Activation of the innate immune system provides broad-spectrum protection against influenza A viruses with pandemic potential in mice

Abstract

The efficacy of a stabilized chemical analog of double-stranded ribonucleic acid (RNA), PIKA, as prophylaxis against infection with 5 different influenza A virus subtypes, including the 2009 swine-origin pandemic H1N1 virus, was evaluated in mice. Intranasal treatment with PIKA resulted in a significant reduction of viral replication in the respiratory tract. The inhibitory effect was mediated by rapid infiltration of immune cells into the lungs, and production of inflammatory cytokines. While TLR3 is important for the optimal production of these inflammatory cytokines, inhibition of viral replication was still observed in TLR3(-/-) mice. In addition, a significant synergistic effect in inhibiting H5N1 virus replication was observed when PIKA was coadministered with oseltamivir. The broad-spectrum protection provided by PIKA makes it an attractive option for prophylaxis from infection with influenza A viruses.

Figure 1. Administration of PIKA inhibited replication of influenza viruses in the respiratory tract of mice

Groups of five mice each were given 100 μg of PIKA in PBS intranasally 6 h before challenge with the indicated viruses. The mice continued to receive PIKA treatment once a day for two additional days. Lungs and NT were harvested on day 3 post-infection. Virus titers in the lungs and NT were determined in MDCK cells. The lower limit of detection was 10^1.5 TCID₅₀ per gram of tissue. The horizontal bars represent the geometric mean of the group. The percent reduction in mean viral titer relative to the control group treated with PBS is shown above each column of data. The `*' symbol indicates that the difference between the groups was statistically significant (p<0.05).

Figure 2. Administration of PIKA protected mice from lethal challenge and initiated influenza-specific antibody responses

(A–B) Groups of five mice each were given PIKA or PBS as previously described and challenged with 4 LD₅₀ of A/Puerto Rico/8/34 intranasally. The mice continued to receive PIKA treatment once a day for two additional days. Weight loss and survival rate was monitored for 2 weeks. (C) Serum samples were collected from PIKA-treated mice on day 14 post-infection and influenza-specific antibody titers were determined by ELISA.

Figure 3. Synergistic anti-viral effect achieved against H5N1 infection in mice by co-administering PIKA with oseltamivir

(A–B) Groups of five mice were given either oseltamivir alone by oral gavage or together with 100 μg of PIKA i.n. 6 hours before challenge with 15 TCID₅₀ of VN04 wt. Mice continued to receive treatment for 2 additional days. NT and lungs were harvested for viral titration on day 3 post-infection. Virus titers in the NT (A) and lungs (B) were determined in MDCK cells. The lower limit of detection was 10^1.5 TCID₅₀ per gram of tissue. The horizontal bars represent the geometric mean of the group. The `*' symbol indicates that the difference between the groups was statistically significant (p<0.05).

Figure 4. Administration of PIKA changed the cellular composition of the lungs

Groups of three mice were given 100 μg of PIKA i.n. in PBS and sacrificed 1, 2 or 3 days later. An additional group received 100 μg of PIKA i.n. once a day for 3 consecutive days and mice were sacrificed 24 hours after the last inoculation. Single-cell suspensions were prepared and the cells were stained with the indicated cellular makers. Eighty thousand events were acquired for each sample. The bars and error bars represent the mean and standard errors of the groups. The `*' symbol indicates that the difference between the groups was statistically significant (p<0.05).

Figure 5. Administration of PIKA induced production of cytokines in the lungs

Groups of three mice each were given 100 μg of PIKA i.n. in PBS and were sacrificed at the indicated time. The lungs were stored at −80°C till all samples were collected and homogenized in 1 mL of RPMI-1640 media. (A–C) To measure the level of different cytokines/ chemokines in the samples, 50 μL of the clarified samples were tested in duplicate using the Bio-plex Protein Array system. (D) For IFN-β, 100 μL of the clarified samples were tested using an ELISA kit. The bars and error bars represent the mean and standard error of the groups.

Figure 6. TLR3 is dispensable in mediating the anti-viral responses of PIKA in mice

(A) Human embryonic kidney (HEK 293 cells) were transfected with an NF-κB-luciferase reporter gene and a β-galactosidase-expressing plasmid with or without co-transfection of the indicated TLR3, MDA-5 or RIG-I receptor-expressing plasmids. Twenty–four hours after transfection, the cells were stimulated with 50 ng of PIKA in plain medium for 6 hours before the cells were lysed and luciferase activity in the lysates was determined. The data were normalized to β-galactosidase activity and expressed as fold-increase relative to expression in cells that were transfected with respective receptor-expressing plasmids without PIKA stimulation. The bar and error bars represent the mean and standard error of 8 replicates and are representative of two independent experiments. (B) Groups of three TLR3^−/− or TLR3^+/+ mice received PIKA or PBS intranasally and were sacrificed 24 hours later. Lung homogenates were prepared and the concentration of various cytokines was determined as previously described. Data are expressed as the fold-increase in PIKA-treated mice over PBS-treated mice. Groups of five TLR3^−/− or TLR3^+/+ mice received PIKA and were challenged with the H7N1 virus as previously described. Virus titers in the NT (C) and lungs (D) were determined in MDCK cells. The horizontal bars represent the geometric mean of the group. The `*' symbol indicates that the difference between the groups was statistically significant (p<0.05).