Influence Of Immune Enhancer On H9N2 Subtype Avian Influenza Vaccine

The H9N2 avian influenza virus give threaten to the poultry industry development. Several H9 subtype vaccines were developed based on the different virus strains in China. However, the poor cross protection efficacy of these vaccines against new emergency variants is challenge to control this disease spread. The reports of the broad spectrum peptide vaccine based on M2e provided another way to prevent disease. Other efficient strategy was addition of immunopotentiator in vaccine to improve immune efficacy. A immunopotentiator (CVC2305) have been screened in previously research. The target of this study was comprehensively assessment the efficacy of immunopotentiator (CVC2305) on H9 subtype influenza vaccine and M2e peptide vaccine.1. The efficacy of the immunopotentiator CVC2305 on the inactivated H9 subtype avian influenza vaccine.This research was explored the efficacy of immunopotentiator CVC2305 on the inactivated H9 subtype avian influenza (AI) vaccine. Three groups of specific pathogen free (SPF) chickens were vaccinated with the H9 subtype AI vaccine combination with CVC2305 (H9-CVC2305 AI vaccine), the H9 subtype AI vaccine only (H9 AI vaccine), respectively, and the remains group was severed as control. The H9 subtype vaccine was prepared with the virus strain of A/Chicken/NJ/02/2001. The CD3+/CD4+and CD3+/CD8+ T lymphocyte subsets were analysis via flow cytometer were at 1、3、5and7 d post vaccination (dpv). The cross presentation of the lymphocyte post vaccination were analysis by indirect immunofluorescence via confocal microscopy. The earlier protection efficacy of the CVC2305 immunopotentiator was assessed by challenge with heterogeneous viruses A/Chicken/SD/01/2011 (SD01/11) strain at 3 dpv. The lymphocyte adoptive transfer assay was carried out to evaluate the efficacy of the CVC2305 immunopotentiator on B19/B19 inbred SPF chicken. The spleen lymphocyte of the donor inbred chickens, which were vaccinated with the H9 AI vaccine only or mixture with the CVC2305 and a control group, were transferred to unvaccinated receptor chicken. The latter were challenged with SD01/11 virus 24 hour post-vaccination. The FACS results indicated that the ratio of CD3+/ CD4+ and CD3+/CD8+T lymphocyte subsets in birds of the H9-CVC2305 AI vaccine group were higher than that of the H9 AI vaccine group, and the ratio was slightly increasing follow time lapse. The birds in the H9-CVC2305 AI vaccine group, the results of the hemagglutinin inhibition antibody titer at 2、3and 4 week post-vaccination, and intensity of fluorescence in cross presentation, heterogeneous virus challenge results at 3dpv, were higher than that of the H9 AI vaccine group. Similar results were showed in the lymphocyte adoptive transfer assay. Birds in H9-CVC2305 group showed higher protection efficacy than that in H9 vaccine.Combined the results together indicated that the immunopotentiator of CVC2305 improve the H9 vaccine efficacy via humoral antibody and cell-mediated immune response2.The efficacy of the immunopotentiator CVC2305 on the M2e(H9) peptide vaccineThe conserved H9 subtype avian influenza virus M2e sequence was selected and peptide were synthesized and prepared as water in oil vaccine (M2e). The addition of CVC2305 in the peptide water in oil severed as another vaccine (M2e-CVC2305). The other control groups were included the H9 subtype commercial avian vaccine (H9 AI vaccine) and blank control. Four groups of 2 week old SPF chicken were immunized with different vaccine recipes, the M2e-CVC2305 and M2e vaccine groups were injection three times with 2 week intervals, the H9 commercial vaccine group was injected only one time at 2 week old, and control group was unvaccinated. The blood was collected at 2 week intervals from the first time vaccination and until 8-week post-first time vaccination. The birds in all groups were challenged with heterogeneous virus (SD01) at 8 week post-first time vaccination. Serum antibody were analysis by several methods, including ELISA detection antibody against M2e, neutralization serum titer against homologous virus (NJ02) in virus-serum neutralization assay on MDCK cell line, serum binding capability against the NJ02 virus infected MDCK cell line with FACS analysis the fluorescence, and serum titer against NJ02 in indirect fluorescence assay. The ELISA antibody titers against M2e in birds of M2e-CVC2305 group were higher than that of M2e group, H9 group and control group. However, the virus-serum neutralization titers against NJ02 in bird of H9 commercial vaccine group were higher than that of M2e-CVC2305 group, that the latter higher than M2e group and control group. Results of the FACS analysis the intensisty of the fluorescence, indirect fluorescence assay and heterogeneous virus challenge showed similar results as the virus-serum neutralization assay. The study indicated that the immune efficacy of M2e peptide vaccine was improved via the immunopotentiator CVC2305, the latter showed prosperity in application of peptide vaccine.