PURPOSE: There is a need to broaden protective coverage of M protein–based vaccines against group A streptococci (GAS) because coverage of the current 30-valent M protein vaccine does not extend to all emm types. An additional GAS antigen and virulence factor that could potentially extend vaccine coverage is M-related protein (Mrp). Previous work indicated that there are three structurally related families of Mrp (MrpI, MrpII, and MrpIII) and peptides of all three elicited bactericidal antibodies against multiple emm types. The purpose of this study was to determine if a recombinant form containing Mrp from the three families would evoke bactericidal antiserum and to determine if this antiserum could enhance the effectiveness of antisera to the 30-valent M protein vaccine. MATERIALS AND METHODS: A trivalent recombinant Mrp (trMrp) protein containing N-terminal fragments from the three families (trMrp) was constructed, purified and used to immunize rabbits. Anti-trMrp sera contained high titers of antibodies against the trMrp immunogen and recombinant forms representing MrpI, MrpII, and MrpIII. RESULTS: The antisera opsonized emm types of GAS representing each Mrp family and also opsonized emm types not covered by the 30-valent M protein–based vaccine. Importantly, a combination of trMrp and 30-valent M protein antiserum resulted in higher levels of opsonization of GAS than either antiserum alone. CONCLUSION: These findings suggest that trMrp may be an effective addition to future constructs of GAS vaccines.
Antibodies ; Humans ; Immune Sera ; Peptides ; Rabbits ; Staphylococcal Protein A* ; Streptococcal Vaccines* ; Streptococcus pyogenes ; Vaccines ; Virulence ; Virulence Factors

BACKGROUND: Erythromycin is currently recommended as an alternative antibiotic for treatment of streptococcal infections in patients allergic to penicillins. Less than 5% of the group A streptococci are known as resistant to erythromycin but the resistance pattern differs among time and region. The purpose of this study was to determine the antimicrobial susceptibility of beta-emolytic streptococcal strains isolated during 1999 in Wonju. METHODS: A total of 107 beta-emolytic streptococci were isolates from the Wonju Christian Hospital during 1999. The susceptibility to penicillin, erythromycin, tetracycline, vancomycin, ceftriaxone, chloramphenicol, and clindamycin was tested with agar dilution method. RESULTS: No beta-emolytic streptococci strain was resistant to penicillin, ceftriaxone and vancomycin. Among beta-emolytic streptococci strains, 20-1%, 18-0% and 14-7% were resistant to tetracycline, erythromycin and clindamycin, respectively. CONCLUSIONS: It appears prudent that active surveillance of the beta-emolytic streptococci for antibiotic resistance be implemented since there are no currently effective vaccines or other methods for controlling the spread of infections due to these virulent organisms.
Agar ; Ceftriaxone ; Chloramphenicol ; Clindamycin ; Drug Resistance, Microbial ; Erythromycin ; Gangwon-do ; Humans ; Penicillins ; Streptococcal Infections ; Tetracycline ; Vaccines ; Vancomycin

BACKGROUND: Erythromycin is currently recommended as an alternative antibiotic for treatment of streptococcal infections in patients allergic to penicillins. Less than 5% of the group A streptococci are known as resistant to erythromycin but the resistance pattern differs among time and region. The purpose of this study was to determine the antimicrobial susceptibility of beta-emolytic streptococcal strains isolated during 1999 in Wonju. METHODS: A total of 107 beta-emolytic streptococci were isolates from the Wonju Christian Hospital during 1999. The susceptibility to penicillin, erythromycin, tetracycline, vancomycin, ceftriaxone, chloramphenicol, and clindamycin was tested with agar dilution method. RESULTS: No beta-emolytic streptococci strain was resistant to penicillin, ceftriaxone and vancomycin. Among beta-emolytic streptococci strains, 20-1%, 18-0% and 14-7% were resistant to tetracycline, erythromycin and clindamycin, respectively. CONCLUSIONS: It appears prudent that active surveillance of the beta-emolytic streptococci for antibiotic resistance be implemented since there are no currently effective vaccines or other methods for controlling the spread of infections due to these virulent organisms.
Agar ; Ceftriaxone ; Chloramphenicol ; Clindamycin ; Drug Resistance, Microbial ; Erythromycin ; Gangwon-do ; Humans ; Penicillins ; Streptococcal Infections ; Tetracycline ; Vaccines ; Vancomycin

OBJECTIVETo investigate the antigenicity of the peptide vaccine HDS from Streptococcus mutans glucosyltransferase and its ability to induce protective immune responses in an experimental rat model of dental caries.

METHODSArtificial antigen HDS-KLH, peptide HDS, glycosyltransferase were injected to immunize rats. Measurement of the specific anti-HDS, GTF IgG or IgA concentration in saliva and serum were undertaken by ELISA among the experimental groups. Gnotobiotic rat model was developed when challenged S. mutans and a caries promoting diet. The jaws of the rats were selected and dyed. The Keyes caries score for each jaw were counted.

RESULTSThe level of serum and salivary specific anti-HDS IgG and IgA in the group immunized by HDS-KLH was significantly higher than that in control group (P < 0.05). The Keyes caries score of GTF, HDS and HDS-KLH immunized group were significantly lower than that of control group, especially lower in smooth tooth surface.

CONCLUSIONArtificial antigen HDS-KLH could induce immune response. As a peptide vaccine, HDS-KLH could reduce the caries incidence in experimental rat model.

Animals ; Dental Caries ; prevention & control ; Glucosyltransferases ; genetics ; immunology ; Male ; Peptides ; immunology ; Rats ; Rats, Sprague-Dawley ; Streptococcal Vaccines ; immunology ; Streptococcus mutans ; enzymology ; genetics ; immunology

OBJECTIVETo investigate the effect of specific anti-streptococcus mutans IgY against streptococcus mutans on dental caries development in rats.

METHODS35 wistar rats were divided into 5 groups: group A received IgY gargle; group B received IgY lyophilized powder; group C received sterilized water as control; group D and E received egg yolk food with or without specific IgY individually. They were all fed with caries-inducing diet 2000#. The number of caries scores was counted by the procedure of Keyes'.

RESULTSThere was a significant lower mean of caries scores in groups treated with IgY lyophilized powder and gargle. By treating with egg-yolk food contained specific IgY, the mean of caries scores decreased comparing with no treatment group.

CONCLUSIONLocal passive immunization with specific anti-streptococcus mutans IgY may be an effective way to prevent the development of dental caries.

Animals ; Antibodies, Bacterial ; administration & dosage ; Dental Caries ; prevention & control ; Female ; Immunization, Passive ; Immunoglobulins ; immunology ; Male ; Rats ; Rats, Wistar ; Streptococcal Vaccines ; immunology ; Streptococcus mutans ; immunology

OBJECTIVEThe purpose of this study is to examine the levels of salivary SIgA and serum IgG induced by pcDNA3-pac and pcDNA3-gtfB immunization, so as to testify the antigenity of the two gene vaccines.

METHODS36 28-day-old Wistar rats were divided into 6 groups, among which 3 experimental groups were vaccinated with pcDNA3-pac, pcDNA3-gtfB or pcDNA3-pac combined with pcDNA3-gtfB, respectively, one positive control was vaccinated with inactive whole cell of S. mutans JBP and other two negative controls were injected with the vector pcDNA3 or PBS buffer, respectively. All vaccines and materials were delivered with 100 micrograms by submandibular gland injection for 3 times. Then the restricted bacterial model of rat was constructed. Following that all rats were fed with cariogenic diet Keyes 2000 for 3 months, saliva and serum samples were collected to assay SIgA or IgG levels by ELASA.

RESULTSThe salivary S-IgA levels both in pcDNA3-pac combined with pcDNA3-gtfB group and inactive S. mutans cell group were higher than others (P < 0.01). In groups of pcDNA3 and PBS buffer, they were lowest (P < 0.01). The serum IgG levels in the three experimental groups and positive control were higher than that in negative control (P < 0.05). It was important that salivary SIgA in groups of gene vaccine and inactive S. mutans vaccination reached its peak at the 11th week after the first inoculation and kept until the end of the study.

CONCLUSIONBoth pcDNA3-pac and pcDNA3-gtfB can express immunogenic protein and induce immune responses of mucosal and humoral immune system in gnobobiotic rats. It is also indicated that the joint gene vaccines immunization is an optimal choice for anticaries strategy.

Animals ; Antibodies, Bacterial ; analysis ; blood ; Dental Caries ; prevention & control ; Female ; Glucosyltransferases ; immunology ; Immunoglobulin A, Secretory ; immunology ; Immunoglobulin G ; analysis ; Male ; Membrane Proteins ; immunology ; Random Allocation ; Rats ; Rats, Wistar ; Saliva ; immunology ; Streptococcal Vaccines ; immunology ; Streptococcus mutans ; immunology ; Vaccination ; Vaccines, DNA ; immunology

OBJECTIVEGene vaccine security is of concern because of the possibility of insertion mutagenesis resulting in inactivation of tumor suppressor or activation of oncogene. The purpose of this study was to examine the potential of anti-caries DNA vaccine pcDNA3-gtfB integrating into the host cell genome.

METHODSAnti-caries DNA vaccine pcDNA3-gtfB was constructed by the previous study. The gtfB gene(904-4,578 bp, genebank M17361) was cloned from Streptococcus mutans GS-5. 36 Wistar rats were divided into 2 groups: submandibular gland-targeted injection(SGT) group and control group. Rats in SGT group were injected with 100 micrograms of plasmid pcDNA3-gtfB, rats in control group with PBS solution. Genomes from submandibular gland, kidney, heart, liver, lung, and brain tissues were isolated later in 12 weeks. Genomes from different tissues were purified by low-melting agarose electrophoresis. Using the purified genomes as template, plasmid integration were examined by PCR(upper primer: 5'-ATATGGTACCATGACCGAAGCGACATCTAAGCAAGA-3', lower primer: 5'-ACTACTCGAGTTAGAACCATTGACCCTG AGCATTGC-3'). The sensitivity level of PCR was determined by adding gradient plasmid copies into genomes in control group.

RESULTSThe examination of 6 tissues failed in revealing any evidence of integration at the sensitivity level that could detect 1 copy integration in 10,000 nuclei.

CONCLUSIONThe potential frequency of plasmid pcDNA3-gtfB integration into host cell genome would not exceed that of the spontaneous mutation. It was indicated that pcDNA3-gtfB was genetically safe as a promising anti-carious DNA vaccine.

Animals ; Antibodies, Antinuclear ; genetics ; immunology ; Cloning, Molecular ; Dental Caries ; prevention & control ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Glucosyltransferases ; genetics ; Plasmids ; genetics ; immunology ; Rats ; Rats, Wistar ; Recombination, Genetic ; Streptococcal Vaccines ; genetics ; Streptococcus mutans ; genetics ; immunology ; Vaccines, DNA ; adverse effects ; genetics ; immunology

OBJECTIVEProtein of Streptococcus mutans is considered as one of the virulence factors due to its ability to mediate the initial attachment of Streptococcus mutans to tooth surface. In this study, an anticaries DNA vaccine pCIA-P was used to immunize rats. The expression of PAc in different tissues in vivo, specific immune response and protection effects against dental caries were observed.

METHODSPlasmid pCIA-P was injected into rats by two different routs: intramuscular injection (i.m.) and targeted salivary gland immunization (TSG). Immunohistochemistry technique was used to detect the expression of PAc. Gnotobiotic rats were vaccinated with pCIA-P by three different approaches: TSG, intramuscular injection and buccal mucosal injection (i.o.). The specific immune responses were evaluated by ELISA and their anticaries effects were evaluated by Keyes caries scores.

RESULTSPAc was expressed in the sarcoplasm and sarcolemma of muscle fibers and submandibular glands, especially strongly positive in duct regions. The levels of serum specific anti-PAc IgG and salivary specific anti-PAc IgA in TSG immunization and buccal mucosal immunization group were significantly higher than those of other groups. The Keyes caries scores of those two groups were significantly lower than those of other groups.

CONCLUSIONThe plasmid pCIA-P could provoke specific immune responses as a novel immunogen. Mucosal immunization with pCIA-P appears to be an effective genetic immunization method against dental caries.

Animals ; Antibodies, Bacterial ; blood ; Bacterial Proteins ; genetics ; immunology ; Dental Caries ; prevention & control ; Germ-Free Life ; Immunization ; Male ; Membrane Glycoproteins ; Rats ; Rats, Wistar ; Streptococcal Vaccines ; immunology ; Streptococcus mutans ; immunology ; Vaccines, DNA ; immunology

To analyze the immunogenicity and protective ability of recombinant IgG-binding protein (EAG) of Streptococcus equi subspecies equi and to evaluate its value when used as equine vaccine antigen, EAG gene was amplified by PCR and inserted into pET-28a vector. The EAG recombinant proteins were expressed and purified to immune mice. The serum antibody and challenge protection were tested. The purified recombinant protein of EAG was 26 kDa, and the protein reacted specifically with positive serum of Streptococcus equi subspecies equi. The mice antibody level for EAG immunization group was 1∶8 100. The immunological protection result showed that the protection rate of the EAG recombinant protein was 90%. The results suggested that the EAG protein has good immunogenicity and immunological protection, and it can effectively increase the humoral immune response and immunological protection of mice.
Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; immunology ; Bacterial Proteins ; immunology ; Bacterial Vaccines ; immunology ; Immunity, Humoral ; Immunoglobulin G ; blood ; Mice ; Polymerase Chain Reaction ; Protein Binding ; Recombinant Proteins ; immunology ; Streptococcal Infections ; prevention & control ; Streptococcus equi ; Vaccination

OBJECTIVETo construct a fusion anti-caries DNA vaccine pGLUA-P carrying GLU fragment from gtfB gene of Streptococcus mutans GS-5 and A-P fragment including the A region and P region of PAc protein from a DNA anti-caries vaccine pCIA-P, and to investigate its expression in prokaryotic and eukaryotic cells.

METHODSThe sequence of GLU fragment in pGLU plasmid was testified by DNA sequencing. The fusion anti-caries DNA vaccine was constructed by ligating A-P fragment from pCIA-P to pGLU. The expression of GLUA-P fusion protein in E. coli BL21 (DE3) was induced by IPTG and checked by SDS-PAGE electrophoresis. pGLUA-P was transfected in vitro to cultured rat primary muscle cells by cation liposome Dosper, and immunohistochemical method was used to test the expression of GLUA-P fusion protein in cells.

RESULTSGLU sequence was identical with relative sequence of GTF-I (GS-5 strain) in Gene Bank. Recombinant eukaryotic expression plasmid pGLUA-P was confirmed to have both GLU and A-P fragment. After pGLUA-P was transferred into E. coli (DE3), it could express a new 115 000 protein by the induce of IPTG. Specific brown products could be found in the cytoplasm of cultured rat primary muscle cells transfected by pGLUA-P.

CONCLUSIONSFusion anti-caries DNA vaccine pGLUA-P is successfully constructed and confirmed by sequencing and enzymes digestion. Fusion GLUA-P protein can be correctly expressed in prokaryotic and eukaryotic cells.

Animals ; Animals, Newborn ; Bacterial Proteins ; genetics ; metabolism ; Cells, Cultured ; Cloning, Molecular ; Dental Caries ; prevention & control ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Gene Expression ; Glucosyltransferases ; genetics ; metabolism ; Membrane Glycoproteins ; Muscle, Skeletal ; cytology ; metabolism ; Plasmids ; genetics ; Rats ; Rats, Wistar ; Recombinant Fusion Proteins ; genetics ; metabolism ; Streptococcal Vaccines ; genetics ; immunology ; therapeutic use ; Streptococcus mutans ; genetics ; immunology ; Transfection ; Vaccines, DNA ; genetics ; therapeutic use