Facilitated intranasal induction of mucosal and systemic immunity to mutans streptococcal glucosyltransferase peptide vaccines.

Synthetic peptide vaccines which are derived from functional domains of Streptococcus mutans glucosyltransferases (GTF) have been shown to induce protective immunity in Sprague-Dawley rats after subcutaneous injection in the salivary gland region. Since mucosal induction of salivary immunity would be preferable in humans, we explored methods to induce mucosal antibody in the rat to the GTF peptide vaccines HDS and HDS-GLU after intranasal administration. Several methods of facilitation of the immune response were studied: the incorporation of peptides in bioadhesive poly(D,L-lactide-coglycolide) (PLGA) microparticles, the use of monoepitopic (HDS) or diepitopic (HDS-GLU) peptide constructs, or the use of mucosal adjuvants. Salivary immunoglobulin A (IgA) responses were not detected after intranasal administration of diepitopic HDS-GLU peptide constructs in alum or after incorporation into PLGA microparticles. However, significant primary and secondary salivary IgA and serum IgG antibody responses to HDS were induced in all rats when cholera holotoxin (CT) or a detoxified mutant Escherichia coli heat-labile enterotoxin (R192G LT) were intranasally administered with HDS peptide constructs in PLGA. Coadministration of LT with HDS resulted in predominantly IgG2a responses in the serum, while coadministration with CT resulted in significant IgG1 and IgG2a responses to HDS. Serum IgG antibody, which was induced to the HDS peptide construct by coadministration with these adjuvants, also bound intact mutans streptococcal GTF in an enzyme-linked immunosorbent assay and inhibited its enzymatic activity. Thus, immune responses which are potentially protective for dental caries can be induced to peptide-based GTF vaccines after mucosal administration if combined with the CT or LT R192G mucosal adjuvant.     

Immunological characteristics of a synthetic peptide associated with a catalytic domain of mutans streptococcal glucosyltransferase.

The immunogenicity of a multiple antigenic peptide construct consisting of four copies of the synthetic 21-mer peptide DANFDSIRVDAVDNVDADLLQ was measured. The composition of this peptide was derived from a sequence in the N-terminal region of mutans streptococcal glucosyltransferases (GTFs) containing an aspartic acid implicated in catalysis. The peptide (CAT) construct was synthesized as a tetramer on a lysine backbone and subcutaneously injected into Sprague-Dawley rats for polyclonal antibody formation or intraperitoneally injected into BALB/c mice, and then spleen cell fused with Sp2/0Ag14 murine myeloma cells for monoclonal antibody formation. The resulting rat antisera and mouse monoclonal antibodies reacted with CAT and with native GTF isozymes from Streptococcus sobrinus and Streptococcus mutans (in enzyme-linked immunosorbent assay and Western blot [immunoblot] analyses). Functional inhibition of the water-insoluble glucan synthetic activity of S. sobrinus GTF-I was demonstrated with an immunoglobulin M anti-CAT monoclonal antibody (> 80% inhibited) and with rat sera (approximately 17% inhibited). The monoclonal antibody preparation also modestly inhibited the water-soluble glucan synthetic activity of an S. mutans GTF mixture. These results suggest that the CAT peptide contains B-cell epitopes that are similar to those of intact mutans streptococcal GTFs and has the potential to elicit antibody that can inhibit GTF function. Thus, sequences within this peptide construct may have value for inclusion in a synthetic dental caries vaccine.     

Antigenicity and immunogenicity of a synthetic peptide derived from a glucan-binding domain of mutans streptococcal glucosyltransferase.

The immunogenicity and antigenicity of a multiply antigenic peptide construct containing four copies of the synthetic peptide TGAQTIKGQKLYFKANGQQVKG were measured in rodents and humans, respectively. The composition of this peptide construct (termed GLU) was derived from a major repeating sequence in the C-terminal region of mutans streptococcal glucosyltransferases that synthesize water-insoluble glucan (GTF-I). The GLU peptide elicited high levels of serum immunoglobulin G antibody to GLU after subcutaneous injection into Sprague-Dawley rats. These antisera also reacted with intact GTF isozymes from Streptococcus sobrinus and Streptococcus mutans (by enzyme-linked immunosorbent assay [ELISA] and Western blot [immunoblot] analyses) and with an 87-kDa glucan-binding protein from S. sobrinus (by Western blot). The synthesis of filter-retained glucan by GTF-Sd of S. sobrinus could be inhibited (30%) by preincubation with anti-GLU rat serum. Splenic and lymph node lymphocytes from rats injected once with S. sobrinus GTF isozymes demonstrated significant proliferation after 5 days of culture with GLU. The GLU peptide reacted with 4 of 29 human parotid saliva samples and 5 of 29 human serum samples (by ELISA). These results suggest that the GLU peptide contains B- and T-cell epitopes that are similar to those of intact mutans streptococcal GTFs and possibly certain other glucan-binding proteins as well. Furthermore, since antibody to this epitope(s) appears to inhibit GTF function, sequences within this peptide construct may have value for inclusion in a synthetic dental caries vaccine.     

The innate immune response to DNA

As a component of all living cells and microbes, it is not surprising that organisms have evolved mechanisms to detect foreign or aberrant DNA and trigger an innate immune response. TLR9 is an endosomal membrane bound receptor that is widely studied and the best understood DNA sensor. However, the existence of TLR9-independent DNA sensing pathways have been recognized for many years. Recently, novel cytosolic DNA sensors have been uncovered that include ZBP1 or DNA-dependent activator of interferon-regulatory factors (DAI) and a DNA sensing inflammasome consisting of the HIN200 protein, absent in melanoma 2 (AIM2). In combination with TLR9, these receptors provide a diverse repertoire of mechanisms to alert the cell to microbial DNA and possibly aberrant host DNA leading to the activation of the innate immune system.     

Pyridine analogs inhibit the glucosyltransferase of Streptococcus mutans.

Soluble glucan synthesis catalyzed by dextransucrase preparations from Streptococcus mutans 6715 were inhibited by pyridoxal-5-phosphate and several other pyridine analogs, including pyridoxine, pyridoxamine, pyridoxamine-5-phosphate, pyridoxal, and 4-pyridoxic acid. Pyridine and pyridine-4-carboxaldehyde were not effective inhibitors of the enzyme. Kinetic analyses suggested that pyridoxal-5-phosphate is a noncompetitive inhibitor of dextransucrase. The inactivation was dependent on time, pyridoxal-5-phosphate concentration, and hydrogen ion concentration. Apparent Ki values were 4.9 mM at pH 7.0 and 4.2 mM at pH 5.5. Dextransucrase activity could be restored by dialysis to remove the inhibitors. Maximum inhibition was observed after a 120-min incubation of the enzyme with pyridoxal-5-phosphate. The pH optima for inhibition by pyridoxal-5-phosphate were 4 and 7. The sucrose-dependent adherence of S. mutans cells to saliva-coated hydroxylapatite beads was also inhibited by pyridoxal-5-phosphate but only marginally by the other pyridine anatogs. In addition, pyridoxal-5-phosphate markedly reduced the rate of acid production by intact S. mutans cells from sucrose or glucose substrates. Another pyridoxal-5-phosphate analog, 2-methyl-5-hydroxypyridine, was also effective in preventing the production of acid by S. mutans from sucrose or glucose. When S. mutans cells were preincubated with pyridoxal-5-phosphate or pyridine analogs, significant reductions in the rate of D-glucose uptake were observed. It is suggested that the inhibition of dextransucrase occurs because of a change iun enzyme conformation which results from the binding of the pyridine derivatives. The results suggest that pyridoxal-5-phosphate or structural analogs may ultimately be useful in reducing the incidence of dental caries.     

Immunogenicity and protective immunity induced by synthetic peptides associated with a catalytic subdomain of mutans group streptococcal glucosyltransferase.

We examined the immunogenicity and induction of protective immunity of two 19-mer sequences (GGY and AND) which overlapped a highly conserved region which has recently been implicated in the enzymatic activity of glucosyltransferases (GTFs) of the mutans group streptococci. These peptides were synthesized as eight-branched constructs on a lysine core. Serum immunoglobulin G (IgG) antibody, induced by subcutaneous (s.c. [salivary gland vicinity]) injection with these peptide constructs, reacted with the inciting antigen, with mutans streptococcal GTFs, and with a 21-mer peptide (CAT) containing an aspartate previously shown to covalently bind sucrose. Several of these antisera also inhibited the ability of Streptococcus sobrinus GTF to synthesize insoluble glucan. Significant levels of salivary IgA antibody were also induced by GGY and AND peptide constructs after s.c. injection. The effect of immunization with the GGY and AND peptide constructs on the cariogenicity of Streptococcus mutans was studied in three experiments by immunization of weanling Sprague-Dawley rats, twice at 7- to 14-day intervals with peptides, S. sobrinus GTF, or phosphate-buffered saline. All rats were then orally infected with S. mutans SJ. After 63-day infection periods, the GGY and AND-injected groups had significant dental caries reductions compared with sham-injected groups in most experiments. These studies support the existence of an additional catalytic subdomain within the sequence defined by the GGY and AND peptides. Furthermore, the epitopes defined in these sequences have significant immunogenicity, can induce immune responses which interfere with GTF-mediated glucan synthesis in vitro, and can protect rats from experimental dental caries.     

Experimental Leishmania major infection suppresses HIV-1 DNA vaccine induced cellular immune response

The AIDS epidemic in the developing world represents a major global crisis and an effective vaccine is imperative. However, many parasites are common in developing countries and can result in a state of chronic immune activation that is polarized towards a Th2 profile and which can potentially impair responses to vaccines or other infectious challenges. In this study we demonstrate that experimental Leishmania major infection of BALB/c mice inhibits responses to a DNA-based HIV-1 gag vaccine. L. major infection in BALB/c results in a polarized Th2 immune response. In this study na?ve BALB/c mice immunized with the HIV-1 gag DNA vaccine mounted a cellular immune response against the vaccine antigen, HIV-1 gag. CD8+ T lymphocytes were able to respond in vitro to HIV-1 gag stimulation and secrete interferon (IFN)-gamma. However, L. major-infected, vaccinated BALB/c mice had a significantly reduced number of IFN-gamma-producing CD8+ T cells following in vitro stimulation with gag antigen. These data suggest that parasitic infection, which results in a Th2 profile, reduces the efficacy of DNA vaccines that are designed to induce antiviral CD8+ T cell responses.     

Immunogenic and protective potential of mutans streptococcal glucosyltransferase peptide constructs selected by major histocompatibility complex class II allele binding

Mutans streptococcal glucosyltransferases (GTF) have been demonstrated to be effective components of dental caries vaccines. We had previously selected peptide subunits of GTF for vaccine development based on putative functional significance and conservation of GTF primary structure among enzyme isoforms. In this study, 20 20-mer linear GTF peptides were synthesized, 17 identified on the basis of the highest potential major histocompatibility complex (MHC) class II-binding activity using computer-generated algorithms (Epimatrix and ProPred) and 3 with previously demonstrated functional significance. The immunoreactivities of these peptides were explored with rodent systems. Sera from GTF-immunized rats, assessed for binding to linear peptides by enzyme-linked immunosorbent assay, demonstrated immunoglobulin G antibody reactivity with peptides 6 and 11 and a T-cell proliferation response to peptides 6, 9, 11, and 16. Multiple antigenic peptide (MAP) constructs were synthesized from promising linear sequences. Rats that were immunized with MAP 7, 11, or 16, respectively, responded well to the immunizing MAP. Most importantly, a robust immune response (antibody and T-cell proliferation) was observed to native GTF following MAP 11 (amino acids 847 to 866; VVINNDKFVSWGITDFEM) immunization. This response inhibited GTF enzyme function. Two dental caries pathogenesis experiments were performed wherein rats were immunized with MAP constructs 11, 16, and/or 11 plus 16, followed by infection with cariogenic Streptococcus sobrinus. In both experiments cariogenic bacterial recoveries were reduced relative to total streptococci in the MAP 11- and MAP 11 plus 16-immunized groups, and the extent of dental caries was also significantly reduced in these groups. Thus, we have identified a peptide with projected avid MHC-binding activity that elicited immunoreactivity with native GTF and demonstrated protection against dental caries infection after immunization, implying that this peptide may be important in a subunit dental caries vaccine.     

Interaction of glucosyltransferase with the cell surface of Streptococcus mutans

The partially purified glucosyltransferase (GTF) fraction synthesizing primarily water-insoluble glucans, GTF-A, and the homogeneous fraction synthesizing water-soluble glucans, GTF-B, were utilized to assess the binding of GTF activity to the cell surface of Streptococcus mutans GS-5. Growth of the cells in either Todd-Hewitt broth or a chemically defined medium did not appear to affect the ability of the cells to bind either enzyme fraction. Heat inactivation of the cells did not singificantly reduce the interaction of the enzymes with the cells. Cell surface glucan molecules appear to be involved in GTF binding to the cells because: (i) dextranase or alpha-1,3-glucanase treatment of the cells markedly reduced enzyme binding; (ii) the inclusion of soluble dextrans in the binding assays reduced both GTF-A and GTF-B binding to the cells; and (iii) pretreatment of the cells or the GTF-B fraction with soluble dextrans before binding significantly reduced enzyme binding to the cells. In addition, enzyme binding appears to require a cell surface protein component because Pronase, but not trypsin, treatment of cells reduced enzyme binding. Furthermore, the removal of a portion of the cell surface GTF-glucan complex with 3 N NaCl appears to provide additional binding sites for the enzymes. These results are interpreted in terms of the mechanism of the conversion of extracellular GTF to the cell-associated form.     

Immunization of rats with synthetic peptide constructs from the glucan-binding or catalytic region of mutans streptococcal glucosyltransferase protects against dental caries.

Previously, we have described peptide constructs from two regions of glucosyltransferase (GTF) of mutans streptococci. A putative catalytic site in the amino-terminal half of the molecule and a repeated glucan-binding site in the carboxyl-terminal half of GTF were the regions upon which sequences were based. The present study explored the effects of immunization with these peptide constructs (called CAT or GLU) and with streptococcal GTFs from Streptococcus sobrinus and S. mutans on immunological, microbiological, and disease parameters. Groups of immunized Sprague-Dawley rats were infected with either 10(8) S. sobrinus 6715 or 10(8) S. mutans SJ32 organisms. Serum immunoglobulin G antibody levels, determined by enzyme-linked immunosorbent assay, to the respective peptide constructs and to the appropriate streptococcal GTF were significantly increased (after immunization) prior to infection and at the end of the experiment. Also, serum antibody from CAT-, GLU-, and S. sobrinus GTF-immunized rats inhibited S. sobrinus GTF-mediated insoluble glucan synthesis (all) and S. mutans GTF-mediated soluble glucan synthesis (all except anti-GLU) from sucrose. Immunization with the CAT or GLU peptide construct resulted in significantly reduced smooth surface and sulcal caries after infection with S. sobrinus. Sulcal dental caries after infection with S. mutans SJ32 were also significantly reduced in CAT- and GLU-immunized rats. Thus, immunization with peptides whose sequences are based on putative functional domains of mutans streptococcal GTF are protective toward a cariogenic S. sobrinus or S. mutans infection.     

The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection

The Drosophila host defense against gram-negative bacteria is mediated by the Imd pathway upon sensing of peptidoglycan by the peptidoglycan recognition protein (PGRP)-LC. Here we report a functional analysis of PGRP-LB, a catalytic member of the PGRP family. We show that PGRP-LB is a secreted protein regulated by the Imd pathway. Biochemical studies demonstrate that PGRP-LB is an amidase that specifically degrades gram-negative bacteria peptidoglycan. In agreement with its amidase activity, PGRP-LB downregulates the Imd pathway. Hence, activation of PGRP-LB by the Imd pathway provides a negative feedback regulation to tightly adjust immune activation to infection. Our study also reveals that PGRP-LB controls the immune reactivity of flies to the presence of ingested bacteria in the gut. Our work highlights the key role of PGRPs that encode both sensors and scavengers of peptidoglycan, which modulate the level of the host immune response to the presence of infectious microorganisms.     

The role of iron in the immune response to bacterial infection

My laboratory has been interested for some time in the influence of iron, a nutrient that is essential for both microbial pathogens and their mammalian hosts, on the course of infectious disease. Our studies indicate that alterations in the expression of host molecules that sequester or transport iron can have direct effects on pathogen growth and can also have an impact on the ability to mount normal immune responses. We have elucidated the mechanistic basis for some of these observations, and have started to apply our findings in strategies to control abnormalities of inflammation and iron metabolism. I will review here what we have learned about the interactions between iron and immunity and discuss the implications of the information that we have acquired.     

Inhibition of Streptococcus mutans glucosyltransferase activity by antiserum to a subsequence peptide.

An antigenic 15-amino-acid peptide sequence (gtfB.1) from the glucosyltransferase B enzyme of the cariogenic bacterium Streptococcus mutans GS-5 was identified previously from the genetic fusion of this sequence to the B subunit of cholera toxin. The resulting chimeric protein was used to raise antiserum in rabbits. This antiserum was shown to recognize the native glucosyltransferase enzyme and to inhibit its activity. The antiserum inhibited the synthesis of water-soluble glucan by approximately 40% and the synthesis of water-insoluble glucan by greater than 90%. The antiserum was shown to partially inhibit fructosyltransferase activity as well. The ability of this antipeptide antiserum to inhibit several enzymes from S. mutans suggests that these enzymes share an epitope related to enzymatic activity.     

Regulation of the immune response to bacterial lipopolysaccharide by adherent cells.

Immune response to bacterial lipopolysaccharide is usually short lived, but it often reappears without additional stimulus in a cyclic fashion. Activated adherent cells, presumably macrophages, were found to have a role in the reduction of the immune response to Escherichia coli O127 lipopolysaccharide. The suppressive activity of the adherent cells was abrogated before renewal of the responsiveness.     

Dengue virus-induced cytotoxic factor suppresses immune response of mice to sheep erythrocytes.

The present study was undertaken to investigate if the suppressed cell-mediated immune responses observed in dengue type 2 virus (DV)-infected mice could be due to the cytotoxic factor (CF) produced in the spleens of DV-infected mice. We have observed that CF given intravenously (i.v.) kills splenic cells and reduces the total cells in the spleen. Mice treated with CF have a significantly depressed immune response to sheep erythrocytes, viz. delayed-type-hypersensitivity as measured by footpad swelling reaction at 24 hr; Jerne's antibody plaque-forming cells in the spleen; and migration inhibition of spleen cells in presence of antigen. These findings are similar to those seen earlier in DV-infected mice.     

Meta-tyrosine modulates the immune response induced by bacterial endotoxins

Sepsis is characterized by an early pro-inflammatory phase followed by compensatory anti-inflammatory mechanisms that lead to a late generalized immunosuppression, period where most deaths occur. Immunotherapy approaches to recover the immunocompetence in sepsis are similar to those used in cancer. Meta-tyrosine (m-Tyr) is a product of oxidative stress present in circulation during the sepsis and cancer-associated pro-inflammatory stages. In this work, considering its potential participation in pro-inflammatory processes, we evaluate the effect of m-Tyr during LPS induced immunosuppression phase in a murine model. In addition, we examine the effect of m-Tyr in a vaccination strategy using a weakly immunogenic tumor model. Our results showed that m-Tyr could prevent the establishment of immunosuppression and rescue the host from an installed immunosuppression induced by LPS. These effects were parallel to the ability of m-Tyr to improve the pro-inflammatory effects induced by LPS and inhibit the anti-inflammatory action of dexamethasone. Also, m-Tyr treatment prevents both the reduction of splenic lymphocytes and the increase of the expression of programmed death ligand-1 in splenic myeloid cells associated with immunosuppression. Besides, treatment with m-Tyr increased the protective effect of an anti-tumor vaccine, suggesting that m-Tyr could improve the immune response. In summary, we suggest that m-Tyr can modulate critical immunological indicators through the inflammatory context, which could improve the management of diseases, such as sepsis and cancer, in which immunosuppression is a significant clinical problem.     

Two unlinked genetic loci interact to control the human immune response to type III group B streptococcal antigen

Serum samples were collected from 30 healthy adult Caucasian volunteers before and after immunization with native type III polysaccharide of group B streptococcus. Serum antibody to this polysaccharide was measured and sera were typed for several Gm and Km(1) allotypes. A significant interactive effect of Gm(23) and Km(1) was found on immune responsiveness to native type III group B streptococcus polysaccharide antigen.     

Monoclonal antibody-mediated modulation of the humoral immune response against mucosally applied Streptococcus mutans

Systemic immunization with antigen coupled to monoclonal antibody (MAb) has been used by several investigators to increase the number of MAb-producing hybridomas against an antigen and to elicit antibodies specific for poorly immunogenic epitopes. This strategy has implications for vaccine design in that protective immunity is not necessarily directed at immunodominant epitopes of pathogens and may be improved by deliberately shifting the immune response toward subdominant epitopes. To our knowledge, no studies to date have addressed the potential for immunomodulatory activity mediated by MAbs bound to mucosally applied antigen. To test whether administration of an exogenous MAb directed against a streptococcal surface protein could influence the humoral immune response, BALB/c mice were immunized orally by gastric intubation or intranasally with Streptococcus mutans alone or S. mutans complexed with a MAb directed against the major surface protein P1. Significant changes in the subclass distribution, as well as the specificity, of anti-P1 serum immunoglobulin G antibodies were demonstrated in groups of mice which received S. mutans coated with the anti-P1 MAb versus those which received S. mutans alone. Alterations in the humoral immune response were dependent on the amount of anti-P1 MAb used to coat the bacteria. In addition, differences in the anti-P1 immune responses were observed between groups of mice immunized via oral versus intranasal routes. In summary, an exogenous MAb complexed with a streptococcal antigen prior to mucosal immunization can influence the immunoglobulin isotype and specificity of the host humoral immune response against the antigen.