Antigen-Capture Elisa For Specific Detection Of2009Pandemic H1N1Influenza Virus And Development Of A Broadly Cross-Protective Swine Influenza Vaccine

Influenza is one of the major threats to global public health. Influenza viruses escape from host immune system and transmit among host species through the accumulation of changes on antigenic sites (antigenic drift) and genetic reassortment of different strains (antigenic shift). The2009swine-origin influenza A H1N1virus (hereafter referred to as pH1N1) resulted in the4th influenza global pandemic in human history. pH1N1transmit more efficiently in humans than that of seasonal influenza viruses, and transmission of pH1N1from human to pigs has also been reported. Although the pH1N1virus is now considered to be post-pandemic, there are still possibilities that the virus recombines with other influenza viruses in pigs then yielding a novel potential epidemic or pandemic strain. It is therefore necessary to establish accurate and specific virus detection and serological diagnostic methods to monitor the spread of virus and the infection state of different hosts, which may provide useful information in predicting the next influenza pandemic.In this study, we established virus-and neutralization antibody-detection methods from the perspective of etiology and serology, respectively. A pH IN1-specific monoclonal antibody (mAb) was first developed based on a baculoviurs surface disply system, the epitope of the mAb was then identified. Results indicated that60KLRG63motif on hemagglutinin (HA) protein is the minimal requirement for the reactivity of the epitope recognized by mAb. Whole virus-positive mouse serum did not reacted with this epitope, suggesting that the epitope is not the immunodominant epitope of HA protein, thus the immune pressure posed by the hosts against the epitope is much less than that against immunodominant epitope, and thereby the identified epitope do not tend to shift. Further, conservation analysis of epitope showed that this epitope was pH1N1-specific, suggesting that the mAb is suitable material for virus-detection. An antigen-capture ELISA (AC-ELISA) method was developed in combinnation with rabbit HA-specific polyclonal antibody. Working dilution of each antibody were optimiezed. Sensitivity assay showed that AC-ELISA was at least8times more sensitive than hemagglutination test. Besides, specificity assay demonstrated that human seasonal H1NI, swine influenza virus (SIV) H1N1and H3N2can not be detected by AC-ELISA. The AC-ELISA was then employed to test the experimental infection samples, it was found that the results of AC-ELISA showed high agreement rate with virus isolation (VI), but was less sensitive than VI. It is conclued that the pH IN1-specific AC-ELISA represents a valuable tool for virus-detection.A recombinant plasmid expressing HA gene of pH1N1A/Califorina/04/2009was co-transfected into human embryonic kidney293T cells with retroviral vector pHIT111and pHIT60to construct Murine leukemia virus (MuLV) pseudotyped virus. The pseudotyped virus infected Madin-Darby canine kidney (MDCK) and293T cells as indicated by the detection of the P-galactosidase as reporter gene. Furthermore, the infections were neutralized by the recombinant pHlN1virus-positive serum, suggesting that the HA-pseudotyped particles can mimic the surface of influenza virions. The pseudotyped virus reported here represents a powerful surrogate for pH1N1-specific neutralization antibody detection, which may be useful in assessing hosts’infection state, screening the antiviral regents, as well as developing vaccines. In addition, we constructed pseudotyped virus with similar infection property with wild type viruses by using the synthetized HA gene which is available in public database, thus, theoretically, other pseudotyped virus can also be constructed based on the method reported here, without the need of viral isolation and identification, especially for highly pathogenic and contagious ones.Pigs play important roles in the ecology of influenza viruses, and could be the source of new strains since pigs express sialic acid receptors for both mammalian and avian strains of influenza viruses on their tracheal epithelial cells. The control of swine influeza (SI) is helpful to reduce the significant health risk for the human population by reducing virus shed and thereby transmission. Vaccination is considered to be the most effective method to control SI. Currently, commercially available swine influenza vaccines are adjuvanted, whole-virus killed vaccines containing of both HI and H3subtypes. Unlike human influenza vaccine, strains used for SI vaccine preparation are not regularly replaced, making the vaccines do not provide consistent protection from infection, because the viruses continuously evolve by antigenic drift and shift. Cross-protective SI vaccine designed based on the conerved viral proteins would be of great help to solve this problem. To this end, we constructed two forms of DNA vaccines, one was made by fusing M2e to consensus H3HA (MHa), which represents the majority of the HA sequences of H3N2viruses, another was made by fusing M2e and a conserved CTL epitope (NP147-155) to consensus H3HA (MNHa). BALB/c mice were immunized twice with the two DNA vaccines via gene gun. It was shown that the two vaccines elicited substantial antibody responses, and MNHa induced more significant T cell-mediated immune response than MHa did. Then two H3N2strains representative of different evolutional clusters were used to challenge the vaccine-immunized mice. Results indicated that both of the two DNA vaccines prevented homosubtypic virus infections completely, suggesting the ehanced T cell-mediated immune response may be non-essential for homologous protection. The vaccines’heterologous protective efficacies were further tested by challenging with a H1N1SI strain and a reassortant pH1N1strain. It was found that MNHa, but not MHa, induces immune responses partially protect against heterologous influenza infection as indicted by the reduced lung viral titer and less lung histopathological damage, suggesting that CTL responses play important roles for heterologous protection. Reults of the current study indicate that strengthening CTL responses are promising ways for universal influenza vaccines development.