| In the20th century there were three influenza pandemics. Influenza, a contagious,acute respiratory disease caused by an influenza virus, is a worldwide pandemic andhazardous infection. And it posed a serious threat to human health. In the "Spanishflu", more than20million people died. Subsequently, pandemic influenza outbreaksin1957and1968, the numbers of infection were up to30%, more than30millionpeople lost their lives. In the recent years, H5N1highly pathogenic avian influenza(HPAI) and2009influenza H1N1flu epidemic broke out, seriously disasters and pub-lic health caused by it needs to be paid close attention.The influenza pandemic appeared because influenza virus is prone to antigenicvariation, infectious and spread rapidly. Vaccination is one of the effective means ofprevention and controlling for influenza. Currently inactivated influenza vaccine andlive attenuated vaccine are available. Commercial trivalent inactivated vaccines sufferfrom limited efficacy in cross-protective immunity and routes of administration, givenby intramuscular injection. In addition, they are unsuitable for high-risk populationand special populations, such as the immune protection of the elderly and children.Undoubtedly the research and development of the nasal vaccine provided a favorableapproach and space for influenza vaccine.Previous studies showed that the respiratory mucosa not only is the site of influ-enza virus infection, but also is immune organs of exogenous antigen to produce im-mune responses against pathogenic micro-organisms infections. Mucosal systemis the first line which organism resists the intrusion of the outside antigen, nasal vac-cination could mimic the natural infection, less antigen dosage and more comprehen-sive immune response. Nasal immunization could cause systemic immunity responseby mucosa administration, thereby strengthening the immune defense mechanismof influenza pathogens, improving the effectiveness of influenza vaccine. Efficacy ofinfluenza vaccine affected by the complexity of the mucosa system, immune responseinhibition of antigen and a small vaccine antigen enter into the mouth and digestivetract, or a small amount of outflow in vitro. Therefore, dose dependence of vaccineantigen is the important issues in the development of the nasal spray vaccine. In this study, the quaternized chitosan hydrogel was used as a delivery system ofthe nasal influenza vaccine, the whole influenza virus and spilt influenza virus wereacted as antigen, and IL-1β, IL-18, IL-33and soluble ViE were acted as adjuvant, weprepared different influenza formulation. Then nasal influenza H1N1vaccine formu-lation were screened and confirmed by animal experiments. These resultsshowed that immunogenicity of influenza vaccine was optimal when we selectthe quaternized chitosan hydrogel as delivery carrier, IL-1β as an adjuvant, and influ-enza split vaccine as antigen. Further we isolated and purified recombinant humanIL-1β protein adjuvant by efficiently expressed in E. coli, and the expression rate, p u-rity and biological activity were detected. At last mice were immun ized with rIL-1βprotein as adjuvant for nasal influeza A (H1N1) vaccine, and its adjuvant effectivenesswere evaluated. Based on these results, nasal trivalent influenza virus vaccines wereprepared and its preliminary immune responses were investigated.The dissertation is divided into three parts.The first part focused on screening and determination of nasal influenza deliverycarrier, adjuvant and vaccine antigen.(1) Vaccine delivery carrier. Influenza A H1N1split virus antigen (4μg HA per mouse) were used, chitosan, quaternized chitosan,chitosan hydrogel and quaternized chitosan hydrogel were acted as vaccine carrier,Balb/c mice were intranasally immunized with or without adjuvant, on0day and oneboost on14days. Effectiveness of influenza vaccines were evaluated by detecting se-rum IgG and inhibition titers (HI), and mucosa sIgA in nasal turbinates and lung lav-age. These results showed that quaternized chitosan hydrogel could induce humoraland cellular immunity much better than chitosan, quaternized chitosan, chitosan hy-drogel. Based on these data, we prepared hydrogel-antigen formulation with differentconcentration ratio1:1,1:2and1:4. The results showed that the effectiveness of vac-cine is optimal when the concentration ratio of hydrogel-antigen was1:1.(2) Vaccineantigen. The quaternized chitosan hydrogel were used as vaccine carrier. Balb/c micewere immunized with the whole influenza A H1N1virus vaccines and split virus vac-cines were used as antigen respectively. Influenza vaccines antigens were determinedby detecting serum IgG and inhibition titers (HI), and mucosa sIgA in nasal turbinatesand lung lavage. The results showed that the immunogenicity of influenza split vac-cine was much better than the whole influenza virus vaccines.(3) Vaccine adjuvants.IL-1β, IL-18, IL-33, soluble ViE were used as an adjuvant, respectively. The quater-nized chitosan hydrogel, influenza split virus antigen and adjuvant mixed. Mice were immunized on0day and one boost on14days, with4μg antigendose,2μgadjuvant,the volume ratio of hydrogel and antigen-adjuvent was1:1. Influenza vaccines adju-vants were determined by detecting serum IgG and inhibition titers (HI), and mucosasIgA in nasal turbinates and lung lavage. The results showed that the immunogenicityof influenza split vaccine combined with IL-1β was much better than, IL-18, IL-33,soluble ViE.In the second part, we prepared and purified the recombinant proteins IL-1β. Weisolated the white blood cells in the peripheral blood of healthy people and extractedthe total RNA, then the human IL-1β gene sequences were amplified by RT-PCR. Theexpression vector pET-24a(+)-IL-1β were constructed and highly expressed in E. coliDH5α. The expression products were isolated and purified by optimizing its expres-sion, synthesis and purification conditions. The final protein concentration, biologicalactivity, efficacy and safety of the adjuvant were evaluated. These results showed thatthe optimal incubation temperature for the synthesis of IL-1β is30°C, the incubationtime is24h, preliminary purification of NaCl elution concentration is20mmol, fi-nal protein concentration is2.25mg/mL, purity is up to94.5%, the biological activi-ty is109/mL. Next we prepared influenza H1N1virus vaccine combined with quater-nized chitosan hydrogel and IL-1β. Meanwhile, commercial IL-1β proteins were usedas control. Mice were intranasally immunized on0day and one boost on14days.IL-1β adjuvants were determined by detecting serum IgG and inhibition titers (HI),and mucosa sIgA in nasal turbinates and lung lavage. The results demonstratedthat IL-1β adjuvant could enhance the immunogenicity of influenza split vaccine.The third part focused on the preparation and evaluation of nasal trivalent influ-enza split vaccine. Based on the first part, we prepared the nasal trivalent influenzasplit vaccine by using quaternized chitosan hydrogel as vaccine carrier, IL-1β adju-vant. Firstly, monovalent influenza H1N1, H3N2and B split vaccine and trivalent in-fluenza vaccine were prepared, and the volume ratio of hydrogel and antigen-adjuventwas1:1. Mice were intranasally immunized on0day and one boost on14days. Serawere collected at0,2and4week, and mucosal samples were taken2week after boostimmunization. Immune response and cross-protection of influenza vaccines were de-termined by detecting serum IgG and inhibition titers (HI), and mucosa sIgA in nasalturbinates and lung lavage. The results showed that the serum IgG and HI titers in tri-valent influenza vaccines were higher than that of monovalent groups. in trivalent in-fluenza vaccines were higher than that of monovalent groups. These data suggested that the immunogenicity of nasal trivalent influenza vaccine was better than intra-muscular vaccine because nasal administration could induce mucosal and cellularimmunity although humoral immunity was less than intramuscular injection.In conclusion, in this paper influenza vaccine formulation were screened and in-vestigated from antigen, adjuvant, delivery carrier and immune response. From thesedata we found that quaternized chitosan hydrogel could induce humoral and cellularimmunity much better than chitosan, quaternized chitosan, chitosan hydrogel; theimmunogenicity of influenza split vaccine was much better than the whole influenzavirus vaccines; immunogenicity of influenza split vaccine combined with IL-1β wasmuch better than, IL-18, IL-33, soluble ViE. Secondly, we prepared and purified therecombinant proteins IL-1β. And biologicalactivity and adjuvant effects were verified.Finally, we found that nasal trivalent influenza vaccine combined with rIL-1β andquaternized chitosan hydrogel is not able to induce mucosal immune response, butalso able to induce humuroal and cellular immune responses. These experiments willcontribute to development of nasal trivanlent influenza vaccine combined with hy-drogel. Furthermore, it will provide a theoretical basis for other nasal vaccine withhydrogel adjuvants. |
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