The effect of different adjuvants on immunogenicity of H1 subtype influenza hemagglutinin virus-like particles

• Main Authors: Hong-ying Chen, 陳宏穎
• Other Authors: Wen-Jen Yang
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/65271751506192337010

碩士 === 國立高雄大學 === 生物科技研究所 === 101 === Adjuvant is an immune stimulator which can assist the antigen presenting cells (APC) to endocytose antigens more easily, and present the antigen to lymphocytes recognized specifically to enhance immune response. Previous studies in our laboratory showed that the structural protein of respiratory tract toxin produced by Actinobacillus pleuropneumonia has the potential of as a mucosal adjuvant. According to the functional domain of RTX (repeats in toxin) family toxin, the full length, N-terminal and C terminal fragment of ApxIIA gene was cloned, expressed and purified, respectively. ApxIIA-N has been showed that had the weakest toxicity and could significantly enhance the titer of IgG1 and IgA, but limited in IgG2a. In this study, a new generation of subunit influenza vaccine called virus-like particles (VLPs) which maintained the structure and immunogenicity of virus but lack of viral genetic materials. The virus-like particles was H1 subtype of hemagglutinin (HA) produced from influenza strain A/PR/8/34(H1N1). Influenza viruses usually infect the upper respiratory tract via the tracheal mucosa of host. Therefore, ApxIIA proteins were used as adjuvant to evaluate the adjuvanticity for VLPs subunit vaccine. In this study, mice were administrated with different dosages of ApxIIA-N, ApxIIA-C by intranasal and intramuscular injections. The HI antibody titers of adjuvant-containing groups via intranasal immunization were over 40 on day 35 after immunization. The results showed that the highest HI titers were reached on day 49 after immunization. On the other hand, intramuscular injection results revealed that HI antibody titers over 40 on day 28 after immunization, and the highest HI titers were reached on day 49 after immunization. Furthermore, the HI antibody titers were still over 40 on day 150 after immunization. The IgG antibody titers of adjuvant-containing groups on day 49 after immunization via intranasal and intramuscular route were 102400 and 204800, respectively. Antibody subtype analyses of IgG1 and IgG2a showed that all of the adjuvant-containing groups via intranasal or intramuscular immunization with increasing in IgG1 levels significantly. It indicates the major pathway induced by adjuvant was Th2. The body weight change of mice between adjuvant-containing groups and control group showed no significant difference. It indicates that the adjuvant was safety for mice. The splenocyte proliferation and Th1 (IL-12, IFN-γ), Th2 (IL-4, IL-6) expression analyses showed that the splenocytes of adjuvant- containing goups were proliferated by specific antigen stimulation and 30μg ApxIIA-N immunized group obtained the strongest proliferation. The results showed that ApxIIA-N could induce immune response toward to Th2 pathway. Analysis of IgA titer in bronchoalveolar lavages fluid (BALF) showed that all ApxIIA adjuvant- containing groups could effectively enhance IgA secretion. All of the results indicated that ApxIIA protein has adjuvant function and could stimulate mucosal immune response. The best immune response could be received through intramuscular immunization with 30μg ApxIIA-N.