Human Monoclonal Antibodies And Epitope-based Peptide Vaccine Research On Hemmagglutinin Protein Of Novel Swine-origin H1N1 Influenza A Virus

Influenza virus, as an ancient virus, presents a significant and persistent threat to public health and killed millions of people worldwide, especially three global influenza pandemic in the last century. In this century, the outbreak of novel swine-origin H1N1 influenza A virus pandemic worldwide, resulted in global panic.Despite the inactivated influenza vaccine developed for control and prevention of this novel virus strain. However, at present the learning for the novel influenza H1N1 virus and its pathogenic mechanism of the immunological research is still poorly understood. Haemegglutinin (HA), as one of the most major surface antigen for viral infection, has a great significance for H1N1 virus research.In this study, recombinant H1N1 HA and NA proteins were expressed in two baculovirus insect expression system. As the basis of the following research:First of all, make human monoclonal antibodies from memory B cells:potent neutralization of 2009 novel H1N1 influenza virus. Using EBV transformed memory B cells, under feeder cells in the presence of CpG, culture the transformed B cells; after limited dilution, screened HA-protein specific antibody-secreting cell clones; 96U-platex 135 were screened totally, that were 12960 B cell samples; the results showed that 45 positive clones were obtained, but 36 of which were died during the culture in vitro. It indicated that EBV-transformed B cells in a large part were short-lived, and only a small part lived for a long survival (>two months). In order to avoid further loss of the positive clone cells, we try to fuse the positive cells and K6H6/B5 using PEG to obtain immortal positive cells. The hybridomas couldn't be obtained in repeated experiments. In the case of failure of fusion experiments,4 pairs of variable region cDNA of light and heavy chain were obtained from positive cell clones by single-cell clone 5'-RACE method, and then genetic engineering antibody was expressed in insect cells by the baculovirus expression system. The result showed that there's no antibody production and insect cells are not suit as host cell to produce antibody. And the better is CHO cells, which become ideal host cells for antibody expression in next step.Second, we research on novel influenza A virus hemmagglutinin-peptide based vaccine. The continuous antigenic drifts and shifts of Influenza virus HA leads to the continuous variation of the virus, and can evade the immune response. Current vaccines only provide immunity to viral isolates similar to the vaccine strain and always based the whole HA proteins, which could not protect against the infection of devise influenza virus strains. At present, the design of epitope-based vaccine is utilizing the linear epitope, including both the B-cell epitope and T-cell epitope. In this part of the study, we provided a novel strategy to mimic conformational epitope as a linear peptide-based vaccine to induce antibody response of mice. The immunogenicity and protective efficiency of the peptides were examined after vaccination using EIISA, haemegglutination inhibition (HI) and neutralization assays. We found that this novel HA peptide-based vaccine could induce strong humoral responses against H1N1 and H5N1 in mice after immunization. This novel peptide is a promising strategy for vaccine design and a candidate for eliciting antibody response against diverse subtypes of influenza virus.In summary, our findings:1. Functional HA and NA proteins were successfully expressed in two distinct bacularvirus expression systems (BaculoGold systema and Bac-to-Bac system), of which the Bac-to-Bac bacularvirus expression system is more suitable for expression of A/Sichuan/1/2OO9(H1N1) HA protein.2.9 HA-specific antibody-secreting cell clones were obtained and 4 pairs of variable region cDNA of light and heavy chains were obtained by 5'-RACE; This is the base of the next genetic engineering antibody production.3. Firstly improved single-cell clone 5'-RACE method has been used to obtain the cDNA of variable region of antibody and more efficient, compare with the single-cell 5'-RACE method.4. Novel HA peptide-based vaccine could induce strong humoral responses against H1NI and H5N1 in mice after immunization. This novel peptide is a promising strategy for vaccine design and a candidate for eliciting antibody response against diverse subtypes of influenza virus.