Recombinant HA1-ΔfliC enhances adherence to respiratory epithelial cells and promotes the superiorly protective immune responses against H9N2 influenza virus in chickens.

H9N2 subtype avian influenza virus (AIV) is an ongoing threat causing substantial loss to the poultry industry and thus necessitating the development of safe and effective vaccines against AIV. Given that inactivated vaccines are less effective in activating the mucosal immune system, we aimed to generate a vaccine that can actively engage the mucosal immunity which is the front line of the immune system. We generated a group of flagellin-based hemagglutinin globular head (HA1) fusion proteins and characterized their immunogenicity and efficacy. We found that Salmonella typhimurium flagellin (fliC) lacking the hypervariable domain (called herein as HA1-ΔfliC) was recognized by TLR5 and induced a moderate innate immune response compared to N-terminus of fliC (HA1-fliC) and C-terminus of fliC (fliC-HA1). The HA1-ΔfliC protein had increased adherence to the nasal cavity and trachea than HA1-fliC and fliC-HA1 and significantly increased the HA-specific sIgA titers. Our in vivo results revealed that chickens treated with HA1-ΔfliC had a significantly reduced level of viral loads in the cloaca and throat compared with chickens treated with inactivated vaccine. Overall, these results revealed that HA1-ΔfliC can protect chickens against H9N2 AIV by eliciting the efficient mucosal immune responses.

Plant-based, adjuvant-free, potent multivalent vaccines for avian influenza virus via Lactococcus surface display.

Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHAH5N6 + H9N2 BLP or a combination of tHAH5N6 BLP and tHAH9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.

Phenotypic and Functional Characteristics of a Novel Influenza Virus Hemagglutinin-Specific Memory NK Cell.

Immune memory represents the most efficient defense against invasion and transmission of infectious pathogens. In contrast to memory T and B cells, the roles of innate immunity in recall responses remain inconclusive. In this study, we identified a novel mouse spleen NK cell subset expressing NKp46 and NKG2A induced by intranasal influenza virus infection. These memory NK cells specifically recognize N-linked glycosylation sites on influenza hemagglutinin (HA) protein. Different from memory-like NK cells reported previously, these NKp46+ NKG2A+ memory NK cells exhibited HA-specific silence of cytotoxicity but increase of gamma interferon (IFN-γ) response against influenza virus-infected cells, which could be reversed by pifithrin-µ, a p53-heat shock protein 70 (HSP70) signaling inhibitor. During recall responses, splenic NKp46+ NKG2A+ NK cells were recruited to infected lung and modulated viral clearance of virus and CD8+ T cell distribution, resulting in improved clinical outcomes. This long-lived NK memory bridges innate and adaptive immune memory response and promotes the homeostasis of local environment during recall response.IMPORTANCE In this study, we demonstrate a novel hemagglutinin (HA)-specific NKp46+ NKG2A+ NK cell subset induced by influenza A virus infection. These memory NK cells show virus-specific decreased cytotoxicity and increased gamma interferon (IFN-γ) on reencountering the same influenza virus antigen. In addition, they modulate host recall responses and CD8 T cell distribution, thus bridging the innate immune and adaptive immune responses during influenza virus infection.

Polyethylenimine-coated PLGA nanoparticles-encapsulated Angelica sinensis polysaccharide as an adjuvant for H9N2 vaccine to improve immune responses in chickens compared to Alum and oil-based adjuvants.

Inactivated H9N2 influenza vaccines required adjuvants to induce strong immune responses to protect poultry from the infections of H9N2 influenza viruses. Recently, positively charged nanoparticles-based adjuvant delivery systems have been extensively investigated as the novel vaccine adjuvant due to the protection antigens and drugs from degradation, promoting antigens and drugs uptake by antigen presenting cells (APCs), and inducing strong humoral and cellular immune responses. In this study, the immunostimulant Angelica sinensis polysaccharide (ASP) was encapsulated into Poly (lactic-co-glycolic acid) PLGA nanoparticles, and the Polyethylenimine (PEI) was coated on the nanoparticles to develop a novel adjuvant (ASP-PLGA-PEI). To further investigate the adjuvant activities of ASP-PLGA-PEI nanoparticles for H9N2 vaccines in chickens and compare the adjuvant activities of nanoparticles adjuvant and conventional adjuvants (Alum and oil-based adjuvant), the H9N2 antigen was incubated with three different adjuvants and then immunized with chickens to evaluate the ability of inducing humoral and cellular immune responses. The results revealed that compared to Alum adjuvant, ASP-PLGA-PEI nanoparticles adjuvant stimulated higher antibody responses, promoted the activation of CD4+ T cells and CD8+ T cells, increased the expression of Th1 cytokines IFN-γ. Compared to oil-based adjuvant (ISA-206), ASP-PLGA-PEI nanoparticles adjuvant induced comparable antibody immune responses at later period after immunization, improved the activation of CD4+ T cells and CD8+ T cells. Therefore, compared to Alum and oil-based adjuvant, the ASP-PLGA-PEI nanoparticles serve as an efficient adjuvant for H9N2 vaccine and have the potential to induce vigorous humoral and cellular immune responses in chickens.

Pulmonary endothelium-derived PD-L1 induced by the H9N2 avian influenza virus inhibits the immune response of T cells.

BACKGROUND: The PD-1/PD-L1 pathway is an inhibitory signaling pathway that maintains the balance between the immune response and immunotolerance, and its overactivation in cancer and viral infections inhibits T cell function. The target cells of various viruses, microvascular endothelial cells (MECs) have been shown to be key regulatory points in immune regulation and virion diffusion in vivo during infection with multiple influenza virus subtypes. Furthermore, avian influenza virus (AIV) infection can induce immunosuppression by causing imbalances in immune responses and immune organ damage. Thus, the aim of this study was to investigate whether the H9N2 virus inhibited the immune function of T cells that migrated across MECs by upregulating PD-L1 expression on MECs. METHODS: The susceptibility of rat pulmonary microvascular endothelial cells (RPMECs) to the H9N2 virus was evaluated by a plaque-forming assay and immunofluorescence staining. Then, we quantified the mRNA and protein levels of PD-L1 in RPMECs induced by H9N2 virus infection using quantitative real-time PCR and flow cytometry. The interaction between the activated T cells and RPMECs infected with the H9N2 virus was revealed using a coculture system. The effect of endothelial-derived PD-L1 on T cell function was investigated by using ELISA and flow cytometry with or without a PD-L1-specific antibody. RESULTS: Surface staining and the plaque-forming assay showed that the H9N2 virus infected and replicated in RPMECs. Both the PD-L1 mRNA level and PD-L1 protein level were upregulated in RPMECs infected with the H9N2 virus. H9N2 virus-induced PD-L1 expression significantly reduced the secretions of IL-2, IFN-γ and granzyme B and perforin expression in T cells. The above data were significantly increased after treatment with an anti-PD-L1 antibody, confirming the above mentioned findings. In addition, the induction of PD-L1 expression decreased the proliferative capacity of the cocultured T cells but did not affect the apoptosis rate of T cells. CONCLUSIONS: Taken together, the results suggest that the H9N2 virus is able to inhibit the T cell immune response by upregulating PD-L1 expression in pulmonary microvascular endothelial cells.

Transcriptomic profile of chicken bone marrow-derive dendritic cells in response to H9N2 avian influenza A virus.

Avian influenza subtype H9N2 infection is a mild but highly contagious disease that is associated with a decrease in the efficacy of vaccine interventions, and an increase in susceptibility to secondary infections in poultry. However, the immune evasion mechanism of H9N2 avian influenza viruses (AIVs) in chickens is poorly understood. Dendritic cells (DCs) are immune cells of major importance, involved in innate immune responses against viruses, but also in the setting of adaptive immune response due to their high ability to present viral antigen. Therefore, in the present study we used high-throughput RNA-sequencing technology at the transcriptome level to identify the differentially expressed genes (DEGs) between chicken DCs infected with H9N2 virus and mock-infected DCs. We identified 4151 upregulated DEGs and 2138 downregulated DEGs. Further enrichment analysis showed that the upregulated DEGs were enriched in the biological processes mainly involved in signal transduction, transmembrane transport, and innate immune/inflammatory responses. In contrast, the downregulated DEGs were associated with the biological processes mainly including metabolic process, and MHC class I antigen processing and presentation. In addition, 49 of these immune-related DEGs were validated by reverse transcription quantitative PCR (RT-qPCR). Collectively, these data suggest that H9N2 virus infection may enhance the signal transduction, and innate immune responses in chicken DCs, but impair their metabolic functions and antigen-presenting responses, which provide helpful insight into the pathogenesis of H9N2 AIVs in chickens and managing this infection in poultry farms.

Evaluation of Immune Responses and Histopathological Effects against Gamma Irradiated Avian Influenza (Sub type H9N2) Vaccine on Broiler Chicken

Abstract Vaccination is a good strategy for the prevention of avian influenza virus. In this research Gamma Irradiated Avian Influenza (Sub type H9N2) Vaccine (GAIV) was prepared by 30 kGy irradiation and used for vaccination of broiler chickens. The purpose was a comparison of immune responses in the two routes of administration for the GAIV vaccine; intranasal and subcutaneously, use of Montanide ISA70 and Trehalose accompanied with irradiated vaccine and compare with formalin vaccine. The Influenza Virus A/Chicken/IRN/Ghazvin/2001/H9N2 was irradiated and used for vaccine formulation, and formalin inactivated AIV was used as conventional vaccine. Chickens were vaccinated by GAIV with and without Trehalose, GAIV and formalin vaccines with ISA70, two routes of administration were intranasal and subcutaneously. All the vaccinated chickens showed a significant increase in antibody titration. The most significant increase of antibody titration was in irradiated vaccine plus Trehalose groups intranasal and subcutaneously. After the first and second intranasal vaccination, the amount of IFN-gamma increased in the irradiated vaccine plus Trehalose group compared to other groups. However, most of the vaccinated groups did not show any significant increase of IFN-α concentration. Histopathological examination revealed lymphocyte infiltration (++), foci dispersed of hemorrhage and edema in intranasal vaccination groups and in addition to these, thickening of alveolar septa was observed in the injection groups. GAIV vaccine can be a good candidate for vaccine preparation, and Trehalose as a stabilizer protects viral antigenic proteins, also makes more absorbance of antigen by the inhalation route. In vaccinated chickens the ulcers in injected vaccines were lower than intranasal vaccines.

The Inducing Roles of the New Isolated Bursal Hexapeptide and Pentapeptide on the Immune Response of AIV Vaccine in Mice.

BACKGROUND: Bursa of Fabricius is the acknowledged central humoral immune organ. The bursal-derived peptides play the important roles on the immature B cell development and antibody production. OBJECTIVES: Here we explored the functions of the new isolated bursal hexapeptide and pentapeptide on the humoral, cellular immune response and antigen presentation to Avian Influenza Virus (AIV) vaccine in mice immunization. METHODS: The bursa extract samples were purified following RP HPLC method, and were analyzed with MS/MS to identify the amino acid sequences. Mice were twice subcutaneously injected with AIV inactivated vaccine plus with two new isolated bursal peptides at three dosages, respectively. On two weeks after the second immunization, sera samples were collected from the immunized mice to measure AIV-specific IgG antibody levels and HI antibody titers. Also, on 7th day after the second immunization, lymphocytes were isolated from the immunized mice to detect T cell subtype and lymphocyte viabilities, and the expressions of co-stimulatory molecule on dendritic cells in the immunized mice. RESULTS: Two new bursal hexapeptide and pentapeptide with amino acid sequences KGNRVY and MPPTH were isolated, respectively. Our investigation proved the strong regulatory roles of bursal hexapeptide on AIV-specific IgG levels and HI antibody titers, and lymphocyte viabilities, and the significant increased T cells subpopulation and expressions of MHCII molecule on dendritic cells in the immunized mice. Moreover, our findings verified the significantly enhanced AIV-specific IgG antibody and HI titers, and the strong increased T cell subpopulation and expressions of CD40 molecule on dendritic cells in the mice immunized with AIV vaccine and bursal pentapeptide. CONCLUSION: We isolated and identified two new hexapeptide and pentapeptide from bursa, and proved that these two bursal peptides effectively induced the AIV-specific antibody, T cell and antigen presentation immune responses, which provided an experimental basis for the further clinical application of the bursal derived active peptide on the vaccine improvement.

Dietary selenium supplementation enhances antiviral immunity in chickens challenged with low pathogenic avian influenza virus subtype H9N2.

Selenium supplementation in poultry feeds has been known to have beneficial effects on the bird health and performance; however antiviral effects of selenium have remained largely unknown. In this study, we have evaluated the effects of supplementation of chicken diets with organic (Selenium Enriched Yeast; SEY) and inorganic selenium (Sodium Selenite; SS) on low pathogenicity avian influenza virus (H9N2) shedding in the cloacal and oropharyngeal swab samples as well as examined the expression of immune related genes. Chickens were fed two doses (High- 0.30 mg/kg of feed; Low- 0.15 mg/kg of feed) of selenium supplementation for 2 weeks followed by low pathogenicity avian influenza virus challenge. Our results showed that the cloacal shedding of virus in all the selenium supplemented groups was significantly lower when compared to the non-supplemented control groups. In addition, the oropharyngeal shedding of virus in chickens fed with organic selenium supplementation was significantly lower than that in the chickens that received either inorganic selenium supplemented feed or controls. Furthermore, the expression of interferon stimulated genes (Viperin, OAS: 2'-5' oligoadenylate synthetase and MDA5: melanoma differentiation-associated gene) in the cecal tonsils was significantly elevated in the selenium treated groups when compared to controls. Additionally, a significantly higher transcription of interferon (IFN)-α, IFN-ß and IFN-γ genes in the cecal tonsils and spleens of chickens receiving SEY-L and SS-H supplemented feed was also observed at post virus challenge time points compared to untreated controls. The results of this study demonstrated that supplementation of chicken diets with selenium, can enhance antiviral defense and thus, may have a beneficial effect in controlling viral infections in poultry.

Potential Use of Genetically Engineered Variable Lymphocyte Receptor B Specific to Avian Influenza Virus H9N2.

The variable lymphocyte receptor (VLR) B of jawless vertebrates functions as a secreted Ab of jawed vertebrates and has emerged as an alternative Ab with a single polypeptide chain. After observing an upregulated VLRB response in hagfish immunized with avian influenza virus (AIV) subtype H9N2, we screened AIV H9N2-specific VLRB using a mammalian expression system. To improve the binding avidity of the Ag-specific VLRB to the Ag, we enabled multimerization of the VLRB by conjugating it with C-terminal domain of human C4b-binding protein. To dramatically enhance the expression and secretion of the Ag-specific VLRB, we introduced a glycine-serine linker and the murine Ig κ leader sequence. The practical use of the Ag-specific VLRB was also demonstrated through various immunoassays, detected by anti-VLRB Ab (11G5). Finally, we found that the Ag-specific VLRB decreased the infectivity of AIV H9N2. Together, our findings suggest that the generated Ag-specific VLRB could be used for various immunoapplications.

A Comparison of Toll-Like Receptor 5 and 21 Ligands as Adjuvants for a Formaldehyde Inactivated H9N2 Avian Influenza Virus Vaccine in Chickens.

Low pathogenic avian influenza virus (AIV) infection in chickens can result in economic losses and has impacts on human health. Poultry vaccination is a tool that can be used to decrease infection and transmission of AIVs. Prior research has demonstrated that Toll-like receptor (TLR) ligands can act as vaccine adjuvants and their addition to inactivated AIV vaccines can enhance immune responses elicited in chickens. The objective of this study was to compare the adjuvant capabilities of TLR5 ligand (flagellin) and TLR21 ligand (CpG ODN 2007) administered either alone or in combination with an intramuscular formaldehyde inactivated H9N2 whole virus vaccine in chickens. Along with the inactivated virus, chickens were administered either a single dose of CpG ODN 2007 (2 or 10 µg), flagellin (0.4 or 2 µg), or a combination of both ligands. An additional group received AddaVax™, an oil emulsion style adjuvant. Chickens were vaccinated twice and serum and lachrymal samples were collected weekly following the primary vaccination, and antibody-mediated immune responses were quantified. Results showed that vaccines containing CpG ODN 2007 induce significantly greater systemic and lachrymal antibody responses than vaccines containing flagellin or AddaVax. Combinations of flagellin and CpG ODN 2007 did not demonstrate inhibitory, additive, or synergistic effects on systemic or lachrymal antibody-mediated immune responses. Additionally, for both flagellin and CpG ODN 2007, a fivefold higher dose of each did not induce significantly higher antibody-mediated immune responses compared with the lesser dose. Future studies should examine the induction of cell-mediated immune responses when flagellin, CpG ODN 2007, or other TLR ligands are administered either alone or combined as adjuvants for inactivated H9N2 AIV vaccines.

Comparison of immunoadjuvant activities of four bursal peptides combined with H9N2 avian influenza virus vaccine.

The bursa of Fabricius (BF) is a central humoral immune organ unique to birds. Four bursal peptides (BP-I, BP-II, BP-III, and BP-IV) have been isolated and identified from the BF. In this study, the immunoadjuvant activities of BPs I to IV were examined in mice immunized with H9N2 avian influenza virus (AIV) vaccine. The results suggested that BP-I effectively enhanced cell-mediated immune responses, increased the secretion of Th1 (interferon gamma)- and Th2 (interleukin-4)-type cytokines, and induced an improved cytotoxic T-lymphocyte (CTL) response to the H9N2 virus. BP-II mainly elevated specific antibody production, especially neutralizing antibodies, and increased Th1- and Th2-type cytokine secretion. BP-III had no significant effect on antibody production or cell-mediated immune responses compared to those in the control group. A strong immune response at both the humoral and cellular levels was induced by BP-IV. Furthermore, a virus challenge experiment followed by H&E staining revealed that BP-I and BP-II promoted removal of the virus and conferred protection in mouse lungs. BP-IV significantly reduced viral titers and histopathological changes and contributed to protection against H9N2 AIV challenge in mouse lungs. This study further elucidated the immunoadjuvant activities of BPs I to IV, providing a novel insight into immunoadjuvants for use in vaccine design.

Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens.

Several types of avian influenza virus (AIV) vaccines exist, including live-attenuated, vectored, and whole inactivated virus (WIV) vaccines. Inactivated vaccines offer some advantages compared to other types of vaccines, including ease of production and lack of ability to revert to a virulent state. However, WIV are poorly immunogenic, especially when these vaccines are delivered to mucosal surfaces. There are several factors that contribute to the immunogenicity of vaccines, one of which is the method used to inactivate viruses. Several methods exist for producing influenza WIVs, including formaldehyde, a chemical that affects protein structures leading to virus inactivation. Other methods include treatment with beta-propiolactone (BPL) and the application of gamma radiation, both of which have less effects on protein structures compared to formaldehyde, and instead alter nucleic acids in the virion. Here, we sought to determine the effect of the above inactivation methods on immunogenicity of AIV vaccines. To this end, chickens were vaccinated with three different H9N2 WIVs using formaldehyde, BPL, and gamma radiation for inactivation. In addition to administering these three WIVs alone as vaccines, we also included CpG ODN 2007, a synthetic ligand recognized by Toll-like receptor (TLR)21 in chickens, as an adjuvant for each WIV. Subsequently, antibody- and cell-mediated immune responses were measured following vaccination. Antibody-mediated immune responses were increased in chickens that received the BPL and Gamma WIVs compared to the formaldehyde WIV. CpG ODN 2007 was found to significantly increase antibody responses for each WIV compared to WIV alone. Furthermore, we observed the presence of cell-mediated immune responses in chickens that received the BPL WIV combined with CpG ODN 2007. Based on these results, the BPL WIV + CpG ODN 2007 combination was the most effective vaccine at inducing adaptive immune responses against H9N2 AIV. Future studies should characterize mucosal adaptive immune responses to these vaccines.

Human Vγ9Vδ2-T Cells Synergize CD4+ T Follicular Helper Cells to Produce Influenza Virus-Specific Antibody.

Human Vγ9Vδ2-T cells recognize nonpeptidic antigens and exert effector functions against microorganisms and tumors, but little is known about their roles in humoral immune response against influenza virus infection. Herein, in the coculture of autologous human B cells, dendritic cells and/or naïve CD4 T cells, and Vγ9Vδ2-T cells, we demonstrated that Vγ9Vδ2-T cells could facilitate H9N2 influenza virus-specific IgG and IgM productions in a CD4 T cell-dependent manner. Vγ9Vδ2-T cells promoted the differentiation of CXCR5+PD1+CD4+ T follicular helper (Tfh) cells, CD19+IgD-CD38++ plasma cells (PCs), and drove B cell proliferation as well as immunoglobulin class switching. Interestingly, Vγ9Vδ2-T cells acquired Tfh-associated molecules such as CXCR5, PD1, CD40L, and ICOS during influenza virus stimulation, especially in the presence of CD4 T cells. Moreover, Vγ9Vδ2-T cells promoted CD4 T cells to secrete IL-13 and IL-21, and neutralizing IL-13 and IL-21 significantly reduced the number of CD19+IgD-CD38++ PCs. Using humanized mice, we further demonstrated that Vγ9Vδ2-T cells could synergize CD4 T cells to produce influenza virus-specific antibody. Our findings provide a greater scope for Vγ9Vδ2-T cells in adaptive immunity, especially for the Tfh development and humoral immune responses against influenza virus infection.

Investigation of antiviral state mediated by interferon-inducible transmembrane protein 1 induced by H9N2 virus and inactivated viral particle in human endothelial cells.

BACKGROUND: Endothelial cells are believed to play an important role in response to virus infection. Our previous microarray analysis showed that H9N2 virus infection and inactivated viral particle inoculation increased the expression of interferon-inducible transmembrane protein 1 (IFITM1) in human umbilical vein endothelial cells (HUVECs). In present study, we deeply investigated the expression patterns of IFITM1 and IFITM1-mediated antiviral response induced by H9N2 virus infection and inactivated viral particle inoculation in HUVECs. Epithelial cells that are considered target cells of the influenza virus were selected as a reference control. METHODS: First, we quantified the expression levels of IFITM1 in HUVECs induced by H9N2 virus infection or viral particle inoculation using quantitative real-time PCR and western blot. Second, we observed whether hemagglutinin or neuraminidase affected IFITM1 expression in HUVECs. Finally, we investigated the effect of induced-IFITM1 on the antiviral state in HUVECs by siRNA and activation plasmid transfection. RESULTS: Both H9N2 virus infection and viral particle inoculation increased the expression of IFITM1 without elevating the levels of interferon-ɑ/ß in HUVECs. HA or NA protein binding alone is not sufficient to increase the levels of IFITM1 and interferon-ɑ/ß in HUVECs. IFITM1 induced by viral particle inoculation significantly decreased the virus titers in culture supernatants of HUVECs. CONCLUSIONS: Our results showed that inactivated viral particle inoculation increased the expression of IFITM1 at mRNA and protein levels. Moreover, the induction of IFITM1 expression mediated the antiviral state in HUVECs.

Recombinant Adenoviruses Displaying Matrix 2 Ectodomain Epitopes on Their Fiber Proteins as Universal Influenza Vaccines.

Influenza is a zoonotic disease that poses severe threats to public health and the global economy. Reemerging influenza pandemics highlight the demand for universal influenza vaccines. We developed a novel virus platform using extracellular domain IV of the matrix 2 protein (M2e), AdC68-F3M2e, by introducing three conserved M2e epitopes into the HI loop of the chimpanzee adenovirus (AdV) fiber protein. The M2e epitopes were expressed sufficiently on the AdV virion surface without affecting fiber trimerization. Additionally, one recombinant adenovirus, AdC68-F3M2e(H1-H5-H7), induced robust M2e-specific antibody responses in BALB/c mice after two sequential vaccinations and conferred efficient protection against homologous and heterologous influenza virus (IV) challenges. We found that the use of AdV with tandem M2e epitopes in fiber is a potential strategy for influenza prevention.IMPORTANCE Influenza epidemics and pandemics severely threaten public health. Universal influenza vaccines have increasingly attracted interest in recent years. Here, we describe a new strategy that incorporates triple M2e epitopes into the fiber protein of chimpanzee adenovirus 68. We optimized the process of inserting foreign genes into the AdC68 structural protein by one-step isothermal assembly and demonstrated that this 225-bp HI loop insertion could be well tolerated. Furthermore, two doses of adjuvant-free fiber-modified AdC68 could confer sufficient protection against homologous and heterologous influenza virus infections in mice. Our results show that AdC68-F3M2e could be pursued as a novel universal influenza vaccine.

Radix isatidis Polysaccharides Inhibit Influenza a Virus and Influenza A Virus-Induced Inflammation via Suppression of Host TLR3 Signaling In Vitro.

Influenza remains one of the major epidemic diseases worldwide, and rapid virus replication and collateral lung tissue damage caused by excessive pro-inflammatory host immune cell responses lead to high mortality rates. Thus, novel therapeutic agents that control influenza A virus (IAV) propagation and attenuate excessive pro-inflammatory responses are needed. Polysaccharide extract from Radix isatidis, a traditional Chinese herbal medicine, exerted potent anti-IAV activity against human seasonal influenza viruses (H1N1 and H3N2) and avian influenza viruses (H6N2 and H9N2) in vitro. The polysaccharides also significantly reduced the expression of pro-inflammatory cytokines (IL-6) and chemokines (IP-10, MIG, and CCL-5) stimulated by A/PR/8/34 (H1N1) at a range of doses (7.5 mg/mL, 15 mg/mL, and 30 mg/mL); however, they were only effective against progeny virus at a high dose. Similar activity was detected against inflammation induced by avian influenza virus H9N2. The polysaccharides strongly inhibited the protein expression of TLR-3 induced by PR8, suggesting that they impair the upregulation of pro-inflammatory factors induced by IAV by inhibiting activation of the TLR-3 signaling pathway. The polysaccharide extract from Radix isatidis root therefore has the potential to be used as an adjunct to antiviral therapy for the treatment of IAV infection.

H9N2 avian influenza virus enhances the immune responses of BMDCs by down-regulating miR29c.

Avian influenza virus (AIV) of the subtypes H9 and N2 is well recognised and caused outbreaks-due to its high genetic variability and high rate of recombination with other influenza virus subtypes. The pathogenicity of H9N2 AIV depends on the host immune response. Dendritic cells (DCs) are major antigen presenting cells that can significantly inhibit H9N2 AIV replication. MicroRNAs (miRNAs) influence the ability of DCs to present antigens, as well as the ability of AIVs to infect host cells and replicate. Here, we studied the molecular mechanism underlying the miRNA-mediated regulation of immune function of mouse DCs. We first screened for and verified the induction of miRNAs in DCs after H9N2 AIVstimulation. We also constructed miR29c, miR339 and miR222 over-expression vector and showed that only the induction of miR29c lead to a hugely increased expression of surface marker MHCII and CD40. Whilst the inhibition of miR29c, miR339 and miR222 in mouse DCs would repressed the expression of DCs surface markers. Moreover, we found that miR29c stimulation not only up-regulate MHCII and CD40, but also enhance the ability of DCs to activate lymphocytes and secrete cytokines IL-6 or TNF-a. Furthermore, we found that Tarbp1 and Rfx7 were targeted and repressed by miR29c. Finally, we revealed that the inhibition of miR29c marvelously accelerated virus replication. Together, our data shed new light on the roles and mechanisms of miR29c in regulating DC function and suggest new strategies for combating AIVs.

Comparative assessment of humoral immune responses of aluminum hydroxide and oil-emulsion adjuvants in Influenza (H9N2) and Newcastle inactive vaccines to chickens.

Context Adjuvants are compounds used in the preparation of inactive vaccines to enhance the immune response. Aluminum hydroxide (alum) is one of the first compounds approved by the Food and Drug Administration, which is used as adjuvants in vaccine products for humans. Montanide ISA 70 is an oil-emulsion adjuvant and is used in poultry inactive vaccines. Objective In this study, the effects of alum adjuvant on the efficiency and induction of immune response in inactive vaccines of Influenza and Newcastle are compared with those of ISA 70. Materials and methods Six groups of 7-d-old specific-pathogen-free chickens were inoculated with 0.3 ml of the prepared vaccines via the subcutaneous route in the neck. Immune response in each group after 7, 14, 21, 31, 41, and 45 d was evaluated using the technique of hemagglutination inhibition. Results The results were compared using SPSS software. Results showed that vaccines containing adjuvant ISA 70 depicted a higher increase in the immune response and adjuvant of 20% alum is similar to adjuvant of ISA 70 in boosting the immune system. There was no statistically significant difference between 10% and 20% alum, but these adjuvants are visibly different from ISA 70. Conclusion In conclusion, alum can be used as an easily accessible, harmless, and effective adjuvant; however, to increase the immune period using the inactive vaccines for poultry, more research would be necessary.

Constitutively Expressed IFITM3 Protein in Human Endothelial Cells Poses an Early Infection Block to Human Influenza Viruses.

A role for pulmonary endothelial cells in the orchestration of cytokine production and leukocyte recruitment during influenza virus infection, leading to severe lung damage, has been recently identified. As the mechanistic pathway for this ability is not fully known, we extended previous studies on influenza virus tropism in cultured human pulmonary endothelial cells. We found that a subset of avian influenza viruses, including potentially pandemic H5N1, H7N9, and H9N2 viruses, could infect human pulmonary endothelial cells (HULEC) with high efficiency compared to human H1N1 or H3N2 viruses. In HULEC, human influenza viruses were capable of binding to host cellular receptors, becoming internalized and initiating hemifusion but failing to uncoat the viral nucleocapsid and to replicate in host nuclei. Unlike numerous cell types, including epithelial cells, we found that pulmonary endothelial cells constitutively express a high level of the restriction protein IFITM3 in endosomal compartments. IFITM3 knockdown by small interfering RNA (siRNA) could partially rescue H1N1 virus infection in HULEC, suggesting IFITM3 proteins were involved in blocking human influenza virus infection in endothelial cells. In contrast, selected avian influenza viruses were able to escape IFITM3 restriction in endothelial cells, possibly by fusing in early endosomes at higher pH or by other, unknown mechanisms. Collectively, our study demonstrates that the human pulmonary endothelium possesses intrinsic immunity to human influenza viruses, in part due to the constitutive expression of IFITM3 proteins. Notably, certain avian influenza viruses have evolved to escape this restriction, possibly contributing to virus-induced pneumonia and severe lung disease in humans. IMPORTANCE: Avian influenza viruses, including H5N1 and H7N9, have been associated with severe respiratory disease and fatal outcomes in humans. Although acute respiratory distress syndrome (ARDS) and progressive pulmonary endothelial damage are known to be present during severe human infections, the role of pulmonary endothelial cells in the pathogenesis of avian influenza virus infections is largely unknown. By comparing human seasonal influenza strains to avian influenza viruses, we provide greater insight into the interaction of influenza virus with human pulmonary endothelial cells. We show that human influenza virus infection is blocked during the early stages of virus entry, which is likely due to the relatively high expression of the host antiviral factors IFITMs (interferon-induced transmembrane proteins) located in membrane-bound compartments inside cells. Overall, this study provides a mechanism by which human endothelial cells limit replication of human influenza virus strains, whereas avian influenza viruses overcome these restriction factors in this cell type.