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The effectiveness of inactivated H9N2 influenza vaccines is doubtful due to changes in antigenic regions of the virus hemagglutinin (HA) protein. One strategy for the development of the efficacious vaccine is the use of nanoparticles that display more immunogenic regions of the influenza virus. In this study, chitosan (CS)-based nanoparticles were developed as a delivery system for intranasal immunization using recombinant H9N2 virus HA1 and nucleoprotein (NP), for the induction of humoral and cellular responses. CS-HA1 and CS-NP nanoparticles were prepared by the ionic gelation method and characterized for their physicochemical properties and shape. The immunogenicity and the protective efficacy were evaluated by measuring antibody titers, T cell proliferation response, CD4 + /CD8 + ratio, and quantitative real-time RT-PCR following intranasal administration of the prepared nanoparticles alone or in combination in chickens compared to an inactivated H9N2 vaccine. The average size, surface charge, and spherical structure of the synthesized nanoparticles showed high quality. Serologic analysis revealed that the immunization of inactivated vaccine groups resulted in strong influenza antibodies, which were significantly ( p  < 0.05) higher compared to the other groups. The vaccinated chickens with CS-HA1+CS-NP developed higher specific anti-influenza antibodies than in those vaccinated with each of rHA1 and rNP. Administration of a combination of the protein-based nanoparticles has stimulated the activation of both CD4 + and CD8 + T cells and induced a significantly higher T cell proliferation. The viral shedding was significantly lower in CS-HA1+CS-NP and inactivated vaccine groups compared with other challenged groups. The data demonstrate the potential of CS-HA1+CS-NP nanoparticles for eliciting specific influenza antibodies and conferring protection in chickens.

Utilizing subunit vaccines is one of the strategies to address influenza infection. Recent innovations have focused on high doses of vaccine antigens and immune enhancers or adjuvant to induce more robust and long-lasting immune responses. Here, an effect of the B cell-activating factor receptor (BAFF-R) to increase the magnitude and durability of immune responses of the recombinant HA1 (rHA1) protein against the H1N1 influenza virus was studied. The HA1 protein and the effector domain of BAFF-R were expressed in the pET-28a (+) vector. Eight-week-old BALB/c mice were equally grouped into five groups (n=20). The 15 and 25 μg/μL of rHA1 were mixed with 2 μg/μL of rBAFF-R and injected three times for vaccinated groups. Three control groups were received normal saline and two concentrations of rHA1. The ability of rBAFF-R in eliciting HA-specific antibody response and stimulating T lymphocyte proliferation to induce the cell-mediated immunity was assayed. Induction of protection was evaluated following the challenge with PR8 strain. Analysis of immune responses showed that the co-administration of rBAFF-R with rHA1 boosted HI responses to the antigen in mice, whilst it was not able to promote the T cell proliferation responses against influenza. Compared to rHA1alone, the rBAFF-R/rHA1 generated efficient protection for the animals. There were no significant differences in eliciting the immune responses in mice immunized with the lower dose of rHA1 than that with the higher dose. The data indicate the rBAFF-R can enhance the primary and memory immune responses to protect against influenza infection.

Immunization with DNA vaccines as a novel alternative to conventional vaccination strategy requires adjuvant for improving vaccine efficacy. The conserved immunogenic HA2 subunit, which harbors neutralizing epitopes is a promising vaccine candidate against influenza viruses. In this study, for the first time we explore the idea of using host interferon inducible Mx protein to increase the immunogenicity of HA2 H9N2 influenza DNA vaccine. The potency and safety of the Mx adjuvanted-HA2 vaccine was evaluated in BALB/c mice by different prime-boost strategies. To assess the effect of the vaccination on the virus clearance rate, mice were challenged with homologous influenza virus. Administration of the adjuvanted vaccine and boosting with the same regimen could effectively enhance both humoral and cellular immune responses in treated mice. These data demonstrated that Mx as host defense peptide can be potentiated for improving influenza vaccine efficacy., (Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Influenza viruses continue to emerge and re-emerge, posing new threats for public health. Control and treatment of influenza depends mainly on vaccination and chemoprophylaxis with approved antiviral drugs. Identification of specific epitopes derived from influenza viruses has significantly advanced the development of epitope-based vaccines. Here, we explore the idea of using HLA binding data to design an epitope-based vaccine that can elicit heterosubtypic T-cell responses against circulating H7N9, H5N1, and H9N2 subtypes. The hemokinin-1 (HK-1) peptide sequence was used to induce immune responses against the influenza viruses. Five conserved high score cytotoxic T lymphocyte (CTL) epitopes restricted to HLA-A*0201-binding peptides within the hemagglutinin (HA) protein of the viruses were chosen, and two HA CTL/HK-1 chimera protein models designed. Using in silico analysis, which involves interferon epitope scanning, protein structure prediction, antigenic epitope determination, and model quality evaluation, chimeric proteins were designed. The applicability of one of these proteins as a heterosubtypic epitopebased vaccine candidate was analyzed.

Utilizing subunit vaccines is one of the strategies to address influenza infection. Recent innovations have focused on high doses of vaccine antigens and immune enhancers or adjuvant to induce more robust and long-lasting immune responses. Here, an effect of the B cell-activating factor receptor (BAFF-R) to increase the magnitude and durability of immune responses of the recombinant HA1 (rHA1) protein against the H1N1 influenza virus was studied. The HA1 protein and the effector domain of BAFF-R were expressed in the pET-28a (+) vector. Eight-week-old BALB/c mice were equally grouped into five groups (n=20). The 15 and 25 μg/μL of rHA1 were mixed with 2 μg/μL of rBAFF-R and injected three times for vaccinated groups. Three control groups were received normal saline and two concentrations of rHA1. The ability of rBAFF-R in eliciting HA-specific antibody response and stimulating T lymphocyte proliferation to induce the cell-mediated immunity was assayed. Induction of protection was evaluated following the challenge with PR8 strain. Analysis of immune responses showed that the co-administration of rBAFF-R with rHA1 boosted HI responses to the antigen in mice, whilst it was not able to promote the T cell proliferation responses against influenza. Compared to rHA1alone, the rBAFF-R/rHA1 generated efficient protection for the animals. There were no significant differences in eliciting the immune responses in mice immunized with the lower dose of rHA1 than that with the higher dose. The data indicate the rBAFF-R can enhance the primary and memory immune responses to protect against influenza infection. [ABSTRACT FROM AUTHOR]

Background: H9N2 avian influenza viruses have the potential to become the next human pandemic threat and next generation vaccine technologies are needed. Current studies introduce nanoparticles as a proper vaccine delivery vehicle for induction of protective immunity. In this study, the efficacy of chitosan nanoparticle-based H9N2 influenza vaccine with and without hemokinin-1 (HK-1) as a molecular adjuvant to induce protective immunity against the virus was examined. Methods: The H9N2 antigen was prepared in MDCK cells and inactivated with formalin. The inactivated antigen alone and in combination with HK-1 was encapsulated into chitosan nanoparticles. Groups of BALB/c mice received chitosan nanoparticle-based H9N2 antigen alone or in combination with HK-1 in a prime/boost platform via eye drop method. To evaluate the efficacy of the adjuvanted-nanovaccine candidate, systemic antibody responses were compared among the groups of animals. Results: Serological analysis indicated that mice receiving the HK-1/H9N2 nanoparticles formulation induced higher antibody titers that were sustained until the end of experiment. However, in the immunized mice, influenza specific antibody titers were comparable to that in the animals which were immunized either with inactivated antigen alone or the H9N2 nanoparticles without HK-1 adjuvant. Conclusion: The data demonstrate the synergy between HK-1 as an adjuvant and chitosan nanoparticles as a delivery antigen/adjuvant carrier in the improvement of influenza immune responses. [ABSTRACT FROM AUTHOR]

Purpose: The direct transmission of avian influenza viruses to human and increasing drug resisted strains posing new threats for public health. Therefore, development of efficient vaccines is needed to generate protective and persistent immunity to the viruses. Methods: Three motifs of Mx protein sequence in human, mouse and poultry located in interferon induced (GTP ase) domain were candidate as biologic adjuvant for enhancing the immune responses against influenza virus. Chimera proteins composed with the conserved HA2 subunit of influenza virus and the Mx motifs named HA2/Mx were modeled and evaluated by in silico analysis includes bioinformatics algorithms in order to explore biological characteristics of these peptides. Results: Amongst the predicted models, HA2/Mx1 peptide showed the better results following protein structures prediction, antigenic epitopes determination and model quality evaluation. Comparative homology modeling was performed with Swiss Model and the model was validated using ProSA. Epitope predictions revealed the construct could induce both B and T cell epitopes that expect a high immune response. Conclusion: Taken together, these data indicate that the HA2/Mx1 chimera peptide can be potentiated for developing an adjuvant-fused influenza vaccine capable of stimulating effective immune response. [ABSTRACT FROM AUTHOR]