A TLR3 ligand that exhibits potent inhibition of influenza virus replication and has strong adjuvant activity has the potential for dual applications in an influenza pandemic

Abstract

The appearance and spread of the H5N1 highly pathogenic avian influenza (HPAI) raise concern of a possible pandemic. Current preventive measures include the development of a pre-pandemic influenza vaccine and stockpiling of neuraminidase inhibitors. However, their benefits can be significantly reduced by mutations in the hemagglutinin or neuraminidase resulting in antigenic changes and the appearance of drug-resistance, respectively. Drugs that target the innate immune system to achieve a 'heightened antiviral' state represent another class of antiviral agents that could contribute to the control and treatment of influenza infection. In this study, PIKA (a stabilized dsRNA) provides broad-spectrum prophylaxis against a number of influenza A viruses. In addition, when PIKA was admixed with influenza vaccine preparations, including a formalin-inactivated whole-virion H5 vaccine, significant adjuvanting effect leading to accelerated viral clearance was observed in a murine model. These biological effects appear to be mediated by the ability of PIKA to promote the maturation of dendritic cells, including up-regulation of co-stimulatory molecules, such as CD80 and CD86, and the induction of various cytokines and chemokines. Toll-like receptor 3 (TLR3) was shown to recognize PIKA in a concentration-dependent manner. The potency and versatility in its activities make PIKA an attractive candidate for use in an influenza pandemic.

Fig. 1

Administration of PIKA reduces pulmonary viral titer in mice challenged with influenza virus. Groups of five mice each were given 100 μg of PIKA in PBS intranasally 6 h before being challenged with 50 PFU of PR8 virus intranasally. (A) One group of mice received PIKA treatment intranasally at 24 and 48 h post-infection and the rest of the mice were left untreated. On day 3 post-infection, the mice were sacrificed and titers of influenza virus in lung homogenates were determined by plaque assay in MDCK cells. Closed circles represent the lung virus titer of individual mice and the line represents 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 when the difference between the two groups was statistically significant (p < 0.05). (B) Mice were given PIKA intranasally at different concentrations 6 h before intranasal challenge with 50 PFU of PR8 virus. The mice received treatment intranasally at 24 and 48 h post-infection. The pulmonary viral titers were determined on day 3 post-infection. (C) Mice were given 100 μg PIKA intranasally 6 h prior to (prophylactic group) or at the time of infection (therapeutic group). The daily groups received the PIKA treatment at 24 and 48 h post-infection while the rest were left untreated. Pulmonary viral titers were determined on day 3 post-infection using MDCK plaque assay. (D) Mice were given 100 μg of PIKA by the indicated route 6 h prior to viral challenge and were treated at 24 and 48 h post-infection. Pulmonary viral titers were determined on day 3 post-infection using MDCK plaque assay.

Fig. 2

PIKA is effective in reducing pulmonary viral titers of different influenza A virus subtypes. Groups of five mice each were given 100 μg of PIKA intranasally 6 h prior to challenge with 50 PFU of PR8 (H1N1) (A), Mem 71 (H3N1) (B) or WS/33 (H1N1) (C). Subsequently, mice received PIKA treatment at 24 and 48 h post-challenge. On day 3 post-infection, the mice were sacrificed and titers of influenza virus in lung homogenates were determined using in MDCK cells. Closed circles represent the lung virus titer of an individual mouse and the line represents the geometric mean titer of the group. The ‘*’ symbol indicates that the difference between the two groups was statistically significant (p < 0.05).

Fig. 3

Intranasal PIKA administration results in a substantial reduction in pulmonary viral titer even when mice are challenged with a high dose of infectious virus or after the establishment of an infection. Groups of five mice each were given 100 μg of PIKA intranasally 6 h prior to challenge with (A) 500 PFU or (B) 5000 PFU of PR8 virus or (C) 10^4.5 PFU of Mem71 virus. Mice received PIKA treatment intranasally at 24 and 48 h post-challenge. On day 3 post-infection, the mice were sacrificed and titers of influenza virus in lung homogenates were determined in MDCK cells. Closed circles represent the lung virus titer of an individual mouse and the line represents the geometric mean titer of the group. (D) Groups of five mice were challenged with 50 PFU of PR8 as previously described and 100 μg of PIKA were given 24 or 48 h post-infection and continued to receive daily treatment. On day 5 post-infection, the titers of influenza virus in lung homogenates were determined. The ‘*’ symbol indicates that the difference between the two groups was statistically significant (p < 0.05).

Fig. 4

PIKA acts as a potent adjuvant by enhancing the immunogenicity of the seasonal influenza vaccine. (A) Groups of four mice were vaccinated with either 50 or 5 μL of Fluvax influenza vaccine (containing 1.5 or 0.15 μg of HA from each subtype) in PBS or in CFA by the subcutaneous route. On day 21, sera were collected and the antibody titer of the sera was determined by an ELISA assay, using Fluvax as the coating antigen. (B) Groups of five mice were vaccinated with 0.5 μL of Fluvax influenza vaccine (containing 15 ng of HA from each subtype) in PBS, either by the subcutaneous route at the base of the tail or by the intranasal route. Some groups received the vaccine with additional adjuvant as indicated in the X-axis. For those that received PIKA as an adjuvant, 100 μg of PIKA was admixed with the vaccine prior to administration. On day 21 post-vaccination, sera were collected and the mice received another boost by the same route and sera were collected on day 42. The antibody titer of the sera was determined by an ELISA assay, using Fluvax as the coating antigen. Each dot represents the antibody titer of an individual mouse. The primary and secondary antibody titers are represented by the black and red dots respectively and the lines represent the geometric mean antibody titer of each group. The ‘*’ symbol indicates that the difference between two groups is statistically significant (p < 0.05). (C) To determine the level of protection mediated by the antibody response, the mice were challenged with 50 PFU of PR8 intranasally 3 weeks after the boosting and the pulmonary viral titer was determined on day 5 post-infection. Closed circles represent the lung virus titer of an individual mouse and the line represents the geometric mean titer of the group of mice. The percent reduction in mean viral titer relative to the PBS control group is shown above each column of data. The ‘*’ symbol indicates that the difference between the two groups was statistically significant (p < 0.05). (D) Serum samples were diluted 1:2000 and the avidity of the antibodies was tested in a urea-displacement ELISA. The O.D. of the urea-treated plates was expressed as a percent of the untreated plates. Data are represented as the mean and standard derivation of five mice.

Fig. 5

PIKA enhances the immunogenicity of formalin-inactivated whole H5 virion vaccine. (A) Groups of five mice were vaccinated with a formalin-inactivated H5 vaccine subcutaneously at the base of the tail (300 ng of total protein) with or without PIKA as an adjuvant. On day 21, sera were collected and the antibody titers against H5 virus were determined by ELISA. (B) The mice were challenged intranasally with 50 PFU of a reverse-engineered H5 influenza virus intranasally to evaluate the degree of protection. The pulmonary viral titer was determined in lung homogenates obtained on day 5 post-infection. (C) Groups of five mice were vaccinated as described in (B) and were boosted on day 21 post-vaccination. On day 42 post-vaccination, the mice were challenged with 50 PFU of the reverse-engineered H5 influenza virus intranasally. The pulmonary viral titer was determined as previously described. The percent reduction in mean viral titer relative to the group treated with PBS is shown above each column of data. The ‘*’ symbol indicates that the difference between the two groups was statistically significant (p < 0.05).

Fig. 6

PIKA interacts with human TLR3. HEK293 cells were transfected with an NF-κB-luciferase reporter gene, with or without co-transfection of a human TLR3-expressing plasmid. Twenty-four hours after transfection, the cells were stimulated with 25 μg per mL of PIKA, 1 μg per mL of LPS or medium alone (A) or 2-fold serially diluted PIKA (B). Six hours after stimulation, the cells were lysed and luciferase activity was determined. The data were normalized with respect to β-galactosidase activity and expressed relative to unstimulated samples. The bars and error bars represent the mean and standard deviation of triplicate samples and are representative of three independent experiments.

Fig. 7

PIKA induces maturation of dendritic cells, with expression of a wide range of immunological genes. Immature murine dendritic cells, D1 cells, were incubated with 500 μg per mL of PIKA or were unstimulated overnight. (A) Total RNA was harvested from the cells and converted into cDNA. The cDNAs derived from PIKA-stimulated DC and unstimulated DC were amplified and labeled with Cy5 and Cy3 dye respectively. The samples were hybridized overnight to a microarray chip and fluorescence signals were measured by an array scanner. Each gene target was printed 4 times on the array and the normalized mean Cy5/Cy3 ratio and coefficient of variation (cv) of the four replicates were determined by the software. (B) RNA was harvested from the DCs and converted into cDNA. The expression level of each cytokine gene was determined by quantitative real-time PCR. The expression level was normalized with beta-actin, a house-keeping gene, and data were expressed relative to unstimulated samples. The bars and error bars represent the mean and standard deviation of triplicate samples and are representative of two independent experiments. (C) Supernatants were harvested from DCs stimulated overnight with either 100 μg of PIKA, 1 μg of LPS or unstimulated. Fifty microliters of the supernatant was used to test for the presence of various cytokines/chemokines in the supernatants using the Bioplex Protein Array system and were measured in duplicate. The bars and error bars represent the mean and standard error.