Saliva-binding Region Research Articles

Enhanced immune response to a dual-promoter anti-caries DNA vaccine orally delivered by attenuated Salmonella typhimurium

The strength of immune responses induced by DNA vaccine is closely associated with the expression level of cloned antigens available to the antigen presenting cells (APCs). To acquire a larger and more persistent amount of antigen, a dual-promoter, which could double the target antigen output through its expression both in prokaryotic and eukaryotic cells, was employed in the constructed anti-caries DNA vaccine with attenuated Salmonella as mucosal delivery vector in this study. Here, both CMV and nirB promoters were included in the plasmid that harbors the genes encoding the functional epitopes of two virulence factors of S. mutans, i.e. the saliva-binding region (SBR) of PAc and the glucan-binding region (GBR) of glucosyltransferase-I (GTF-I). Delivered by attenuated Salmonella Typhimurium strain SL3261, the anti-caries vaccine was administered intragastrointestinally to BALB/c mice for evaluation of the effectiveness of this immune regime. Specific anti-SBR and anti-GBR antibodies were detected in the serum and saliva of experimental animals by week 3 after immunization. These immune responses were further enhanced after a booster vaccination at week 16. However, in mice receiving Salmonella expressing SBR and GBR under the control of nirB alone these antibody responses were significantly (P<0.01) lower. The serum IgG subclass profiles suggested a Th1/Th2-mixed but Th2 biased immune response to the cloned antigens, which was further confirmed by a significant increase in the Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-10) cytokines in splenocytes of immunized mice upon stimulation with SBR or GBR. To further determine the protective efficacy of these responses, a challenge test with S. mutans strain UA159 was performed in mice after the second immunization. Following challenge, mice immunized with Salmonella expressing SBR and GBR under the control of the CMV-nirB promoter showed a significant (P<0.01) reduction in the number of S. mutans in the dental plaque compared to the empty vector-immunized or unimmunized mice, and the reduction was also significant at weeks 3–8 (P<0.05) post-challenge when compared with those receiving Salmonella clones with nirB promoter alone. These results provide evidence for the effectiveness of a dual-promoter strategy in the anti-caries DNA vaccine when employing attenuated Salmonella as delivering vehicle for mucosal immunization.

Identification and Characterization of Intestinal Antigen-Presenting Cells Involved in Uptake and Processing of a Nontoxic Recombinant Chimeric Mucosal Immunogen Based on Cholera Toxin Using Imaging Flow Cytometry

Intragastric immunization with recombinant chimeric immunogen, SBR-CTA2/B, constructed from the saliva-binding region (SBR) of Streptococcus mutans antigen AgI/II and the A2/B subunits of cholera toxin (CT) induces salivary and circulating antibodies against S. mutans that protect against dental caries. We previously found that SBR-CTA2/B activated dendritic cells (DC) in the Peyer's patches (PP) and mesenteric lymph nodes (MLN). To identify the cells involved in the intestinal uptake of SBR-CTA2/B and the initiation of immune responses, mice were immunized intragastrically with fluorescein-labeled SBR-CTA2/B or SBR, and intestinal cells were examined by imaging flow cytometry after fluorescent staining for cell surface markers. SBR-CTA2/B was preferentially taken up by CD103(+) DC in the PP and by both CD103(+) and CD11c(+) DC in intestinal lamina propria (LP), whereas SBR was taken up to a lesser extent by PP CD11c(+) DC, within 2 to 16 h. By 16 h, CD103(+) and CD11c(+) DC containing fluorescein-labeled SBR-CTA2/B were found in MLN and showed upregulation of the chemokine receptor CCR7. Large numbers of SBR-CTA2/B-containing DC were found interacting with CD4(+) (T helper) cells, which costained for nuclear transcription factors T-bet or RORγt, identifying them as Th1 or Th17 cells. In contrast, SBR-containing CD11c(+) DC interacted preferentially with GATA3(+) (Th2) cells. No SBR- or SBR-CTA2/B-containing DC were found interacting with Foxp3(+) (T regulatory) cells. We conclude that the coupling of SBR to CTA2/B enhances its immunogenicity by promoting uptake by DC in both PP and LP and that these antigen-containing DC migrated to MLN and interacted preferentially with Th1 and Th17 cells to induce active immune responses.

Contribution of a Streptococcus mutans Antigen Expressed by a Salmonella Vector Vaccine in Dendritic Cell Activation

A Salmonella vector vaccine expressing the saliva-binding region (SBR) of the adhesin AgI/II of Streptococcus mutans has been shown to induce a mixed Th1/Th2 anti-SBR immune response in mice and to require Toll-like receptor 2 (TLR2), TLR4, and MyD88 signaling for the induction of mucosal anti-SBR antibody responses. Since dendritic cells (DC) are critical in innate and adaptive immunity, the present study assessed the role of SBR expression by the vector vaccine in DC activation. Bone marrow-derived DC from wild-type and TLR2, TLR4, and MyD88 knockout mice were stimulated with Salmonella vector BRD509, the SBR-expressing Salmonella vector vaccine BRD509(pSBRT7), or SBR protein, and the DC responses to different stimuli were compared by assessing costimulatory molecule expression, cytokine production, and signaling pathways. The DC response to both BRD509(pSBRT7) and BRD509 was dependent mainly on TLR4. BRD509(pSBRT7) and BRD509 induced upregulation of CD80, CD86, CD40, and major histocompatibility complex class II (MHC II) expression. Lower levels of interleukin-10 (IL-10) and IL-12p40 were produced by BRD509(pSBRT7)-stimulated DC than by BRD509-stimulated DC. Furthermore, BRD509(pSBRT7)-stimulated DC showed decreased p38 phosphorylation compared to that induced by DC stimulated with BRD509. However, BRD509(pSBRT7)-treated DC produced a higher level of IL-6 than BRD509-stimulated cells. The low IL-12p40 and high IL-6 cytokine profile expressed by BRD509(pSBRT7)-stimulated DC may represent a shift toward a Th2 response, as suggested by the increased expression in Jagged-1. These results provide novel evidence that a heterologous protein expressed by a Salmonella vector vaccine can differentially affect DC activation.

Uptake and disposition of recombinant chimeric immunogens constructed from Streptococcus mutans SBR-CTA2/B at mucosal immune inductive sites (100.35)

Abstract Purpose: To investigate cellular pathways of uptake and processing of the recombinant chimeric immunogen SBR-CTA2/B consisting of the saliva-binding region (SBR) of S. mutans AgI/II coupled to cholera toxin A2/B subunits, in comparison with SBR alone. Methods: Amnis ImageStream technology was used to characterize cells from lamina propria (LP), Peyer’s patch (PP), and mesenteric lymph nodes (MLN) of mice before and 2, 4, 16, and 48h after intragastric immunization with FITC-labeled proteins. Dendritic cells (DC) were stained for phenotypic markers and the migration receptor CCR7 using contrasting fluorochromes. Results: LP CD103+, CD11c+, and especially CD103/CD11c double+ DC took up SBR-CTA2/B through 4h, after which the proportion of antigen-containing DC stayed level until 16h, although the number of DCs continued to increase. DCs in PP captured SBR-CTA2/B through 16h. The proportion of LP DCs containing SBR peaked at 2h and declined by 16h. CCR7 expression by CD11c+ LP DCs taking up SBR-CTA2/B rose at 2h and dropped at 4h, whereas CCR7 expression by DC taking up SBR declined from 0h levels. In MLN the number of CD11c+/CD4+ cell doublets dramatically increased at 48 h after SBR-CTA2/B immunization. Conclusions: Compared to SBR alone, the chimeric protein SBR-CTA2/B enhanced the functional activation of DCs in LP and PP, in which most antigen uptake occurred within the first few hours. DCs loaded with protein may then migrate to MLN and intercommunicate with T cells.

Characterization of antigen-presenting cells induced by intragastric immunization with recombinant chimeric immunogens constructed from Streptococcus mutans AgI/II and type I or type II heat-labile enterotoxins

Intragastric (i.g.) immunization with recombinant chimeric proteins constructed from the saliva-binding region (SBR) of Streptococcus mutans surface antigen AgI/II and the A2/B subunits of enterobacterial heat-labile enterotoxins has been successfully used to induce salivary and circulating antibodies against S.mutans that have protective potential against dental caries. To investigate the mode of action of these vaccine constructs, mice were immunized i.g. with chimeric proteins constructed from SBR and cholera toxin (CT) or the type II enterotoxins of Escherichia coli, LT-IIa and LT-IIb. Antigen-presenting cells (APC) in Peyer's patches (PP) and mesenteric lymph nodes (MLN) were characterized by flow cytometry. Compared with immunization with SBR alone, chimeric proteins SBR-LTIIaA2/B and SBR-LTIIbA2/B increased the number of B cells and macrophages in PP and diminished B cell numbers in MLN, whereas SBR-CTA2/B diminished the numbers of B cells and macrophages in PP and MLN. Immunization with all three chimeric proteins led to upregulation of MHC class II molecules and co-stimulatory receptors CD40, CD80, and CD86 especially on dendritic cells in PP and also on APC in MLN. The results provide a molecular basis for the enhanced immune responses induced by chimeric proteins compared with uncoupled antigen, and for differential responses to chimeric proteins based on CT or type II enterotoxins.

Role of Toll-like receptors in host responses to a virulence antigen of Streptococcus mutans expressed by a recombinant, attenuated Salmonella vector vaccine

In the present study, we investigated the role of Toll-like receptors (TLRs) in host responses to the saliva-binding region (SBR) of Streptococcus mutans expressed by a recombinant, attenuated Salmonella vaccine. C57BL/6 wild type (wt), TLR2 −/−, TLR4 −/− and MyD88 −/− mice were immunized by the intranasal route on days 0, 18 and boosted on day 98 with Salmonella typhimurium BRD 509 containing a plasmid encoding SBR. Serum and saliva samples were collected throughout the experiment and assessed for antibody activity by ELISA. Evidence is provided that the induction of a serum IgG2a (Th1-type) anti-SBR antibody response involved TLR2 signaling, whereas the anti- Salmonella response involved signaling through TLR4. The adaptor molecule MyD88 was not essential for the induction of a primary Th1-type response to SBR or Salmonella, but was necessary for a secondary response to SBR. Furthermore, the absence of TLR2, TLR4 or MyD88 resulted in enhanced Th2-type serum IgG1 anti-SBR and anti- Salmonella responses. Mucosal IgA responses to SBR were TLR2-, TLR4- and MyD88-dependent, while IgA responses to Salmonella were TLR4- and MyD88-dependent.

Immunogenicity of Salmonella vector vaccines expressing SBR of Streptococcus mutans under the control of a T7-nirB (dual) promoter system

The purpose of the present study was to determine if a Salmonella vector expressing the cloned saliva-binding region (SBR) of Streptococcus mutans or SBR linked to the A2 and B subunits of cholera toxin (CTA2/B) under the control of both the T7 and nirB promoters (T7-nirB dual promoter) was more effective in inducing mucosal and systemic anti-SBR antibody responses than Salmonella clones expressing the same antigens but under the control of either the nirB or T7 promoter. Mice were immunized by the intranasal route on days 0, 18 and 320 with Salmonella enterica serovar Typhimurium strain BRD 509 containing one of six plasmids encoding SBR or SBR-CTA2/B under the control of the T7-nirB, T7, or nirB promoter. Serum, saliva and vaginal wash samples were collected throughout the experiment and assessed for antibody activity by ELISA. Evidence is provided that Salmonella clones expressing SBR or SBR-CAT2/B under the control of either the T7 or T7-nirB promoter induced a high and persistent mucosal and systemic anti-SBR antibody response. All Salmonella clones induced good anti-SBR responses following the boost on day 320.

Induction and recall of immune memory by mucosal immunization with a non-toxic recombinant enterotoxin-based chimeric protein.

Previous reports have suggested that peroral delivery of antigens chemically coupled to non-toxic recombinant enterotoxin B subunits, such as the cholera toxin B subunit (CTB), induces tolerance to the antigen that may be abrogated by the toxic enzyme activity of intact enterotoxins, such as cholera toxin (CT). The aim of this study was to examine the immunogenicity of a genetically coupled protein composed of the saliva-binding region (SBR) of the Streptococcus mutans surface antigen AgI/II and the non-toxic A2 and B subunits of CT (SBR-CTA2/B) compared with that of recombinant SBR admixed with CT (SBR + CT) and SBR chemically coupled to recombinant CTB (SBR-CTB) following peroral delivery by intragastric (i.g.) immunization. The results showed that i.g. immunization with SBR-CTA2/B, like SBR + CT, induced antigen-specific serum immunoglobulin G (IgG) and salivary IgA antibodies, and sensitized splenic T cells. Comparison studies with SBR-CTB produced serum IgG but not salivary IgA titres and failed to sensitize splenic cells. Immunization with SBR-CTA2/B via the intranasal route also primed for the recall of antigen-specific memory antibody responses 6 months later. These findings show that SBR-CTA2/B is an immunogenic, not tolerogenic, chimeric protein that can induce and recall antigen-specific memory responses upon mucosal immunization.

Role of B7 costimulatory molecules in mediating systemic and mucosal antibody responses to attenuated Salmonella enterica serovar Typhimurium and its cloned antigen.

The purpose of the present study was to evaluate the ability of an attenuated Salmonella enterica serovar Typhimurium vaccine strain to up-regulate B7-1 and B7-2 on antigen-presenting cells and to examine the functional roles these costimulatory molecules play in mediating immune responses to Salmonella and to an expressed cloned antigen, the saliva-binding region (SBR) of antigen I/II. In vitro stimulation of B cells (B220+), macrophages (CD11b+), and dendritic cells (CD11c+) with S. enterica serovar Typhimurium induced an up-regulation of B7-2 and, especially, B7-1 expression. The in vivo functional roles of B7-1, B7-2, and B7-1/2 were evaluated in BALB/c wild-type and B7-1, B7-2, and B7-1/2 knockout (KO) mice following intranasal immunization with the Salmonella expressing the cloned SBR. Differential requirements for B7-1 and B7-2 were observed upon primary and secondary immunizations. Compared to wild-type controls, B7-1 and B7-2 KO mice had reduced mucosal and systemic anti-Salmonella antibody responses after a single immunization, while only B7-1 KO mice exhibited suppressed anti-Salmonella antibody responses following the second immunization. Mucosal and systemic antibody responses to SBR were reduced following the primary immunization, whereas a compensatory role for either B7-1 or B7-2 was observed after the second immunization. B7-1/2 double KO mice failed to induce detectable levels of mucosal or systemic immunoglobulin A (IgA) or IgG antibody responses to either Salmonella or SBR. These findings demonstrate that B7-1 and B7-2 can play distinct as well as redundant roles for mediating mucosal and systemic antibody responses, which are likely dependent upon the nature of the antigen.

Enhanced immunogenicity of a genetic chimeric protein consisting of two virulence antigens of Streptococcus mutans and protection against infection.

The saliva-binding region (SBR) of the cell surface antigen I/II (AgI/II) and the glucan-binding region (GLU) of the glucosyltransferase enzyme of Streptococcus mutans have been implicated in the initial adherence of S. mutans to saliva-coated tooth surfaces and the subsequent sucrose-dependent accumulation of S. mutans, respectively. Here, we describe the construction and characterization of a genetic chimeric protein consisting of the two virulence determinants SBR and GLU (SBR-GLU). The effectiveness of this construct in inducing mucosal and systemic immune responses to each virulence determinant following intranasal immunization was compared to that of each antigen alone or an equal mixture of SBR and GLU (SBR+GLU) in a mouse model. Furthermore, the ability of antibodies induced to SBR-GLU to protect against S. mutans infection was also investigated. Immunization of mice with the chimeric protein SBR-GLU resulted in significantly enhanced (P < 0.001) levels of serum immunoglobulin G (IgG) anti-SBR antibody activity compared to those in the SBR and SBR+GLU groups. The SBR-GLU-immunized mice also demonstrated a significant (P < 0.05) increase in salivary and vaginal IgA antibody responses to SBR and GLU. Analysis of the serum IgG subclass responses to SBR in mice immunized with SBR alone indicated a mixed IgG1 and IgG2a response. A preferential IgG1 response compared to an IgG2a anti-GLU response was induced in mice immunized with GLU alone. Similarly, a preferential IgG1 response was also induced to SBR when GLU was present in either a mixed or conjugated form. Finally, a significant reduction (P < 0.05) in S. mutans colonization was observed only in mice immunized with the SBR-GLU chimeric protein. Taken together, our results indicate that the chimeric protein SBR-GLU significantly enhanced mucosal immune responses to SBR and GLU and systemic immune responses to SBR. The ability of SBR-GLU to induce responses effective in protection against colonization of S. mutans suggests its potential as a vaccine antigen for dental caries.

Dependence of bacterial protein adhesins on toll-like receptors for proinflammatory cytokine induction.

Toll-like receptors (TLRs) are important signal transducers that mediate inflammatory reactions induced by microbes through pattern recognition of virulence molecules such as lipopolysaccharide (LPS) and lipoproteins. We investigated whether proinflammatory cytokine responses induced by certain bacterial protein adhesins may also depend on TLRs. In differentiated THP-1 mononuclear cells stimulated by LPS-free recombinant fimbrillin (rFimA) from Porphyromonas gingivalis, cytokine release was abrogated by monoclonal antibodies (MAbs) to CD14 and TLR4 but not to TLR2. Similar experiments using anti-beta2 integrin MAbs suggested that beta2 integrins (CD11/CD18) also play a role in cytokine induction by rFimA or native fimbriae. Minor fimbriae (distinct from the fimA-encoded major fimbriae) of P. gingivalis induced proinflammatory cytokine release in a CD14- and TLR2-dependent mode. Cytokine induction by BspA, a leucine-rich repeat protein from Bacteroides forsythus, depended heavily on CD14 and TLR2. We also found that the ability of the streptococcal protein AgI/II to stimulate cytokine release depended partially on CD14 and TLR4, and the AgI/II segment that possibly interacts with these receptors was identified as its N-terminal saliva-binding region. When THP-1 cells were exposed to rFimA for 24 h, surface expression of CD14 and CD18 was decreased and the cells became hyporesponsive to cytokine induction by a second challenge with rFimA. However, tolerance induction was abolished when the THP-1 cells were pretreated with rFimA in the presence of either anti-CD14 MAb or anti-TLR4 MAb. Induction of cross-tolerance between rFimA and LPS correlated with downregulation of the pattern recognition receptors involved. Our data suggest that the CD14-TLR2/4 system is involved in cytokine production and tolerance induction upon interaction with certain proinflammatory bacterial protein adhesins.

Inhibitory Effect of Bovine Milk Lactoferrin on the Interaction between a Streptococcal Surface Protein Antigen and Human Salivary Agglutinin

Human whole saliva induces aggregation of Streptococcus mutans cells via an interaction between a surface protein antigen (PAc) of the organism and salivary agglutinin. Bovine milk inhibits the saliva-induced aggregation of S. mutans. In this study, the milk component that possesses inhibitory activity against this aggregation was isolated and found to be lactoferrin. Surface plasmon resonance analysis indicated that bovine lactoferrin binds more strongly to salivary agglutinin, especially to high molecular mass glycoprotein, which is a component of the agglutinin, than to recombinant PAc. The binding of bovine lactoferrin to salivary agglutinin was thermostable, and the optimal pH for binding was 4.0. To identify the saliva-binding region of bovine lactoferrin, 11 truncated bovine lactoferrin fragments were constructed. A fragment corresponding to the C-terminal half of the lactoferrin molecule had a strong inhibitory effect on the saliva-induced aggregation of S. mutans, whereas a fragment corresponding to the N-terminal half had a weak inhibitory effect. Seven shorter fragments corresponding to lactoferrin residues 473-538 also showed a high ability to inhibit the aggregation of S. mutans. These results suggest that residues 473-538 of bovine lactoferrin are important in the inhibition of saliva-induced aggregation of S. mutans.

Induction of Protective Immunity againstStreptococcus mutansColonization after Mucosal Immunization with AttenuatedSalmonella entericaSerovar Typhimurium Expressing anS. mutansAdhesin under the Control of In Vivo-InduciblenirBPromoter

The purpose of the present study was to evaluate the effectiveness of an attenuated Salmonella enterica serovar Typhimurium vaccine strain expressing the saliva-binding region (SBR) of the Streptococcus mutans antigen I/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2 and B subunits (CTA2/B) and under the control of the anaerobically inducible nirB promoter, in inducing a protective immune response against S. mutans infection. BALB/c mice were immunized by either the intranasal or the intragastric route with a single dose of 10(9) or 10(10) Salmonella CFU, respectively. The Salmonella vaccine strain expressing an unrelated antigen (fragment C of tetanus toxin [TetC]) was also used for immunization as a control. Samples of serum and secretion (saliva and vaginal washes) were collected prior to and following immunization and assessed for antibody activity by enzyme-linked immunosorbent assay. Anti-SBR antibodies were detected in the serum and saliva of experimental animals by week 3 after immunization. A booster immunization at week 17 after the initial immunization resulted in enhanced immune responses to the SBR. The serum immunoglobulin G subclass profiles were indicative of T helper type 1 responses against both the vector and the SBR antigen. To determine the effectiveness of these responses on the protection against S. mutans infection, mice were challenged after the second immunization with a virulent strain of S. mutans which was resistant to tetracycline and erythromycin. Prior to the challenge, mice were treated for 5 days with tetracycline, erythromycin, and penicillin. S. mutans was initially recovered from all of the challenged mice. This bacterium persisted at high levels for at least 5 weeks in control TetC-immunized or nonimmunized mice despite the reappearance of indigenous oral organisms. However, mice immunized with Salmonella clones expressing SBR or SBR-CTA2/B demonstrated a significant reduction in the number of S. mutans present in plaque compared to the control groups. These results provide evidence for the effectiveness of the Salmonella vector in delivering the SBR antigen for the induction of mucosal and systemic immune responses to SBR. Furthermore, the induction of a salivary anti-SBR response corresponded with protection against S. mutans colonization of tooth surfaces.

Recombinant antigen-enterotoxin A2/B chimeric mucosal immunogens differentially enhance antibody responses and B7-dependent costimulation of CD4(+) T cells.

The ADP-ribosylating enterotoxins, cholera toxin (CT) and the Escherichia coli heat-labile toxin (LT-IIa), have been shown to enhance mucosal and systemic antibody (Ab) responses to coadministered antigens. The purpose of the present study was to compare the ability of the nontoxic A2/B subunits of these toxins, which have distinct targeting properties, to augment the immunogenicity of a genetically coupled protein antigen. Structurally similar chimeric proteins were generated by genetically replacing the toxic A1 subunit of CT or LT-IIa with the saliva-binding region (SBR) from the streptococcal adhesin AgI/II. Intranasal immunization of BALB/c mice with either chimeric protein induced significantly higher plasma and mucosal anti-SBR immunoglobulin A (IgA) and IgG Ab responses than SBR alone. Moreover, compared to SBR-LT-IIaA2/B, SBR-CTA2/B elicited significantly higher levels of plasma IgG1 and salivary IgA anti-SBR Ab responses. Ex vivo and in vitro experiments revealed that SBR-CTA2/B selectively up-regulated B7-2 expression on murine B cells isolated from both the nasal associated lymphoid tissue, cervical lymph nodes, and spleen. In contrast, SBR-LT-IIaA2/B had little effect on B7-1 or B7-2 expression on B220(+), CD11b(+), or CD11c(+) cells. Analysis of the functional costimulatory activity of SBR-CTA2/B-treated B cells revealed a significant enhancement in anti-CD3-stimulated CD4(+) T-cell proliferative responses, and this proliferation was significantly reduced by treatment with anti-B7-2 but not with anti-B7-1 or isotype control Abs. Thus, SBR-CTA2/B and SBR-LT-IIaA2/B exhibit distinct patterns of antibody responses associated with differential effects on B7-2 expression and subsequent costimulatory effects on CD4(+) T cells.

Construction and characterization of a Salmonella enterica serovar typhimurium clone expressing a salivary adhesin of Streptococcus mutans under control of the anaerobically inducible nirB promoter.

Attenuated Salmonella enterica serovar Typhimurium has been used for targeted delivery of recombinant antigens to the gut-associated lymphoid tissues. One potential problem associated with this vaccine approach is the likelihood of in vivo instability of the plasmid constructs caused by constitutive hyperexpression of the heterologous immunogen. The aim of this study was to generate and characterize an expression system encoding the saliva-binding region (SBR) of Streptococcus mutans antigen I/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2/B subunits (CTA2/B), under the control of the inducible nirB promoter. This promoter is activated in an anaerobic environment and within macrophages, which are the primary antigen-presenting cells involved in phagocytosis and processing of Salmonella. The gene encoding the chimeric SBR-CTA2/B was amplified by PCR using primers containing appropriate restriction sites for subcloning into pTETnirB, which contains the nirB promoter. The resulting plasmid was introduced into serovar Typhimurium by electroporation. Production of the SBR-CTA2/B chimeric protein under anaerobic conditions was verified by enzyme-linked immunosorbent assay of whole-cell lysates on plates coated with G(M1) ganglioside and developed with antibodies to SBR. Similar procedures were followed for cloning the gene encoding SBR in serovar Typhimurium under nirB control. Anaerobic expression of SBR was confirmed by Western blotting of whole-cell lysates probed with anti-SBR antibodies. The resulting serovar Typhimurium strains were administered by either the oral or the intranasal route to mice, and colonization was assessed by microbiologic analysis of dissociated spleens, Peyer's patches (PP), and nasal tissues. High numbers of the recombinant strains persisted in PP and spleen for at least 21 days following oral challenge. A single intranasal administration of the Salmonella clones to mice also resulted in the colonization of the nasal tissues by the recombinant bacteria. Salmonellae were recovered from nasal lymphoid tissues, superficial lymph nodes, internal jugular lymph nodes, PP, and spleens of mice for at least 21 days after challenge. This study provides quantitative evidence for colonization by Salmonella strains expressing a recombinant protein under the control of the inducible nirB promoter in PP or nasal tissues following a single oral or nasal administration of the bacteria, respectively.

Effectiveness of liposomes possessing surface-linked recombinant B subunit of cholera toxin as an oral antigen delivery system.

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.

Oral immunization with the saliva-binding region of Streptococcus mutans AgI/II genetically coupled to the cholera toxin B subunit elicits T-helper-cell responses in gut-associated lymphoid tissues.

Mice immunized intragastrically (i.g.) with a genetically constructed chimeric protein consisting of the saliva-binding region (SBR) of Streptococcus mutans AgI/II coupled to cholera toxin (CT) A2 and B subunits (CTA2/B) develop serum immunoglobulin G (IgG) and mucosal IgA antibody responses against AgI/II that are enhanced by the coadministration of CT as an adjuvant. To investigate the development of antigen-specific T cells in the gut-associated lymphoid tissues, mice were immunized i.g. with SBR, SBR-CTA2/B, or SBR-CTA2/B plus CT. AgI/II-specific T cells in Peyer's patches (PP), mesenteric lymph nodes (MLN), and spleen were assayed by lymphoproliferation and flow cytometry for the expression of T-cell surface markers, and cytokine mRNA expression was evaluated by reverse transcription-PCR. T-cell responses were consistent with antibody responses but were detectable after the first immunization. Proliferative responses of PP and MLN cells upon stimulation with AgI/II in vitro were low and delayed in mice given SBR alone, and these cells displayed a mixed type 1 and 2 (or Th0) pattern of cytokine expression. Immunization with SBR-CTA2/B resulted in greater AgI/II-specific proliferative responses in PP cells and an increase in the proportion of CD4+ T cells. Coadministration of CT with SBR-CTA2/B led to greater proliferative responses especially in the MLN cells, which then showed an increase in CD4+ cells. Immunization with SBR-CTA2/B (with or without CT) skewed the cytokine expression pattern in PP and MLN cells toward Th2. The results indicate that T helper cells were induced in gut-associated lymphoid tissues by i.g. immunization with SBR-CTA2/B, concomitantly with and prior to the appearance of circulating and mucosal antibodies.

Saliva-binding region of Streptococcus mutans surface protein antigen.

A 190-kDa surface protein antigen (PAc) of Streptococcus mutans binds to human salivary components. For detection of specific binding of the PAc protein to human salivary components, a simple sandwich assay was used. Microtiter plates precoated with recombinant PAc (rPAc), PAc fragments, or S. mutans whole cells were allowed to react with human whole saliva and then were incubated with biotinylated rPAc. The biotinylated rPAc bound to salivary components was detected by use of alkaline phosphatase-conjugated streptavidin and p-nitrophenylphosphate. In this assay, the binding of whole cells of S. mutans and purified rPAc to salivary components was confirmed. For determination of a saliva-binding region of the PAc molecule, 14 truncated PAc fragments were constructed by use of the polymerase chain reaction and an expression vector, pAX4a+. The binding of these truncated PAc fragments to human salivary components was determined by the sandwich assay. Among the truncated PAc fragments, fragments corresponding to residues 39 to 864 and residues 39 to 1000 of PAc showed a high ability to bind to salivary components. Shorter recombinant fragments corresponding to residues 39 to 217, residues 200 to 481, residues 470 to 749, and residues 688 to 864 did not exhibit any binding ability. The fragment that corresponds to a proline-rich repeating region (residues 828 to 1000) bound directly to the PAc protein. These results suggest that residues 39 864 of the PAc molecule are important in the binding of the surface protein to human salivary components, and the proline-rich repeating region of the PAc protein may contribute to spontaneous self-aggregation of the PAc protein.