Trivalent M-related protein as a component of next generation group A streptococcal vaccines

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.

Immunogenicity and prediction of epitopic region of antigen Ag I/II and glucosyltransferase from Streptococcus mutans / 华中科技大学学报（医学）（英德文版）

The levels of Streptococcus (S.) mutans infections in saliva were evaluated and a comparison for specific antibody levels among children with different levels of S. mutans infection was made. The promising epitopic regions of antigen AgI/II (PAc) and glucosyltransferase (GTF) for potential vaccine targets related to S. mutans adherence were screened. A total of 94 children aged 3-4 years were randomly selected, including 53 caries-negative and 41 caries-positive children. The values of S. mutans and those of salivary total secretory immunoglobulin A (sIgA), anti-PAc and anti-Glucan binding domain (anti-GLU) were compared to determine the correlation among them. It was found the level of s-IgA against specific antigens did not increase with increasing severity of S. mutans infection, and the complete amino acid sequence of PAc and GTFB was analyzed using the DNAStar Protean system for developing specific anti-caries vaccines related to S. mutans adherence. A significantly positive correlation between the amount of S. mutans and children decayed, missing, and filled teeth index was observed. No significant difference was detected in specific sIgA against PAc or GLU between any two groups. No significant correlation was found between such specific sIgA and caries index. A total of 16 peptides from PAc as well as 13 peptides from GTFB were chosen for further investigation. S. mutans colonization contributed to early children caries as an important etiological factor. The level of sIgA against specific antigens did not increase with increasing severity of S. mutans infection in children. The epitopes of PAc and GTF have been screened to develop the peptide-based or protein-based anti-caries vaccines.

Febre reumática – patogênese e vacina / Rheumatic fever – pathogenesis and vaccine

Há um amplo espectro de doenças causadas por estreptococos do grupo A (GAS), e estas são consideradas um problema de saúde pública em países em desenvolvimento, com aproximadamente 600 milhões de casos/ano. As infecções causadas por GAS podem ocasionar doenças invasivas como faringite e pioderma com seqüelas auto-imunes graves como a febre reumática (FR) e glomerulonefrite. A FR acomete principalmente crianças e jovens, inicia com poliartrite na maioria dos casos. Tem como sequelas principais a coreia de Sydenham e a doença reumática cardíaca (DRC), considerada a forma mais grave da doença e caracterizada por lesões cardíacas valvares progressivas e permanentes, que necessitam de cirurgias cardíacas para correção valvular, com alto custo para os Sistemas de Saúde, no mundo. A presente revisão descreve os principais mecanismos desencadeadores das lesões reumáticas no coração e o desenvolvimento da vacina contra o Streptococcus pyogenes para prevenção das principais sequelas decorrentes das streptococcias.

Group A streptococci (GAS) infections are considered a public health problem in developing countries, with about 600 million cases per year and are responsible for an wide spectrum of diseases, mainly invasive diseases as pharyngitis and pyoderma that lead to rheumatic fever (RF) and glomerulonephritis autoimmune sequelae. RF affects children and young adults, and presents differentmanifestations such as rheumatic heart disease (RHD), Sydenham chorea, erythema marginatum, subcutaneousnodules. RHD is considered the most serious complications leading to cardiac valvular lesions characterized byprogressive and permanent heart-valve damage, which entails high cost to the Healthy System around the world. In the present review we described the mechanisms that lead to rheumatic heart lesions and the development of avaccine against Streptococcus pyogenes.

[Effects of beta-glucan on the growth, survival, and the efficacy of Anti-streptococcus iniae vaccine in rainbow trout [onchorhynchus mykiss]]

Glucans are complex polysaccharide components of yeast and fungal cell walls. This compounds can stimulate the fish immune system. The present study investigated the effects of Beta glucan[Macrogard] on growth, survival and some immunological parameters of rainbow trout [10 mg] and also on the efficiency of Streptococcus vaccine. Treatments of macrogard [1 g/kg food/day for 42 days], macrogard [macrogard 1 g/kg diet] with 15 minutes bathin vaccine, vaccine bath without macrogard and control were examined in a 42 daysperiod. In all treatments the fish growth [weight] showed a significant increase [p<0.05] compared to the control, while there was no significant difference among the treatments. The lyzosyme level of the blood serum showed a significant difference between control and the other treatments, the highest level was observed in the macrogaurd with vaccine treatment [2.7 micro g/ml] and the lowest level was observed in the control[0.5 micro g/ml]. Leukocytes count also had a significant difference between the control [3.3%Nuetrophil] and other treatments [6.6% Nuetrophil] [p<0.05]. The result of survival of fish challenged with Streptococcus iniae via bath route was 55%, while those for vaccine treatment, macrogard plus vaccine, macrogard every day and macrogard every 10 days interval were 75%, 86%, 60 and and 55%, respectively. The results of this study showed that supplement of macrogard has positive effect on growth and non-specific immune responses of rainbow trout and can enhance efficacy of anti-streptococcus iniae vaccine in this fish

Profilaxis de sepsis neonatal por Streptococcus Agalactiae (grupo B) basada en vacunas: revisión de la literatura / Prophylaxis of early neonatal sepsis Streptococcus Agalactiae (group B) based on vaccines: literature review

El Streptococcus agalactiae o grupo B (SGB), es el principal agente de sepsis neonatal precoz. A pesar de los intentos de prevención de esta infección, aún no se logra la efectividad esperada. Es por esto que se ha intentado desarrollar una vacuna que pueda prevenir la mayoría de las patologías que esta bacteria produce, incluyendo la sepsis neonatal precoz y tardía. De esta manera se evitarían las limitaciones actuales de la profilaxis antibiótica. Los intentos de crear una vacuna han incluido la utilización de polisacáridos del SGB tanto puros como asociados a proteínas como el toxoide tetánico. También, se han usado proteínas específicas de la cápsula que tienen potencial efectividad como factores inmunogénicos. Las vacunas conjugadas son las más estudiadas en la actualidad, habiendo completado estudios clínicos en fase II, tanto en adultos sanos como en embarazadas. Al ser la sepsis neonatal una complicación grave aún no controlada óptimamente, la creación de una vacuna contra este patógeno sería de gran impacto en salud pública. Se presentan los diferentes tipos de vacunas desarrolladas y el estado de avance en el que se encuentran.

Streptococcus agalactiae or group B, is the mayor causing agent of early onset neonatal sepsis. Although mayor prevention strategies have been made, the expected effectiveness hasn't been achieved. That's why efforts have been made to develop a vaccine that can prevent most of the diseases these bacteria can produce, including early and late onset neonatal sepsis. These way, actual antibiotic prophylaxis limitations can be avoided. Attempts include the utilization of Streptococcus group B polysaccharides in their pure state or combined with proteins as tetanic toxoid. Specific capsule proteins have been used also because of their potential effectiveness as inmunogenic factors. Overall vaccines conjugated ones are the most studied, having completed phase II clinical trials in healthy adults and pregnant women. Neonatal sepsis is a severe complication that has not been controlled yet, so the creation of a vaccine against this pathogen would be of great impact in public health. We introduce now the different developed vaccines and their state of progress.

Reflexiones sobre la prevención de la sepsis neonatal debida a Streptococcus agalactieae (SGB) / Guidelines for the prevention of perinatal group B streptococcal sepsis

Considering the incidence of neonatal sepsis we can observe that group B streptococcus emerged as the predominant pathogen. Studies of prevention of neonatal infection have focused on gropu B streptococcus because of its greater prevalence.

Vacunas para la prevención de enfermedades por streptococcus pneumoniae (S. pn) / Vaccines for the prevention of diseases by streptococcus pneumoniae (S. pn)

Las infecciones causadas por S.pn. son un importante problema de salud pública en la infancia en todo el mundo. Es la principal causa de infecciones respiratorias altas (otitis media, sinusitis), bajas (neumonía), bacteremia oculta y meningitis agudas. Adicionalmente, el problema de la creciente resistencia a antimicrobianos ha puesto una variable más al complejo escenario clínico. El estudio de la epidemiología de las infecciones por streptococcus pneumoniae resulta particularmente importante para conocer el real impacto de medidas de prevención a través de medidas sociales, uso de terapia antimicrobiana empírica y prevención a través de vacunas. En estos aspectos resultan particularmente importantes dos iniciativas en desarrollo: la vigilancia latinoamericana de los serotipos mas prevalentes en la región (Sistema Regional de Vigilancia para Vacunas, SIREVA)(8,9) y los estudios locales del Centro para Desarrollo de Vacunas (CVD-Chile) dirigido por la Dra. Rosanna Lagos Z. (1,2,3).

Production of monoclonal antibodies against Streptococcus mutans antigens

Several studies have been conducted in the last decades aiming to obtain an anti-caries vaccine, however some studies have demonstrated cross reactivity between Streptococcus mutans surface antigens and the human cardiac tissue. In this work, the reactivity of five anti-Streptococcus mutans monoclonal antibodies (MoAb) (24A, 56G, C8, E8 and F6) was tested against oral streptococci, cardiac antigens and skeletal and cardiac myosins, aiming to evaluate the specificity of these MoAb. The hybrid producers of immunoglobulins of the IgG2b class were cloned by limit dilution and expanded in vivo. MoAb were tested by ELISA. The hybrid 24A reacted with S. mutans CCT 1910, S. salivarius CCT 0365 and S. pyogenes T23. No reactivity difference was observed among the tested species. Cross reactivity with heart and cardiac myosin was not confirmed and only reaction with myosin of skeletal muscle was observed (p = 0.0381). The hybrid 56G reacted with all the tested microorganisms and there was statistically significant difference between S. mutans and S. pyogenes T23 (p < 0.001). This hybrid also reacted with myosin of skeletal muscle (p = 0.0095). C8, E8 and F6 presented low reactivity against oral streptococci strains and no reactivity against cardiac antigens. The data of this study showed that the 24A and 56G anti-S. mutans MoAb presented reactivity with S. pyogenes and S. salivarius, reinforcing the occurrence of common antigens between these species. The tested MoAb presented low cross-reactivity with myosin of skeletal muscle, but anti-heart activity could not be confirmed.

Diversos estudos foram realizados nas últimas décadas com o intuito de se obter uma vacina anticárie dentária, mas alguns trabalhos têm demonstrado reatividade cruzada entre antígenos de superfície de Streptococcus mutans e tecido cardíaco humano. Neste trabalho, foi testada a reatividade de cinco anticorpos monoclonais (AcMo) anti-Streptococcus mutans (24A, 56G, C8, E8 e F6) contra estreptococos orais, antígenos cardíacos e miosinas esquelética e cardíaca, no intuito de avaliar a especificidade desses AcMo. Os híbridos produtores de imunoglobulinas da classe IgG2b foram clonados por diluição limite e expandidos in vivo. Os AcMo foram testados por ELISA. O híbrido 24A reagiu com S. mutans CCT 1910, S. salivarius CCT 0365 e S. pyogenes T23. Nenhuma diferença de reatividade foi detectada entre as espécies analisadas. Reatividade cruzada com coração e miosina cardíaca não foi confirmada, existindo somente reação com miosina de músculo esquelético (p = 0,0381). O híbrido 56G reagiu com todos os microrganismos testados e houve diferença estatisticamente significante entre S. mutans e S. pyogenes T23 (p < 0,001). Este híbrido também reagiu com miosina de músculo esquelético (p = 0,0095). C8, E8 e F6 apresentaram baixa reatividade contra cepas de estreptococos orais e nenhuma reatividade com antígenos cardíacos. Os dados deste trabalho demonstraram que os AcMo 24A e 56G anti-S. mutans reagiram com S. pyogenes e S. salivarius, confirmando a existência de antígenos comuns entre essas espécies. Esses AcMo avaliados apresentaram baixa reatividade cruzada com miosina de músculo esquelético, porém a atividade anticoração não foi confirmada.

Vacinas em desenvolvimento: estreptococo do grupo B, herpes-zóster, HIV, malária e dengue / Vaccines under development: group B streptococcus, herpes-zoster, HIV, malaria and dengue

OBJETIVOS: As vacinas contra o estreptococo B, o herpes-zóster, o HIV, a malária e a dengue, selecionadas por critérios de comercialização iminente ou devido a problemas específicos para sua obtenção, foram objeto de uma revisão sobre o estado atual do seu desenvolvimento. FONTE DOS DADOS:Foi realizada revisão da literatura através da MEDLINE no período de 1996 a 2006, sobre a epidemiologia e imunologia das doenças, analisando tanto os maiores problemas para a obtenção de uma vacina como o estado atual dos estudos, com ênfase para os que estavam em fase mais adiantada. SíNTESE DOS DADOS: Cada uma das cinco doenças escolhidas apresenta problemas específicos para o desenvolvimento de uma vacina. No entanto, a maioria deles já foi ou está em vias de ser resolvido, permitindo prever que uma vacina - ou vacinas - eficaz e segura estará disponível em futuro próximo. CONCLUSÕES:Apesar dos problemas enfrentados para o desenvolvimento dessas vacinas, os avanços da biologia molecular e da imunologia permitiram superar a maioria deles, abrindo a perspectiva para a obtenção de novas vacinas.

Emergência de Streptococcus pneumoniae resistente à penicilina em Salvado, Bahia: um estudo epidemiológico e molecular / Emergence of Streptococcus pneumoniae resistant to penicillin Salvado, Bahia: an epidemiological study and molecular

As infecções pelo S. pneumoniae persistem como uma das principais causas de morbimortalidade, apesar da disponibilidade de antibioticoterapia apropriada. Em 1995, foi implantado uma rede de vigilância para identificar pneumococos não susceptível à penicilina em dois diferentes grupos populacionais: 1) pacientes com meningite pneumocócica e 2) indivíduos saudáveis na comunidade. No período de dezembro de 1995 a agosto de 2002, o Hospital Couto Maia atendeu 477 pacientes com meningite pneumocócica identificados através da cultura de líquor positiva para S. pneumoniae, 453 (95%) tiveram os isolados caracterizados quanto à susceptibilidade e sorotipagem. Cepas não susceptíveis à penicilina (CIM 0,1 - 1,0 μg/ml) foram isoladas em 59 (13%) indivíduos com meningite pneumocócica. Os sorotipos mais prevalentes foram 14 (14%), 3 (10%), 19F (8%), 6B (8%), 6A (7%), 23F (6%), 4 (5%), 18C (5%) e 8 (4%), sendo que reduzida sensibilidade à penicilina foi associada aos sorogrupos 14 (38), 6 (9), 23 (7) e 19 (5). A provável taxa de cobertura da vacina heptavalente conjugada foi de 70% entre pacientes <5 anos e 91% entre aqueles com isolados não susceptíveis à penicilina. Tipagem de cepas pela reação de polimerase em cadeia (PCR) do elemento repetitivo BOX A, demonstou que os isolados de sorotipo 14 não susceptíveis à penicilina tinham um padrão clonal relacionado e quando comparados com isolados de outras cidades brasileiras tiveram um padrão similar. O segundo grupo populacional foram residentes de 39 domicílios selecionados randomicamente em uma comunidade urbana. Neste grupo, a colonização nasofaringeana foi investigada em 262 indivíduos, 95 (36%) estavam colonizados por pneumococos. Destes, 9 (9,5%) tiveram isolados não susceptíveis à penicilina. Os sorotipos mais prevalentes foram 19F (13%), e 6A (11%), seguidos do sorotipo 14 (7%), 23F (7%), 18C (6%) e 19A (5%). A cobertura teórica da vacina conjugada heptavalente nesta comunidade seria de 49% para todos...

Construction and expression of traceable DNA vaccine for prevention of caries / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Streptococcus mutans has been proved as a causative bacteria of human dental caries. The surface protein antigen is one of the important pathogenic factors. The A region of the surface protein antigen pac gene (pacA) can enrich T-cells and B-cells epitope. In this study, a DNA vaccine carrying pacA and gfp gene (a reporter gene) for caries prevention was constructed. The DNA vaccine was liable to be traced in vitro and in vivo.</p><p><b>METHODS</b>The fragment of pacA (1.3 kb) was amplified by PCR with the plasmid pPC41 as template, and inserted into a pEGFP-C1 vector. The recombinant plasmid produced was named as pEGFPC1-pacA. After the COS1 cell line was transfected by the recombinant plasmid, the expression of gfp was detected by observing the green fluorescence and measuring the fluorescence intensity, and the expression of pacA was detected by RT-PCR.</p><p><b>RESULTS</b>Restricted analyzing, sequencing and PCR technique were employed to identify the recombinant plasmid. The phase and orientation of the pacA gene inserted into the vector pEGFPC1 were correct and no changes of their open reading frames were discovered. The transfected COS1 carrying green fluorescent protein (GFP) was observed; the GFP expression level of transfected cells was higher than that of controlled cell. The transcript of pacA gene was confirmed by RT-PCR.</p><p><b>CONCLUSION</b>Construction of the recombinant plasmid was successful. The gfp gene and pacA gene in the plasmid was transcribed and expressed simultaneously in the transfected cells. Moreover, detection of GFP is simple, safe and effective for living cells.</p>

Study on potential anti-caries DNA vaccine pcDNA3-gtfB integration into host cell genome / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Gene 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.</p><p><b>METHODS</b>Anti-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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>

Construction of plant expression plasmid of chimera SBR-CT delta A1 / 华西口腔医学杂志

<p><b>OBJECTIVE</b>The purpose of this study is to construct plant expression plasmid containing the gene encoding chimera SBR-CT delta A1.</p><p><b>METHODS</b>The target gene fragment P2, including the gene-encoded chimera SBR-CT delta A1 (3,498-5,378 bp), was obtained by standard PCR amplification. The PCR products were ligated with pGEM-easy vector through TA clone to form plasmid pTSC. The plasmid pTSC and plasmid pPOKII were digested by restricted endonuclease BamHI and KpnI, and the digested products were extracted and purified for recombination. Then the purified P2 and plasmid pPOKII were recombined by T4 DNA ligase to form recombinant plasmid pROSC; inserting bar gene into the plasmid and form pROSB plasmid. The recombined plasmids were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing.</p><p><b>RESULTS</b>P2 gene was linked to pPOKII plasmid and formed recombinant plasmid pROSC. The DNA sequence and orientation were corrected. And bar gene was inserted into pPOSC and form recombinant plasmid pROSB.</p><p><b>CONCLUSION</b>Plant expression vector pROSC and pROSB containing the gene encoding chimera SBR-CT delta A1, which may provide useful experiment foundation for further study on edible vaccine against caries have been successfully constructed.</p>

The study on specific anti-Streptococcus mutans IgY against dental caries in rats / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of specific anti-streptococcus mutans IgY against streptococcus mutans on dental caries development in rats.</p><p><b>METHODS</b>35 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'.</p><p><b>RESULTS</b>There 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.</p><p><b>CONCLUSION</b>Local passive immunization with specific anti-streptococcus mutans IgY may be an effective way to prevent the development of dental caries.</p>

The study of peptide vaccine HDS from Streptococcus mutans glucosyltransferase / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Artificial 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Artificial antigen HDS-KLH could induce immune response. As a peptide vaccine, HDS-KLH could reduce the caries incidence in experimental rat model.</p>

An experimental study on PAc and GTF gene vaccines of Streptococcus mutans against rats caries: antibody levels in saliva and serum / 华西口腔医学杂志

<p><b>OBJECTIVE</b>The 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.</p><p><b>METHODS</b>36 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Both 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.</p>

Intranasal immunization against dental caries with plasmid DNA encoding pac gene of Streptococcus mutans in gnotobiotic rats / 中华口腔医学杂志

<p><b>OBJECTIVES</b>To assess the efficacy of plasmid DNA encoding pac gene of Streptococcus mutans (S. mutans) intranasally immunized in gnotobiotic rats and to compare the effect of two different delivery systems.</p><p><b>METHODS</b>Sprague Dawley rats, infected with S. mutans at 20 days of age, were intranasally immunized with plasmid pCIA-P (group A), Dosper-DNA complex (group B), Bupivacaine-DNA complex (group C). Control rats were either immunized with plasmid pCI (group D), distilled water (group E) or immunized intramuscularly (group F). All the rats were boosted 2 weeks later. ELISA determined the antibodies against the vaccines. Keyes caries score was used to evaluate the anti- caries effectiveness of the vaccines at the terminal study.</p><p><b>RESULTS</b>As for the antibody reactions, there were significantly (P < 0.01) differences between rats immunized with DNA vaccine and non-immunized rats. And rats in group B and C had the significantly (P < 0.01) higher level of specific salivary anti-PAc IgA antibodies and rats (group B, C, F) had the significantly (P < 0.01) higher specific serum anti-PAc IgG responses to DNA vaccine. Keyes scores of rats (group B and C) were significantly (P < 0.01) lower than others.</p><p><b>CONCLUSIONS</b>Intranasal immunization with plasmid pCIA-P encoding pac gene successfully reduces the caries and appears to be a promising approach against dental caries. Cationic liposome Dosper and local anesthetic bupivacaine could enhance the efficacy of DNA vaccine.</p>

Construction and cellular expression of GTF-PAc fusion anti-caries DNA vaccine / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>GLU 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.</p><p><b>CONCLUSIONS</b>Fusion 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.</p>