Inhibition Of Streptococcus Mutans Biofilm Formation By Secretory Immunoglobulin A (S-IgA) Antibodies Induced By Targeted Fusion Anti-caries DNA Vaccine

Dental caries is one of the most common infectious diseases in the word. Streptococcus mutansfS.mutansJh the major etiological agent of dental caries. Plaque is the incipient factor of caries.As the typical bacteria biofilmjt is made up of Streptococci,Actino?nycetes,Lactobacilli,other microorganism cell and extracellular polysaccharides matrix. All kinds of bacteria exist in the three-dimensional structure which is surrounded by extracellular polymer substances matrix.They conglutinate,adhere and fix on the surface of teeth and other interfaces. As the preponderant bacteria of biofilm,S.mutans play important part in the formation、 development and muture of plaque.The enzyme of S.mutans has ruling action in the sugar metabolizing.It can produce acid and can live in acid environment, which have important impact in the acidification of plaque and the enamel demineralization. A cell surface protein antigen (PAc) mediate sucrose-independent adherence of S.mutans to tooth surface. PAc contribute to the adherence and accumulation of organism in dental plaque,so it is considered to be one of the important virulence factor of S.mutans. Another important virulence factor of it is GTFs,one region of which is glucan-binding domain((GLU).GLU is responsible for the composition of glucan. Glucans mediate sucrose-dependent adherence of S.mutans,making S.mutans adhere on tooth surface tightly and form compact dental plaque.An important approach to prevent dental caries would be consider PAc and GLU as the candidate antigen to control the infection of S.mutans by immune method. Our group have build the DNA plasmid pGJA-P which encode the signal peptide and extracellular regions of human CTLA4, CH2and CH3regions of IgA1,the GLU gene and A-P fragment of S.mutanspac gene. A new targeted anti-caries DNA plasmid pGJA-P/VAXl was successfully constructed join the pGJA-P with the pVAXl,the only vector authorized by Food and Drug Administration in clinical trials. It is proved that the vaccine can stimulate the secretion of IgA (secretory IgA,S-IgA) in saliva and the results of caries scores also prove the truth the DNA vaccine has the effect of anti-caries.The purpose of this study is to detect whether the immunity effect of pGJA-P/VAXl is that the S-IgA antibody interfere the formation of dental plaque. Study the kinetics of the antibody responses generated following targeted anti-caries DNA plasmid immunization.Use the S-IgA to influence the biofilm cultivated by UA140:: Φ(mutAp-rfpI),then use the CLSM to determine the depth of biofilm and CFU to determine the effect of S-IgA on the adherence ratio of S.mutans and the possible mechanism of the effect at the same time;the effect of S-IgA antibodies on the adhesion of S. mutas MT8148onto S-HA beads was examined;in additional,we study inhibition of Streptococcus mutans biofilm formation by S-IgA antibodies induced by DNA vaccine in vivo through measure the depth of biofilm, calculating the adherence bacterial number by CFU and observing the growth condition of S.mutans on tooth of rats by CLSM.The present study consists of three parts:Part one: Inhibition of Streptococcus mutans biofilm formation by secretory immunoglobulin A (S-IgA) antibodies induced by targeted fusion anti-caries DNA vaccine in vitro.Experiment Ⅰ: Kinetics of the antibody responses generated following targeted anti-caries DNA plasmid immunization.Method:Four-to-six-week-old female Wistar rats,8per group, were immunized with pGJA-P/VAX (Group A) or pVAXl (Group B) by the intranasal route at week0and week2, respectively.Bupivacaine:DNA complexes were prepared by adding bupivacaine hydrochloride to the aqueous DNA solution using a fast mixing method. SD rats were immunized with Bupivacaine:pGJA-P/VAX1complex or Bupivacaine:pVAXl complex intranasally. Saliva samples were collected for ELISA.Result:The salivary IgA response of pGJA-P/VAX1i.n. immunized group were significantly higher than those of the pVAXl i.n. immunized group (p<0.01) and the level of SIgA arrived climax in10weeks after first immunization. Conclusion: The targeted fusion DNA vaccine pGJA-P/VAX1markedly enhance the magnitude of mucosal specific antibody response via the intranasal route. Expriment II:Preparation of S. mutans bio filmsMethod:HA discs were placed in the wells of24-well polystyrene cell culture plate and incubated under agitation for4hours at37℃with0.5ml of sterile saliva. After that,the saliva were removed and replaced with0.8ml of TSB+0.15%(w/v) sucrose and0.8ml of sterile saliva.Each well was inoculated at37℃with S.mutans UA140::Φ(mufAp-rfp1)0.2ml and culture plates were incubated aerobically at37℃for16h,the HA discs were dip-washed twice in the PBS and swirled gently to remove loosely adherent bacteria to harvest the adherent cells.each disc were transferred to a sterile50-ml polypropylene tube containing1ml of PBS and vortexed vigorously for2min.Viable counts were carried by this dilusion.SEM was used to determine(ⅰ) the surface of HA disc,(ⅱ)the formation of bacterial biofilms,(ⅲ)the efficacy of the resuspension of protocol.Result:saliva pellicle was formed evenly on the HA disc bacterial biofilms can be easily observed under the SEM,the resuspension of protocol was satisfactory because few bacterial can be seen on the HA disc and can be used for CFU.ConclusioniThis method can be used in our experiment. Experiment Ⅲ:The mechanism of the effect of S-IgA on the adherence of S.mutans onto HA diskMethod:Use different designs to find out the mechanism of the effect of S-IgA on S.mutans biofilm. group Ⅰ:acquired pellicle was formed by saliva from groups A and B under agitation, replaced with BHI, sucrose, S. mutans and sterile blank saliva,biofilms were grown for16h before the cells were harvested and enumerated, and the depth of the biofilms was measured by confocal; group II:acquired pellicles were formed on HA disks with blank saliva,then the saliva was replaced with BHI, sucrose, S. mutans and sterile saliva from experimental or control groups.S.mutans biofilms were cultivated for16h, cells were recovered and enumerate;groupIII:S. mutans biofilms were cultivated with blank saliva for16h transferred to new wells containing sterile saliva from experimental or control groups, viable bacteria counts and biofilm measurement were carried out. Result:More S-IgA could be seen from the pellicle formed by saliva from group A compared with the pellicle formed by saliva from group B, there were significant differences in the number of cells recovered from the biofilm(41.79%lesser) and the depth of the biofilm (41.02%lesser)(P<0.01); bacteria count obtained from HA disks (27.4%less) and measured in the experimental group (22.81%less) were less than biofilms obtained in the control group (P<0.01); S-IgA could assemble S. mutans together or cover the surface of S.mutans; while there is no significant differences between treatment on mature biofilm(P>0.05). Conclusion:the mechanism of effect of S-IgA on biofilm is to effect the early adherence of S.mutans by forming plaque;and prevent the adherence of S.mutans later by joining with protein on the surface of S.mutans,while have no effect on mature bioflimPart Two:The effect of S-IgA antibodies on the adhesion of S. mutans MT8148onto HA beadsExperiment Ⅳ:The effect of S-IgA antibodies on the adhesion of S. mutans MT8148onto S-HA beadsMethod:Four-to-six-week-old female Wistar rats,8per group, were immunized with pGJA-P/VAX (Group A) or pVAX1(Group B) by the intranasal route at week0and week2, respectively.Bupivacaine:DNA complexes were prepared by adding bupivacaine hydrochloride to the aqueous DNA solution using a fast mixing method. Saliva samples were collected at2,4,6,8,10,12,14, and16weeks after the first immunization and ELISA to measure SIgA/total TgA.5mg of HA beads were equilibrated in buffered KC1and cultivated with4-week,10-week, and16-week saliva, and serial dilutions (1:2;1:4;1:8;1:16;1:32) of10-week saliva of immunized mouse and unimmunized mouse and PBS was used as control. Result:The IgA anti-Pac and anti-Glu responses in rats peaked at10weeks, The adherence of S. mutans in saliva of experiment rats was significantly lower than that in the control group (p<0.001). The S-IgA antibodies in10week has the strongest inhibition of the adhesion of S. mutans cells, but there were no significant differences between the4th,10th, and16th weeks, the lowest dilution of saliva with effect was1:16, and reduced the adherence activity of S. mutans to approximately8.7%.Conchision:the salivary sample containing specific S-IgA has the consistent effect through out the experiment and also shown to inhibit the binding of S. mutansin a dose-dependent manner.Part Three:Inhibition of Streptococcus mutans biofilm formation by secretory immunoglobulin A (S-IgA) antibodies induced by targeted fusion anti-caries DNA vaccine in vivoExperiment V:Inhibition of Streptococcus mutans biofilm formation by secretory immunoglobulin A (S-IgA) antibodies induced by targeted fusion anti-caries DNA vaccine in vivo.Method:At the9th week after immunization, remove the biofilm on the surfaces of tooth of rats by mechanism methods, broken neck death after4h behind the last cleaing, removed the mandibular molars of rats,observe the fluorescence intensity on the tooth after co-cultivated the tooth with anti-RAT IgA antibody (Rhodamine Conjugated) by confocal laser microscope to observe whether SIgA is involved in the formation of the acquired membrane.48h after putting S.mutans onti mouth of rats,saliva and mandibular molar were collected;S. mutans adhesion amount were observes on the left maxillary molar using scanning electron microscopy; the comparison of the amount of S. mutans adhesion on tooth surface were taken from the left mandibular molars by confocal laser microscope; bacterial count were carried out by right maxillary molar oscillation surface; S-IgA in saliva and S. mutans adhesion were observed by confocal; and testing UA140::Φ(mutAp-gfp1)/total Streptococcus in saliva Results:fluorescence intensity in the experimental group is higher than control group,showing SlgA involved in the formation of the acquired membrane; scanning electron microscopy (SEM)showed experimental tooth surface had more S. mutans bacteria than the control group; there is significant differences between the biofilm thickness and bacterial amount in the control group and experimental group (P<0.01); and S. mutans clouds grow more in the saliva in the experimental group whereas the S. mutans in saliva of control group grown as scattered distribution; S-IgA can closely combine with S. mutans,wrapped in the surface of the bacteria; During the experiment the experimental group significantly reduced the percentage of UA140::Φ(mutAp-gfpI)/total Streptococcus (P<0.01),and the trend has been maintained to the end of the experiment.Conclusion: targeted fusion anti-caries DNA vaccine could induce the production of S-IgA and can be significantly reduced adhesion of Streptococcus mutans on the tooth surface in vIvo.