| Background and objective Pertussis is a highly contagious respiratory disease.The causative agent is a bacterium named Bordetella pertussis(B.pertussis).Vaccines are an effective means to control pertussis.The incidence of pertussis has been increasing in recent years,even in developed countries where vaccination is widespread,which is called pertussis reemergence.It is possible that natural infection immunity or the currently used acellular pertussis vaccine(ap)are not ideal in inducing protection against infection as well as immune persistence.In addition,the vaccine selection pressure led to the continuous emergence of pertussis epidemic strains with different genotypes may have further weakened the protective efficacy of the vaccine.Based on the above background,this study started with the laboratory testing of pertussis to isolate new pertussis epidemic strains.The genome,infection and immune characteristics of pertussis strains,and the level of immune response caused by infection were measured to investigate the differences between epidemic strains and vaccine strains and the effects of these differences on the recurrence of pertussis.Meanwhile,the in vivo gene expression levels were examined to explore the molecular mechanism of the infection difference among different strains.Incomplete protection of the acellular pertussis vaccine against epidemic strain induction was observed in this study.We therefore developed a fractional dose of the intradermal DTaP-sIPV vaccine and evaluated its protection against epidemic strain infection as well as the dose-sparing effect.To explore the reason for pertussis reemergence and the research basis for the development of new pertussis vaccines or target genes.Methods Firstly,the isolation,identification,infection characteristics and immune mechanism of pertussis clinical epidemic strains were investigated.Pertussis epidemic strains were isolated by laboratory testing and bedside sampling.Phylogenetic tree and colollinearity analysis were performed on the whole genome of pertussis isolates to identify homologous relationships between epidemic strains,vaccine strains and international epidemic strains.Then,the methods of antigen content detection,Western Blot,protein N terminal sequencing and inter-molecular binding-affinity assay were used to explore the differences in antigen expression and the antigenicity of key proteins.The colonization characteristics of infection,host immune response and immune memory protection were detected by aerosol infection model,Cytometric Bead Array(CBA)cytokines detection,pathological evaluation,and flow cytometry,respectively.Cross-infection verified the difference in the actual protection effect.Gene expression differences in infection states of different strains were examined by in vivo bacterial isolation and RNA-seq to explore the molecular mechanisms of different colonization tendencies between strains.Based on the observations on the protective efficacy of aP vaccine,we developed a dose-sparing intradermal DTaP-sIPV vaccine and evaluated its immune and protective efficacy against infection with epidemic strains.First,in the rat model,serum antibody levels induced by 1/5,1/10 dose of intradermal immunization or full-dose intramuscular immunization were determined by ELISA.Second,the types of immune responses induced by different vaccines were explored by cytokine detection and lung transcriptome sequencing.Finally,the protective efficacy of the intradermal vaccine against epidemic strain infection was evaluated by epidemic strain aerosol attack infection.Results 1)Four pertussis epidemic strains(BP-1,BP-2,BP-3,BP-4)were isolated.The results of genome-wide analysis showed large gene col linearity,SNP homology,and allelic differences between epidemic and vaccine strains,and epidemic strains showed stronger evolutionary potential for genomic rearrangements.2)An amino acid deletion from 18 to 391 was found in the PRN coding region of the BP-L2 strain.We verified that BP-L2 was able to express the remaining PRN,which retained the ability to bind the antibody but had less binding affinity than the complete PRN of BP-L1 or CS strain.3)BP-L1 and BP-L2 have the advantage of colonization in the lungs and trachea,respectively,and are able to cause significantly higher cell and humoral immune responses at higher colonization sites than CS strains,as well as higher levels of immune memory response.4)In the lungs,aP vaccine induced better immune protection than infection immunity,which induced by epidemic strains.However,the advantage of colonization in the lungs and trachea of the different epidemic strains were still evident in cross-infection and post-immune infection.5)In vivo RNA-seq results showed that the expression of prn and bipA in mice infected with BP-L2 strain was significantly lower and the relA was significantly higher,however,the above gene expression level of the BP-L1 group showed the opposite trend.Infection differences may be related to prn expression levels and biofilm-associated bipA,relA.6)Intradermal immunization of 1/5 dose of DTaP-sIPV vaccine was able to induce serum antibody levels and seroconversion rates comparable to full-dose intramuscular immunization.The 1/5 dose of intradermal immunization was able to induce a significant Th1/Th2/Th17 type immune response in the lungs and thus have a stronger protective efficacy against the pertussis epidemic strain infection.Conclusion 1)Existing epidemic strains have a colonization tendency to the lung or upper respiratory tract beyond the genotype,which may cause alternating transmissible or pathogenic strains and aggravating pertussis reemergence.This difference in colonization is related to the structural changes of the key antigen PRN as well as the expression level upon infection in vivo.2)Existing epidemic strains infection can elicit stronger immune memory responses,but exhibit relatively weaker cross-immune protection,which may account for the emergence and coexistence of multiple epidemic strains.3)Intradermal dose-sparing DTaP-sIPV can induce greater immune protection against infection with 5-fold dose-sparing effect,which is an ideal strategy to alleviate the pertussis reemergence and IPV shortage.In this study,a more comprehensive joint study of pertussis epidemic strains,from clinical isolation to infection immune characteristics,so that the molecular mechanism was explored.For the first time,the infection and immune characteristics of pertussis epidemic strains were analyzed jointly with the in vivo transcriptome,which can more accurately reflect the real differences and internal mechanisms between different epidemic strains than the simple genotyping studies or pathogenic microbiology studies.In addition,intradermal vaccination of 1/5 doses of DTaP-sIPV vaccine were found to induce better protection against infection by epidemic strains,providing an important theoretical basis and a new vaccine candidate strategy. |
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