary results in BI0.M mice. Persistence in the reticuloendothelial system is believed to be important in determining vaccine efficacy (Collins, 1968a; O'Callaghan et al., 1988; Sigwart et al., 1989). The present results suggest that factors other than persistence of the vaccine and virulence of the challenge as measured by in vivo net growth rates or LDs0 determine vaccine efficacy in this model. Further work on
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the specificity o f protection and the immune response induced by effective and noneffective vaccines could yield useh:l informe*~<,o on the ~ e c h a n i s m s o f immunity to salmonellae and the requ~remems for an eti¢ctive vaccine strain. Acknowledgements.
This work was supported by a grant from the Medical Research Council (UK).
References.
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