RESUMEN
Passage in human blood of group A streptococcal isolate 64p was previously shown to result in the enhanced expression of M and M-related proteins. Similarly, when this isolate was injected into mice via an air sac model for skin infection, organisms recovered from the spleens showed both increased expression of M and M-related proteins and increased skin-invasive potential. We show that these phenotypic changes were not solely the result of increased transcription of the mRNAs encoding the M and M-related gene products. Rather, the altered expression was associated with posttranslational modifications of the M and M-related proteins that occur in this strain, based on the presence or absence of another virulence protein, the streptococcal cysteine protease SpeB. The phenotypic variability also correlates with colony size variation. Large colonies selected by both regimens expressed more hyaluronic acid, which may explain differences in colony morphology. All large-colony variants were SpeB negative and expressed three distinct immunoglobulin G (IgG)-binding proteins in the M and M-related protein family. Small-colony variants were SpeB positive and bound little IgG through their M and M-related proteins because these proteins, although made, were degraded or altered in profile by the SpeB protease. We conclude that passage in either human blood or a mouse selects for a stable, phase-varied strain of group A streptococci which is altered in many virulence properties.
Asunto(s)
Antígenos Bacterianos , Cápsulas Bacterianas/fisiología , Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/biosíntesis , Cisteína Endopeptidasas/biosíntesis , Streptococcus pyogenes/patogenicidad , Proteínas Portadoras/biosíntesis , Humanos , Peso Molecular , Fenotipo , Streptococcus pyogenes/metabolismo , VirulenciaRESUMEN
Liposomes appear to be a promising oral antigen delivery system for the development of vaccines against infectious diseases, although their uptake efficiency by Peyer's patches in the gut and the subsequent induction of mucosal immunoglobulin A (IgA) responses remain a major concern. Aiming at targeted delivery of liposomal immunogens, we have previously reported the conjugation via a thioether bond of the GM1 ganglioside-binding subunit of cholera toxin (CTB) to the liposomal outer surface. In the present study, we have investigated the effectiveness of liposomes containing the saliva-binding region (SBR) of Streptococcus mutans AgI/II adhesin and possessing surface-linked recombinant CTB (rCTB) in generating mucosal (salivary, vaginal, and intestinal) IgA as well as serum IgG responses to the parent molecule, AgI/II. Responses in mice given a single oral dose of the rCTB-conjugated liposomes were compared to those in mice given one of the following unconjugated liposome preparations: (i) empty liposomes, (ii) liposomes containing SBR, (iii) liposomes containing SBR and coadministered with rCTB, and (iv) liposomes containing SBR plus rCTB. Three weeks after the primary immunization, significantly higher levels of mucosal IgA and serum IgG antibodies to AgI/II were observed in the rCTB-conjugated group than in mice given the unconjugated liposome preparations, although the latter mice received a booster dose at week 9. The antibody responses in mice immunized with rCTB-conjugated liposomes persisted at high levels for at least 6 months, at which time (week 26) a recall immunization significantly augmented the responses. In general, mice given unconjugated liposome preparations required one or two booster immunizations to develop a substantial anti-AgI/II antibody response, which was more prominent in the group given coencapsulated SBR and rCTB. These data indicate that conjugation of rCTB to liposomes greatly enhances their effectiveness as an antigen delivery system. This oral immunization strategy should be applicable for the development of vaccines against oral, intestinal, or sexually transmitted diseases.
Asunto(s)
Antígenos Bacterianos/inmunología , Toxina del Cólera/inmunología , Vacunas contra el Cólera/inmunología , Vacunas Conjugadas/inmunología , Adhesinas Bacterianas/inmunología , Administración Oral , Animales , Anticuerpos Antibacterianos/inmunología , Toxina del Cólera/administración & dosificación , Vacunas contra el Cólera/administración & dosificación , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Inmunoglobulina A Secretora , Inmunoglobulina G/inmunología , Intestinos/inmunología , Liposomas/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Saliva/inmunología , Streptococcus mutans/inmunología , Vacunas Conjugadas/administración & dosificación , Vagina/inmunologíaRESUMEN
An avirulent Salmonella typhimurium vaccine strain expressing a streptococcal protein adhesin and a similar clone which produces the same streptococcal antigen linked to the cholera toxin (CT) A2 and B subunits (CTA2/B) were compared for the ability to induce antibody responses to the expressed heterologous antigen after oral or intranasal immunization of mice. Expression of cloned immunogens in these systems is temperature regulated, being optimal at 37 degrees C, and the two clones under comparison were shown to produce similar levels of the streptococcal antigen. Both clones were found to stimulate high levels of serum immunoglobulin G (IgG) and mucosal IgA antibodies to the cloned immunogen. A consistent trend was observed toward higher mucosal IgA but lower serum IgG responses in the case of the S. typhimurium vector that coexpressed CTA2/B, a potential mucosal adjuvant, regardless of the route of administration. Also noteworthy was the capacity of these antigen delivery systems to induce anamnestic systemic and secretory responses to the cloned immunogen 15 weeks after the primary immunization, despite preexisting immunity to the Salmonella vectors. These antibody responses were sustained for at least 7 months following the booster immunization, at which time the secretory IgA antibody levels were significantly higher in mice given the Salmonella clone that coexpressed CTA2/B. Although the serum IgG response against the Salmonella vector was characterized by a high IgG2a/IgG1 ratio (indicative of the T helper type 1 [Th1]/Th2 profile), a mixed IgG1 and IgG2a pattern was observed for the carried heterologous antigen, which displayed a dominant IgG1 response when administered as a purified immunogen. Our findings indicate that the recombinant streptococcal antigen and CTA2/B are strong immunogens when expressed by the antigen delivery system used in this study and suggest that CTA2/B may have an additional immunoenhancing activity in the mucosal compartment besides its ability to target antigen uptake into the mucosal inductive sites. CTA2/B may thus be useful as an S. typhimurium-cloned adjuvant for coexpressed protein antigens.
Asunto(s)
Antígenos Bacterianos/inmunología , Toxina del Cólera/inmunología , Inmunidad Mucosa , Salmonella typhimurium/inmunología , Animales , Formación de Anticuerpos , Ratones , Proteínas Recombinantes/inmunologíaRESUMEN
A major adhesin from the oral pathogen Streptococcus mutans has been shown to be mucosally immunogenic upon genetic fusion with the cholera toxin A2/B subunits. To take advantage of the ability of Salmonella typhimurium to deliver cloned antigens to the mucosal inductive sites that would obviate the need for antigen purification, we expressed this chimeric construct in an attenuated S. typhimurium strain under the control of bacteriophage T7 transcription. Residual expression of the temperature-regulated T7 RNA polymerase at 30 degrees C allowed production of the chimeric protein at 2-3% of the total soluble protein, but it was increased five to six times following induction at 37 degrees C. Oral administration of a single dose of 10(9) recombinant Salmonella to mice resulted in serum IgG and salivary IgA antibody responses to Salmonella, cholera toxin, and the streptococcal adhesin, which were generally enhanced after a booster immunization.
Asunto(s)
Adhesinas Bacterianas/biosíntesis , Toxina del Cólera/biosíntesis , Vectores Genéticos/biosíntesis , Vectores Genéticos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Salmonella typhimurium/inmunología , Salmonella typhimurium/metabolismo , Streptococcus mutans/metabolismo , Adhesinas Bacterianas/metabolismo , Administración Oral , Animales , Toxina del Cólera/genética , Vectores Genéticos/administración & dosificación , Intubación Gastrointestinal , Ratones , Ratones Endogámicos BALB C , Unión Proteica/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/biosíntesis , Salmonella typhimurium/genéticaRESUMEN
Here we report the effectiveness of various liposome vaccines containing Streptococcus mutans glucosyltransferase (GTF) in protecting against dental caries after oral immunization. Rats were immunized by gastric intubation of the appropriate liposome vaccine at weaning and boosted 3 times. Rats were infected with S. mutans following initial immunization and fed cariogenic diet (Diet 305). Saliva and serum were collected during the study and assessed for antibody activity by enzyme-linked immunosorbent assay. Mandibles were removed on day 47 and assessed for S. mutans levels and then for caries. Animals immunized with sonicated, filtered and microemulsified GTF liposome preparations had decreased levels of dental caries compared with control animals given empty liposomes. Rats given dehydrated/rehydrated or purified liposomal GTF also had significantly less caries than control group (GTF alone). Because of economy, ease of preparation and efficiency in amount of antigen used, filtered, dehydrated/ rehydrated and purified liposomal GTF preparations are most practical for use in further assessing the efficacy of liposomal GTF in oral immunization.
Asunto(s)
Vacunas Bacterianas/inmunología , Caries Dental/prevención & control , Glucosiltransferasas/inmunología , Streptococcus mutans/inmunología , Análisis de Varianza , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Vacunas Bacterianas/administración & dosificación , Caries Dental/inmunología , Caries Dental/microbiología , Placa Dental/microbiología , Relación Dosis-Respuesta a Droga , Portadores de Fármacos , Vida Libre de Gérmenes , Glucosiltransferasas/administración & dosificación , Inmunidad Mucosa , Liposomas , Tamaño de la Partícula , Ratas , Ratas Endogámicas F344 , Saliva/inmunología , Streptococcus mutans/enzimologíaRESUMEN
Several immunoadjuvant systems have been proposed to enhance mucosal immune responses of orally administered purified antigens. Cholera toxin (CT) or its B subunit (CTB) have been found to promote immune responses to antigens when they are co-administered via mucosal routes. Oral administration of antigens incorporated into liposomes has also been shown to result in enhanced mucosal immune responses. Here, we describe the covalent coupling of CT and CTB to small unilamellar liposomes for targeting these vesicles to Peyer's patch M cells, following their oral administration. Conjugation was done by means of a thioether bond using succinimidyl(4-N-maleimidomethyl)cyclohexane-1-carboxylate to modify the dipalmitoylphosphatidyl-ethanolamine constituent of liposomes and N-succinimidyl-3-(2-pyridyldithio)propionate to thiolate CT or CTB. The biological activity of CT or CTB bound to liposomes was confirmed by a hemagglutination assay using GM1-enriched human erythrocytes. Furthermore, oral administration of CT-conjugated liposomes to rats resulted in the induction of serum IgG and salivary IgA anti-CT responses. CT-conjugated liposomes may prove to be a useful system for targeted delivery and immunoenhancement of weakly immunogenic antigens.