Study On The Supplement That Enhances Heterogeneous Protection Of Avian Influenza Inactivated Vaccine

H5N1, a highly pathogenic avian influenza virus(HPAIV), poses a significant threat to poultry and human health. Vaccination is the most effective strategy for preventing and controlling avian influenza. The currently available inactivated influenza vaccines, which induce strong neutralizing antibodies specific to the highly variable surface glycoprotein hemagglutinin(HA), provide poor protection against heterologous strains. In the past few years, the Chinese Government has introduced a series of H5N1 influenza vaccines derived from different H5 HA phylogenetic clades to control HPAIV H5N1, but it cannot keep up with the speed of viral mutation. Therefore, it is of high priority to develop a supplement that enhances heterogeneous protection of inactivated vaccine.It has been reported that a combination of multiple conserved B- and T-lymphocyte epitopes in a single construct should stimulate both the humoral and cellular immune responses. The region of HA corresponding to amino acid 76–130 of the H5N1 subtype demonstrated the high degree of conservation in this sequence. A synthetic peptide vaccine based on amino acids 76–130 of the HA2 subunit afforded protection to mice against multiple subtypes of influenza viruses. The ectodomain of matrix 2(M2e) of the influenza A virus, a highly conserved antigen, has been extensively studied in universal vaccine research. Nucleoprotein amino acids 55–69(NP55–69) and 380–393(NP380–393) stimulate helper T lymphocytes(Th cells) and cytotoxic T lymphocytes(CTLs), respectively, and contributed to cross-protection. Although epitope vaccines have been widely demonstrated to elicit broad immune responses, strategies based on epitopes alone have shown limited protection against H5N1 HPAIV challenge. For these reasons, coimmunization with an epitope antigen and an inactivated H5N1 vaccine might be a feasible strategy with which to counter heterologous H5N1 viruses.In this study, we we chose four highly conserved antigen epitopes: HA2 76–130, M2 e, NP55–69 and NP380–393(HMNN). The HMNN gene containing flexible linker sequence was synthesized and cloned into downstream of the polyhedrin promoter(PPH) in the p Fast Bac-VSV-G baculovirus transfer vector to generate p Fast-G-HMNN. Recombinant baculovirus(BV-HMNN) were produced according to the manufacturer’s manual using the Bac-to-Bac? Baculovirus Expression System.In order to assess the protection efficacy that coimmunization with recombinant baculovirus and an inactivated H5N1 vaccine against heterologous virus. SPF chickens were immunized intramuscularly(i.m.) with 0.1 m L(1010 pfu/m L) BV-HMNN and 0.3 m L Re-6 inactivated vaccine corresponding to clade 2.3.2.1(Re-6+BV-HMNN). The other two groups were immunized with wild type baculovirus plus Re-6 inactivated vaccine(Re-6+BV-WT) or Re-6 inactivated vaccine(Re-6). For negative control, chickens were immunized with PBS alone(Mock). Three weeks after a single immunization, the chickens were challenged with A/goose/guangdong/14079/2013(GD/14079, H5N1) from clade 2.3.4.4. The results indicate that BV-HMNN antigen significantly enhance M2e-specific antibodies not influence HI titers when coimmunization with oil-based inactivated Re-6 vaccine. The survival rate of the Re-6+BV-HMNN immunized chickens was 100% while the Re-6 group showed only 50% survival protection against lethal challenge with heterologous GD/14079 virus. In the viral shedding, at 3、5 and 7 day post infection(dpi), no virus was isolated from the oropharyngeal or cloacal swabs of the chickens immunized with Re-6+BV-HMNN. However, in the Re-6 group, one chicken was detected viral shedding at 5 dpi, and three chickens at 7 dpi. On day 3 after viral challenge, oropharyngeal and cloacal viral shedding was detected in the unvaccinated chickens(PBS group), a peaking at 5 dpi. Chickens immunized with Re-6+BV-WT showed improved protection, decreased viral titer compared with that afforded the Re-6 group.In conclusion, our results suggest that BV-HMNN has potential utility as a supplement to inactivated H5N1 vaccines which has great significance for the prevention of H5N1 HPAI and the influenza pandemic.