The Study Of Integrated Immunity Elicited By A Novel Pneumococcal Polysaccharide Conjugate Vaccine With HBV Surface Antigen In Mice

Bacterial pneumonia caused by Streptococcus pneumoniae is a global infectious disease that endangers human health.Streptococcus pneumoniae causes a variety of diseases,such as pneumonia,meningitis,sinusitis,otitis media and bacteremia etc.in the whole population through invasive or non-invasive infections,especially in children and the elderly.Pneumococcal polysaccharide protein conjugate vaccines have made great success in prevention and control of infections and diseases caused by streptococcus pneumoniae.Conjugate vaccine has become the main development and application direction of pneumococcal vaccine because of its better antibody enhancement effect than simple polysaccharide vaccine and advantages of immune memory and group protection.Conjugation of polysaccharide and effective carrier protein is the key to develop highly effective pneumococcal polysaccharide conjugate vaccine.At present,there are only a few protein carrier used in pneumococcal polysaccharide conjugate vaccine,including tetanus toxoid(TT),diphtheria toxoid(DT),diphtheria toxin mutant crossreacting material 197(CRM197)and haemophilus influenzae,protein D(PD).These carrier proteins are also used in many other conjugate vaccines.Repeated vaccination with same carrier protein antigen will lead to the carrier-induced-epitopic suppression(CIES)and weaken the immune response to polysaccharide.On the other hand,the immunogenicity of carrier protein may be changed since its antigen epitope is damaged through detoxification,conjugation and other chemical processes.The immune effect of the carrier protein of vaccine dosen’t clearly listed in the product manual.Therefore,there is an urgent need to develop a safe and effective new carrier protein conjugate vaccine to control the risk of CIES,and to induce an effective immune response against polysaccharide and protein carrier,which is very necessary and important for polysaccharide protein conjugate vaccine,especially for pneumococcal polysaccharide protein conjugate vaccine.Finally,the goal of double protection-effect with one immunization is achieved.In this study,a pathogen-associated protein,hepatitis B virus surface antigen(HBsAg),was used as a new carrier protein to conjugate with pneumococcal polysaccharide.The corresponding protein antigens and polysaccharide antigens were used to immunize mice.The level of specific antibody,cellular immune response and early gene expression profile of capsular polysaccharide and HBsAg induced at different time points after immunization were compared.The results showed that the new pneumonia conjugate vaccine with HBs as carrier cojugate with type 33F pneumonia capsular polysaccharide(PN33Fps)could stimulate the specific humoral and cellular immune responses to the two antigens in mice,respectively.Compared with mice immunized with two single antigens,this novel conjugate vaccine elicite stronger humoral and cellular immunity against polysaccharides(PN33Fps)and proteins(HBs).This result suggested that double protection-effect with one immunization of this new conjugate vaccine with one injection is enhanced with the increase of immunization times.This result suggested the double protective effect of this new conjugate vaccine by one immunization,and the immune effect was enhanced with the increase of the number of immunization.However,even with boosted immunization,antibody level and antibody positive conversion could not be induced by the same content of polysaccharide immunized in mice.In addition,the spleen tissue was collected to analyze the differential characteristics of gene expression profiles in the early transcriptome of mice after immunization.It was found that the novel pneumococcal conjugate vaccine with hepatitis B surface protein as carrier induced more expression of immune related genesand showed different changes from single antigen,especially in the differentiation and development of immune cells.This difference may be due to characteristic expression of some immune signal molecules in innate and adaptive immune cells stimulated by different antigens,which is related to the production of stronger antibody level and cellular immune response after polysaccharide and protein conjugating Monoclonal antibody cell of specific B lymphocytes against pneumonia 33F polysaccharide antigen and hepatitis B surface protein antigen were isolated from splenic lymphocytes of mice immunized with this novel hepatitis B surface protein-conjugated pneumococcal polysaccharide conjugate vaccine by cell fusion technology.Identification of antibody secretion ability and characteristics of isolated specific B lymphocyte monoclonal cell lines showed that the selected cell lines had the ability to continuously secrete specific IgG1 antibodies.It was further verified that this novel pneumococcal conjugate vaccine with hepatitis B virus surface protein as carrier protein induced the differentiation and development of specific B lymphocytes in mice and the production of antibodies in serum.In short,the research from humoral immunity,cellular immunity to molecular level suggests that this novel conjugated vaccine elicite good immune response in mice.On the basis of the study on the immune effect of this novel pneumococcal conjugate vaccine with hepatitis B surface protein as carrier,we also evaluated the preliminary safety of the immunized mice.By observing and studying the weight of mice at different time after immunization and the histopathology of the main organs,the results showed that the mice immunized with this conjugate vaccine did not show significant pathological changes and the weight continued to increase during the immune period campared with the control group,which showed that the preliminary safety of the conjugate vaccine was predictable.In conclusion,the results showed that this novel pneumococcal polysaccharide conjugate vaccine conjugated with hepatitis B surface protein had good immune effect and safety in mice.It was suggested that conjugate vaccine using hepatitis B surface antigen as carrier can not only promote the immune effect of polysaccharides,but also induce immune response against hepatitis B surface protein in mice.It was proved that the HBsAg protein can be used as a new carrier of pneumococcal conjugate vaccine which can get the double immune effects with one inoculation in mice.The research on the early humoral,cellular and molecular immune mechanism of conjugate vaccine provides a new way for the research and development of conjugate vaccine.The monoclonal antibody cell lines of pneumonia 33F polysaccharide and hepatitis B surface protein isolated from mice immunized with the cojugate vaccine and the application of the antibody secreted by themestablish a foundation for the quality evaluation of the development of conjugate vaccine in the later.