Ribonucleic Acid-Protein Fractions of Virulent Salmonella typhimurium as Protective Immunogens

Mice were injected with virulent Salmonella typhimurium SR-11 subfractions containing varied amounts of ribonucleic acid (RNA) and protein or with living attenuated S. typhimurium RIA. In these mice, maximal resistance to lethal infection by 1,000 or 5,000 median lethal doses of S. typhimurium SR-11 was seen 2 to 3 weeks after immunization. The S. typhimurium RIA vaccine and a crude ethanol-precipitated RNA fraction (E-RNA) prepared from lysates of S. typhimurium SR-11 were the most efficient immunogens inducing protection against salmonellosis. The contribution of the components present in the E-RNA fractions to host protection against lethal salmonella infection was also examined. RNA-rich fractions (P-RNA) prepared from lysates of the virulent salmonellae contained several bands of protein when examined by disc electrophoresis. P-RNA fractions stimulated protective immunity in mice to infection with S. typhimurium SR-11 but to a much lesser degree than did the E-RNA fractions or strain RIA vaccine. Protein-rich fractions (NP), separated from E-RNA by salt precipitation, exhibited the same number and distribution of protein bands by disc electrophoresis as did the parent E-RNA fractions. Mixtures of either bovine liver soluble RNA or various synthetic polynucleotides and NP were examined, as was NP fraction alone, for the ability to confer protection in mice to challenge infections by the virulent strain of salmonella. Polyadenylic-uridylic acid plus NP conferred significant protective immunity to challenge infections in mice immunized with this mixture, being nearly as effective an immunogen as were the E-RNA fractions of S. typhimurium SR-11 or the attenuated S. typhimurium RIA.     

Variability of protection in inbred mice induced by a ribosomal vaccine prepared from Salmonella typhimurium.

Ribosomal vaccines prepared from Salmonella typhimurium were effective immunogens in A/J inbred mice and C3H/HeTex, inbred mice. However, ribosomal vaccines were not protective in C57BL/6J inbred mice. A/J mice were protected against lethal challenge by attenuated S. typhimurium live-cell, ribosomal, phenol, and heat-killed vaccines. C3H/HeTex mice were protected by live-cell, ribosomal, and phenol vaccines but not the heat-killed vaccine. Only the live-cell vaccine gave significant protection in the C57BL/6J inbred mice. A comparison of the kinetics of infection in sham-immunized mice and mice immunized with ribosomes showed that ribosome preparations elicited protection against Salmonella infection in mice inherently sensitive and resistant to Salmonella.     

Immunization of mice against Plasmodium vinckei with a combination of attenuated Salmonella typhimurium and malarial antigen.

Infection with the blood stage of the malaria parasite Plasmodium vinckei is uniformly lethal in mice. We found that immunization of BALB/c mice with a combination of killed P. vinckei antigens and an attenuated (aroA) Salmonella typhimurium strain induces high levels of protection against challenge with live P. vinckei. This is especially significant because, in our previous studies, immunization of mice with killed P. vinckei antigens and adjuvants such as Bordetella pertussis, complete Freund adjuvant, and saponin failed to induce protective immunity. Immunization with attenuated S. typhimurium alone did not provide any nonspecific immunity. In vivo depletion of CD4+ T cells in the mice immunized with attenuated S. typhimurium and P. vinckei antigens caused the loss of their immunity. Expression of this immunity required the presence of a spleen. These results support our previous hypothesis that a blood stage malaria vaccine may need both induction of CD4+ T cells specific for the parasite and modification of the spleen with a vaccine vehicle. Therefore, attenuated Salmonella strains such as the one used in this study, when expressing recombinant malarial antigens, might fulfill this requirement.     

Immunity to Salmonella typhimurium infection in C3H/HeJ and C3H/HeNCrlBR mice: studies with an aromatic-dependent live S. typhimurium strain as a vaccine

Immunization with avirulent Salmonella typhimurium strain SL3235, a smooth, aroA- derivative, was shown to induce high levels of resistance to challenge with virulent S. typhimurium in innately hypersusceptible C3H/HeJ mice and inherently resistant C3H/HeNCrlBR mice. Strain SL3235 is one of a class of avirulent aroA- derivatives made from various strains and species of Salmonella that are being considered as vaccine candidates for cattle and humans. This paper supports their efficacy and potential utility in this regard. In C3H/HeJ mice, immunity against over 1,000 50% lethal doses of virulent S. typhimurium was evident as early as 3 days after immunization and persisted for at least 7 months. Further, the vaccine was effective over a broad spectrum of doses, ranging from 10(4) to 10(6) organisms. Infection with SL3235 led to marked splenomegaly in both mouse strains. The relationship of splenomegaly to the growth kinetics and colonization by SL3235 in the spleens of infected C3H/HeJ and C3H/HeNCrlBR mice was followed. SL3235 initially multiplied slowly in the spleens of both mouse strains and then was rapidly cleared. Less multiplication was seen in the resistant C3H/HeNCrlBR mice than in C3H/HeJ mice. Maximum splenomegaly occurred after clearance of the organism had begun. Protection against virulent S. typhimurium persisted after virtually all of the SL3235 vaccine strain had been cleared from the spleen. Cross-protection against Listeria monocytogenes was evident, but had a later onset, waned by 21 days, and was not detectable by 1 month after vaccination. Demonstration of this cross-protection is consistent with the interpretation that SL3235 induces cellular immunity. One-week immune spleen cells adoptively transferred anti-S. typhimurium and anti-L. monocytogenes immunity. T cell-enriched fractions were ineffective in adoptive transfer, as were spleen cells taken 2 weeks or later after immunization. Protective capacity was in the adherent cell fraction and seemed to be associated with macrophages. Evidence for induction of a population of sensitized T cells was obtained by using a peritoneal exudate T-lymphocyte proliferation assay on peritoneal T lymphocytes collected 1 to 3 months after SL3235 infection.     

Cellular aspects of the longer-lasting immunity against mouse typhoid infection afforded by the live-cell and ribosomal vaccines.

In order to compare the potential of salmonella vaccines prepared from Salmonella typhimurium to provide the longer-lasting protection from the aspects of cell-mediated immunity, groups of mice were immunized with optimal doses of the following preparations: live cells, ribosome-rich extract, acetone-killed cells, and heat-killed cells. At various intervals post-immunization, mouse peritoneal macrophages and splenic T cells were tested for biological activities. The capacity of each vaccine to confer mouse protection against a lethal challenge with S. typhimurium correlated with the degree of macrophage activation engendered by each of them in the early stage of immunization. In the late stage of immunization, the level of mouse protection conferred by each vaccine was found to be based on the capacity of T cells to respond to salmonella antigens, which correlated with the degree of adoptive immunity by T cells. The live-cell and ribosomal vaccines were superior to killed-cell vaccines in inducing the cell-mediated protection. Thus, the longer-lasting immunity provided by the live-cell and ribosomal vaccines can be accounted for by the fact that T cells of mice immunized with both vaccines have the persistent reactivity to salmonella antigens.     

Protection against Salmonella typhi infection in mice after immunization with outer membrane proteins isolated from Salmonella typhi 9,12,d, Vi.

The current studies were undertaken to assess the ability of the outer membrane proteins (OMPs) of Salmonella typhi to induce protection against challenge with the bacteria in mucin. OMPs were isolated as described by Schnaitman (J. Bacteriol. 108:553-556, 1971) and were found to be contaminated with approximately 4% lipopolysaccharide (LPS). Immunization with as little as 30 micrograms of OMPs conferred 100% protection to mice challenged with up to 1,000 50% lethal doses (LD50) of two strains of S. typhi (9,12,d, Vi and Ty2). In addition, 30% protection against challenge with up to 500 LD50 of Salmonella typhimurium was achieved. Immunization with LPS at doses equivalent to those found in the OMPs was considerably inferior to the OMPs in the induction of an immune status. Moreover, LPS was effective only when the challenge was performed with S. typhi 9,12,d, Vi (40% protection to 100 LD50). An antiserum raised in rabbits reacted mainly against the bands of the molecular weights corresponding to the so-called porins contained in the OMP preparation as shown by Western blotting (immunoblotting). This rabbit antiserum protected 100% of mice against challenge with 100 LD50 of either strain of S. typhi and 80% of mice against challenge with the same LD50 of S. typhimurium. These results indicate the usefulness of OMPs in the induction of active immunity against S. typhi in mice.     

Delayed-type hypersensitivity and immunity to Salmonella typhimurium

Studies were carried out to correlate immunity and expression of delayed-type hypersensitivity (DTH) in mice of the C3H lineage immunized with an avirulent strain of Salmonella typhimurium (strain SL3235). This strain belongs to a class of aroA- organisms which are being considered as vaccine strains for humans and veterinary use. In a systematic study, the relationship between the mouse strain and the immunizing dose of strain SL3235 on the development of protective immunity and DTH was examined. It was found that in hypersusceptible C3H/HeJ and C3HeB/FeJ mice, several doses of strain SL3235 afforded protection against intravenous challenge doses as high as 1,300 50% lethal doses. Despite these significant levels of immunity to challenge, mice of these two strains never mounted significant DTH responses following immunization with the doses of strain SL3235 tested, which spanned 3 orders of magnitude. Nonresponsiveness was not due to antigen overload, as all of the mouse strains were comparably colonized with strain SL3235 at the time of DTH elicitation. Further, it was found that the ability of responsive C3H/HeNCrlBR mice to display DTH was dependent on the immunizing dose of strain SL3235 and that a dosage could be found that resulted in increased resistance to challenge in these mice without a concomitant display of DTH. Thus, while both induction of protective immunity and DTH were vaccine dosage dependent in the responsive mouse strain (C3H/HeNCrlBR), DTH was a less sensitive measure of protective immunity than survival. Vaccine dosages ranging over three orders of magnitude failed to yield positive footpads to the Salmonella elicitin in the nonresponsive mice. The data suggest that caution should be observed in interpreting Salmonella DTH tests that are used as screens of immune status to typhoid fever in humans, as the extent of discordance between immunity and DTH in humans is unknown.     

Immune responses in BALB/c mice following immunization with aromatic compound or purine-dependent Salmonella typhimurium strains.

Two near isogenic strains of Salmonella typhimurium HWSH, stably mutated in either the aroA gene affecting the biosynthesis of aromatic compounds, or the purA gene affecting the biosynthesis of purines, were administered intravenously as live attenuated vaccines to BALB/c mice. HWSH aroA-immunized mice were well protected against intravenous (i.v.) challenge with wild-type virulent HWSH for at least 10 weeks, whereas HWSH purA-immunized mice were unprotected. Furthermore, HWSH aroA-immunized mice could also control a heterologous challenge with virulent Listeria monocytogenes at 7 and 14 days post-immunization, whereas mice receiving a similar dose of HWSH purA could not. Increasing the i.v. dose of HWSH purA compared to HWSH aroA induced some resistance to L. monocytogenes. Induction of early anti-S. typhimurium resistance by HWSH aroA immunization appeared slightly later than the anti-L. monocytogenes resistance. Mice immunized with either vaccine were able to mount S. typhimurium-specific T-cell proliferative responses and produced anti-S. typhimurium humoral antibodies in their serum. The antibody titre was greater in those mice immunized with the aroA mutant.     

Mechanism of protective immunity induced by porin-lipopolysaccharide against murine salmonellosis.

Investigations were undertaken to characterize the protective immunity induced by porin-lipopolysaccharide (LPS) against Salmonella typhimurium infection in mice. Mice immunized with porin-LPS showed higher levels of antiporin immunoglobulin G than mice which received porin alone. Further, T cells from porin-LPS-immunized mice showed an augmented proliferative response to porin in vitro compared with the response of T cells from porin-injected animals. The passive transfer of anti-LPS antibodies conferred significant protection (17%), while antiporin serum failed to protect mice against lethal challenge, indicating the protective ability of anti-LPS antibodies. However, the transfer of serum obtained from porin-LPS-immunized mice resulted in better protection (30%) than did anti-LPS or antiporin antibodies alone. In contrast to LPS, monophosphoryl lipid A completely failed to induce protection against lethal infection. However, comparable to the effect of LPS, injection of porin with monophosphoryl lipid A enhanced antibody response and the protective ability of porin (81.25%). The transfer of T cells from porin-LPS-immunized mice provided higher levels of protection (47%) against lethal challenge than did T cells from porin-immunized mice (23%). The combination of T cells and serum from porin-immunized mice transferred 36% protection. However, a combination of T cells and serum from porin-LPS-immunized mice conferred the highest level of protection (92%), which was reflected by the number of survivors (100%) in the porin-LPS-immunized group. These results demonstrate that besides the protective effect of anti-LPS antibodies, the ability of LPS to augment humoral and cell-mediated immune responses to porin confers effective protection against Salmonella infection.     

Protective immunity induced in mice by immunization with high-molecular-weight polysaccharide from Pseudomonas aeruginosa.

A high-molecular-weight alkali-labile polysaccharide (PS) isolated from the slime of immunotype 1 Pseudomonas aeruginosa was tested for its ability to protect mice from lethal challenge with the live, homologous organism. Intraperitoneal (i.p.) injection of 10 to 25 microgram of the PS protected 60 to 70% of the mice against challenge with up to 50 50% lethal dose units. Although single immunization of mice with up to 250 microgram of PS effected protective levels of only 70%, two successive immunizations provided 100% protection. Subcutaneous and intravenous immunization with PS also provided protection to i.p. challenges with immunotype 1 P. aeruginosa, but not to i.p. challenge with immunotype 4 P. aeruginosa. Although lipopolysaccharide (LPS) was found to be more immunogenic than PS in out studies, contamination of the alkali-labile PS with LPS did not account for the protection seen. Alkali treatment (0.1 N NaOH, 37 degrees C, 2 h) of the PS destroyed its protective effectiveness, while similarly treated LPS retained its capacity for inducing immunity in mice. Adsorption and passive protection studies with sera raised to either PS or a mixture of PS and LPS indicated that antibody directed to the alkali-labile PS antigen was capable of contributing to the protection of mice against challenge with P. aeruginosa.     

Ribosomal Vaccines II. Specificity of the Immune Response to Ribosomal Ribonucleic Acid and Protein Isolated from Salmonella typhimurium

Ribosomal proteins isolated from Salmonella typhimurium were effective in inducing immunity in mice. This immunity was specific since animals immunized with ribosomal proteins from S. typhimurium were not protected against challenge with S. cholerae-suis or S. enteritidis. Immunity was evident as early as 5 days after immunization. Ribosomal ribonucleic acid (RNA) failed to provide any effective immunity in mice. Polyinosinic acid: polycytidylic acid (poly I: C) induced a rapid, short-lived immunity to all three Salmonella species. In contrast, ribosomal RNA failed to elicit any rapid nonspecific response to infection.     

Immunity conferred by Aro- Salmonella live vaccines

The specificity of protection conferred by Aro- salmonellae was studied in BALB/c mice challenged 3 months after intravenous (i.v.) vaccination, more than 1 month after the vaccine had been cleared. Oral challenge showed better protection than i.v. challenge. Salmonella typhimurium aroA SL3261 conferred very good protection against wild-type S. typhimurium C5 (over 10,000 x LD50). Cross protection experiments were performed using S. typhimurium, S. enteritidis and S. dublin for vaccination and challenge, including variants of S. typhimurium and S. enteritidis of similar virulence differing in the main LPS antigen (O-4 or O-9). Salmonella typhimurium aroA conferred solid protection against S. typhimurium (O-4), but no protection against wild-type S. enteritidis (O-9). However challenge with LPS variant strains showed that although protection was generally better to strains of the homologous LPS type, specificity of protection was determined more by the parent strain background (S. typhimurium or S. enteritidis) of the challenge than by O-factors 4 or 9, suggesting that other antigens are involved. The nature of the protective antigen(s) in this model is unclear, but it does not appear to be the main O-specific antigen. A S. enteritidis Se795 aroA vaccine gave good protection against wild-type S. enteritidis Se795 2 weeks after vaccination, but much less at 3 months (approximately 10-200 x LD50), although the persistence of the S. enteritidis aroA vaccine in the liver and spleen was similar to that of the S. typhimurium vaccine, and the wild-type Se795 challenge strain was of similar virulence to S. typhimurium C5. A S. dublin aroA vaccine conferred similar protection against wild-type S. dublin (approximately 300 x LD50).     

Immunity induced by live attenuated Salmonella vaccines

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 x LD50). The specificity of cross protection was studied using S. typhimurium, 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 LD50). 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 type S. dublin (approx 300 x LD50).     

Infection-Immunity in Tularemia: Specificity of Cellular Immunity

The relationship between hypersensitivity and cellular resistance to infection with facultative intracellular parasites was studied in mice by using infection-immunity in tularemia as a model system. Delayed hypersensitivity to antigenic fractions of Francisella tularensis was first detected 6 to 7 days after immunization with viable F. tularensis vaccine, at which time immunity against challenge infection developed. Both immunity and delayed-type sensitivity reached maximal levels by 9 to 10 days. Immediate hypersensitivity occurred after immunization with both viable and nonviable tularemia vaccines but could not be correlated with resistance since nonviable antigens were not protective. Attempts to relate resistance to F. tularensis with nonspecific immunity factors were unsuccessful. Immunization of mice with BCG vaccine stimulated protection against infection with F. novicida and Salmonella typhimurium but provided no protection against infection with F. tularensis. Moreover, viable tularemia vaccine, while inducing marked protection against challenge with specific organisms, afforded no protection against infection with S. typhimurium or S. enteritidis. It is concluded that cellular immunity in tularemia involves an immunologically specific component.     

Immunization against experimental murine salmonellosis with liposome-associated O-antigen.

Partially delipidated Salmonella typhimurium (O-1,4,5,12) lipopolysaccharide was incorporated into small multilamellar liposomes composed of either naturally occurring or synthetic phospholipids. Vaccination of mice with the liposome-lipopolysaccharide complexes induced a cellular response specific for O-1,4,5,12 determinants, as determined by the development of a delayed-type hypersensitivity reaction. The liposome-lipopolysaccharide vaccines were significantly more effective, compared with other nonviable vaccines tested, in protecting mice against a lethal intravenous challenge infection with virulent S. typhimurium. Protection afforded by the liposome-lipopolysaccharide vaccines was comparable to that conferred by a live S. typhimurium vaccine. Results suggest that liposome-induced modulation of the host immune response in favor of cell-mediated immunity may be more efficacious in preventing diseases in which cell-mediated immunity is of prime importance.     

Mice vaccinated with a non-virulent, aromatic-dependent mutant of Salmonella choleraesuis die from challenge with its virulent parent but survive challenge with Salmonella typhimurium

BALB/c mice given a live vaccine of an aroA mutant of Salmonella choleraesuis by intraperitoneal (i.p.) injection were not protected against i.p. challenge with its virulent parental strain but were protected against i.p. challenge with either of two virulent strains of Salmonella typhimurium (O [1], 4, [5], 12). Vaccination with a live vaccine of S. typhimurium aroA protected against challenge with S. typhimurium but not with S. choleraesuis. Intraperitoneal administration of either aroA strain evoked high levels of serum antibody against the homologous lipopolysacharide (LPS) as determined by an enzyme immunoassay. Sera from vaccinated mice also reacted with heterologous LPS but at dilutions at least seven-fold lower than homologous endpoint titres. The vaccination schedule employed with either live-vaccine strain conferred an equal degree of resistance to challenge with Listeria monocytogenes. After mixed infection of mice with equal numbers of virulent S. typhimurium and S. choleraesuis by the i.p. route, the former was isolated in numbers at least 50,000 times greater than the latter from the liver and spleen between days 1 and 5. When mice were vaccinated i.p. with S. choleraesuis aroA, L. monocytogenes or P. multocida before mixed infection, neither serotype showed more than a slight predominance in the liver and spleen during the same period. Thus, in relative terms, vaccination with S. choleraesuis aroA or inoculation with unrelated bacteria suppressed the growth of virulent S. typhimurium in mice but allowed virulent S. choleraesuis to multiply. These results clearly show that S. choleraesuis 38(1) can multiply to kill immunised BALB/c mice.     

Protection and immune responses induced by attenuated Salmonella typhimurium UK-1 strains

We previously reported that Salmonella typhimurium SR-11 mutants with deletion mutations in the genes encoding adenylate cyclase (cya) and the cAMP receptor protein (crp) are avirulent and protective in mice. Salmonella typhimurium UK-1 is highly virulent for chicks (oral LD50 of 3x10(3) CFU) and mice (oral LD50 of 8.5x10(3) CFU) and is capable of lethal infections in pigs, calves and horses. We postulated that attenuated derivatives of this lethal strain would probably induce a higher level of protective immunity than achieved with attenuated derivatives of less virulent S. typhimurium strains such as SR11. To test this hypothesis, we have constructed S. typhimurium UK-1 Deltacya-12Deltacrp-11 mutant strain chi3985 and its virulence plasmid cured derivative chi4095 to investigate their avirulence and immunogenicity in mice. We found that the mutants are avirulent and able to induce protective immune responses in BALB/c mice. These mutant strains retained wild-type ability to colonize the gut associated lymphoid tissue but reach and persist in spleen and liver at a significantly lower level than the wild-type parent strain. Mice survived oral infection with >1x10(9) CFU of chi3985 (the equivalent to 10(5) 50% lethal doses of wild-type S. typhimurium UK-1) and were fully protected against challenge with 10(5)times the LD50 of the wild-type parent. Immunized mice developed a high level of serum IgG titre to Salmonella LPS and delayed-type hypersensitivity (DTH) response to S. typhimurium outer membrane proteins. Compared to the virulence plasmid-containing strain chi3985, the virulence plasmid cured DeltacyaDeltacrp mutant strain chi4095 was more attenuated and less protective, as some mice immunized with chi4095 died when challenged with the wild-type UK-1 strain. This work demonstrates that S. typhimurium UK-1 Deltacrp Deltacya mutant strain may be a potential live vaccine to induce protective immunity against Salmonella infection or to deliver foreign antigens to the immune system.     

Protective effects of a supernatant factor from Salmonella typhimurium on Salmonella typhimurium infection of inbred mice.

A supernatant factor prepared from 48-h cultures of Salmonella typhimurium has been used to immunize mice against subsequent challenge with normally lethal doses of S. typhimurium. The mouse strains used, C57BL and BALB/c, were sensitive to S. typhimurium with 50% lethal doses of less than 50 organisms. Two doses of supernatant factor, given intraperitoneally 20 days apart, protected mice against a subcutaneous challenge dose 10 days later of 100 50% lethal doses of S. typhimurium, resulting in 50 to 80% survival. The viable counts were reduced initially in organs of immunized mice compared with controls, and the multiplication of bacteria was delayed, although the final levels found in the organs would normally have been lethal. Protection obtained was specific for S. typhimurium in that no increased survival was shown after Salmonella enteritidis challenge of immunized mice. Although lipopolysaccharide was demonstrated in the supernatant factor, lipopolysaccharide alone did not protect challenged mice. Supernatant factor produced delayed-type hypersensitivity reactions in mice sensitized with nonlethal doses of Salmonella. The nature of the active factor, found to be partially protein, has yet to be elucidated.     

Comparison of the abilities ofSalmonella typhimurium rpoS,aroAandrpoS aroAstrains to elicit humoral immune responses in BALB/c mice and to cause lethal infection in athymic BALB/c mice

Salmonella typhimurium rpoS and rpoS aroA mutants are effective live vaccines in the murine model of salmonellosis (Coynault et al., Mol. Microbiol. 1996; 22: 149-60). Here, we further investigate the characteristics of these vaccines. The systemic humoral response induced by S. typhimurium rpoS, aroA and rpoS aroA vaccine candidates against S. typhimurium LPS was studied by ELISA. In BALB/c mice, the rpoS aroA strain induced a systemic anti-LPS humoral response similar to that induced by the rpoS and aroA strains. The virulence of aroA and rpoS aroA vaccines in nude (nu/nu) BALB/c mice was also compared. Salmonella typhimurium aroA and rpoS aroA vaccines both produced slowly progressing lethal infections in athymic mice inoculated i.p. but the rpoS aroA strain was more attenuated than the aroA strain, as determined by time to death and bacterial counts in spleens. Finally, a rpoS mutant of Salmonella dublin conferred protection in mice against an oral challenge with a wild-type strain of S. dublin whereas a rpoS mutant of S. typhimurium did not. This suggests that the protection provided by the S. typhimurium rpoS vaccine is serotype-dependent.     

Immunogenicity of Ribosomal Vaccines Isolated from Group A, Type 14 Streptococcus pyogenes

A ribosomal preparation isolated from group A, type 14 Streptococcus pyogenes protected mice against a challenge of 1,000 mean lethal doses. Optimal immunization consisted of subcutaneous administration of 326 μg of protein with Freund incomplete adjuvant. Animals immunized with higher or lower antigen concentrations showed decreased survival. Freund incomplete adjuvant was necessary to insure optimum immunization. Immunochemical studies showed no M protein or C carbohydrate present in the ribosomal preparation. Mice optimally immunized with the type 14 ribosomal preparation were protected against heterologous challenge with M types 2, 5, 8, and 12. Protein-free ribonucleic acid extracts isolated from the type 14 ribosomal preparation did not protect mice against homologous challenge. But ribonucleic acid-free protein extracts showed 100% protection in mice after homologous challenge.