1. Synergistic Inhibition of Avian Influenza (H5N1) by Poly I: Poly C12U Combined with Oseltamivir or Zanamivir D. Strayer 1, W. Carter 1, W. Mitchell 2, D. Smee 3, H. Hasegawa 4 ; 1 Hemispherx Biopharma, Inc., Philadelphia, PA, 2 Vanderbilt University, Nashville, TN, 3 Utah State University, Logan, UT, 4 National Institute of Infectious Diseases, Tokyo, Japan

2. Avian influenza (H5N1) is a major, global public-health concern. Since antiviral monotherapy can rapidly lead to drug resistant virus, it will be important to identify well- tolerated, synergistic drug combinations active against (H5N1) influenza. Ampligen®, poly I: poly C12U, is a bi- functional, double-stranded (ds) RNA (see Figure 1) with both antiviral and immunomodulatory activities. DsRNAs are powerful toll-like receptor 3 (TLR3) agonist in the induction of innate immune responses (see Figure 2). Phase II/III clinical trials of Ampligen® for other viral indications show that it is generally well tolerated. Since Ampligen® and oseltamivir/zanamivir exhibit antiviral activity against influenza virus, but by different mechanisms, Ampligen® in combination with each was studied. Background of Ampligen ® Synergy Studies

3. Materials and Methods for In Vitro Synergy Studies Virus and Cells: Influenza A/Duck/MN/1525/81 (H5N1) was propagated in MDCK cells. The cells were passaged in MEM containing 5% fetal bovine serum. Virus studies were performed in MEM without serum, but containing 50 µg/ml gentamicin, 10 units/ml of trypsin and 1 µg/ml of EDTA. Compounds: Ampligen was provided frozen in ampules. Oseltamivir carboxylate and Zanamivir were obtained from the R.W. Johnson Pharmaceutical Research Institute (Raritan, NJ). The compounds were prepared in cell culture medium shortly before applying to cells. Antiviral assays: Oseltamivir or zanamivir (each either used alone or in combination with Ampligen) were applied to cells 18 hours prior to virus infection. Shortly before infection the medium was replaced with fresh compounds. Three microwells (in 96-well plates) were used for infection and 2 wells were uninfected to serve as toxicity controls. Three days later the virus-induced cytopathic effect (CPE) was at 100% in the untreated cultures. The plates were examined microscopically for percent CPE in infected wells. After recording the values, the wells received 0.1 ml of a 0.034% neutral red PBS solution for 2 hours. After the 2-hour period the cells were rinsed 2x with PBS to remove unincorporated dye followed by aspiration to dryness. Later, the dye in each plate was eluted with 50:50 Sorenson’s citrate buffer (pH 4.2)/ethanol. The optical density of each well was read with an ELISA plate reader at 450 nm. A computer program converted readings to percentage of cell viability. Calculations of antiviral activity: For this study, we reported actual percent CPE observed at each concentration of inhibitor (or drug combination) by both visual and neutral red methods. Calculation of Synergy: The combination index was calculated base on the method of Chou and Talalay (J. Biol. Chem.; 252:6438 (1977)) using CalcuSyn Software.

4. Both Ampligen® and the neuraminidase inhibitors exhibited dose responses in the inhibition of the cytopathic effect (CPE) of avian H5N1 influenza virus infection. Direct observation and vital dye uptake measurements of the inhibition of CPE were similar in magnitude. Ampligen® at 32 and 100 μg/ml was effective in reducing or eliminating CPE. Oseltamivir alone was effective in reducing CPE at 0.1 and 0.32 μg/ml, but not effective at < 0.032 μg/ml. Zanamivir reduced or eliminated CPE at 0.032 - 0.32 μg/ml. The combination of Ampligen® plus oseltamivir showed synergistic inhibition of CPE at Ampligen® to oseltamivir ratios of 32:1 (Combination Index (CI) = 0.16 to 0.31 for ED90 to ED50). Zanamivir in combination with Ampligen® demonstrated strong to very strong synergy (CI = 0.01 at ED50 to CI = 0.14 at ED90) at an Ampligen® to zanamivir ratio of 3:1. These cell culture studies show that Ampligen® combined with either oseltamivir or zanamivir, synergistically inhibits CPE of avian influenza A/Duck/MN/1521/81(H5N1) infection of MDCK cells. Results of Ampligen ® Synergy Studies

5. Effect of Combination of Ampligen and Oseltamivir Carboxylate on an Influenza A/Duck/MN/1525/81 (H5N1) Infection in MDCK Cells as Determined by Cytopathic Effect Inhibition Assay Percent Cytopathic Effect Ampligen Oseltamivir Carboxylate (µg/ml) (µg/ml) 0.32 0.1 0.032 0.01 0.0032 0 100 0 0 0 0 0 0 32 0 0 0 0 0 75 10 0 0 0 58 63 100 3.2 13 29 100 71 100 1.0 13 50 100 0.32 13 50 100 0 25 54 100

6. Percent Cell Destruction Ampligen Oseltamivir Carboxylate (µg/ml) (µg/ml) 0.32 0.1 0.032 0.01 0.0032 0 100 0 0 0 0 0 7 32 0 0 0 0 0 58 10 0 0 0 43 48 97 3.2 0 0 31 96 59 100 1.0 0 0 50 100 88 100 0.32 9 9 65 100 95 100 0 23 34 95 100 Effect of Combination of Ampligen and Oseltamivir Carboxylate on an Influenza A/Duck/MN/1525/81 (H5N1) Infection in MDCK Cells as Determined by Neutral Red Uptake Assay

7. Ampligen: Oseltamivir Ratio Combination Index (CI) † ED50ED75ED90 32:10.310.210.16 100:10.310.180.12 320:10.330.21.015 1000:10.260.21.018 3200:10.320.260.22 † Combination Indices < 0.9 Indicate Synergism Synergy of Ampligen and Oseltamivir

8. Percent Cytopathic Effect Ampligen Zanamivir (µg/ml) (µg/ml) 0.32 0.1 0.032 0.01 0.0032 0 100 0 0 0 0 0 0 32 0 0 0 0 0 0 10 0 0 0 0 0 0 3.2 0 0 0 0 0 54 1.0 0 0 0 0 0 100 0.32 0 0 0 0 33 67 0 0 0 42 88 100 Effect of Combination of Ampligen and Zanamivir on an Influenza A/Duck/MN/1525/81 (H5N1) Infection in MDCK Cells as Determined by Cytopathic Effect Inhibition Assay

9. Percent Cell Destruction Ampligen Zanamivir (µg/ml) (µg/ml) 0.32 0.1 0.032 0.01 0.0032 0 100 0 0 0 0 0 0 32 0 0 0 0 0 0 10 0 0 0 0 0 0 3.2 0 0 0 0 0 40 1.0 8 0 0 0 10 78 0.32 8 0 0 0 22 67 0 0 0 41 83 96 100 Effect of Combination of Ampligen and Zanamivir on an Influenza A/Duck/MN/1525/81 (H5N1) Infection in MDCK Cells as Determined by Neutral Red Uptake Assay

10. Ampligen: Zanamivir Ratio Combination Index (CI) † ED50ED75ED90 3:1<0.01 0.14 10:10.020.080.32 32:10.330.34.035 100:10.110.17.028 320:10.080.160.31 † Combination Indices < 0.9 Indicate Synergism Synergy of Ampligen and Zanamivir

11. Range of CISymbolDescription < 0.1+++++Very strong synergism 0.1 – 0.3++++Strong synergism 0.3 – 0.7+++Synergism 0.7 – 0.85++Moderate synergism 0.85 – 0.90+Slight synergism 0.90 – 1.10+Nearly additive 1.10 – 1.20-Slight antagonism 1.20 – 1.45- Moderate antagonism 1.45 – 3.3- - -Antagonism 3.3 – 10- - Strong antagonism > 10- - - - -Very strong antagonism Recommended Symbols and Interpretation of Combination Index (CI) for Describing Synergism or Antagonism in Drug Combination Studies a a The combination index method is based on that described by Chou an Talalay (Adv. Enz. Regul.; 22: 27-55 (1984)).

12. Background for Ampligen ® Vaccine Adjuvant Study The respiratory tract mucosal immune system is usually the first immunological barrier against influenza virus infection. The influenza virus causes annual epidemics of influenza by altering the antigenic properties of its surface hemagglutinin. Inactivated vaccines against the influenza virus have been administered parenterally to induce serum anti-HA IgG antibodies that are protective against homologous virus infection but are less effective against heterologous virus infection. In contrast, a number of studies have shown that the mucosal immunity acquired by natural infection, which is mainly due to the secreted form IgA is more effective and cross- protective against virus infections than systemic immunity induced by parenteral vaccines. Double-stranded RNA (dsRNA), like Ampligen, acts as a molecular mimic associated with viral infection, because most viruses produce dsRNA during their replication. It has also been shown that mammalian toll-like receptor 3 (TLR3) recognizes dsRNA (see Figures 1 and 2) and activates the NF-  B pathway, resulting in activation of alpha/beta interferon (IFN-α/β), which enhances the primary antibody response against subcutaneous immunization of soluble materials. The adjuvant activity of IFN-α/β seems to play an important role in bridging the gap between innate and adaptive immunity. In a prior study, it was demonstrated that the mucosal adjuvant activity of intranasal administration of synthetic dsRNA [poly (I:C)] with inactivated influenza virus HA vaccine induced cross-protection immune responses against homologous and heterologous variant influenza virus infection (J. Virol.; 79: 2910 (2005)). This study evaluated a well-characterized dsRNA (Ampligen) as a vaccine adjuvant.

13. Materials and Methods for In Vivo Study of Ampligen ® as a Vaccine Adjuvant Immunization with vaccine and virus challenge: Female BALB/c mice, age 6 to 8 weeks at the time of immunization were used. Five mice for each experiment group were anesthetized with diethyl ether and immunized primarily by dropping 5 μl of phosphate-buffered saline (PBS) containing 0.5 μg of A/Vietnam (H5N1) vaccine with or without 5 μg Ampligen into each nostril. Three weeks later, they were reimmunized in the same manner with or without the same adjuvant. Two weeks after the second immunization, the immunized mice were challenged by upper respiratory tract infection with 100 pfu of A/Vietnam (H5N1) or A/Hong Kong (H5N1) virus. Measurement of virus titer and antibodies: Three days after virus challenge nasal wash and serum specimens were collected for measurement of virus titer and antibodies.

14. Results of Ampligen ® Vaccine Adjuvant Study The activity of Ampligen as a vaccine adjuvant was demonstrated in mice by both intranasal (IN) and subcutaneous (SC) administration routes using vaccination with influenza A/Vietnam (H5N1) vaccine. Augmentation of anti-A/Vietnam IgA in nasal wash and anti-A/Vietnam IgG in serum is shown. Virus challenge studies show both homologous (A/Vietnam) and heterologous variant (A/Hong Kong) anti-viral activity.

15. * : p < 0.01 Anti-A/VN IgA in nasal wash ( U/ml ) Anti-A/VN IgG in serum ( U/ml ) 0 200 400 0 3 2 4 5 6 1 A/VN virus titer in nasal wash ( PFU / ml ; 10 n ) 0 3 2 1 4 < 1* Activity of Ampligen as a Vaccine Adjuvant: Vaccination with A/Vietnam (H5N1) and Challenge with A/Vietnam (H5N1) Vaccination Route AMP (μg) A/VN (μg) IN SC 10- - 11-11

16. Anti-A/VN IgA in nasal wash ( U/ml ) Anti-A/VN IgG in serum ( U/ml ) 2004000324561 A/HK virus titer in nasal wash ( PFU / ml ; 10 n ) 0 32 10 4 Activity of Ampligen as a Vaccine Adjuvant: Intranasal Vaccination with A/Vietnam (H5N1) and Challenge with A/Hong Kong (H5N1) A/Vietnam Vaccine Ampligen (μg) Challenge A/Hong Kong ++- 10- +++

17. Figure 1Figure 2 Structure of dsRNA Ampligen (poly I∙ poly C12U). DsRNAs specifically bind to activate TLR3. Structure of Toll-like Receptor 3 (TLR3) (Adapted from PNAS 102, 10976, 2005) a proposed dsRNA recognition site. TLR3 is found in high concentration in human airway epithelial cells. Structure of Ampligen ® and TLR 3

18. CONCLUSIONS Cell culture studies show that Ampligen combinedwith either oseltamivir or zanamivir, synergisticallyinhibit CPE of avian influenza (H5N1) infection ofMDCK cells. Studies in mice show that Ampligen given intranasallywith A/Vietnam (H5N1) influenza vaccine increasemucosal IgA anti-influenza antibodies. Virus challenge with influenza A/Vietnam (H5N1) ofmice vaccinated with influenza A/Vietnam showreduced virus titers in nasal wash when Ampligen isincluded as an intranasal adjuvant. Virus challenge with A/Hong Kong (H5N1) of micevaccinated with influenza A/Vietnam (H5N1) showheterologous cross-protection with reduced virustiters when Ampligen is used as an intranasaladjuvant. Ampligen has been generally well-tolerated in priorclinical testing for anti-viral/cancer indications utilizingover 75,000 administered doses. These studies suggest a new and potentially pivotalrole of dsRNA therapeutics in improving the efficacy ofthe present standards of care for influenzaprevention/treatment.