Immunoadjuvant Effect Of Hemagglutinating Virus Of Japan Envelope (HVJ-E)

Newcastle disease and avian influenza are globally important infectious diseases in poultry, which quickly assume epizootic proportions and serious ecomomic losses if strict, effective control measures are not implemented. Now, NDV and LPAI H9N2can be prevented and controlled by the use of vaccines. However, the commonly used adjuvants for killed vaccines (e.g.oil-emulsion and aluminum) ordinarily result in strong local reactions, including pain, inflammation and so on, or failure to augment the immunogenicity of antigen. So, it is necessary to study and develop immunoadjuvants with high efficiency and low toxicity.Hemagglutinating virus of Japan (HVJ) is known as Sendai virus, which is a kind of enveloped virus with hemagglutination and efficient membrane fusion ability. Hemagglutinating virus of Japan envelope (HVJ-E), which derived from HVJ by UV radiation, has a dual immunomodulatory effects, it can promote both innate and adaptive immunities. A few studies have demonstrated that HVJ-E can stimulate dendritic cells (DC) into maturation and promote IFNs, IL-6and other cytokines secretion. Furthermore, IL-6can inhibit the activation of regulatory T cells and promote effector T cells and NK cells production, reducing IL-10, TGF and other immunosuppressive cytokines production, which make it possess the immunostimulatory property. However, the immunoadjuvant effect of HVJ-E on vaccines has not been reported to date. So, to explore the adjuvant property of HVJ-E, the murime dendritic cell maturation was verified in the current experiment, and the killed vaccines against NDV and AIV (H9N2subtype) were co-inoculated into SPF chickens with HVJ-E, and the specific antibodies to the viruses, the cytokines production as well as the post-challenge virological profiles (virus shedding from oropharyngeal swabs and cloacal swabs) were assessed. The main studies are as follows:(1) DC maturation treated by HVJ-E:Murine DCs derived from bone (tibia and femur) marrow were generated from C57BL/6mice. The filtrate was washed and cells (2×106) sedimented by centrifugation were seeded in6-well plates in3mL of culture medium supplemented with50ng/ml of recombinant mouse GM-CSF and IL-4. Five days later, nonadherent and loosely adherent clusters of proliferating dendritic cells were used in subsequent experiments as immature dendritic cells. DC was stimulated by HVJ-E for48h, cytokine secretion in the dendritic cell supernatants was measured by ELISA, and the expression level of DC surface molecules was measured by FCM. The results indicated that the expression levels of IFN-β, IL-6, TNF-α and IFN-γ were significant higher in HVJ-E treated group vs. PBS control (p<0.05), meanwhile, higher expression level of the co-stimulatory molecules of CD40, CD80, MHC II and CD11c on DC surface were also observed. These results demonstrated that HVJ-E can promote DCs maturation and cytokines secretion, which may play active roles in immunization.(2) Immunoadjuvant effect of HVJ-E on the inactivated NDV vaccine:SPF chickens were randomly divided into four groups of twenty, the first group was immunized with killed vaccine, the second group was co-inoculated with1.5×109HVJ-E and killed virus, the third group was injected with1.5×109HVJ-E and the last group was immunized with PBS. Chicken blood samples were collected from the wing vein on day7,14and21post-immunization for sera separation and antibody detection. Three weeks after the vaccination, all chickens were challenged with the strong NDV. Then oropharyngeal and cloacal swabs were taken on day2,4,7and10post challenge for virus isolation. The results showed that the group immunized with HVJ-E and killed vaccine had higher titers than the killed vaccine group at every time-point post-immunization (p<0.05), what is more, the challenge results indicated that the chickens co-immunization of without virus shedding.(3) Immunoadjuvant effect of HVJ-E on the inactivated H9subtype Avian Influenza virus vaccine:SPF chickens were randomly divided into four groups and immunized as previously described. Blood samples were collected from the wing vein on day7,14and21post-immunization to prepare sera for antibody and cytokines determination. Blood with anticoagulant from chickens were sampled at day14and21post-immunization for PBMCs isolation, the total RNA was immediately extracted after PBMCs were isolated, then the IFN-y and IFN-(3expression levels were compared among the immunized groups using semi-quantitative RT-PCR. Three weeks after the vaccination, all chickens were challenged with the AIV H9subtype virus by intranasal administration. Oropharyngeal and cloacal swabs taken on day2,4,7and10post challenge were used for virus isolation. The results showed that, at7day post immunization, specific antibodies of chickens were detectable both in the HVJ-E added vaccine and killed vaccine group, and no difference was observed between them. While at day14and21post immunization, antibody titers in the group immunized with HVJ-E and killed vaccine had higher titers than those in the killed vaccine group (p<0.05). Meanwhile, the results were revealed that the cytokines secretion in sera and mRNA transcriptional level in HVJ-E and HVJ-E+KV group were higher than those in PBS and KV injected group (p<0.05). Furthermore, the challenge results indicated that chickens in the HVJ-E+KV group were completely protected. Taken together, these results indicated that HVJ-E could be used as an adjuvant for killed vaccines.