Zhonghua Jiang

Expression of mouse beta-defensin-3 in MDCK cells and its anti-influenza-virus activity

Influenza (flu) pandemics have presented a threat to human health in the past century. Because of outbreaks of avian flu in humans in some developing countries in recent years, humans are more eager to find a way to control flu. Mammalian beta-defensins (β-defensins) are associated primarily with mucosal and skin innate immunity. Previous studies have demonstrated antimicrobial properties of a variety of defensin peptides. We have identified the presence of mouse β-defensin 1, 2, and 3 genes (Mbd-1, 2, and 3) in trachea and lung tissues by RT-PCR before and after infection with influenza virus. We constructed a eukaryotic expression plasmid containing Mbd-3, pcDNA 3.1(+)/MBD-3, and the plasmid was introduced into Madin–Darby canine kidney (MDCK) cells by transfection. The expression of Mbd-3 in MDCK cells was verified by immunofluorescence test, RT-PCR, and Western blot. The pcDNA 3.1(+)/MBD-3 plasmid was injected into mice to observe its effect against influenza A virus (IAV) in vivo. Mouse β-defensin genes could be expressed in trachea and lung tissues before IAV infection, but expression of Mbd-2 and Mbd-3 was increased significantly after IAV infection. The survival rate of mice with MBD-3 against IAV challenge was 71.43%, and MDCK cells with MBD-3 could clearly inhibit IAV replication. The results demonstrated that mouse β-defensins possess anti-influenza virus activity, suggesting that mouse β-defensins might be used as agents to prevent and treat influenza.

Antiviral activity of recombinant mouse β-defensin 3 against influenza A virus in vitro and in vivo.

Background: Influenza causes significant morbidity and mortality. Mammalian β-defensins are small peptides of about 4.5–6 kDa in mass and are effectors of the innate immune response with potent antimicrobial activity. In this paper, we focused on the anti-influenza A activity of the recombinant mouse β-defensin 3 (rMBD-3) in vivo and in vitro. Methods: The rMBD-3 peptide was added to Madin-Darby canine kidney (MDCK) cells at different stages of influenza A virus (IAV) A/PR/8/34 (H1N1) infection and its virus inhibitory properties were determined. Mice were infected with IAV and treated with rMBD-3 peptide from 12 h post-infection. The effect of rMBD-3 peptide was determined by pulmonary viral load, pathology and mortality. In addition, the expression of interleukin (IL)-12, interferon (IFN)-γ and tumour necrosis factor (TNF)-α genes in mice with or without rMBD-3 treatment was determined by semi-quantitative reverse transcriptase PCR. Results: rMBD-3 was shown to protect MDCK cells against IAV infection and had a major role in inhibition of adsorption and uptake by cells infected with IAV. Following the addition of 100 μg/ml rMBD-3 to MDCK cells medium, approximately 80% of cells were protected from infection in vitro. rMBD-3 given by tail vein injection (10 mg/kg/day) was the most effective method to improve the survival rate of the mice. Treatment with rMBD-3 was found to up-regulate IFN-γ and IL-12 gene expression, but reduced expression of the TNF-α gene. Conclusions: These results demonstrate that rMBD-3 possesses anti-influenza virus activity both in vivo and in vitro that might be of therapeutic use.

Immunogenicity of multiple-epitope antigen gene of HCV carried by novel biodegradable polymers.

In order to develop a promising vaccine candidate utilizing a combined approach to induce both antibody production and T-cell activity, the DNA fragment containing MA of HCV with five conserved epitopes was synthesized. Two types of HCV vaccine candidates (the DNA type and DNA/polymers) were constructed using MA. PLA-PEG-PLA and PLGA-PEG-PLGA were synthesized and used as micelles with encapsulated plasmid pcDNA3.1(+)-MA. The preparation of copolymers, the cloning and analysis of recombinant plasmid DNA, in vitro expression, and immunogenicity in transgenic mice were evaluated in detail. The results indicated that even single immunization and oral immunization with DNA/polymers achieved satisfying immune responses in vivo tests. As biodegradable and nontoxic triblock copolymers, the novel copolymers demonstrated a great advantage, as they made long-term and single-immunizing vaccines possible; in addition, the copolymers showed a better adjuvant effect and scarcely any side effects.

Antifungal activity of recombinant mouse beta-defensin 3.

Aims:  To identify the presence of mouse β‐defensin 3 (Mbd3) (the human homologue of β‐defensin 2) in different tissues and to define the antimicrobial properties of recombinant MBD3 (rMBD3) against a panel of human pathogens.

Association of TNF-α Gene Promoter Polymorphisms With Susceptibility of Cervical Cancer in Southwest China

Objective: Variations in the tumor necrosis factor-alpha (TNF-α) gene may lead to changes in the level of TNF-α associated with the susceptibility to cancer. This study is designed to determine the association of TNF-α promoter polymorphisms with the susceptibility of cervical cancer among women living in Southwest China. Methods: Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing, we analyzed the genotype and allele distributions of 4 single-nucleotide polymorphisms (SNP) of TNF-α gene in 239 cervical cancer patients and 110 controls. Results: Compared to controls, cervical cancer patients show a significant increase in the frequency of GA genotype at −308 G/A, and a significantly decreased frequency of the CT genotype. A significant decrease in the frequency of the T allele at −857 C/T ( P <0.05) was also found. No significant differences of SNP genotype and allele at TNF-α-863 C/A and −238 G/A were observed between the 2 groups. Conclusion: An SNP at TNF-α −857 C/T and −308 G/A, but not −863 C/A or −238 G/A, were significantly associated with an increased risk of cervical cancer in the studied population. * TNF-α : tumor necrosis factor-alpha PCR-RFLP : polymerase chain reaction-restriction fragment length polymorphism SNP : single-nucleotide polymorphism HPV : human papillomavirus EGF : epidermal growth factor CIN : cervical intraepithelial neoplasia EDTA : ethylenediaminetetraacetic acid OR : odds ratios CI : confidence interval ICC : invasive cervical cancer

Immune effects against influenza A virus and a novel DNA vaccine with co-expression of haemagglutinin- and neuraminidase-encoding genes.

The high variability of influenza virus causes difficulties in the control and prevention of influenza, thus seeking a promising approach for dealing with these problems is a hot topic. Haemagglutinin (HA) and neuraminidase (NA) are major surface antigens of the influenza virus, and provide effective protection against lethal challenges with this virus. We constructed a DNA vaccine (pHA-IRES2-NA) that co-expressed both HA and NA, and compared its protective efficacy and immunogenic ability with that of singly expressed HA or NA, or a mixture of the two singly expressed proteins. Our findings showed that both HA and NA proteins expressed by pHA-IRES2-NA could be detected in vivo and in vitro. The protection of DNA vaccines was evaluated by serum antibody titres, residual lung virus titres and survival rates of the mice. In the murine model, immunization of pHA-IRES2-NA generated significant anti-HA and anti-NA antibody, increased the percentage of CD8(+) cells and gamma interferon-producing CD8(+) cells and the ratio of Th1/Th2 (T helper) cells, which was comparable to the effects of immunization with HA or NA DNA alone or with a mixture of HA and NA DNA. All the mice inoculated by pHA-IRES2-NA resisted the lethal challenge by homologous influenza virus and survived with low lung virus titre. In addition, previous studies reported that co-expression allowed higher-frequency transduction compared to co-transduction of separated vector systems encoding different genes. The novel HA and NA co-expression DNA vaccine is a successful alternative to using a mixture of purified HA and NA proteins or HA and NA DNA.

Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus

Influenza (flu) pandemics have exhibited a great threat to human health throughout history. With the emergence of drug-resistant strains of influenza A virus (IAV), it is necessary to look for new agents for treatment and transmission prevention of the flu. Defensins are small (2–6 kDa) cationic peptides known for their broad-spectrum antimicrobial activity. Beta-defensins (β-defensins) are mainly produced by barrier epithelial cells and play an important role in attacking microbe invasion by epithelium. In this study, we focused on the anti-influenza A virus activity of mouse β-defensin 1 (mBD1) and β defensin-3 (mBD3) by synthesizing their fusion peptide with standard recombinant methods. The eukaryotic expression vectors pcDNA3.1(+)/mBD1-mBD3 were constructed successfully by overlap-PCR and transfected into Madin-Darby canine kidney (MDCK) cells. The MDCK cells transfected by pcDNA3.1(+)/mBD1-mBD3 were obtained by G418 screening, and the mBD1-mBD3 stable expression pattern was confirmed in MDCK cells by RT-PCR and immunofluorescence assay. The acquired stable transfected MDCK cells were infected with IAV (A/PR/8/34, H1N1, 0.1 MOI) subsequently and the virus titers in cell culture supernatants were analyzed by TCID50 72 h later. The TCID50 titer of the experimental group was clearly lower than that of the control group (p < 0.001). Furthermore, BALB/C mice were injected with liposome-encapsulated pcDNA3.1(+)/mBD1-mBD3 through muscle and then challenged with the A/PR/8/34 virus. Results showed the survival rate of 100% and lung index inhibitory rate of 32.6% in pcDNA3.1(+)/mBD1-mBD3group; the TCID50 titer of lung homogenates was clearly lower than that of the control group (p < 0.001). This study demonstrates that mBD1-mBD3 expressed by the recombinant plasmid pcDNA3.1(+)/mBD1-mBD3 could inhibit influenza A virus replication both in vitro and in vivo. These observations suggested that the recombinant mBD1-mBD3 might be developed into an agent for influenza prevention and treatment.

A novel DNA vaccine expressing the Ag85A-HA2 fusion protein provides protection against influenza A virus and Staphylococcus aureus

Secondary pneumonia due to Staphylococcus aureus (S. aureus) causes significant morbidity and mortality. The aim of the research was designed a novel DNA vaccine encoding the Mycobacterium tuberculosis secreted antigen Ag85A fused with the influenza A virus (IAV) HA2 protein to provide protection against both influenza and secondary infection with S. aureus. The DNA vaccine vector efficiently expressed the encoded antigen in mammalian cells, as determined by RT-PCR, Western blotting and immunofluorescence analysis. Mice were immunized with the vaccine by intramuscular injection before challenge with IAV and S. aureus. The pulmonary and the splenocyte culture IFN-γ levels were significant higher in immunized mice than their respective controls. Although the antibody titer in the HI test was low, the sera of mice immunized with the novel vaccine vector were effective in neutralisation assay in vitro. The vaccine could reduce the loss of body weight in mice during IAV challenge. Both Western blotting and RT-PCR showed that the vaccine markedly enhanced toll like receptor 2 (TLR2) expression in splenocytes after the secondary infection with S. aureus. The survival rate of mice with high TLR2 expression (pEGFP/Ag85A-HA2 or iPR) was significantly increased compared with mice immunized with pEGFP/HA2 after challenge with S. aureus. However, the pulmonary IL-10 concentration and S. aureus titer were significantly decreased in immunized mice, and expression of TLR2 was increased after challenge with S. aureus. These results demonstrated that Ag85A could strengthen the immune response to IAV and S. aureus, and TLR2 was involved in the host response to S. aureus.

Effect of influenza A virus non-structural protein 1(NS1) on a mouse model of diabetes mellitus induced by Streptozotocin

Type 1 diabetes (T1D) is a chronic autoimmune disease caused by proinflammatory autoreactive T cells that mediate the selective destruction of insulin-producing β cells via both direct and indirect mechanisms. Many immune cells and proinflammatory cytokines are involved in the pathogenesis of autoimmune diabetes. Immune intervention is effective for the prevention and treatment of T1D by blocking the autoimmune assault to β cells. The non-structural protein 1(NS1) of influenza A viruses is a non-essential virulence factor encoded on segment 8 that has multiple accessory functions, including suppression of innate immunity and adaptive immunity, inhibition of apoptosis and activation of phosphoinositide 3-kinase (PI3K). This research investigated whether the expression of NS1 can prevent and treat diabetes mellitus induced by Streptozotocin (STZ). The NS1 expressing plasmid pEGFP-C2/NS1 was constructed and injected intramuscularly to both thighs of mice. Its effect on mice was observed. Intramuscular delivery of pEGFP-C2/NS1 resulted in reduction in hyperglycemia and diabetes incidence, with an increase in insulin. pEGFP-C2/NS1 could also increase glycogen and regulated serum cytokine levels. In addition, by comparison to the mice treated with empty vector pEGFP-C2, ameliorative insulitis was observed in the mice treated with recombinant plasmid pEGFP-C2/NS1. This result suggests that the expression of NS1 is effective for the prevention and treatment of diabetes mellitus induced by STZ in a mouse model.

Expression of mouse beta defensin 2 in Escherichia coli and its broad-spectrum antimicrobial activity

Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34oC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95%). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86% at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.