Development of an Inactivated H7N9 Subtype Avian Influenza Serological DIVA Vaccine Using the Chimeric HA Epitope Approach

DIVA vaccine is a useful tool for eradicating avian influenza, especially for highly pathogenic avian influenza. Several different DIVA strategies have been proposed for avian influenza inactivated whole-virus vaccine, involving the neuraminidase (NA), nonstructural protein 1 (NS1), matrix protein 2 ectodomain (M2e), or HA2 gene.

AS03-adjuvanted H7N9 inactivated split virion vaccines induce cross-reactive and protective responses in ferrets

Human infections with avian H7N9 subtype influenza viruses are a major public health concern and vaccines against H7N9 are urgently needed for pandemic preparedness. In early 2013, novel H7N9 influenza viruses emerged in China that caused about 1600 human cases of infection with a high associated case fatality rate. In this study, two H7N9 split virion vaccines with or without AS03 adjuvant were tested in the naive ferret model. Serological analyses demonstrated that homologous hemagglutination inhibition and microneutralization antibody titers were detectable in the ferrets after the first immunization with the AS03-adjuvanted vaccines that were further boosted by the second immunization. In addition, heterologous antibody titers against older H7 subtype viruses of the North American lineage (H7N7, H7N3) and newer H7 subtype viruses of the Eurasian lineage (H7N9) were detected in the animals receiving the AS03-adjuvanted vaccines. Animals receiving two immunizations of the AS03-adjuvanted vaccines were protected from weight loss and fever in the homologous challenge study and had no detectable virus in throat or lung samples. In addition, microscopic examination post-challenge showed animals immunized with the AS03-adjuvanted vaccines had the least signs of lung injury and inflammation, consistent with the greater relative efficacy of the adjuvanted vaccines. In conclusion, this study demonstrated that the AS03-adjuvanted H7N9 vaccines elicited high levels of homologous and heterologous antibodies and protected against H7N9 virus damage post-challenge.

Sequencing of clinical samples reveals that adaptation keeps establishing during H7N9 virus infection in humans

The H7 subtype avian influenza viruses (AIV) have a much longer history and their adaptation through evolution pose continuous threat to humans 1. Since 2013 March, the novel reasserted H7N9 subtype have transmitted to humans through their repeated assertion in the poultry market. Through repeated transmission, H7N9 gradually became the second AIV subtype posing greater public health risk after H5N1 2,3. After infection, how the virus tunes its genome to adapt and evolve in humans remains unknown. Through direct amplification of H7N9 and high throughput (HT) sequencing of full genomes from the swabs and lower respiratory tract samples collected from infected patients in Shenzhen, China, we have analyzed the in vivo H7N9 mutations at the level of whole genomes and have compared with the genomes derived by in vitro cultures. These comparisons and frequency analysis against the H7N9 genomes in the public database, 40 amino acids were identified that play potential roles in virus adaptation during H7N9 infection in humans. Various synonymous mutations were also identified that might be crucial to H7N9 adaptation in humans. The mechanism of these mutations occurred in a single infection are discussed in this study.

Designing a chimeric subunit vaccine for influenza virus, based on HA2, M2e and CTxB: a bioinformatics study

Background Type A influenza viruses are contagious and even life-threatening if left untreated. So far, no broadly protective vaccine is available due to rapid antigenic changes and emergence of new subtypes of influenza virus. In this study, we exploited bioinformatics tools in order to design a subunit chimeric vaccine from the antigenic and highly conserved regions of HA and M2 proteins of H7N9 subtype of influenza virus. We used mucosal adjuvant candidates, including CTxB, STxB, ASP-1, and LTB to stimulate mucosal immunity and analyzed the combination of HA2, M2e, and the adjuvant. Furthermore, to improve the antigen function and to maintain their three-dimensional structure, 12 different linkers including six rigid linkers and six flexible linkers were used. The 3D structure model was generated using a combination of homology and ab initio modeling methods and the molecular dynamics of the model were analyzed, either. Results Analysis of different adjuvants showed that using CtxB as an adjuvant, results in higher overall vaccine stability and higher half-life among four adjuvant candidates. Fusion of antigens and the CTxB in the form of M2e-linker-CTxB-linker-HA2 has the most stability and half life compared to other combination forms. Furthermore, the KPKPKP rigid linker showed the best result for this candidate vaccine among 12 analyzed linkers. The changes in the vaccine 3D structure made by linker insertion found to be negligible, however, although small, the linker insertion between the antigens causes the structure to change slightly. Eventually, using predictive tools such as Ellipro, NetMHCpan I and II, CD4episcore, CTLpred, BepiPred and other epitope analyzing tools, we analyzed the conformational and linear epitopes of the vaccine. The solubility, proteasome cleavage sites, peptidase and potential chemical cutters, codon optimization, post translational modification were also carried out on the final vaccine. Conclusions It is concluded that M2e-Linker-CTxB-Linker-HA2 combination of chimeric vaccine retains its 3D structure and antigenicity when KPKPKP used as linker and CTxB used as adjuvant.

Evaluation of the Viricidal Efficacy of Four Chemical Disinfectants against Emerged H7N9 Avian Influenza Virus in China

Background: Establishment of scientific disinfection measures in domestic poultry farms and live-poultry markets (LPMs) are critical to prevent the spread of H7N9 subtype avian influenza that outbreaks in poultry and infection in humans. The current study was aimed to evaluate the efficacy of four common disinfectants to inactivate H7N9 subtype avian influenza virus (AIV). Methods: According to the disinfection technical specification, the average inactivation logarithmic values of four disinfectants against the epidemic H7N9 subtype AIV, including deciquam solution, glutaral and benzalkonium bromide solution, sodium dichloroisocyanurate powder, and peroxyacetic acid solution, were systematically evaluated. Result: Our data showed that incubation of 0.005% (w/v) deciquam solution for 30 min contact time, or 0.05% (v/v) glutaral and benzalkonium bromide solution for 10 min, or 0.24 g/L sodium dichloroisocyanurate powder for 30 min, or 0.05% (v/v) peroxyacetic acid solution for 10 min, can completely inactivate H7N9 subtype AIV. These results suggested that glutaral and benzalkonium bromide solution as well as peroxyacetic acid solution at recommended concentrations can be effectively used to cut off the spread of H7N9 subtype AIV in poultry farms and LPMs, however, deciquam solution and sodium dichloroisocyanurate powder need to increase their working concentration.