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.     

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.     

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.     

Protection against murine listeriosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the naturally somatic antigen superoxide dismutase.

A recombinant (r)-Salmonella typhimurium aroA vaccine strain was constructed which secretes the naturally somatic protein of Listeria monocytogenes, superoxide dismutase (SOD), by the HlyB/HlyD/TolC export machinery. Vaccine efficacy of the SOD-bearing carrier strain was compared with that of the p60-secreting construct, S. typhimurium p60s (J. Hess, I. Gentschev, D. Miko, M. Welzel, C. Ladel, W. Goebel, and S. H. E. Kaufmann, Proc. Natl. Acad. Sci. USA 93:1458-1463, 1996). Vaccination of mice with both constructs induced protection against a lethal challenge with the intracellular pathogen, L. monocytogenes. While the somatic listerial antigen, SOD, is immunologically uncharacterized, the naturally secreted protein of L. monocytogenes, p60, is known to be highly immunogenic. Our data emphasize the high vaccine potential of r-Salmonella constructs secreting antigens of somatic or secreted origin. Moreover, they suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated Salmonella spp. as the carrier is capable of potentiating the immune response against foreign proteins independent from their immunogenicity in and display by the natural host.     

Salmonella typhimurium aroA mutants as carriers of the Escherichia coli heat-labile enterotoxin B subunit to the murine secretory and systemic immune systems

We investigated the ability of Salmonella typhimurium vaccines to deliver heterologous antigens to the systemic and secretory immune systems of the mouse, while retaining their immunogenicity against salmonellosis. S. typhimurium SL3261, an avirulent aroA mutant, or SL3261 carrying plasmid pBRD026, a pBR322 derivative encoding the gene for Escherichia coli LT-B were used to immunize BALB/c mice orally. Both immunizing strains invaded the mononuclear phagocyte system of the mice, grew slowly until approximately day 14 post-infection, and then were rapidly cleared. No salmonellae were detected in livers, spleens, mesenteric lymph nodes or Peyer's patches by day 42. Mice immunized with either strain and challenged orally with the virulent parent strain, SL1344, several weeks after clearing the immunizing organism, were protected against the lethal S. typhimurium infection. Mice infected with SL3261 (pBRD026) developed substantial levels of IgG and IgA anti-LT-B antibodies 14 days post-infection in both serum and gut samples. The sera neutralized the effects of LT in an in vitro Vero cell assay. Thus, aroA mutants of S. typhimurium can deliver a heterologous antigen from a different enteric pathogen to the murine systemic and secretory immune systems without altering their efficacy against salmonellosis.     

Listeria monocytogenes p60 supports host cell invasion by and in vivo survival of attenuated Salmonella typhimurium.

The extracellular protein p60 is a major virulence factor of the intracellular bacterium Listeria monocytogenes. Its roles in pathogen survival in vivo and host cell invasion in vitro were studied. To this end, Salmonella typhimurium SL7207 was used as carrier for secreted p60-HlyA fusion protein by Escherichia coli HlyB and HlyD transport proteins. C57BL/6 mice infected intravenously with this strain suffered from increased bacterial numbers in livers and spleens compared with the p60-nonexpressing control strain, but only transiently. In vitro experiments showed that p60 promotes invasion of recombinant S. typhimurium SL7207 p60 into hepatocytes and resting macrophages independent from complement. Moreover, the uptake of wild-type L. monocytogenes EGD and L. monocytogenes BUG 8, an internalin-deficient strain, into hepatocytes was partially blocked by anti-p60 antibodies. The impaired invasion of dissociated bacterial chains of L. monocytogenes RIII, a p60 expression mutant, into hepatocytes and macrophages was partially restored by addition of p60- or p60-HlyA-enriched bacterial supernatants. These data suggest that the L. monocytogenes surface-associated proteins, p60 and internalin, act in concert to achieve optimal uptake into nonprofessional phagocytes and macrophages. Together, these experiments reveal a substantial impact of p60 on cell invasion and virulence and thus emphasize the importance of the intracellular habitat for survival of L. monocytogenes in the host.     

Immunization with live aroA recombinant Salmonella typhimurium producing invasin inhibits intestinal translocation of Yersinia pseudotuberculosis.

The Yersinia pseudotuberculosis inv gene encodes invasin, a 103-kDa outer membrane protein that allows bacteria to enter mammalian cells. The gene was subcloned into the attenuated aroA mutant of Salmonella typhimurium SL3261. Invasin was produced by the recombinant Salmonella strain and increased the ability of microorganisms to translocate from the intestinal lumen to the mesenteric lymph nodes. Specific antibodies for invasin were detected in sera and intestinal secretions of mice following oral immunization with the live Inv+ Salmonella strain. The immunization strongly inhibited intestinal translocation of Y. pseudotuberculosis when this pathogen was inoculated to mice but failed to prevent Yersinia dissemination from the gut lymphoid tissue.     

Listeria monocytogenes, but not Salmonella typhimurium, elicits a CD18-independent mechanism of neutrophil extravasation into the murine peritoneal cavity.

This study shows that extravasation of neutrophils into the peritoneal cavities of mice in response to intraperitoneal (i.p.) inoculation of wild-type Listeria monocytogenes requires the participation of leukocyte adhesion molecules that are different from those involved in neutrophil recruitment in response to i.p. inoculation of Salmonella typhimurium. In the case of S. typhimurium, extensive neutrophil influx could be essentially abolished by treating mice with either anti-CD11b or anti-CD18 monoclonal antibodies, whereas the same monoclonal antibodies failed to prevent neutrophil accumulation in the peritoneal cavity in response to inoculation of L. monocytogenes. On the other hand, i.p. inoculation of a listeriolysin-negative strain of L. monocytogenes induced a CD11b-dependent neutrophil influx. The possibility that wild-type L. monocytogenes, by virtue of its ability to inhabit the cytosol of the cells it infects, induces the expression of endothelial cell adhesion molecules in the microvasculature of the peritoneal cavity to which neutrophils adhere via leukocyte adhesion molecules distinct from beta-2 integrins is discussed.     

Evaluation of Salmonella typhimurium strains harbouring defined mutations in htrA and aroA in the murine salmonellosis model.

Derivatives of the mouse-virulent Salmonella typhimurium strain SL1344 were constructed harbouring defined mutations in htrA, aroA or htrA aroA combined. When administered orally or intravenously to BALB/c mice, all the mutants were found to be highly attenuated. All mutants were able to confer significant protection against lethal challenge with SL1344 after a single oral dose of live organisms. SL1344 htrA mutants persisted in livers and spleens at a lower level than SL1344 aroA mutants after intravenous administration. SL1344 htrA aroA mutants persisted at an even lower level and were cleared from the livers and spleens of mice within 21 days of intravenous administration. Thus htrA and htrA aroA mutants can be considered as potential oral vaccines against salmonellosis.     

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).     

Expression and immunogenicity of an Echinococcus granulosus fatty acid-binding protein in live attenuated Salmonella vaccine strains.

Fatty acid-binding proteins (FABPs) are candidate molecules for vaccines against several parasitic platyhelminths. A FABP from the cestode Echinococcus granulosus (EgDf1) was expressed in Salmonella vaccine strains as a C-terminal fusion to fragment C of tetanus toxin (TetC) by using expression vector pTECH. The fusion protein was equally expressed in several attenuated vaccine strains derived from bacteria with different genetic backgrounds and different attenuating mutations. Single-dose immunization experiments with the aroA Salmonella typhimurium strain SL3261 carrying the pTECH-EgDf1 construct were conducted with mice, using both the intravenous and the oral routes. Surprisingly, the antibody response to EgDf1 and the antigen-specific cytokine production in spleen cells were stronger in mice immunized orally. Furthermore, immune mouse sera strongly reacted with fixed sections of the worm's larval stage. Analysis of the isotype distribution of the specific anti-EgDf1 antibodies showed similar production of immunoglobulin G1 (IgG1) and IgG2a together with specific IgA antibodies. In addition, stimulation of spleen cells from mice immunized with the different constructs with either Salmonella lysate, TetC, or EgDf1 showed that, together with Th1-related cytokines (gamma interferon and interleukin 2 [IL-2]), significant levels of a Th2 cytokine (IL-5) were produced specifically, indicating a Th2 component to the response to the Salmonella carrier and to the recombinant antigens. Salmonellae expressing the TetC-rEgDfl fusion are currently under evaluation as potential vaccines against E. granulosus.     

Control by H-2 genes of the Th1 response induced against a foreign antigen expressed by attenuated Salmonella typhimurium.

Attenuated salmonellae represent an attractive vehicle for the delivery of heterologous protective antigens to the immune system. Here, we have investigated the influence of the genetic background of the host which regulates the growth and elimination of Salmonella cells on the cellular response induced against a foreign antigen delivered by an aroA Salmonella strain. We have tested CD4+ T-cell responses (cell proliferation and cytokine production) in various mouse strains following immunization with Salmonella typhimurium SL3261 expressing a high level of the recombinant Escherichia coli MalE protein. We were able to detect a CD4+ T-cell response against the recombinant MalE protein only in a restricted number of mouse strains, whereas all mice produced good levels of anti-MalE immunoglobulin G antibodies. The Ity gene did not play a major role in these differences in T-cell responses, since both Ity-resistant and -susceptible strains of mice were found to be unresponsive to MalE delivered by recombinant salmonellae. In contrast, when B10 congenic mice were used, a correlation was established between MalE-specific T-cell unresponsiveness and H-2 genes. The discrepancies described in this paper in the ability of various strains of mice to develop an efficient Th1 response against a recombinant antigen displayed by a live Salmonella vaccine underscore the difficulties that can be encountered in the vaccination of human populations by such a strategy.     

Oral vaccination of mice against tetanus by use of a live attenuated Salmonella carrier.

A Salmonella typhimurium aroA mutant has been used as a live carrier to immunize mice against tetanus. Plasmid pTETtac4, which expresses a 50-kilodalton fragment of tetanus toxin (fragment C) under the control of the tac promoter, was introduced into SL3261 aroA. When used as a live vaccine and administered orally or intravenously, this strain was able to induce protective immunity in mice against a lethal tetanus toxin challenge. When plasmid pTETtac2, which contains the lacI gene, was used, no immunity was obtained, indicating that the expression of fragment C was repressed in vivo. We believe that this is the first example of a successful oral vaccination that uses an attenuated bacterial carrier to deliver a protective antigen derived from tetanus toxin.     

Expression of the Yersinia pestis capsular antigen (F1 antigen) on the surface of an aroA mutant of Salmonella typhimurium induces high levels of protection against plague.

The caf operon from Yersinia pestis encoding the structural subunit (caf1), the molecular chaperone (caf1M), the outer membrane anchor (caf1A), and the regulatory protein (caf1R) was cloned into Salmonella typhimurium SL3261 aroA. The recombinant Salmonella organisms were encapsulated when cultured at 37 degrees C but not when cultured at 28 degrees C. Oral inoculation of mice with the recombinant Salmonella induced predominantly an immunoglobulin G2a response to F1 antigen, and isolated T cells showed a recall response to soluble or Salmonella-associated F1 antigen. Mice immunized with S. typhimurium SL3261 aroA expressing F1 antigen intracellularly developed lower antibody responses to F1 antigen and showed a T-cell recall response only to Salmonella-associated F1 antigen. Mice immunized orally with two doses of the recombinant Salmonella which expressed F1 antigen on the surface were protected against 10(7) 50% lethal doses (LD50) of virulent Y. pestis given by the subcutaneous route of challenge, whereas mice immunized with the recombinant Salmonella expressing F1 antigen intracellularly were only partially protected against 10(5) LD50 of Y. pestis.     

Antibodies against listerial protein 60 act as an opsonin for phagocytosis of Listeria monocytogenes by human dendritic cells

Human-monocyte-derived dendritic cells (MoDC) are very efficient in the uptake of Listeria monocytogenes, a gram-positive bacterium which is an important pathogen in humans and animals causing systemic infections with symptoms such as septicemia and meningitis. In this work, we analyzed the influence of blood plasma on the internalization of L. monocytogenes into human MoDC and compared the uptake of L. monocytogenes with that of Salmonella enterica serovar Typhimurium and Yersinia enterocolitica. While human plasma did not significantly influence the uptake of serovar Typhimurium and Y. enterocolitica by human MoDC, the efficiency of the uptake of L. monocytogenes by these phagocytes was strongly enhanced by human plasma. In plasma-free medium the internalization of L. monocytogenes was very low, whereas the addition of pooled human immunoglobulins resulted in the internalization of these bacteria to a degree comparable to the highly efficient uptake observed with human plasma. All human plasma tested contained antibodies against the 60-kDa extracellular protein of L. monocytogenes (p60), and anti-p60 antibodies were also found in the commercially available pooled immunoglobulins. Strikingly, in contrast to L. monocytogenes wild type, an iap deletion mutant (totally deficient in p60) showed only a minor difference in the uptake by human MoDC in the presence or the absence of human plasma. These results support the assumption that antibodies against the listerial p60 protein may play an important role in Fc-receptor-mediated uptake of L. monocytogenes by human MoDC via opsonization of the bacteria. This process may have a major impact in preventing systemic infection in L. monocytogenes in immunocompetent humans.     

Antibody responses in the lungs of mice following oral immunization with Salmonella typhimurium aroA and invasive Escherichia coli strains expressing the filamentous hemagglutinin of Bordetella pertussis.

The filamentous hemagglutinin (FHA) of Bordetella pertussis was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, and in a strain of Escherichia coli harboring Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness for epithelial cells. Expression of FHA in these strains did not interfere with their ability to invade Henle cells. Immunoglobulins A and G specific for FHA were detected in lung washes of mice following oral immunization with the live recombinant organisms; antibody levels were significantly higher than those in mice immunized with killed bacteria administered orally or intraperitoneally. Live oral vaccines carrying protective antigens of B. pertussis may be an important alternative to new-generation component vaccines against whooping cough.     

Salmonella typhimurium aroA, htrA, and aroD htrA mutants cause progressive infections in athymic (nu/nu) BALB/c mice.

Athymic (nu/nu) BALB/c mice and their euthymic (nu/+) littermates were inoculated intravenously with live attenuated vaccine strains of Salmonella typhimurium. All strains caused progressive infections in the athymic mice but not in their euthymic littermates. Athymic mice given strain SL3261, an aroA derivative of SL1344, in doses between log 4.7 and 5.7 CFU were all severely ill and were killed by weeks 4 to 5. Athymic mice given log 4.7 CFU of a derivative of S. typhimurium C5 carrying a mutation in htrA, encoding a stress protein, were ill and were killed by week 7 in one experiment but survived to week 13 in another. Athymic mice given log 4.6 CFU of a C5 aroD htrA double mutant were ill and were killed at week 7. Athymic mice given SL3261 had high bacterial counts in the reticuloendothelial system at 4 weeks. Athymic mice given SL3261 or C5 htrA made immunoglobulin G3 (IgG3) (and to a lesser extent IgM) antibody to lipopolysaccharide (LPS), whereas euthymic mice made IgM, IgG1, IgG2a, IgG2b, and IgG3 anti-LPS antibodies. The results indicate that both aroA and htrA strains will produce slow, progressively lethal infections in athymic mice, that the htrA strain is more attenuated than the aroA strain as measured by time to death in this model, and that IgG3 anti-LPS antibody alone cannot suppress the progress of infections by very attenuated strains in athymic mice.     

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).     

Effect of the new immunostimulator HAB 439 on cell-mediated immunity against intracellular bacteria.

The isoxazoline derivative HAB 439 was tested for its enzyme inhibiting potency and was found to be an inhibitor of aminopeptidase B (IC50 = 22.5 micrograms/ml). In further immunopharmacological experiments its efficacy to stimulate cell-mediated immunity was evaluated. HAB 439 was shown to stimulate DTH-reaction against Salmonella typhimurium and Listeria monocytogenes. HAB 439 protected animals against infection by reducing the bacterial load in livers and spleens and by decreasing the mortality rate. Treatment with the antibiotic ampicillin induced a decreased DTH-reaction in mice which was demonstrated to be due to a reduction of the antigen to be presented to the immune system and not to immune suppression. HAB 439 restored the impaired immune response to S. typhimurium and L. monocytogenes in a dose-dependent way. Restoration of DTH was shown to lead to an improvement of protection in ampicillin-treated mice which were challenged with the intracellular bacteria.     

Yersinia enterocolitica aroA mutants as carriers of the B subunit of the Escherichia coli heat-labile enterotoxin to the murine immune system

Plasmid p5F which directs the expression of the Escherichia coli heat-labile enterotoxin B subunit (LT-B) from the ptac promoter was introduced into the attenuated Yersinia enterocolitica O:8 aroA mutant strain YAM.1. YAM.1 (p5F) expressed high levels of cell-associated and secreted LT-B in a stable fashion when grown on normal laboratory medium. The strain was used as a live oral vaccine in BALB/c mice and vaccinated mice developed high levels of gut-associated and systemic antibodies to both LT-B and the lipopolysaccharide (LPS) of the vaccine strain. Anti-LT-B and anti-LPS responses in the sera were predominantly of the IgG class whereas gut-associated antibodies were predominantly IgA. ELISPOT assays carried out on selected tissues prepared from vaccinated mice showed significant numbers of cells synthesising IgG and IgA antibodies to LT-B. These results show that Y. enterocolitica aroA mutants can be used effectively as carriers of heterologous antigens to the murine immune system.