Preparation And Efficacy Evaluation Of P6 Protein Conjugated With Haemophilus Influenzae Type B Capsular Polysaccharide

Haemophilus influenzae(Hi)is a common Gram-negative bacterium with pleomorphic characteristics such as short rods,bulbous rods and filaments.It can cause meningitis,pneumonia,otitis media,septicaemia,sinusitis and acute epiglottitis,among which Haemophilus influenzae type b,Hib and Nontypeable Haemophilus influenzae(NTHi)are the most common infections.With application of the Hib conjugate vaccines,the incidence of Hib related diseases have been controlled,but NTHi has become one of the main opportunistic pathogens leading to acute otitis media in children and lung disease in adults.Meanwhile,the drug resistance of NTHi increases with long-term antibiotics treatments.So the research and development of vaccine for preventing NTHi appears especially important.Currently,the most common protein carrier for Hib conjugate vaccines is tetanus toxoid,but the kind of vaccine has little effect on non-capsulated NTHi.Most researchers have used the outer membrane proteins as vaccine candidates to prevent and control NTHI infection.The outer membrane proteins P4,P5,P6,P26,Protein D,Protein E,and HAPs have potential advantages.Among them,the outer membrane protein P6,which has good conservatism and immunogenicity,and is present in all the Hi strains,is studied more.Rats immunized with P6 can produce anti-P6 antibodies with bactericidal activity.A large number of early work of our group also showed that the outer membrane protein P6 has protective effects in mice.Therefore,it’s expected to prevent both Hib and NTHI infection by using P6 protein as carrier to conjugate with Hib capsular polysaccharide(PRP).Moreover,it’s significant for the development of new anti-Hi vaccine in the future.Firstly,the seed batch of Haemophilus influenzae type b ATCC 10211 was identified.Then,the refined polysaccharides were extracted and purified from Haemophilus influenzae type b through the steps of enrichment,precipitation,nucleic acid removal,phenol extraction and dialysis.The refined polysaccharides were qualified by structural and chemical identification.The recombinant plasmid p ET-28a(+)/P6 was constructed by using known P6 gene sequence.The freezing bacteria containing the recombinant plasmids was amplified,induced by IPTG,and purified by Ni-Sepharose FF affinity chromatography to get P6 protein.The PRP-P6 conjugate vaccine was prepared by binding P6 protein with PRPs which were activated by 1-Cyano-4-Dimethylaminopyridine tetrafluoroborate under a specific p H value.BALB/c mices were randomly divided into 4 groups,including the adjuvant(FA)group,FA+P6 group,FA+PRP group and FA+PRP-P6 group,which were immunized by nasal drops to evaluate the mucosal immunity effect of PRP-P6 conjugate vaccine.Specific P6 and PRP IgG antibodies in serum of immunized mice and specific P6 and PRP IgA antibodies in nasal cavity and lung lavage fluid of mices were detected by ELISA.The results showed that: the levels of specific PRP IgA and IgG antibodies in FA+PRP-P6 group were significantly higher than those in FA+PRP group and FA group,but the levels of specific PRP IgG antibodies in FA+PRP group showed no significant difference with those in FA group,and the IgA antibodies in nose and lung were increased.The level of specific P6 IgA antibody in FA+PRP-P6 group was significantly higher than that in FA+P6 group,while the level of specific P6 IgG antibody was similar to that in FA+P6 group,but the levels of specific P6 IgG antibody in both groups were higher than that in FA+P6 group,and the differences were significant.These results suggest that the conjugate vaccine can simultaneously induce two kinds of specific antibodies,and it can simultaneously induce humoral immunity and mucosal immunity.The cytokines IFN-γ,IL-2,IL-4,IL-5 and IL-17 A in the supernatant of splenic lymphocyte culture were detected by the cytokine kit.The results showed that the secretion of IL-2,IFN-γ,IL-4,IL-5 and IL-17 in the FA+PRP-P6 group was higher than that in the FA group after the stimulation of P6 protein.At the same time,the levels of IL-2,IFN-γ,IL-4 and IL-5 in FA+PRP-P6 group had no significant difference with those in FA+P6group,suggesting that the conjugate vaccine group had a similar trend in cellular immunity compared with P6 alone.PRP stimulated spleen lymphocytes in each group,and IFN-γ,IL-2 and IL-4 secreted in FA+PRP group were not significantly different from those in FA group,suggesting that capsular polysaccharide alone could not induce effective cellular immune response in mice.The levels of cytokines in FA+PRP-P6 group were significantly increased compared with that in FA group,indicating that the PRP-P6 conjugate vaccine could induce a strong cellular immune response.NTHi(ATCC 49247)and Hib(ATCC 10211)were used to test nasal challenge of immunized mice.The pathological changes of nasal mucosa and lung tissue were observed by HE staining.The inflammatory response in lung tissues of immunized mice FA+P6 group and FA+PRP-P6 group after NTHi challenge was significantly weaker than that in FA group,and the inflammatory response in nasal and lung tissues of immunized mice FA+PRP-P6 group after Hib challenge was also significantly weaker than that in other groups,while the pathological changes of nasal mucosa were not obvious in all groups.In this study,the PRP-P6 conjugate vaccine was successfully prepared,which could induce effective humoral and cellular immune responses through nasal mucosal immunization,as well as strong mucosal immunity.The pathological changes of lung tissue in immune-protective experiments showed that PRP-P6 had a certain preventive effect on Hib and NTHI infection.