Bivalent Hemagglutinin Cleavage-Site Peptide Vaccines Protect Chickens from Lethal Infections with Highly Pathogenic H5N1 and H5N6 Avian Influenza Viruses.

BACKGROUND: Outbreaks of highly pathogenic avian influenza viruses cause huge economic losses to the poultry industry worldwide. Vaccines that can protect chickens from infections caused by various variants of highly pathogenic H5Nx avian influenza viruses are needed owing to the continuous emergence of new variants. We previously showed that vaccines containing the H5 cleavage-site peptide from clade 2.3.4.4. H5N6 avian influenza virus protects chickens from infection with homologous clade 2.3.4.4. H5N6 avian influenza virus, but not from infection with the heterologous clade 1 H5N1 avian influenza virus. Therefore, we developed bivalent peptide vaccines containing H5 cleavage sites of viruses from both clades to protect chickens from both H5N1 and H5N6 avian influenza viruses. METHODS: Chickens were vaccinated with two doses of a combined peptide vaccine containing cleavage-site peptides from clade 1 and clade 2.3.4.4. highly pathogenic H5N1 and H5N6 avian influenza viruses and then challenged with both viruses. The infected chickens were monitored for survival and their tracheae and cloacae were sampled to check for viral shedding based on the median tissue culture infectious dose of 50 (log10TCID50/mL) in Madin-Darby canine kidney cells. RESULTS: Antibody production was induced at similar levels in the sera of chickens immunized with two doses of the combined peptide vaccines containing cleavage-site peptides from highly pathogenic H5N1 and H5N6 avian influenza viruses. The immunized chickens were protected from infection with both H5N1 and H5N6 avian influenza viruses without viral shedding in the tracheae and cloacae. CONCLUSIONS: Dual-peptide vaccines containing cleavage-site peptides of both clades can protect chickens from highly pathogenic avian influenza virus infections.

Differential Protection of Chickens against Highly Pathogenic H5 Avian Influenza Virus Using Polybasic Amino Acids with H5 Cleavage Peptide.

BACKGROUND: Highly pathogenic H5Nx viruses cause avian influenza, a zoonotic disease that can infect humans. The vaccine can facilitate the prevention of human infections from infected poultry. Our previous study showed that an H5 cleavage-site peptide vaccine containing the polybasic amino acid RRRK could protect chickens from lethal infections of the highly pathogenic H5N6 avian influenza virus. METHODS: Chickens immunized with the various polybasic amino combinations (RRRK, RRR, RR, R, RK, and K) of H5 cleavage-site peptides were challenged with highly pathogenic H5N6 avian influenza viruses. The challenged chickens were monitored for survival rate, and viral titers in swabs and tissue samples were measured in Madin-Darby canine kidney (MDCK) cells using the median tissue culture infectious dose 50 (log10 TCID50/mL). RESULTS: Most H5 cleavage-site vaccines containing various combinations of polybasic amino acids protected chickens from lethal infection. Chickens immunized with the RK-containing peptide combination of the H5 cleavage site were not protected. CONCLUSIONS: The polybasic amino acids (RRRK) of H5 cleavage cleavage-site peptide vaccines are important for protecting chickens against HP H5N6 avian influenza virus. The H5 cleavage cleavage-site peptide containing RK did not protect chickens against the virus.

Comparison of the Pathogenicity of SARS-CoV-2 Delta and Omicron Variants by Analyzing the Expression Patterns of Immune Response Genes in K18-hACE2 Transgenic Mice.

BACKGROUND: The recently emerged variants of the severe acute respiratory coronavirus 2 (SARS-CoV-2) pose a threat to public health. Understanding the pathogenicity of these variants is a salient factor in the development of effective SARS-CoV-2 therapeutics. This study aimed to compare the expression patterns of genes involved in immune responses in K18-hACE2 mice infected with the wild-type, Delta, and Omicron SARS-CoV-2 variants. METHODS: K18-hACE2 mice were intranasally infected with either wild-type (B.1), Delta (B.1.617.2), or Omicron (B.1.1.529) variants. On day 6 post-infection, lung, brain, and kidney tissues were collected from each variant-infected group. The mRNA expression levels of 39 immune response genes in all three groups were compared by RT-qPCR. Viral titers were measured using the median tissue culture infectious dose (TCID50) assay and expressed as Log10 TCID50/0.1 g. The statistical significance of the differences in gene expression was determined by one-way analysis of variance (ANOVA) (alpha = 0.05). RESULTS: The expression of toll-like receptors (TLRs) was upregulated in the lung and brain tissues of the wild-type- and Delta-infected groups but not in those of the Omicron-infected group. The highest expression of cytokines, including interleukin (IL)-1α, IL-1ß, IL-17α, interferon, and tumor necrosis factors, was observed in the lungs of mice infected with the wild-type variant. Additionally, CCL4, CCL11, CXCL9, and CXCL10 were upregulated (>3-fold) in wild-type-infected mice, with markedly higher expressions in the brain than in the lungs. Most of the apoptotic factors were mainly expressed in the brain tissues of Omicron-infected mice (caspase 8, caspase 9, p53, Bax, Bak, BCL-2, and Bcl-XL), whereas neither the lung nor kidney showed more than 3-fold upregulation of these apoptotic factors. CONCLUSIONS: Collectively, our findings revealed that the wild-type SARS-CoV-2 variant exhibited the highest pathogenicity, followed by the Delta variant, then the Omicron variant.

Evaluation of Efficacy of Oil Adjuvanted H5N6 Inactivated Vaccine against Highly Pathogenic H5N6 and H5N1 Influenza Viruses Infected Chickens.

BACKGROUND: Over the last 20 years, circulating highly pathogenic (HP) Asian H5 subtype avian influenza viruses have caused global pandemics in poultry and sporadic infections in humans. Vaccines are a desirable solution to prevent viral infections in poultry and reduce transmission to humans. Herein, we investigated the efficacy of an oil-adjuvanted inactivated H5N6 vaccine against highly pathogenic H5N6 and H5N1 influenza virus infections in chickens. METHODS: The polybasic amino acid cleavage site depleted HA gene and NA gene of A/Waterfowl/Korea/S57/2016 (clade 2.3.4.4) (H5N6) was assembled with the rest of the A/PR/8/34 (H1N1) genes to construct the vaccine virus. The vaccine virus was propagated in fertilized eggs, partially purified using a tangential flow filtration (TFF) system, and inactivated using formalin. The chickens were intramuscularly immunized with 384 HA, 192HA, and 96HA units of oil-adjuvanted inactivated H5N6 vaccine. Antibody titer, survival rate, and lung pathology were evaluated against the homologous H5N6: A/waterfowl/Korea/S57/2016 (clade 2.3.4.4) and heterologous H5N1: A/Hong Kong/213/2003 (clade 1) viruses 12 and 4 weeks post-vaccination (p.v.), respectively. Data were statistically analyzed using the Mann-Whitney U test. RESULTS: The 384HA (n = 10) and 192HA (n = 5) antigen-immunized chickens showed 100% survival after lethal infections with homologous H5N6, and no virus shedding was observed from tracheal and cloacal routes. All chickens that received the 384HA vaccine survived the challenge of heterologous H5N1 after 4 weeks of immunization. The chickens that received the 384HA vaccine showed mean HI titers of 60 and 240 after 12 and 4 weeks of vaccination, respectively, against HP H5N6, whereas a mean HI titer of 80 was observed in sera collected 4 weeks after vaccination against HP H5N1. CONCLUSIONS: Our findings indicate that one dose of 384HA oil-adjuvanted inactivated H5N6 vaccine can induce a long-lasting immune response against both homologous H5N6 and heterologous H5N1 infections in chickens.

Gene expression pattern differences in primary human pulmonary epithelial cells infected with MERS-CoV or SARS-CoV-2.

Coronaviruses such as MERS-CoV and SARS-CoV-2 infect the human respiratory tract and can cause severe pneumonia. Disease severity and outcomes are different for these two infections: the human mortality rate for MERS-CoV and SARS-CoV-2 is over 30% and less than 10%, respectively. Here, using microarray assay, we analyzed the global alterations in gene expression induced by MERS-CoV or SARS-CoV-2 infections in primary human pulmonary epithelial cells. Overall, the number of differentially expressed genes was higher in human lung cells infected with MERS-CoV than in cells with SARS-CoV-2. Out of 44,556 genes analyzed, 127 and 50 were differentially expressed in cells infected with MERS-CoV and SARS-CoV-2, respectively (> 2-fold increase, compared to uninfected cells). Of these, only eight genes, including the one coding for CXCL8, were similarly modulated (upregulated or downregulated) by the two coronaviruses. Importantly, these results were virus-specific and not conditioned by differences in viral load, and viral growth curves were similar in human lung cells infected with both viruses. Our results suggest that these distinct gene expression profiles, detected early after infection by these two coronaviruses, may help us understand the differences in clinical outcomes of MERS-CoV and SARS-CoV-2 infections.

Higher virulence of swine H1N2 influenza viruses containing avian-origin HA and 2009 pandemic PA and NP in pigs and mice.

Pigs are capable of harbouring influenza A viruses of human and avian origin in their respiratory tracts and thus act as an important intermediary host to generate novel influenza viruses with pandemic potential by genetic reassortment between the two viruses. Here, we show that two distinct H1N2 swine influenza viruses contain avian-like or classical swine-like hemagglutinins with polymerase acidic (PA) and nucleoprotein (NP) genes from 2009 pandemic H1N1 influenza viruses that were found to be circulating in Korean pigs in 2018. Swine H1N2 influenza virus containing an avian-like hemagglutinin gene had enhanced pathogenicity, causing severe interstitial pneumonia in infected pigs and mice. The mortality rate of mice infected with swine H1N2 influenza virus containing an avian-like hemagglutinin gene was higher by 100% when compared to that of mice infected with swine H1N2 influenza virus harbouring classical swine-like hemagglutinin. Further, chemokines attracting inflammatory cells were strongly induced in lung tissues of pigs and mice infected by swine H1N2 influenza virus containing an avian-like hemagglutinin gene. In conclusion, it is necessary for the well-being of humans and pigs to closely monitor swine influenza viruses containing avian-like hemagglutinin with PA and NP genes from 2009 pandemic H1N1 influenza viruses.

The 2009 pandemic H1N1 influenza virus is more pathogenic in pregnant mice than seasonal H1N1 influenza virus.

Pregnant women can experience high mortality, high rates of abortion, and severe pneumonia when infected with pandemic influenza viruses. In this context, the severity of the 2009 pandemic H1N1 influenza virus compared with seasonal H1N1 influenza virus is not clear. Presently, in a mouse model of pregnancy, the 2009 pandemic H1N1 influenza virus killed up to 60% of pregnant mice and caused abortion in up to 40%, whereas a circulating seasonal H1N1 influenza virus did not cause any deaths or abortions. Higher viral titers and levels of inflammatory cytokines and chemokines such as interleukin (IL)-1α, IL-6, granulocyte colony-stimulating factor, RANTES, monocyte chemotactic protein, and KC (CXCL1), were detected in the lungs of pregnant mice infected with the 2009 pandemic H1N1 influenza virus, compared with the seasonal H1N1 influenza virus. The results of our study with pregnant mice suggest that the observed higher pathogenesis in pregnant women infected with the 2009 pandemic H1N1 influenza virus than the seasonal H1N1 influenza virus may be due to higher viral replication, elevated induction of inflammatory chemokines, and reduced progesterone.

Induction of inflammatory cytokines and toll-like receptors in human normal respiratory epithelial cells infected with seasonal H1N1, 2009 pandemic H1N1, seasonal H3N2, and highly pathogenic H5N1 influenza virus.

Respiratory epithelial cells are one of main targets for infections caused by influenza viruses. Recently, the induction of proinflammatory cytokines and toll-like receptors (TLRs) in normal human bronchial/tracheal epithelial cells infected with seasonal H1N1, 2009 pandemic H1N1, seasonal H3N2, or highly pathogenic H5N1 influenza virus were studied to understand the pathogenesis and early immune responses. The cells were productively infected with the viruses. Among the inflammatory cytokines tested, interleukin (IL)-8 was predominantly induced in virus-infected cells. Among the chemokines tested, interferon-γ-inducible protein-10 (IP-10) and growth-related oncogene-α (GRO-α) were predominantly induced in virus-infected cells. TLR-5 was predominantly induced in cells infected with seasonal H1N1, pandemic H1N1, or H5N1 influenza virus, and TLR-3 was predominantly induced in cells infected with seasonal H3N2 influenza virus. Taken together, the results suggest that IL-8, IP-10, and GRO-α are predominantly induced in respiratory epithelial cells infected with influenza A viruses, and that TLR-5 and TLR-3 are involved in the stimulation of virus-infected respiratory epithelial cells.

Induction of inflammatory cytokines and Toll-like receptors in chickens infected with avian H9N2 influenza virus.

H9N2 influenza virus is endemic in many Asian countries and is regarded as a candidate for the next human pandemic. Knowledge of the induction of inflammatory responses and toll-like receptors (TLRs) in chickens infected with H9N2 is limited. Here, we show that H9N2 induces pro-inflammatory cytokines such as transforming growth factor-beta 3; tumor necrosis factor-alpha; interferon-alpha, -beta, and gamma; and TLR 1, 2, 3, 4, 5, 7, and 15 in trachea, lung, and intestine of infected chickens. In the lung, TLR-15 was dominantly induced. Taken together, it seems that H9N2 infections efficiently induce inflammatory cytokines and TLRs in trachea, lung and intestine of chickens.

Pandemic H1N1 influenza virus causes a stronger inflammatory response than seasonal H1N1 influenza virus in ferrets.

A 2009 H1N1 influenza virus pandemic, which had its origin in swine, caused severe illness and mortality in humans. Inflammatory responses may be responsible for pathogenesis caused by infection with influenza viruses. To better understand the pathogenic mechanism, clinical signs and inflammatory responses in ferrets infected with the pandemic H1N1 were compared with those caused by seasonal H1N1 influenza virus. Ferrets infected with the 2009 pandemic H1N1 virus displayed higher body temperatures, greater reduction in body weight, and higher viral titers in the tracheae and lungs. Levels of inflammatory cytokines, including interleukin-6, interferon-alpha, and tumor necrosis factor-alpha, were higher in the lungs of ferrets infected with the 2009 pandemic H1N1. The data support the idea that increased pathogenesis caused by the 2009 pandemic H1N1 influenza virus may have been partially mediated by a higher induction of pro-inflammatory cytokines in the lungs of affected humans or animals.

Single dose of oil-adjuvanted inactivated vaccine protects chickens from lethal infections of highly pathogenic H5N1 influenza virus.

The highly pathogenic H5N1 influenza viruses are endemic in poultry in many countries, but continuously infect humans and cause human mortality. H5N1 influenza viruses have been regarded as a pandemic candidate. In a pandemic event by this virus, the protection of poultry with an effective vaccine will help to greatly reduce the spread of this virus to humans since it easily infects poultry. Here we showed that immunization with one dose of oil-adjuvanted inactivated H5N1 vaccine could protect chickens from lethal infection by highly pathogenic H5N1 influenza virus until 12 weeks post-immunization. The complete protection of chickens depended on the amount of HA antigens in the vaccine. Complete homologous protection required over 1.25 µg of HA antigens and complete heterologous protection required over 5.0 µg of HA antigens. The bivalent H5N1 inactivated vaccine composed of 1.25 µg of each antigen from clade 1 and clade 2.3.4 H5N1 influenza virus completely protected chickens from the lethal challenge of both viruses. When we determined the induction of antibody subtypes in tissues including nasal cavity, trachea, and lungs, the IgG subtype of antibody was induced more than the IgM or IgA subtype of antibody. Taken together, our results suggest that one dose of oil-adjuvanted inactivated H5N1 vaccine could provide chickens with sterile immunity against the homologous highly pathogenic H5N1 influenza virus.

Alveolar macrophages are indispensable for controlling influenza viruses in lungs of pigs.

Alveolar macrophages constitutively reside in the respiratory tracts of pigs and humans. An in vivo role of alveolar macrophages in defending against influenza viruses in mice infected with a reassorted influenza virus, 1918 HA/NA:Tx/91, was reported, but there has been no report on an in vivo role of alveolar macrophages in a natural host such as a pig using currently circulating human influenza virus. Here we show that in vivo depletion of alveolar macrophages in pigs by dichloromethylene diphosphonate (MDPCL2) treatment results in 40% mortality when pigs are infected with currently circulating human H1N1 influenza viruses, while none of the infected control pigs died. All infected pigs depleted of alveolar macrophages suffered from more severe respiratory signs than infected control pigs. Induction of tumor necrosis factor alpha in the infected pigs depleted of alveolar macrophages was significantly lower than that in the lungs of infected control pigs, and the induction of interleukin-10, an immunosuppressive cytokine, significantly increased in the lungs of infected pigs depleted of alveolar macrophages compared to infected control pigs. When we measured antibody titers and CD8(+) T lymphocytes expressing gamma interferon (IFN-gamma), lower antibody titers and a lower percentage of CD8(+) T lymphocytes expressing IFN-gamma were detectable in MDPCL2-treated infected pigs than in phosphate-buffered saline- and liposome-treated and infected pigs. Taken together, our findings suggest that alveolar macrophages are essential for controlling H1N1 influenza viruses in pigs.

No apoptotic deaths and different levels of inductions of inflammatory cytokines in alveolar macrophages infected with influenza viruses.

Influenza viruses are reported to infect mainly the respiratory tract epithelium of hosts. Our studies in a pig model show that influenza A viruses infect alveolar macrophages that constitutively reside in the respiratory tract, without causing apoptosis. Tumor necrosis factor alpha was the inflammatory cytokine most highly induced in these macrophages. In vivo, alveolar macrophages infected with human H3N2 influenza virus showed greater expression of tumor necrosis factor alpha than did alveolar macrophages infected with human H1N1 influenza virus. Induction of specific inflammatory cytokine such as TNF-alpha is a polygenic trait that involves the HA and NA genes. Markedly elevated expression of tumor necrosis factor alpha may be responsible for the high mortality rate caused by H3N2 influenza virus infection in elderly patients.

The NS1 gene of H5N1 influenza viruses circumvents the host anti-viral cytokine responses.

The H5N1 influenza viruses transmitted to humans in 1997 were highly virulent, but the mechanism of their virulence in humans is largely unknown. Here we show that lethal H5N1 influenza viruses, unlike other human, avian, and swine influenza viruses, are resistant to the anti-viral effects of interferons and tumor necrosis factor alpha The nonstructural (NS) gene of H5N1 viruses is associated with this resistance. Pigs infected with recombinant human H1N1 influenza virus that carried the H5N1 NS gene experienced significantly greater and more prolonged viremia, fever, and weight loss than did pigs infected with wild-type human H1N1 influenza virus. These effects required the presence of glutamic acid at position 92 of the NS1 molecule. These findings may explain the mechanism of the high virulence of H5N1 influenza viruses in humans and provide insight into the virulence of 1918 Spanish influenza.

Lethal H5N1 influenza viruses escape host anti-viral cytokine responses.

The H5N1 influenza viruses transmitted to humans in 1997 were highly virulent, but the mechanism of their virulence in humans is largely unknown. Here we show that lethal H5N1 influenza viruses, unlike other human, avian and swine influenza viruses, are resistant to the antiviral effects of interferons and tumor necrosis factor alpha. The nonstructural (NS) gene of H5N1 viruses is associated with this resistance. Pigs infected with recombinant human H1N1 influenza virus that carried the H5N1 NS gene experienced significantly greater and more prolonged viremia, fever and weight loss than did pigs infected with wild-type human H1N1 influenza virus. These effects required the presence of glutamic acid at position 92 of the NS1 molecule. These findings may explain the mechanism of the high virulence of H5N1 influenza viruses in humans.

Tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells.

Previous studies have associated influenza virus-induced expression of inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), with influenza pathogenesis in the human respiratory tract and have suggested that alpha and beta interferons are the first cytokines recruited to counteract such infection. However, we report here that TNF-alpha has powerful anti-influenza virus activity. When infected with influenza virus, cultured porcine lung epithelial cells expressed TNF-alpha in a dose-dependent manner. Expression of TNF-alpha was induced only by replicating virus. TNF-alpha showed strong antiviral activity against avian, swine, and human influenza viruses, and the antiviral effect of TNF-alpha was greater than that of gamma or alpha interferon. These findings suggest that TNF-alpha serves as the first line of defense against influenza virus infection in the natural host.