Efficient production of hemagglutinin stem region from Pichia pastoris for development of universal influenza vaccine

• Main Authors: Shih-Chi Wang, 王世吉
• Other Authors: Chi-Huey Wong
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/36q8ru

博士 === 國立陽明大學 === 生化暨分子生物研究所 === 107 === More than 500,000 people worldwide are killed annually by seasonal influenza. The disease causes an economic burden for millions. Vaccination has been the most effective method to protect human from influenza virus-induced morbidity and mortality, especially in elders and infants. The traditional influenza vaccines are based on the influenza viruses produced in eggs. The major limitation of method is the time-consuming production process and the safety concern for persons with chicken egg protein allergy. An outbreak of novel influenza virus A/California/04/2009 in 2009 revealed the disadvantage of the traditional influenza vaccine. How to produce influenza vaccine in more economic and efficient way is very important. The specific aim of this thesis is development of hemagglutinin (HA) stem based universal vaccine. For this purpose, we produced H1N1 A/Brisbane/59/2007 HA stem region that is highly conserved region by Pichia expression system. We further use fed-batch fermentation to increase the productivity of stem region. The yield is able to reach 100 mg/L. This way is more rapid and economic. We also modify the glycoforms of HA stem region to compare the structure and stability. The immunogenicity of HA stem protein in various glycoforms was further investigated and compared. Immunizing mice with HA stem protein (the fully glycosylated form or mono-glycosylated form) which can elicit the cross-reactive antibodies to bind H1 and H5 protein. The cross-reactive antibodies also can neutralize the H1N1 and H5N1 virus. The mono-glycosylated form of the HA stem produced in yeast, together with the glycolipid C34 as the adjuvant, can elicit greater antibody-dependent cellular cytotoxicity (ADCC) responses through FcγRⅣ. In the animal challenge study, immunization with HA stem region can offer cross protection against infections with H1N1 and H5N1 virus. The strategy may help establishing a platform to produce an effective and economic influenza vaccine to prevent influenza pandemic. This way provides alternative option apart from traditional influenza vaccine.