~) INSTITUT PASTEUR/ELsEVIER Res. MicrobioL Paris 1990 1990, 141, 757-764

I M M U N I T Y I N D U C E D BY LI V E A TTEN U A TED S A L M O N E L L A

VACCINES

C.E. Hormaeche(t) ('), H.S. Joysey(l), L. Desilva(l), M. Izhar( l ) and 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 im- munized 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. typhimuri- urn, Salmonella enteritidis and Salmonella dublin for vaccination and challenge, in- cluding 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. typhimu- rium SL3261 gave very good protection against S. typhimurium C5 (0-4), but no pro- tection 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 in- volved. The nature of the antigen(s) responsible for protection in this model is un- clear, 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 persis- tence 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.

(') Corresponding author.

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 at- tenuated vaccines suggest that cellular immunity is important in human typhoid (Mur- phy et al., 1989).

Salmonellae wi:h mutations in genes of the aromatic pathway are effective as vac- cines 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 lipopolysac- charide (LPS) and other antigens (Acharya et al., 1987; Eisenstein et al., 1984; Fier- er 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 ex- pressing 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. en- teritidis 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 pro- tected 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.

L I V E S A L M O N E L L A V A C C I N E A N D I M M U N I T Y 7 5 9

10

§8 =,

6 ,o ,,,..

< - - -

" • ! , i . ! , I .

0 1 2 3 4 5 DAYS

FIG. 1. - - Growth o f S. typh imur ium C5 (solid symbols) and S. enteritidis Se795 (open sym- bols) in livers (circles) and spleens (triangles) o f BALB mice.

~z 6

o 5

tD < 4

o 3

"J 2

1

0 1 '0 2'0 3'0 4'0

DAYS FIG. 2. - - Growth o f S. typhimur ium SL3261 (solid symbols) and S. enteritidis Se795aroA

in livers (circles) and spleens (triangles) o f BALB/c mice.

760 C.E. H O R M A E C H E E T AL.

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. enteriti- dis 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 par- ent 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 vac- cination, protection against S. enteritidis was at best 200 x the LDs0 in controls. Pro- tection 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 ki- netics 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 (ta- ble IV). Both S. typhimurium SL3261 and S. enteritidis Se795 aroA conferred equal- ly 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 vacci- nation 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.

log 10 LDs0 Challenge strain LPS Vacc. Cont. Protect.

S. typhimurium SLI344(a) 1,4,5,12 >4.9 <2.1 yes S. typhimurium C5 1,4,5,12 4.38 < 1.9 yes S. typhimurium C5-04(b) 1,4,5,12 4.0 <2.3 yes S. typhimurium C5-09(b) 1,9,12 3.83 2.2 yes S. enteritidis Se795 1,9,12 <2.3 <2.3 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. typhimurium C5.

TABLE II. - - Vaccination with S. typhimurium 8L3261 aroA: oral challenge.

log 10 LDs0 log 10 Challenge strain LPS Vacc. Controls protection

S. typhimurium C5-04(a) 1,4,5,12 10.59 5.76 4.83 $. typhimurium C5-09(a) 1,9,12 9.17 5.94 3.23 S. enteritidisSe795 1,9,12 < 6.17 < 6.37 ? S. enterMdis Se795-04(b) 1,4,12 8.14 6.72 1.42 S. enteritidis Se795-09(b) 1,9,12 6.63 7.04 ?

BALB/c mice vaccinated i.v. with 106 organism~, oral cha)le,ge at 3 months. Sister transductants expressing different LPS 0 antigens on an S. typhimurium CSp) or S. enteriti-

dis 8e795(b) background.

L I V E S A L M O N E L L A VACCINE A N D I M M U N I T Y 761

Proteetien conferred by S. dublin aroA.

The protection conferred by S. dublin aroA against its virulent parent was a hun- dredfold 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. typhimuri- um C5-09, but there was no immunity to C5-04.

TABLE I I I . - - Vaccination with S. typhimurium Se795 areA: oral challenge.

log 10 LDs0 log 10 Challenge strain LPS Vacc. Controls protection

S. enteritidis Se795 1,9,12 8.35 5.9 2.36 S. enteritidis Se795-04(b) 1,4,12 7.8 6.58 1.22 S. enteritidis Se795-09(b) 1,9,12 8.04 6.99 1.05 S. typhimurium C5-04(a) 1,4,5,12 6.74 6.04 0.7 S. typhimurium C5-09(a) 1,9,12 7.84 5.50 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. typhimurium C5(~) or S. enteriti-

dis Se795(b) background.

TABLE IV. - - Vaccination with S. typhimurium SL3261 and S. enteritidis Se795 aroA: oral challenge at 14 days.

log 10 log 10 Vaccine Challenge strain LDs0 protection

S. typhimurium SL3261 S. typhimurium C5-04 > 10.8 >5 S. enteritidis Se795 10.39 4.72

S. enteritidis Se795 aroA S. typhimurium C5-04 9.7 3.9 S. enteritidis Se795 > 10.9 > 5.23

None S. typhimurium C5-04 5.8 S. enteritidis Se795 5.67

BALB/c mice were vaccinated i.v. with approx. 106 organisms and challenged orally 14 days later.

TABLE V . - - Vaccination with S. dublin aroA: oral challenge.

log 10 LDso log 10 Challenge strain LPS Vacc. Controls protection

S. dublin 1,9,12 8.74 <6.31 >2.43 S. typhimurium C5-04(a) 1,4,5,12 6.25 6.93 none S. typhimurium C5-09(a) 1,9,12 g.80 6.98 1.81

BALB/c mice were vaccinated i.v. with approx. 10~ organisms and challenged orally 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 de- termined 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 ca- pacity for mice.

There is evidence obtained with different experimental models for both LPS and non-LPS antigens conferring protection in salmonellosis. LPS antigens are impor- tant 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 be- tween S. typhimurium and S. enteritidis, which decreased as the vaccine was cleared, has been described (Collins, 1968b). Calves challenged orally three weeks after im- munization 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 prolifera- tive 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 vacci- nation with aroA S. typhimurium (Eisenstein eta/., 1984; Killar and Eisenstein, 1985).

The present results show that vaccination with aroA salmonellae confers short- lived non-speciflc protection which is followed by solid but species-specific immuni- ty 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 adapta- tion 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. typhimu- rium 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 of LPS-specific protection.

It is not clear why the S. enteritidis and S. dublin vaccines gave much less protec- tion 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'Calla- ghan 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

L I V E S A L M O N E L L A V A C C I N E A N D I M 3 ~ U N I T Y 763

the specificity o f protect ion and the immune response induced by effective and non- effective vaccines could yield useh:l informe*~<,o on the ~echanisms of 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).

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