Preparation Of Mannose-modified Chitosan Microspheres Loading Non-typeable Haemophilus Influenzaeouter Membrane Proteinp6 Andimmune Responses In BALB/c Mice

Haemophilus influenzae(Hi),a conditional pathogen colonizated in human nasopharynx,can be divided into encapsulated strains and non-encapsulated strains,of which the latter are designated non-typeable(NTHi).NTHi,as a major pathogen that causes enormous global morbidity in two clinical diseases:otitis media in children and acute exacerbations with chronic obstructive pulmonary disease in adults,has been increasingly focused by scholars all over the world.There is an urgent need to design and develop effective vaccines for NTHi,due to the lack of effective vaccines as well as the spread and prevalence of NTHi worldwide.NTHi outer membrane protein plays an important role in its infection,pathogenic mechanism and interaction with host cells,can cause protective immunity of the host,and is listed as NTHi candidate vaccine targeting antigen.Among them,outer membrane protein P6 has become the focus of many scientists because of its highly conservation.Nasal mucosal immunity is an effective immune route to prevent NTHi respiratory tract infections,but the immune response is weak caused by the simple P6 protein.Therefore,an effective carrier and adjuvant are still needed to enhance its immune effect.Chitosan microspheres have been used as protein and nucleic acid transport carriers for mucosal immune pathways due to their adhesion,high permeability,and phagocytosis by mucosal M cells.In addition,with the development of the targeting technology with antigen presenting cells(APC),the mannose receptor(MR) has been used in delivery systems of various vaccines.In this study,To improve the antigen uptake efficiency of APC based on the protein transport function of chitosan microspheres and mannose-targeted MR-mediated endocytosis,mannose-modified chitosan microspheres loading with P6 protein was prepared,thereby enhancing the immune effect of P6 protein.The method of gene cloning was used to obtain a P6 nucleotide sequence length of 462 bp from the strain NTHi ATCC49247.According to the bioinformatics software and website analysis,the P6 gene in H.influenzae ATCC49247 and nontypeable H.influenzae had a nucleotide similarity of 99.17-100% and an amino acid similarity of 96.17-100%.The secondary structure of hydrophilic protein is mainly composed ofα-helix and random coil.Recombinant plasmid PGEX-6p2/P6 was constructed,and the recombinant protein GST-P6 was successfully expressed in the prokaryotic expression system.A molecular weight of 16 kDa was available after the tag GST removed with Pre Scission protease.Under the catalysis of sodium cyanoborohydride,the hydroxy group of mannose was connected to the free amino group of chitosan to obtain a mannose-modified chitosan derivative.The substitution degree of the free amino group of chitosan was calculated to be 18.75% measured by ninhydrin colorimetry.Chitosan microspheres(CMs) and mannose-modified chitosan microspheres(MCMs) were prepared by the ionotropic gelation process.After then,the protein P6 was loaded onto the microsphere at 25°C,per milliliter CMs bed volume can be loaded with 7.13±0.39 mg P6,while MCMs can be loaded with 9.52±0.29 mg.Scanning electron microscopy presented a regular spherical dispersion,and the average particle sizes of P6-CMs and P6-MCMs were 463.7±15.1 nm and 590.4±16.2 nm,respectively.The P6 release rate of the microsphere vaccine was measured in vitro at 37°C,and the sustained release effect of the microsphere vaccine was clearly observed,it indicated that the release rate of the mannose-modified microsphere vaccine was higher than that of the unmodified one.Nasal mucosal immunity was used to evaluate the protective immune responsses of the microsphere vaccines.After nasal drip test on BALB/c mice,the indirect ELISA was used to detect the serum IgG antibody level to evaluate the systemic humoral immunity.Evaluation of mucosal humoral immunity showed that the microsphere vaccine also effectively stimulated systemic immunity through the mucosal immune pathway.The IgA and IgG antibody levels of the microsphere vaccine group were significantly higher than those of the pure P6 group and the mannose-modified microsphere vaccine group.IgA and IgG antibody levels were significantly higher in the unmodified group.In cellular immunity,IFN-γ,IL-2,IL-4,IL-5,and IL-17a were detected in spleen lymphocyte culture supernatants by indirect ELISA.The results showed that IFN-γand IL in the P6-MCMs group-2,IL-5 and IL-17a factor levels were significantly higher than other groups,while IL-4 factor levels were significantly increased in the P6-CMs group,suggesting that P6-MCMs can cause mixed Th1/Th2 responses,while P6-CMs Can cause Th2response.Flow cytometry detection of spleen lymphocyte subgroups CD3~+,CD4~+and CD8a~+ showed that the cell subtypes CD3~+,CD3~+CD4~+and CD3~+CD8~+ in the P6-MCMs group were significantly higher than those in the control group and the P6 group alone,suggesting that P6-MCMs In addition to MHC class Ⅰ antigen presentation,it also participates in MHC class Ⅱ antigen presentation.After nasal toxic effects were performed on mice with bacteria NTHi ATCC49247,HE staining was used to observe pathological changes in nasal mucosa and lung tissues.The inflammatory cells in nasal mucosa and lung tissues of immunized mice were significantly less In the control group,the tissue lines of the immune group were cleared and tended to be normal.The above research shows that the mannose-modified chitosan microspheres loading Haemophilus influenzae P6 protein was successfully prepared,transnasal mucosal immunity can induce effective systemic humoral immunity and mucosal immunity,induce mixed Th1/Th2,Th17cell immune response,and can stimulate the immune response of CD4+-mediated Th cells and CD8+-mediated cytotoxic T lymphocyte(CTL) through two presentation pathways of MHC Ⅰ/MHC Ⅱ.The significantly immune effect of P6 MCMs suggests that P6 MCMs target the mannose receptor on the surface of APC in vivo,thereby improving the ability of APC antigen presentation.Immunoprotective experiments show that P6-MCMs plays a certain preventive role on the infection of non-typeable Haemophilus influenzae.