Alpha C protein as a carrier for type III capsular polysaccharide and as a protective protein in group B streptococcal vaccines

The alpha C protein, a protective surface protein of group B streptococci (GBS), is present in most non-type III GBS strains. Conjugate vaccines composed of the alpha C protein and type III capsular polysaccharide (CPS) might be protective against most GBS infections. In this study, the type III CPS was covalently coupled to full-length, nine-repeat alpha C protein (resulting in III-alpha9r conjugate vaccine) or to two-repeat alpha C protein (resulting in III-alpha2r conjugate vaccine) by reductive amination. Initial experiments with the III-alpha9r vaccine showed that it was poorly immunogenic in mice with respect to both vaccine antigens and was suboptimally efficacious in providing protection in mice against challenge with GBS. Therefore, modified vaccination protocols were used with the III-alpha2r vaccine. Female mice were immunized three times with 0.5, 5, or 20 microgram of the III-alpha2r vaccine with an aluminum hydroxide adjuvant and bred. Ninety-five percent of neonatal mice born to dams immunized with the III-alpha2r vaccine survived challenge with GBS expressing type III CPS, and 60% survived challenge with GBS expressing wild-type (nine-repeat) alpha C protein; 18 and 17%, respectively, of mice in the negative control groups survived (P, <0.0001). These protection levels did not differ significantly from those obtained with the type III CPS-tetanus toxoid conjugate vaccine and the unconjugated two-repeat alpha C protein, which protected 98 and 58% of neonates from infection with GBS expressing type III CPS or the alpha C protein, respectively. Thus, the two-repeat alpha C protein in the vaccine was immunogenic and simultaneously enhanced the immunogenicity of type III CPS. III-alpha vaccines may be alternatives to GBS polysaccharide-tetanus toxoid vaccines, eliciting additional antibodies protective against GBS infection.     

Protection from group B streptococcal infection in neonatal mice by maternal immunization with recombinant Sip protein

The protective potential of antibodies directed against group B streptococcus (GBS) Sip surface protein was determined by using the mouse neonatal infection model. Rabbit Sip-specific antibodies administered passively to pregnant mice protected their pups against a GBS lethal challenge. In addition, active immunization with purified recombinant Sip protein of female CD-1 mice induced the production of specific antibodies that also confer protection to the newborn pups against GBS strains of serotypes Ia/c, Ib, II, III, and V. These data confirm that Sip-specific antibodies can cross the placenta and conferred protective immunity against GBS infections.     

Deletion of Repeats in the Alpha C Protein Enhances the Pathogenicity of Group B Streptococci in Immune Mice

The alpha C protein is a protective surface-associated antigen of group B streptococci (GBS). The prototype alpha C protein of GBS (strain A909) contains nine identical tandem repeats, each comprising 82 amino acids, flanked by N- and C-terminal domains. Clinical isolates of GBS show variable numbers of repeats with a normal distribution and a median of 9 to 10 repeats. Here, we show that escape mutants of GBS expressing one-repeat alpha C protein were 100-fold more pathogenic than GBS expressing wild-type nine-repeat alpha C protein in neonatal mice whose dams were immunized with antiserum elicited to nine-repeat alpha C protein (50% lethal doses of 1.6 × 10³ and 1.8 × 10⁵, respectively; P = 0.0073). There was no difference in pathogenicity in nonimmune mice. Enzyme-linked immunosorbent assay inhibition showed that nine-repeat but not one-repeat alpha C protein is readily available for antibody binding on the surface of intact GBS. Immune electron microscopy studies with antibodies to the capsular polysaccharide (CPS) and to the alpha C protein demonstrated localization of the nine-repeat alpha C protein and the CPS at similar distances from the cell wall. The one-repeat alpha C protein was visualized poorly and only in close proximity to the cell wall, thus suggesting that antibody binding to the protein was hindered by CPS or other cell surface components. We concluded that deletion in the repeat region of the alpha C protein enhanced the pathogenicity of GBS in immune mice by (i) loss of a protective (conformational) epitope(s) and (ii) loss of antibody binding to the alpha C protein due to a decrease in antigen size relative to cell wall components and/or CPS.     

Recombinant group B streptococcus Beta C protein and a variant with the deletion of its immunoglobulin A-binding site are protective mouse maternal vaccines and effective carriers in conjugate vaccines

Immunogenic vaccines against group B Streptococcus (GBS) have been created by coupling the GBS capsular polysaccharides (CPS) to carrier proteins. The GBS beta C protein (BCP) serves as an effective carrier while inducing protective immunity against BCP-expressing strains. BCP also binds human immunoglobulin A (IgA), a characteristic that may be undesirable for use in humans. Here, we examined the immunogenicity and protective efficacy of a recombinant GBS BCP (rBCP), an rBCP modified to eliminate its IgA-binding site (rBCP(DeltaIgA)), and their corresponding GBS serotype III CPS conjugates (III-rBCP and III-rBCP(DeltaIgA)). Deletion of the IgA-binding site or conjugation to CPS did not alter antigenic BCP epitopes. Recombinant proteins and conjugates elicited specific, high-titered IgG in mice. Antisera to rBCP, rBCP(DeltaIgA), III-rBCP, and III-rBCP(DeltaIgA) opsonized GBS strains A909 (Ia/BCP(+)) and H36B (Ib/BCP(+)) for killing by HL-60 cells; antiserum to III-rBCP and III-rBCP(DeltaIgA) also opsonized strain M781 (III/BCP(-)). Vaccination of female mice with either rBCP or rBCP(DeltaIgA) protected approximately 40% of their pups challenged with GBS strain A909. Pups born to III-rBCP- or III-rBCP(DeltaIgA)-vaccinated dams survived at rates of 56% and 66%, respectively. Over 90% of pups born to dams that received the type III CPS conjugates survived challenge with GBS strain M781. In summary, rBCP and rBCP(DeltaIgA) proteins and the conjugates containing them were immunogenic in mice, inducing both CPS- and protein-specific functional IgG. These results suggest that the rBCP(DeltaIgA) could be used as a carrier to augment the immunogenicity of the CPS while expanding coverage to GBS strains bearing BCP.     

Protection of C3H/He mice from experimental Borrelia burgdorferi infection by immunization with a 110-kilodalton fusion protein.

A 110-kDa Borrelia burgdorferi fusion protein, Escherichia coli expressing the fusion protein, transformed E. coli lacking the fusion protein insert, and lyophilized whole B. burgdorferi bacteria were compared for immunogenicity in C3H/He mice. Immunized mice were challenged with a variety of isolates from the United States or the European isolate P/Gau 3 weeks following the last inoculation. An average of 76.7% of the mice immunized with 25 micrograms of lyophilized whole B. burgdorferi cells were protected from infection, while 60% of the mice immunized with the 110-kDa fusion protein were protected. Whole E. coli bacteria expressing the fusion protein protected 57.7% of immunized mice against experimental challenge. Lower levels of protection occurred in mice challenged with the European isolate than in those challenged with isolates originating from the United States. These results demonstrate the potential of the 110-kDa fusion protein for use as a component of a subunit vaccine for prevention of Lyme borreliosis.     

Antigenic relationship between the common antigen (OEP) of Pseudomonas aeruginosa and Vibrio cholerae.

Antibodies were found by the OEP-passive hemagglutination test to cross-react with the common antigen (OEP) of Pseudomonas aeruginosa in sera of rabbits immunized with two serotype (Inaba and Ogawa) strains of Vibrio cholerae. The titer in the OEP-passive hemagglutination reaction rose later than did the agglutinin titer and reached a peak of 640 to 1,280. The titers of OEP antibody formation in rabbits immunized with V. cholerae were almost the same as that of P. aeruginosa. The common antigen of P. aeruginosa was confirmed to exist serologically in both strains of V. cholerae as determined by the indirect fluorescent antibody test and the agar gel precipitin test. Passive immunization with the V. cholerae immune rabbit serum significantly protected mice against P. aeruginosa infection. Purified antibodies cross-reacting with the OEP of P. aeruginosa derived from the V. cholerae immune rabbit sera by OEP-coupled affinity chromatography protected mice against P. aeruginosa infection as compared with the control group, which was injected with 100 microgram of immunoglobin G not containing OEP antibody. The purified antibodies (2.5 microgram per mouse) protected animals challenged with approximately 10,000 50% lethal doses in the control group. Consequently, the common antigen (OEP) of P. aeruginosa proved to be a common antigen of V. cholerae both serologically and in possessing infection protective properties.     

Phenotypic diversity in the alpha C protein of group B streptococci.

Group B streptococci (GBS) is the leading cause of neonatal sepsis and meningitis. C proteins are an immunologically important group of surface-associated antigens in GBS that remain incompletely characterized. Two C proteins have been designated alpha and beta on the basis of protease susceptibility. We recently used a monoclonal antibody to describe a protective epitope of the GBS alpha (or trypsin-resistant) C protein in the prototype Ia/c GBS strain. In the present study, we examined 51 GBS isolates for expression of C-protein alpha and beta antigens. The alpha antigen, as detected with monoclonal antibody in sodium dodecyl sulfate (SDS) extracts, appears as a heterogeneous series of proteins spaced 8 kDa apart on SDS-polyacrylamide gel electrophoresis, but has a maximum molecular mass that varies among strains from 62.5 to 167 kDa. By immunoblotting with human immunoglobulin A, polyclonal antiserum, or monoclonal antibody, the beta antigen, in contrast, appears as a single protein of molecular mass between 124 and 134 kDa. The amount of alpha antigen expressed by each strain was quantified by enzyme immunoassay inhibition and was found to vary markedly from strain to strain. The susceptibility of strains of GBS to opsonization and killing by human polymorphonuclear leukocytes in the presence of either complement alone or complement with alpha-specific monoclonal antibody was examined. Strains expressing the alpha antigen were less readily killed in the absence of specific antibody than were alpha-negative strains. Killing in the presence of alpha-specific monoclonal antibody was found to correlate directly with the maximum molecular mass of the alpha antigen and with the quantity of antigen on the bacterial cell surface. Isolates of GBS that express the alpha C protein vary widely in the quantity and molecular mass of the alpha antigen produced, and this heterogeneity appears to have biologic importance.     

Cloned alpha and beta C-protein antigens of group B streptococci elicit protective immunity.

Streptococcus agalactiae (group B streptococci [GBS]) is the leading cause of neonatal sepsis and meningitis in the United States. The surface-associated C proteins of GBS play a role in immunity, but their number, size, structure, function, and virulence properties have not been well characterized. A recombinant library of DNA fragments from GBS strain A909 (type Ia/C) was prepared in the plasmid pUX12, a specially constructed Escherichia coli expression vector. The library was screened with a rabbit antiserum shown to be protective for passive immunity to GBS infection in a mouse lethality model. Clones were divided into two distinct groups on the basis of DNA-DNA cross-hybridization, restriction enzyme analysis, and the expression of antigenic proteins in E. coli. A characteristic clone from each group was chosen for further study. Clone pJMS23 expresses gene products that biochemically and immunologically correspond to the trypsin-resistant, C-protein alpha antigen. Clone pJMS1 expresses a gene product that binds to immunoglobulin A and is similar to the trypsin-sensitive, C-protein beta antigen. Antisera raised in rabbits against E. coli containing each of the plasmid clones were able to elicit protective immunity in mice challenged by GBS strains carrying the C proteins but not by non-C-protein-bearing strains. Southern blot analysis shows no DNA homology between the clones, and there is no immunological cross-reactivity between the antigens they express. Therefore, pJMS23 and pJMS1 encode two different C proteins that define unique protective epitopes.     

Anti-idiotypic Vaccination against Group B Streptococci

We describe the antigenic properties of an anti-idiotypic single chain fragment variable (scFv) recombinant antibody mimicking the type III capsular polysaccharide of group B streptococci (GBS), an important cause of neonatal sepsis. This scFv could compete with the nominal antigen for binding to specific mouse or rabbit antibodies. Moreover, the scFv elicited, in mice, the production of antibodies which reacted against the type III polysaccharide and passively protected neonatal pups from GBS disease. Maternal immunization with the scFv also protected neonatal mice against GBS infection. Next, the scFv was expressed on the surface of the commensal bacterium Streptococcus gordonii. Intravaginal inoculation of mice with these recombinant bacteria induced significant elevations in serum titers of anti-GBS type III antibodies. Therefore, the expression scFv in commensal bacteria may be a convenient and effective way of delivering anti-idiotypic vaccines.     

Variation in repeat number within the alpha C protein of group B streptococci alters antigenicity and protective epitopes.

Variable expression of repeating units of the protective alpha C proteins among clinical isolates of group B streptococci (GBS) may have implications for vaccine development. In this study, alpha C protein genes containing various numbers of repeats (1,2,9, and 16) were cloned in a T7 overexpression vector in Escherichia coli. Expression was induced by isopropyl-beta-D-thiogalactopyranoside, and proteins were purified by anion-exchange, gel filtration, or affinity chromatography or by isoelectric focusing. Rabbits were immunized with purified 1-,2-,9-, or 16-repeat proteins. All proteins appeared to be highly immunogenic. Enzyme-linked immunosorbent assay inhibition with 9-repeat protein as the coating antigen and 9-repeat-antigen-elicited antiserum showed that a 200-fold-higher concentration of 1-repeat antigen than of 9- or 16-repeat antigen was required for 50% inhibition of antibody-antigen binding. The concentration of 2-repeat antigen required for 50% inhibition was intermediate relative to the concentrations of 1- and 9-repeat antigens. These results suggested that antibodies to 9-repeat antigen recognized predominantly a conformational epitope(s) contained in proteins with higher numbers of repeats (9 or 16) but lost considerable binding affinities for an epitope(s) contained in alpha C proteins with fewer repeats (1 or 2). Similar results were obtained with antiserum to 16-repeat antigen. However, antibodies to 1- and 2-repeat antigens recognized 1-,2-,9-,and 16-repeat antigens with equal binding affinities. This finding suggested that 1- and 2-repeat-elicited antibodies recognized an epitope(s) on individual repeats. Loss of repeating units from the alpha C proteins may result in decreased protection because the loss of epitopes (including conformational epitopes) gives the microorganisms the opportunity to escape host antibodies. If 1- and 2-repeat-elicited antibodies bind all alpha C proteins with equal affinity, regardless of their repeat number, they may prevent GBS strains with fewer repeats from escaping host immunity. Protection data obtained with antisera to the proteins with different repeat numbers support this hypothesis: mouse pups challenged with GBS strain A909 were better protected when immunized with 1- or 2-repeat-elicited antiserum (76 and 75%, respectively) than when immunized with 9- or 16-repeat-elicited antiserum (41 and 48%, respectively).     

Immunogenicity and protective efficacy of the alpha C protein of group B streptococci are inversely related to the number of repeats.

Infection by group B streptococci (GBS) is an important cause of bacterial disease in neonates. Alpha C protein is a protective cell surface-associated protein of GBS. This protein contains a repeat region flanked by N and C termini. Variable expression of tandem repeating units of alpha C proteins had been found among clinical isolates of GBS. We examined the effect of the number of repeats on the immunogenicity of the alpha C protein and its ability to elicit protection from GBS infection in a neonatal mouse model. Mice were immunized with purified alpha C proteins of constructs containing various numbers of repeats (n = 1, 2, 9, and 16) and the N- and C-terminal regions. Both the N-terminal and the repeat regions contain protective and opsonic epitopes. Antibody responses to the alpha C protein constructs with various numbers of repeats were tested with enzyme-linked immunosorbent assay plates coated with either native, nine-repeat alpha C protein or "repeatless" N-terminal antigen. An inverse relationship was found between the number of repeats and the immunogenicity of the alpha C protein; this effect was most pronounced on titers of antibody to the N-terminal region. An inverse relationship was also observed between the number of repeats and protective efficacy, i.e., mouse dams immunized with 5 microg of one- or nine-repeat alpha C protein transferred protective immunity to 65 or 11% of their pups, respectively (P < 0.0001). Thus, the presence of multiple repeats appears to lessen the antibody response to the complete alpha C protein, and especially the antibody response to its N-terminal region, and suggests a mechanism whereby repeat elements contribute to the evasion of host immunity.     

Anti-idiotypic vaccination against group B streptococci

We describe the antigenic properties of an anti-idiotypic single chain fragment variable (scFv) recombinant antibody mimicking the type III capsular polysaccharide of group B streptococci (GBS), an important cause of neonatal sepsis. This scFv could compete with the nominal antigen for binding to specific mouse or rabbit antibodies. Moreover, the scFv elicited, in mice, the production of antibodies which reacted against the type IlI polysaccharide and passively protected neonatal pups from GBS disease. Maternal immunization with the scFv also protected neonatal mice against GBS infection. Next, the scFv was expressed on the surface of the commensal bacterium Streptococcus gordonii. Intravaginal inoculation of mice with these recombinant bacteria induced significant elevations in serum titers of anti-GBS type III antibodies. Therefore, the expression scFv in commensal bacteria may be a convenient and effective way of delivering anti-idiotypic vaccines.     

The putative R1 protein of Streptococcus agalactiae as serotype marker and target of protective antibodies

The streptococcal R1 protein was studied by means of anti-R1 antibodies prepared by appropriate cross-absorption of rabbit antiserum raised against the group B streptococcal (GBS) strain ATCC 12403 (D136C), serotype III/R1. The protein was a ladder-forming antigen according to banding patterns in immunoblotting, similar to several other GBS proteins, and was susceptible to digestion by both pepsin and trypsin. Antibody-based testing revealed that 10% of Norwegian GBS isolates expressed the R1 protein, most frequently capsular antigen type V strains (72%) and less frequently type III strains (3%). None of 132 GBS strains from Zimbabwe, including 39 type V strains, expressed the R1 protein. R1-specific rabbit antibodies showed protective activity in mice challenged with a GBS type V/R1 strain. The results show that the R1 protein is an important GBS serotype marker in strains from certain geographical areas, notably for the subtyping of capsular type V strains, and that this protein is a target of protective antibodies.     

Homotypic protection against rotavirus-induced diarrhea in infant mice breast-fed by dams immunized with the recombinant VP8* subunit of the VP4 capsid protein

The outer capsid proteins VP4 and VP7 induce neutralizing antibody against rotavirus. We have investigated in a mouse model the protection mediated by immunization with VP8*, the amino-terminal tryptic fragment of VP4. BALB/c female mice immunized with simian rotavirus SA11 VP6 and VP8* proteins expressed in Escherichia coli were mated with seronegative males. Litters were orally challenged with the SA11 strain (P5B[2], G3) or with the murine rotavirus strain EDIM (P10[16], G3) to verify the degree of protection against diarrhea induced in the newborns. Only those pups born to dams immunized with VP8* did not develop diarrhea after having been orally challenged with the SA11 strain. Pups born to naive dams but foster nursed by VP8*-immunized dams did not develop diarrhea after having been orally infected with the SA11 strain, but they suffered diarrhea when challenged with the EDIM strain. These results support the concepts that (1) VP8* is a highly immunogenic polypeptide that induces effective homotypic protection against disease in pups born to dams immunized with this antigen and (2) in newborn mice the protection against disease is mediated by neutralizing secretory antibodies present in the milk rather than by serum antibodies transferred through the placenta to the offspring.     

Isolation of a C (Ibc) protein from group B Streptococcus which elicits mouse protective antibody

The C (Ibc) proteins of group B Streptococcus (GBS) have been shown to induce mouse protective antibodies when present as immunogens on whole organisms. However, characterization of specific proteins responsible for inducing protection has not been reported. We have grown type Ic GBS in a dialysate of Todd Hewitt broth and analyzed the proteins extruded into the broth. Multiple proteins of varying size were visualized by SDS-PAGE. Ultrafiltration was used to separate the GBS components by molecular weight (MW) into 2 pools, those below 30,000 MW but above 10,000 MW (P10) and those above 30,000 MW (P30). The P10 contained 4 major proteins, including a 14,000 MW protein. Balb-c mice were immunized with the P10 fraction and the antisera used in mouse protection studies. This immune sera protected 100% of mice against challenge with type Ib GBS and protection was not altered by prior absorption of the sera with type Ia or Ib capsular polysaccharide. The P10 was fractionated by column chromatography and eluted proteins examined by SDS-PAGE and Western blot with the mouse protective antisera elicited to the P10. There was one major immunologically reactive protein at 14,000 MW which eluted in a partially purified form from the column. The 14,000 MW protein was reisolated from preparative SDS-PAGE gels and used to elicit antiserum in a rabbit. In mouse protection studies this rabbit antiserum protected mice against subsequent challenge with type Ib GBS (89% protection). Surface antigens were extracted from 125I-labelled type Ic GBS and immunoprecipitated with antiserum to the 14,000 MW protein. The 14,000 MW protein and multiple higher molecular weight proteins were immunologically cross-reactive suggesting the presence of shared epitopes. Thus the 14,000 MW protein from type Ic GBS that is antigenic and elicits mouse protective antibodies against the heterologous type Ib GBS fulfills the criteria for a C protein of GBS.     

Characterization of two distinct opsonic and protective epitopes within the alpha C protein of the group B Streptococcus.

Group B Streptococcus (GBS) is a major cause of neonatal sepsis, meningitis in early infancy, postpartum endometritis, and serious invasive infections in adults in the United States. We previously cloned, sequenced, and characterized the alpha antigen gene, bca, and showed that the alpha C protein of GBS is a trypsin-resistant, surface-associated polypeptide that contains a signal sequence, a unique N terminus, nine identical tandem repeats, and a C-terminal membrane anchor structure. Polyclonal antiserum raised to the recombinant alpha C protein and an opsonic monoclonal antibody, 4G8, raised to the native protein from GBS have been shown to be protective in a mouse model. The binding site of 4G8 has now been localized to the tandem repeat region of the alpha C protein. To determine whether the N terminus of the alpha C protein contains additional opsonic and/or protective epitopes, the sequence corresponding to the alpha C protein N terminus was subcloned into a pET vector, the expressed peptide from Escherichia coli was purified by Ni2+ affinity chromatography, and rabbit polyclonal antibodies were raised to the purified recombinant peptide. Antibodies to the alpha C protein N terminus were shown to be opsonic by an in vitro opsonophagocytosis assay. In addition, 69% of newborn mouse pups from mothers passively immunized with the antiserum to the recombinant N-terminal polypeptide of the alpha C protein were protected against lethal challenge with GBS A909. These data indicate that at least two distinct regions of the alpha C protein, the N terminus and the tandem repeat region, contain opsonic and protective epitopes.     

Recombinant group B Streptococcus alpha-like protein 3 is an effective immunogen and carrier protein

Conjugate vaccines against pathogens of multiple serotypes are optimized when all components induce functional antibody, resulting in broadened coverage. While most clinical studies of vaccines against group B Streptococcus (GBS) have evaluated conjugates composed of capsular polysaccharide (CPS) coupled to tetanus toxoid, conjugates prepared with GBS proteins as carriers have also been efficacious in animals. Here, we report that recombinant GBS alpha-like protein 3 (rAlp3) is both a strong immunogen and a viable carrier protein for type III CPS. The type III CPS-specific immunoglobulin G (IgG) titer rose from <100 to 64,000 among mice that received type III CPS coupled to rAlp3 (III-rAlp3) compared with an absence of a specific response among mice that received an uncoupled mixture. Most (94%) newborn pups born to III-rAlp-vaccinated dams survived challenge with viable type III GBS, compared with 43% survival among those born to dams that received the uncoupled mixture (P < 0.0001). A tricomponent conjugate of type III CPS, rAlp3, and a GBS recombinant beta C protein lacking its IgA binding site (III-rAlp3-rBCP(DeltaIgA)) provided protection against a serotype III strain and a serotype Ia strain bearing beta C protein. High-titered anti-rAlp3 rabbit serum opsonized Alp3-containing strains of two GBS serotypes (types V and VIII) and invasive type III strains bearing the cross-reactive Rib protein for in vitro killing by human peripheral blood leukocytes. Thus, the potential exists for the inclusion of rAlp3 in a GBS vaccine formulated to provide multiserotype coverage.     

Paucibacillary tuberculosis in mice after prior aerosol immunization with Mycobacterium bovis BCG

To develop a murine model of paucibacillary tuberculosis for experimental chemotherapy of latent tuberculosis infection, mice were immunized with viable Mycobacterium bovis BCG by the aerosol or intravenous route and then challenged six weeks later with virulent Mycobacterium tuberculosis. The day after immunization, the counts were 3.71 +/- 0.10 log(10) CFU in the lungs of aerosol-immunized mice and 3.65 +/- 0.11 and 4.93 +/- 0.07 log(10) CFU in the lungs and spleens of intravenously immunized mice, respectively. Six weeks later, the lungs of all BCG-immunized mice had many gross lung lesions and splenomegaly; the counts were 5.97 +/- 0.14 and 3.54 +/- 0.07 log(10) CFU in the lungs and spleens of aerosol-immunized mice, respectively, and 4.36 +/- 0.28 and 5.12 +/- 0.23 log(10) CFU in the lungs and spleens of intravenously immunized mice, respectively. Mice were then aerosol challenged with M. tuberculosis by implanting 2.37 +/- 0.13 log(10) CFU in the lungs. Six weeks after challenge, M. tuberculosis had multiplied so that the counts were 6.41 +/- 0.27 and 4.44 +/- 0.14 log(10) CFU in the lungs and spleens of control mice, respectively. Multiplication of M. tuberculosis was greatly limited in BCG-immunized mice. Six weeks after challenge, the counts were 4.76 +/- 0.24 and 3.73 +/- 0.34 log(10) CFU in the lungs of intravenously immunized and aerosol-immunized mice, respectively. In contrast to intravenously immunized mice, there was no detectable dissemination to the spleen in aerosol-immunized mice. Therefore, immunization of mice with BCG by the aerosol route prior to challenge with a low dose of M. tuberculosis resulted in improved containment of infection and a stable paucibacillary infection. This model may prove to be useful for evaluation of new treatments for latent tuberculosis infection in humans.     

Experimental vaccination against group B streptococcus, an encapsulated bacterium, with highly purified preparations of cell surface proteins Rib and alpha.

Encapsulated bacteria cause some of the most common diseases in humans. Although the polysaccharide capsules of these pathogens have attracted the most attention with regard to vaccine development, recent evidence suggests that bacterial surface proteins may also be used to confer protective immunity. We have analyzed this possibility in group B streptococcus (GBS), an encapsulated bacterium that is the major cause of invasive bacterial disease in the neonatal period. Previous work has shown that the majority of GBS strains causing invasive infections express the Rib protein, and that most strains lacking Rib express a protein designated alpha. Here we report that active immunization with highly purified preparations of Rib or alpha protected mice against lethal infection with strains expressing the corresponding protein. Vaccination with the Rib protein protected against two strains of capsular type III and two strains of type II, and vaccination with the alpha protein protected against one strain of type II and one strain of type Ib. The mice vaccinated with Rib or alpha showed a good immunoglobulin G response to the immunogen. These data suggest that a vaccine against GBS disease may be based on cell surface proteins and support the notion that proteins may be used for immunization against encapsulated bacteria.     

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.