The Experimental Research On Safty And Protective Efficacy Of A Series Of Transformation Defected Noncapsulated Streptococcus Pneumoniae Strains As Attenuated Live Vaccine Candidates

ObjectiveStreptococcus pneumoniae is a major human pathogen which usuallycolonizes on upper respiratory tract and causes both life-threatening diseasessuch as pneumonia, sepsis, and meningitis and milder but common diseases,like sinusitis and otitis media. Considering that antibiotic resistance strainshave been arising frequently, It’s urgent to develop a vaccine with highsafety and broad protective efficacy to restrain pneumococci infection.The disadvantages of pneumococcal polysaccharide vaccine(PPV)andpneumococcal conjugate vaccine(PCV)are high cost、 limited serotypecoverage、serotype replacement，etc. For that reason, this study focus ondeveloping a series of serotype-independent pneumococcal vaccines.Given Streptococcus pneumoniae stabile genetic by knockout genecomE(controls the development of genetic competence). These strains losttheir ability to obtain exogenous gene, therefore hard to reply their virulences. Then, explore the series of mutants as viable attenuated livevaccine candidates of safety and protective efficacy.MethodsThe comE-deletion mutants were constructed by LFH-PCRtechnology. Then, sent for gene sequencing and counted rates oftransformation in vitro. The evaluation of safety of mutants for micethrough following aspects: the survival rate experiments, colonizationexperiment and lung HE staining tissue sections observation. The evaluationof protective efficacy through following aspects: the survival rateexperiments after challenged with D39of the lethal dose; Protectionagainst19F nasopharyngeal colonization by detecting colonize loads; Theserum IgG and its subtypes、cytokines secreted by mice spleen cells weredetected by ELISA.ResultsThe comE-deletion mutants were successfully constructed. In vitro, therates of transformation are range from1.4%to2.5%in wild bacteriagroups, while no transformant was observed in comE-deletion mutantsgroups.The average survival time of mice which nasopharyngeal infectedwith wild D39is1.5d. On the contrary, all of BALB/c mice were alivewhich were infected with the same dose of D39Δcps、D39ΔcpsΔcomE、R6、R6ΔcomE, survival rate is100%. As for these groups, the numbers of bacteria in nasopharyngeal lavage fluid and lung tissue(homogenate) weresignificantly lower than that of the positive control D39group. Furthermore,these four bacteria can not dectect in blood and brain. Observe the lungtissue sections in the different time points after nasopharyngeal infection, wefound that the pulmonary inflammation of mice given comE-deletionmutants is lighter than that of wild group, correspondingly. Challengedafter72hours, lung inflammation of all four groups was recovered.Separately Mucosal vaccination with D39Δcps、D39ΔcpsΔcomE、R6、R6ΔcomE, the mice of experimental groups could secrete high level ofspecifically serum IgG(the main subtypes: IgG1、IgG2b)and cytokines IL-4、IL-10and IL-17A. The colonize loads of nasopharyngeal and lung in fourgroups of vaccines are significantly lower than that of control group after19F infection. In active protection, the survival rate of mice in fourexperimental groups was60%、70%、70%、50%, a significant statisticaldifference with the control group.ConclusionNoncapsulated Streptococcus pneumoniae R6、D39Δcps andtransformation defected noncapsulated strains R6ΔcomE、D39ΔcpsΔcomEdo not have the ability to cause mice diseases, can effective againstStreptococcus pneumoniae colonization and fatal infection after immuningmice. We suggest that there were no effect on protection of noncapsulatedStreptococcus pneumoniae strains after defected transformation, but can make the bacteria cannot obtain exogenous genes and virulence, the safetywas improved. The study provides a new idea for developing high safety oflive bacteria vaccine.