Immunostimulatory Motifs Enhance Antiviral siRNAs Targeting Highly Pathogenic Avian Influenza H5N1

Highly pathogenic avian influenza (HPAI) H5N1 virus is endemic in many regions around the world and remains a significant pandemic threat. To date H5N1 has claimed almost 300 human lives worldwide, with a mortality rate of 60% and has caused the death or culling of hundreds of millions of poultry since its initial outbreak in 1997. We have designed multi-functional RNA interference (RNAi)-based therapeutics targeting H5N1 that degrade viral mRNA via the RNAi pathway while at the same time augmenting the host antiviral response by inducing host type I interferon (IFN) production. Moreover, we have identified two factors critical for maximising the immunostimulatory properties of short interfering (si)RNAs in chicken cells (i) mode of synthesis and (ii) nucleoside sequence to augment the response to virus. The 5-bp nucleoside sequence 5′-UGUGU-3′ is a key determinant in inducing high levels of expression of IFN -α, -β, -λ and interleukin 1- β in chicken cells. Positioning of this 5′-UGUGU-3′ motif at the 5′- end of the sense strand of siRNAs, but not the 3′- end, resulted in a rapid and enhanced induction of type I IFN. An anti-H5N1 avian influenza siRNA directed against the PB1 gene (PB1-2257) tagged with 5′-UGUGU-3′ induced type I IFN earlier and to a greater extent compared to a non-tagged PB1-2257. Tested against H5N1 in vitro, the tagged PB1-2257 was more effective than non-tagged PB1-2257. These data demonstrate the ability of an immunostimulatory motif to improve the performance of an RNAi-based antiviral, a finding that may influence the design of future RNAi-based anti-influenza therapeutics

Induction of IFN-β in chicken DF-1 cells by siRNAs.

<p>IFN-β mRNA levels induced in DF-1 cells by siRNAs (20 pmol) or polyI:C (0.8 µg). Each value is the mean + standard deviation (3 replicates) and representative of data from 3 experiments. <i>a</i> = <i>p</i><0.001 between M-592 and PA-2087 after 8 h. <i>b</i> = <i>p</i><0.001 between PA-2087 and PB2-2240 after 8 h.</p

Immunostimulatory properties of T7-siRNAs and c-siRNAs in chicken cells.

<p>IFN-β mRNA levels were measured in DF-1 cells transfected with T7-siRNAs (white bars, 20 pmol) or c-siRNAs (black bars, 20 pmol) at (A) 8 h or (B) 24 h. <i>a</i> = <i>p</i><0.05 between paired white bars (T7-siRNA) and black bars (c-siRNA). (C) IFN-β mRNA levels in DF-1 cells induced by isPB1-2557 (chemically synthesized, 20 pmol) or 2Me-isPB1-2257 (chemically-synthesized, 20 pmol). <i>a</i>. = <i>p</i><0.001 between isPB1-2257 and 2Me-isPB1-2257 at 24 h. (D) IFN-β mRNA levels in DF-1 cells transfected with T7-siRNAs (20 pmol) untreated (white bars) or treated with shrimp alkaline phosphatase (SAP) (black bars). Data shows IFN-β mRNA levels 8 h post-transfection. <i>a</i>. = <i>p</i><0.05 between levels of IFN-β in cells transfected with untreated siRNAs or SAP-treated siRNAs. Results are representative of 3 separate experiments.</p

Inhibition of H5N1 influenza virus growth by <i>5′-UGUGU-3′</i>-tagged PB1-2257.

<p>(A) IFN-β mRNA levels in HD-11 cells transfected with c-siRNAs (all 20 pmol). Data shows IFN-β mRNA levels 8 h post-transfection. <i>a</i>. = <i>p</i><0.01 between levels of IFN-β in cells transfected with uPB1-2257 versus PB1-2257. (B) TCID₅₀ of H5N1 virus growth in HD-11 cells transfected with c-siRNAs (5 pmol, 96 well plate) or polyI:C (0.1 µg). Cells were transfected with siRNAs and infected with H5N1 virus 24 h later at dilutions of virus of 10^-1to 10⁻⁷. Cells were rinsed with fresh medium before the addition of virus. TCID₅₀ values were measured 72 h post-infection. Each value is the mean + standard deviation (4 replicates). <i>a</i> = <i>p</i><0.05 between virus levels in cell supernatants of cells treated with PB1-2257 or uPB1-2257. (C) Viability of HD-11 cells 24 h post-transfection with c-siRNAs (5 pmol, 96 well plate) as measured by Alamar blue. Results are representative of 3 separate experiments.</p

siRNAs used in this study

<p>siRNAs used in this study</p

QRT-PCR primers and probes used in this study

<p>QRT-PCR primers and probes used in this study</p