Predominant Antigenic Enitopes And Their Immunogenicity Of Hap Adhesin Of Of Nontypeable Haemophilus Influenzae

Objective:Haemophilus infuenzaeis a kind of Gram-negative small bacillus that can colonize on human nasal pharynx and upper respiratory tract to act as the major pathogen of infectious diseases such as meningitis,pneumonia and acute tympanitis.According to the presence or absence of polysaccharide capsule,all H.influenzae strains can be classified into capsule-presented and capsule-absent groups.The capsule-presented H.influenzae strains are further divided into six serotypes using capsular polysaccharide antibodies and called typeable H.influenzae(THi)while the capsule-absent H.influenzae strains can not be typing by capsular polysaccharide antibodies and called nontypeable H.influenzae(NTHi).The inoculation of capsular polysaccharide vaccine of H.influenzae provides an effective immunoprotection against infection of THi in human body,but the vaccine has no immunoprotective effect against infection of capsule-absent NTHi.More importantly,since the immunoselection of the capsular polysaccharide vaccine,in the recent years NTHi gradually replaces THi to become the predominant strain in clinic,which caused that not only the infection rate but also the drug resistance rate of NTHi strains are continuously increased year by year.Vaccination is an important strategy to prevent and control the prevalence and infection of NTHi strains.However,untill now,no commercial NTHi vaccine products have been available at home and abroad.Hap protein of H.influenzae has been reported as an adherence factor that located on the outer membrane and expressed by both NTHi and THi.Moreover,Hap protein of H.influenzae can induce human body to produce immunoprotective mucosal S-IgA and serum IgG.Epitope is a short peptide segment that determines immunogenicity in antigenic molecule.Epitope peptide-based multiple antigenic peptide(MAP)vaccine is the main approach in development of novel genetic engineering vaccines.Therefore,investigation of the distribution and sequence conservation of Hap adhensin-encoding gene(hap)in NTHi isolates,screening and identification of the predominant T-and B-cell(T-B)-combined antigenic epitopes in Hap protein and their immunogenicity can establish a solid foundation for further developing MAP vaccines of NTHi.Methods:The sequence conservation of hap genes of NTHi strains and T-B-combined antigenic epitopes in Hap adhensin were predicted using bioinformatic softwares.PCR was used to amplify the 156 bp segment at 5’-end and 855 bp segment at 3’-end of hap gene from NTHi isolates(hap-5’-156 和 hap-3’-855)for sequencing.Phage display systems of seven T-B-combined antigenic epitopes from the 55-aa segment at N-terminal and 285-aa segment at C-terminal of Hap adhensin(Hap-N52 and Hap-C285)were constructed.Western Blot assay and ELISA were applied to determine the antigenicity and immunoreactivity of different T-B-combined epitope peptides displayed by recombinant phage PⅢ proteins(rPⅢ).Results:The product of hap gene was predicted as a membrane superficial protein of NTHi.In the different NTHi isolates,the sequences of 156 bp segment at 5’-end and 855 bp segment at 3’-end of hap genes were relatively conserved but many mutations could be found in the sequences at middle region in the hap genes.All the 56 NTHi isolates were detectable for both the hap-5’-156 and hap-3’-855 segments with 92.3%-100%nucleotide and amino acid sequence identities.Hap-N5-24 in the Hap-N52 segments as well as Hap-C4-27,Hap-C114-129,Hap-C150-173,Hap-C200-227 and Hap-C241-267 in the Hap-C285 segments were predicted as the T-B-combined antigenic epitopes with higher scores and lower mutations.The Western Blot assay and ELISA confirmed that the rPⅢ-displayed Hap-C4-27 and Hap-C150-173 epitope peptides presented stronger hybridization bands with NTHi antisera,and 96.9%(63/65)and 92.3%(60/65)of serum samples from NTHi-infected children were positive for antibodies against the Hap-C4-27 and Hap-C150-173 epitope peptides,respectively.Conclusion:The hap gene has an extensive distribution in NTHi isolates and the sequences of 156-bp segment at its 5’-end and 855-bp segment at its 3’-end are conserved.The Hap-C4-27 and Hap-C150-173 are the predominant T-B-combined antigenic epitopes of Hap adhesin that can be used as the epitope candidates for developing MAP vaccines against NTHi.