Zhongpeng Zhao

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.

Dysfunction of IL-10-producing type 1 regulatory T cells and CD4+CD25+ regulatory T cells in a mimic model of human multiple sclerosis in Cynomolgus monkeys

CD4(+)CD25(+) Treg and IL-10(+) Tr1 cells play a major role in controlling autoimmunity by suppressing self-reactive T cells. Dysfunction of Tregs appears to be a critical factor in the pathogenesis of autoimmune diseases. Multiple sclerosis (MS) is an inflammatory demyelinating disorder of CNS, where CD4(+) T cells result in nervous tissue damage. The aim of this study was to investigate the protective role of Treg and Tr1 cells in a mimic model of human MS in Cynomolgus monkeys. This study indicated the suppressive capacity of Tregs from MS monkeys was impaired compared with naive controls. The population of CD4(+)CD25(+) Tregs was decreased in acute stage of MS. However, they showed a restored function and percentage in remitting monkeys. In stable phase, CD4(+)CD25(+) Tregs differentially expressed elevated level of CD62P cell adhesion molecule which contributes to the mechanism by which Treg cells inhibit CD4(+) T cell responses. On the other hand, the percentage of CD4(+)IL-10(+) Tr1 and suppressive function of Tr1 cells were found reduced in MS monkeys. IL-10 secretion was diminished almost 9-fold in active MS, and recovered in active MS. This deficit in IL-10 secretion was specific to CD3/CD46, but not to CD3/CD28 stimulation. The concentrations of IFN-gamma secreted by CD3/CD46-activated T cells were also not affected. These results demonstrate that Tregs are dysfunctional in Cynomolgus monkey with MS. Loss of regulatory function appears to be an important factor in the pathogenesis of MS. Hence, to develop new approaches for induction of Tregs in vivo may be beneficial for the clinical treatment in autoimmune diseases.

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

Protection of mice from Brucella infection by immunization with attenuated Salmonella enterica serovar typhimurium expressing A L7/L12 and BLS fusion antigen of Brucella.

This study describes the potential use of attenuated Salmonella enterica serovar Typhimurium Strains (S. typhimurium) to express and deliver a L7/L12 and BLS fusion antigen of Brucella as a vaccination strategy to prevent Brucella infection in mice. S. typhimurium X4072 that contained a pTrc99A-BLS-L7/L12 plasmid, designated X4072bl, can deliver a L7/L12 and BLS fusion antigen expressed by the bacterium itself, while S. typhimurium X4550 that contained an asd-pVAX1-BLS-L7/L12 (asd-pBL) plasmid, designated X4550bl, can deliver the antigen to be expressed in target eukaryotic cells. When orally administered to BALB/c mice, both attenuated carrier strains were able to elicit mucosal and systemic immunity, which induced protection against B. abortus 544 infection in mice. The immunogenicity and protective efficacy of X4072bl and X4550bl were compared with a recombinant BLS-L7/L12 fusion protein vaccine (rBL) and a pVAX1-BLS-L7/L12 DNA vaccine (pBL) in this study. When rBL and pBL were intramuscularly injected into mice, both vaccines could also elicit comparable humoral and cellular immune responses, but not mucosal immunity, which therefore induced lower protection. Taken together, Salmonella-based subunit vaccines are a promising vaccine strategy in the prevention of Brucella infection.

Immunogenicity and protective efficacy of a live attenuated vaccine against the 2009 pandemic A H1N1 in mice and ferrets.

A novel 2009 influenza A (H1N1) virus was transmitted from humans to humans worldwide. The live attenuated monovalent A H1N1 vaccine (LAMV) for intranasal administration has shown promising immunogenicity and safety in clinical trials and for human use, but the experimental data based on LAMV is incomplete. In this study, using reverse genetic technology, we produced a cold-adapted (ca), live attenuated BJ/AA ca that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2009 pandemic A H1N1 isolate, A/Beijing/501/2009 virus (BJ501), and the remaining six internal gene segments from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). BJ/AA ca exhibited phenotypes of temperature sensitivity (ts), ca, and attenuation (att). The candidate BJ/AA ca was immunogenic in mice and induced strong mucosal secretory IgA (sIgA) in the respiratory tract. Two dosages of intranasal immunization induced robust HI antibodies and offered efficient protection against challenge by the wild-type (wt) 2009 pandemic A H1N1 (A/Beijing/501/2009 or A/California/07/2009) in mice and ferrets. These results support the evaluation of this vaccine made from a wt strain isolated in China for clinical trials.

Identification of immunoreactive proteins of Brucella melitensis by immunoproteomics

Infection with Brucella causes brucellosis, a chronic disease in humans, which induces abortion and sterility in livestock. Among the different Brucella species, Brucella melitensis is considered the most virulent and is the predominant species associated with outbreaks in China. To date, no safe human vaccine is available against Brucella infection. The currently used live vaccines against Brucella in livestock induce antibodies that interfere with the diagnosis of field infection in vaccinated animals, which is harmful to eradication programs. However, there is as yet no complete profile of immunogenic proteins of B. melitensis. Towards the development of a safer, equally efficacious, and field infection-distinguishable vaccine, we used immunoproteomics to identify novel candidate immunogenic proteins from B. melitensis M5. Eighty-eight immunoreactive protein spots from B. melitensis M5 were identified by Western blotting and were assigned to sixty-one proteins by mass spectrometry, including many new immunoreactive proteins such as elongation factor G, F0F1 ATP synthase subunit beta, and OMP1. These provide many candidate immunoreactive proteins for vaccine development.

The 2009 pandemic A/Wenshan/01/2009 H1N1 induces apoptotic cell death in human airway epithelial cells

In 2009, a novel swine-origin H1N1 influenza virus emerged in Mexico and quickly spread to other countries, including China. This 2009 pandemic H1N1 can cause human respiratory disease, but its pathogenesis remains poorly understood. Here, we studied the infection and pathogenesis of a new 2009 pandemic strain, A/Wenshan/01/2009 H1N1, in China in human airway epithelial cell lines compared with contemporary seasonal H1N1 influenza virus. Our results showed that viral infection by the A/Wenshan H1N1 induced significant apoptotic cell death in both the human nasopharyngeal carcinoma cell line CNE-2Z and the human lung adenocarcinoma cell line A549. The A/Wenshan H1N1 virus enters both of these cell types more efficiently than the seasonal influenza virus. Viral entry in both cell lines was shown to be mediated by clathrin- and dynamin-dependent endocytosis. Therefore, we discovered that the 2009 pandemic H1N1 strain, A/Wenshan/01/2009, can induce apoptotic cell death in epithelial cells of the human respiratory tract, suggesting a molecular pathogenesis for the 2009 pandemic H1N1.

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.