Improved Whole Gamma Irradiated Avian Influenza Subtype H9N2 Virus Vaccine Using Trehalose and Optimization of Vaccination Regime on Broiler Chicken

Abstract

Gamma (γ)-radiation can target viral genome replication and preserve viral structural proteins compared to formalin inactivation. Thus, a stronger immunity could be induced after the inoculation of the irradiated virus. In this study, γ-irradiated low-pathogenic avian influenza virus-H9N2 (LPAIV-H9N2) was used to immunize the broiler chicken in two formulations, including γ-irradiated LPAIV-H9N2 with 20% Trehalose intranasally (IVT.IN) or γ-irradiated LPAIV-H9N2 plus Montanide oil adjuvant ISA70 subcutaneously (IV+ISA.SC) in comparison with formalin-inactivated LPAIV-H9N2 vaccine intranasally (FV.IN) or formalin-inactivated LPAIV-H9N2 plus ISA70 subcutaneously (FV+ISA.SC). Two vaccination regimes were employed; the first one was primed on day 1 and boosted on day 15 (early regime), and the second one was primed on day 11 and boosted on day 25 (late regime). A challenge test was performed with a live homologous subtype virus. Virus shedding was monitored by quantifying the viral load via RT-qPCR on tracheal and cloacal swabs. Hemagglutination inhibition (HI) antibody titration and stimulation index (SI) of the splenic lymphocyte proliferation were measured, respectively, by HI test and Cell Proliferation assay. Cytokine assay was conducted by the RT-qPCR on antigen-stimulated spleen cells. The results of the HI test showed significant increases in antibody titer in all vaccinated groups, but it was more evident in the IVT late vaccination regime, reaching 5.33 log2. The proliferation of stimulated spleen lymphocytes was upregulated more in the IVT.IN vaccine compared to other vaccines. The mRNA transcription levels of T-helper type 1 cytokines such as interferon-gamma (IFN-γ) and interleukin 2 (IL-2) were upregulated in all vaccinated groups at the late regime. Moreover, IL-6, a pro-inflammatory cytokine was upregulated as well. However, upregulation was more noticeable in the early vaccination than the late vaccination (p< 0.05). After the challenge, the monitoring of virus shedding for the H9 gene represented an extremely low viral load. The body weight loss was not significant (p > 0.05) among the vaccinated groups. In addition, the viral load of <100.5 TCID50/ml in the vaccinated chicken indicated the protective response for all the vaccines. Accordingly, the IVT vaccine is a good candidate for the immunization of broiler chicken via the intranasal route at late regime.

Influenza Virus Inactivated by Heavy Ion Beam Irradiation Stimulates Antigen-Specific Immune Responses

“…In fact, CD8 T cells were stimulated following vaccination of mice with the irradiated virus, recognizing the MHC class I-restricted ASNETNMETM peptide of the nucleoprotein. Notably, this finding is in line with the finding of Motamedi Sedeh et al that cell-mediated immune responses stimulated following immunization with irradiated vaccine candidates were prior to those observed following vaccination using chemically inactivated viruses [48]. Similar results were obtained when chickens were immunized to compare irradiated and formalin-treated vaccine candidates [43].…”

Vaccination with recombinant Lactococcus lactis expressing HA1-IgY Fc fusion protein provides protective mucosal immunity against H9N2 avian influenza virus in chickens

“…At present, inactivated H9N2 vaccines have demonstrated high efficacy in protecting against clinical disease, but variable results have also been observed in reducing the mortality and the level of viral shedding in chickens. It was reported that H9N2 AIV causes no death in chicken after intranasal challenge in control group [ 46 ] and no virus shedding in inactivated vaccine vaccination group [ 47 ]. However, other studies have shown that H9N2 AIV can cause death in chicken after intranasal challenge [ 48 – 50 ], and intranasal challenge of H9N2 virus resulted in different degrees of virus shedding in the inactivated vaccine group [ 46 , 50 – 52 ], which was consistent with our results.…”

Immune Responses Following Administration of Whole Gamma Irradiated SARS-CoV-2 Vaccine Stabilized With Disaccharide Trehalose on Syrian Hamster Model

“…The BrdU kit allows accurate assessment of cell proliferation by measuring BrdU incorporation into newly synthesized cellular DNA. Absorbance was measured at 450 nm, and the stimulation index (SI) was calculated as the mean OD of three wells corresponding to cells stimulated with an antigen divided by the mean OD of the triple-negative control wells [13,17,19].…”