Penghui Yang

IL-17 response mediates acute lung injury induced by the 2009 Pandemic Influenza A (H1N1) Virus

The 2009 flu pandemic involved the emergence of a new strain of a swine-origin H1N1 influenza virus (S-OIV H1N1) that infected almost every country in the world. Most infections resulted in respiratory illness and some severe cases resulted in acute lung injury. In this report, we are the first to describe a mouse model of S-OIV virus infection with acute lung injury and immune responses that reflect human clinical disease. The clinical efficacy of the antiviral oseltamivir (Tamiflu) administered in the early stages of S-OIV H1N1 infection was confirmed in the mouse model. Moreover, elevated levels of IL-17, Th-17 mediators and IL-17-responsive cytokines were found in serum samples of S-OIV-infected patients in Beijing. IL-17 deficiency or treatment with monoclonal antibodies against IL-17-ameliorated acute lung injury induced by the S-OIV H1N1 virus in mice. These results suggest that IL-17 plays an important role in S-OIV-induced acute lung injury and that monoclonal antibodies against IL-17 could be useful as a potential therapeutic remedy for future S-OIV H1N1 pandemics.

Monoclonal antibody against CXCL-10/IP-10 ameliorates influenza A (H1N1) virus induced acute lung injury.

Monoclonal antibody against CXCL-10/IP-10 ameliorates influenza A (H1N1) virus induced acute lung injury

Multiple-Clade H5N1 Influenza Split Vaccine Elicits Broad Cross Protection against Lethal Influenza Virus Challenge in Mice by Intranasal Vaccination

Background The increase in recent outbreaks and unpredictable changes of highly pathogenic avian influenza (HPAI) H5N1 in birds and humans highlights the urgent need to develop a cross-protective H5N1 vaccine. We here report our development of a multiple-clade H5N1 influenza vaccine tested for immunogenicity and efficacy to confer cross-protection in an animal model. Methodology/Principal Findings Mice received two doses of influenza split vaccine with oil-in-water emulsion adjuvant SP01 by intranasal administration separated by two weeks. Single vaccines (3 µg HA per dose) included rg-A/Vietnam/1203/2004(Clade 1), rg-A/Indonesia/05/2005(Clade 2.1), and rg-A/Anhui/1/2005(Clade 2.3.4). The trivalent vaccine contained 1 µg HA per dose of each single vaccine. Importantly, complete cross-protection was observed in mice immunized using trivalent vaccine with oil-in-water emulsion adjuvant SP01 that was subsequently challenged with the lethal A/OT/SZ/097/03 influenza strain (Clade 0), whereas only the survival rate was up to 60% in single A/Anhui/1/2005 vaccine group. Conclusion/Significance Our findings demonstrated that the multiple-clade H5N1 influenza vaccine was able to elicit a cross-protective immune response to heterologous HPAI H5N1 virus, thus giving rise to a broadly cross-reactive vaccine to potential prevention use ahead of the strain-specific pandemic influenza vaccine in the event of an HPAI H5N1 influenza outbreak. Also, the multiple-clade adjuvanted vaccine could be useful in allowing timely initiation of vaccination against unknown pandemic virus.

Angiotensin-converting enzyme 2 (ACE2) mediates influenza H7N9 virus-induced acute lung injury

Since March 2013, the emergence of an avian-origin influenza A (H7N9) virus has raised concern in China. Although most infections resulted in respiratory illness, some severe cases resulted in acute respiratory distress syndrome (ARDS), which is a severe form of acute lung injury (ALI) that further contributes to morbidity. To date, no effective drugs that improve the clinical outcome of influenza A (H7N9) virus-infected patients have been identified. Angiotensin-converting enzyme (ACE) and ACE2 are involved in several pathologies such as cardiovascular functions, renal disease, and acute lung injury. In the current study, we report that ACE2 could mediate the severe acute lung injury induced by influenza A (H7N9) virus infection in an experimental mouse model. Moreover, ACE2 deficiency worsened the disease pathogenesis markedly, mainly by targeting the angiotensin II type 1 receptor (AT1). The current findings demonstrate that ACE2 plays a critical role in influenza A (H7N9) virus-induced acute lung injury, and suggest that might be a useful potential therapeutic target for future influenza A (H7N9) outbreaks.

Enhanced Influenza VLP vaccines comprising matrix-2 ectodomain and nucleoprotein epitopes protects mice from lethal challenge.

The matrix protein 2ectodomain (M2e) of the influenza A virus is a rational target antigen candidate for the development of a universal influenza virus-like particle (VLP) vaccine. In this study, a recombinant M2 protein with three tandem copies of M2e (3M2e), nucleoprotein (NP) epitopes and hepatitis B virus core (HBc), were expressed in Escherichia coli and purified by column chromatography. Mice immunized with 3M2e-NP-HBc in combination with an oil-in-water SP01 adjuvant produced robust M2e specific antibodies and cellular immune responses. Most importantly, the 3M2e-NP-HBc VLP vaccine provided enhanced protection against a lethal challenge with pandemic 2009 H1N1 and HPAI H5N1 virus through increased survival rates, a significant decrease in viral replication, and obvious alleviation of histopathological lung changes in challenged mice. Our results imply that a cellular immune response to NP is a plausible mechanism mediating this enhanced protection. These findings suggest that 3M2e-NP-HBc VLP has great potential as the basis development of a broadly protective influenza vaccine.

Response of mice and ferrets to a monovalent influenza A (H7N9) split vaccine.

In early spring 2013, the emergence of the influenza A (H7N9) virus in humans in Eastern China raised concerns of a new influenza pandemic. Development of a safe and effective H7N9 influenza vaccine is urgently needed. To this end, we first synthesized the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A (H7N9) virus A/AnHui/1/2013. Using reverse genetics, we rescued a reassortant virus (H7N9/PR8) that contained the HA and NA genes from wild-type H7N9 and six genes encoding internal proteins from the A/Puerto Rico/8/34 (PR8) virus. Next, the pathogenicity of the reassortant virus was evaluated both in vivo and in vitro. We found that the virus was non-pathogenic in mice and was stable after serial passaging in eggs. Furthermore, we found that a monovalent influenza A (H7N9) split vaccine prepared from the virus was immunogenic in mice and ferrets. When given intramuscularly, the vaccine (two doses of at least 15-µg) completely protected mice from normally lethal wild-type H7N9 virus challenge. In summary, our H7N9 vaccine, developed over a short time, is a potential candidate for further clinical evaluation and human use.

Corticosteroid treatment ameliorates acute lung injury induced by 2009 swine origin influenza A (H1N1) virus in mice.

Background The 2009 influenza pandemic affected people in almost all countries in the world, especially in younger age groups. During this time, the debate over whether to use corticosteroid treatment in severe influenza H1N1 infections patients resurfaced and was disputed by clinicians. There is an urgent need for a susceptible animal model of 2009 H1N1 infection that can be used to evaluate the pathogenesis and the therapeutic effect of corticosteroid treatment during infection. Methodology/Principal Findings We intranasally inoculated two groups of C57BL/6 and BALB/c mice (using 4- or 6-to 8-week-old mice) to compare the pathogenesis of several different H1N1 strains in mice of different ages. Based on the results, a very susceptible 4-week-old C57BL/6 mouse model of Beijing 501 strain of 2009 H1N1 virus infection was established, showing significantly elevated lung edema and cytokine levels compared to controls. Using our established animal model, the cytokine production profile and lung histology were assessed at different times post-infection, revealing increased lung lesions in a time-dependent manner. In additional,the mice were also treated with dexamethasone, which significantly improved survival rate and lung lesions in infected mice compared to those in control mice. Our data showed that corticosteroid treatment ameliorated acute lung injury induced by the 2009 A/H1N1 virus in mice and suggested that corticosteroids are valid drugs for treating 2009 A/H1N1 infection. Conclusions/Significance Using the established, very susceptible 2009 Pandemic Influenza A (H1N1) mouse model, our studies indicate that corticosteroids are a potential therapeutic remedy that may address the increasing concerns over future 2009 A/H1N1pandemics.

Characterization of the 2009 pandemic A/Beijing/501/2009 H1N1 influenza strain in human airway epithelial cells and ferrets.

Background A novel 2009 swine-origin influenza A H1N1 virus (S-OIV H1N1) has been transmitted among humans worldwide. However, the pathogenesis of this virus in human airway epithelial cells and mammals is not well understood. Methodology/Principal Finding In this study, we showed that a 2009 A (H1N1) influenza virus strain, A/Beijing/501/2009, isolated from a human patient, caused typical influenza-like symptoms including weight loss, fluctuations in body temperature, and pulmonary pathological changes in ferrets. We demonstrated that the human lung adenocarcinoma epithelial cell line A549 was susceptible to infection and that the infected cells underwent apoptosis at 24 h post-infection. In contrast to the seasonal H1N1 influenza virus, the 2009 A (H1N1) influenza virus strain A/Beijing/501/2009 induced more cell death involving caspase-3-dependent apoptosis in A549 cells. Additionally, ferrets infected with the A/Beijing/501/2009 H1N1 virus strain exhibited increased body temperature, greater weight loss, and higher viral titers in the lungs. Therefore, the A/Beijing/501/2009 H1N1 isolate successfully infected the lungs of ferrets and caused more pathological lesions than the seasonal influenza virus. Our findings demonstrate that the difference in virulence of the 2009 pandemic H1N1 influenza virus and the seasonal H1N1 influenza virus in vitro and in vivo may have been mediated by different mechanisms. Conclusion/Significance Our understanding of the pathogenesis of the 2009 A (H1N1) influenza virus infection in both humans and animals is broadened by our findings that apoptotic cell death is involved in the cytopathic effect observed in vitro and that the pathological alterations in the lungs of S-OIV H1N1-infected ferrets are much more severe.

Influenza virus vaccine expressing fusion and attachment protein epitopes of respiratory syncytial virus induces protective antibodies in BALB/c mice.

Respiratory syncytial virus (RSV) is an important viral pathogen that causes life-threatening respiratory infections in both infants and the elderly; no vaccines are at present available. In this report, we examined the use of influenza virus as a vehicle for production of an experimental RSV vaccine. We used reverse genetics to generate a recombinant influenza A virus with epitopes from the RSV fusion (F) and attachment (G) proteins (rFlu/RSV/F+G) in the influenza virus nonstructural (NS1) protein gene. Expression of RSV F+G epitope proteins was confirmed by Western blotting, and no changes in viral morphology were evident following examination by electron microscopy. BALB/c mice immunized intranasally with rFlu/RSV/F+G showed viral-specific antibody responses against both influenza and RSV. Total IgG, IgG1, IgG2a and IgA were measured in mice immunized with rFlu/RSV/F+G, revealing robust cellular and mucosal immune responses. Furthermore, we found that rFlu/RSV/F+G conferred protection against subsequent influenza and RSV challenges, showing significant decreases in viral replication and obvious attenuation of histopathological changes associated with viral infections. These findings suggest that rFlu/RSV/F+G is a promising vaccine candidate, which should be further assessed using cotton rat and primate models.

Immunization with a Live Attenuated H7N9 Influenza Vaccine Protects Mice against Lethal Challenge

The emergence of severe cases of human influenza A (H7N9) viral infection in China in the spring of 2003 resulted in a global effort to rapidly develop an effective candidate vaccine. In this study, a cold-adapted (ca), live attenuated monovalent reassortant influenza H7N9 virus (Ah01/AA ca) was generated using reverse genetics that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2013 pandemic A H7N9 isolate, A/Anhui/01/2013 virus (Ah01/H7N9); the remaining six backbone genes derived from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). Ah01/AA ca virus exhibited temperature sensitivity (ts), ca, and attenuation (att) phenotypes. Intranasal immunization of female BALB/c mice with Ah01/AA ca twice at a 2-week interval induced robust humoral, mucosal, and cell-mediated immune responses in a dose-dependent manner. Furthermore, the candidate Ah01/AA ca virus was immunogenic and offered partial or complete protection of mice against a lethal challenge by the live 2013 influenza A H7N9 (A/Anhui/01/2013). Protection was demonstrated by the inhibition of viral replication and the attenuation of histopathological changes in the challenged mouse lung. Taken together, these data support the further evaluation of this Ah01/AA ca candidate vaccine in primates.