Study For Immune Enhancer For H9N2Subtype Avian Influenza Inactivated Vaccine

The avian influenza viruses (AIVs) subtype H9N2caused enormous economic losses in chicken-breeding industry in China. Several H9subtype AIVs vaccines have been developed. However, AIVs subtype H9remains co-circulating and new mutants emergence continuously. Besides renewal of virus vaccine candidate, some other techniques may adapted to enhance the ready-made vaccine efficacy.Immunopotentiators are bioactive material to improve vaccine efficacy. Numerous Immunopotentiators have been developed in human vaccines. The Immunopotentiators are capable of stimulating the innate immunity and adaptive immunity, and provide a new way to develop AIVs subtype H9vaccines.1. Screening of Immunopotentiators for the Avian Influenza(H9N2)Eight groups of twenty14-day old SPF chickens were included in this trial. Six types of Immunopotentiators were mixed with AIVs subtype H9vaccine (strain NJ/02) and vaccinated chickens, respectively. One out of two remaining group birds were only immunized H9subtype of AIVs vaccine (NJ/02), and another group as control. Bleeding at2-week,3-week and4-week post-vaccination (wpv), and serum titers were measured by hemagglutinin inhibition (HI) test. The chickens were challenged with virus strain SD01containing107.0EID50. Oropharyngeal and cloacal swab samples were collected at3-,5-and7-day post-challenge (dpc) to detect virus shedding. Both swab samples of each bird were pooled and inoculated three10-day old embryonated chicken eggs for virus isolation.HI titers of bird serum from immunopotentiators containing vaccine groups were significantly higher than that of the conventional vaccine group. Moreover, efficacy on HI titers of immunopotentiators VA1, VA2and VA6group were superior to that of other three groups. Virus shedding isolation from six immunopotentiators containing vaccine groups were lower than the conventional vaccine group. Efficacy on inhibtion virus shedding of VA1, VA2and VA6were still superior to that of other three groups. Taken together, six immunopotentiators for avian influenza subtype H9N2inactivated vaccine were developed, and efficacy of VA1, VA2and VA6better than other three types.2. Establishment of Real-Time PCR to detect IFN-y, IL-4and IL-2Primers of chicken ACTB, IFN-γ, IL-4and IL-2were designed according to gene sequences published in GenBank. The target gene fragments were got by RT-PCR from chicken peripheral lymphocytes, and connected with pMD19-T vector after sequencing to verify its correctness.The plasmids were used as the standard sample of the Real-Time PCR.Primers of chicken ACTB, IFN-γ, IL-4, and IL-2for Real-Time PCR were designed, the primers concentration,annealing temperature and plasmid template concentration were optimized respectively. These is no significant effect on the melting curves and amplification curves of amplification product with0.2-1.0ul20pmol/ul primer.The the amplifiction efficiency of the ACTB, IFN-Y, IL-4and IL-2are92.1%and100.2%,98.0%,103.0%respectively. Their correlation coefficient R2was greater than0.99. So, the Real-Time PCR reaction system established possess good amplification efficiency, specificity, reproducibility, stability and sensitivity for the template concentration range of10-10copies/ul. Therefore, the Real-Time PCR reaction system protocol is as follow:the template concentration of the10-10copies/ul, theprimer concentration of0.5ul (20pmol/ul), the annealing temperature of60℃, and40cycles.3. Effect of immunopotentiator VA5on expression of IFN-γ、IL-4and IL-2The spleen lymphocytes were isolated from the SPF chickens post vaccination with AIV subtype H9vaccine mixed with immunopotentiators VA5, and cutured with PHA stimulation.Then to optimize detection time expression of IFN-y, IL-4and IL-2were detected at2h,4h,6h,8h,10h,12h and24h after PHA stimulation respectively.SPF chickens of14days were devided into three grougs. The birds of the first group were immunized with AIV subtype H9inactivited vaccine mix with VA5immunopotentiator. The2th group was conventional AIV vaccine control.The3th group was blank control. The expression of cytokines at1,35,7and10days post vaccination were determined.the proliferation activity of the spleen lymphocytes were detected simultaneity.IFN-y and IL-2optimal detection time are at6hour, and IL-4at12hour after PHA stimulation. IFN-y expression of immunopotentiator VA5containing vaccine group peaked at the fifth day after immunization,which is30times quantitatively more than the conventional vaccine group. Up-regulating of IL-4expression is at the third days postvaccination, which is significantly higher than that of conventional vaccine group. IL-2reached quantitatively a peak until the third day after immunizationand, and then gradually declined, but it is still significantly higher than the conventional vaccine group. Lymphocyte proliferation of the VA5immunopotentiator containing vaccine group were much stronger than conventional vaccine group from the third days to10th days postvaccination.To sum up, the VA5immunostimulants can improve remarkably the immunity of AIV subtype H9inactivated vaccine,because immunostimulants can promote animal cellular and humoral immune effectively and efficiently.