Inhibition Of Highly Pathogenic Avian Influenza Virus (H5N1) Production By Antisense Oligonucleotides Specific For Viral PA And NP Genes

Objective:In this study, six antisense oligonucleotides (AS ODNs) targeting the PA gene or NP gene of highly pathogenic avian influenza virus (HPAIV) H5N1 were designed and evaluated for their ability to inhibit HPAIV replication in vitro and vivo.Methods:In accordance with AS ODNs design principles, we designed three AS ODNs (PA4, PA492 and PA1203) targeting the PA gene and three AS ODNs(NP267, NP628 and NP749) targeting the NP gene of H5N1 HPAIV, and the PA492 was complete match the 2009 H1N1 by BLAST. To evaluate their potential antiviral effect in MDCK cells, virus titer assay(hemagglutination assay and TCID50 test), indirect immunofluorescence assay(IFA) and real time PCR were carried out. Furthermore, the antiviral effect was detected by survival rate, weight loss, lung index and virus titers of lung in mice.Results:These results showed that at six h post-transfection, we achieved 94.4%of transfection efficiency with 4.0μM AS ODNs and all the six AS ODNs could inhibit viral replication. PA492 targeting the PA gene and NP628 targeting the NP gene had the most optimal antiviral effect and the viral replication was 29 fold and 17 fold lower than the viral control, respectively; PA492 and NP628 could partial protect the mice from lethal H5N1 influenza virus challenge and improve the surviral rate of infected mice, and the survival rate was 50% and 37.5%, and the PA492 also could partial protect the mice from lethal H1N1 influenza virus challenge and the survial rate was 80%. Moreover, the liposome mediated PA492 and NP628 had the lower lung index and virus titers in the infected mice compared with virus control, and showed that the viral replication was inhibited significantly at different time points post-infection. These results provided a potential method for the use of AS ODNs in prophylaxis and treatment of H5N1 avian influenza virus or H1N1 influenza virus.Conclusions:Our results showed that AS ODNs targeting of the AIV NP or PA gene could potently inhibit AIV H5N1 reproduction, thus, formulating a candidate for an emergent therapeutic drug for the pathogenic H5N1 influenza virus infection. Background:In recent years, frequent outbreaks of H5N1-subtype avian influenza virus (AIV) in poultry and humans have become serious health and economic concerns in China. AIV is composed of three transmembrane proteins:hemagglutinin (HA), neuraminidase (NA) and matrix protein 2 (M2). The HA protein is the principal immunogen on the envelope of AIV and is crucial for the infectivity of AIV. The HA protein is abundant on the surface of the infected cells, and is currently the main target of specific antibodies. Single-chain fragment variable (scFv) format antibody is a small antibody engineered by connecting the gene fragments for the variable regions of the heavy and light chains of an immunoglobin with a linker. The resulting scFv antibody usually retains the affinity and specificity of its parent antibody. Human scFvs have been shown to have potent neutralizing activity against AIV. The scFv antibody has many advantages in diagnostic and therapeutic applications because the scFv is small molecule and has targeting drug-delivery agent potential. Protamine, a major component of salmon sperm nucleus, contains many basic arginine residues. Protamine can bind to and deliver DNA into cells efficiently and be used for gene therapy. The 22 amino-acid form of truncated protamine (tP) containing the arginine-enriched domain usually retains protamine's ability to bind to and deliver DNA. Accordingly, a fusion protein of scFv specifically against AIV and tP may facilitate delivery of genetic materials, such as the AIV-specific siRNA, into the AIV-infected cells, inhibiting the AIV replication.Methods:The hemagglutinin antigen (HA) of avian influenza virus (AIV) is an immunogen abundant on the surfaces of infected cells, and can be used as a target for specific antibodies to clear viral infection. Protamine has been demonstrated to deliver DNA into cells effectively. Accordingly, a fusion protein of anti-HA single chain fragment variable (scFv) and truncated protamine (tP) may be used as a vehicle for delivering the anti-AIV siRNA into the AIV-infected cells for gene therapy. To test this hypothesis, we constructed a novel recombinant plasmid, pET28-scFv-tP, by connecting the genes for anti-H5N1 AIV HA-specific scFv with synthesized oligonucleotides encoding the 22 amino acids of human tP and a linker. Furthermore, the recombinant scFV-tP was expressed, refolding and purified. To determine whether the recombinant scFv-tP retained the capacity to recognize the specific antigen of H5N1 virus, and characterization of its bioactivity by cell based ELISA and indirect immunofluorescence assay. To determine whether the generated recombinant scFv-tP and scFv could bind to DNA, the plasmid DNA was mixed with different doses of scFv-tP, and their mobilizing behaviors were characterized by EMSA. To further determine whether scFv-tP was capable of mediating DNA transfection, the transfection efficacy of EGFP-expressing cells was examined, and the AIV-infected cells were transfected with FITC-ODNs mixed with scFv-tP and the transfection efficacy was determined by FACS analysis.We examined whether the scFv-tP could deliver the plasmid expressing the NP-specific siRNA into the AIV-infected MDCK cells and enhance the anti-AIV activity of a NP-specific siRNA against the viral replication. We examined whether the scFv-tP could enhance the antiviral activity of AS-ODNs. MDCK cells were infected with AIV and transfected with the PA492 in scFv-tP by viral titers, quantitative RT-PCR and indirect immunofluorescence (IFA) assays. To evaluate the scFv-tP-mediated targeted delivery of AS ODNs in vivo, FITC-PA492 was injected into AIV-infected mice alone or combined with scFv-tP fusion protein. To assess whether the inhibition of viral replication by PA492 treatment was adequate for protection, the surviral rate and weight loss were tested. To further determine the antiviral activity of scFv-tP-mediated PA492 in mice, the lung index and viral titers of lung were detected on days 2,4, and 6.Results:The recombinant scFV-tP was expressed and purified, with a yield of 7-8 mg of scFv-tP and a purity of> 92% from 1 L of bacterial culture. Characterization of its bioactivity revealed that scFv-tP recognized HA, similar to its scFv control, in a dose-dependent manner and that the scFv-tP, but not its scFv control, bound to DNA and delivered plasmid and oligonucleotide DNA into the AIV-infected MDCK cells effectively. More importantly, transfection with the mixture of the scFv-tP and plasmid for the NP-specific siRNA significantly inhibited the replication of AIV in MDCK cells, as compared with that transfection with the scFv-plasmid mixture, even with the plasmid in liposome. The results showed that scFv-tP fusion protein improved the antiviral effect of PA492 in MDCK cells by In addition, fluorescein isothiocyanate labeled-AS ODNs were delivered into the lung of AIV infected mice using the scFv-tP mediated transfection, and could partial protect the mice from lethal H5N1 influenza virus challenge. Moreover, the fusion protein mediated PA492 had a lower lung index and viral titers in the infected mice as compared with the liposome method.Conclusion:Our data demonstrated that the recombinant scFv-tP retained the functions of both scFv and tP, and might be potentially used for delivering genetic materials for targeting therapy of AIV infection, and provided a potential method for using anti-HA fusion protein for the targeted delivery of AS ODNs against AIV H5N1.