Synergistic Inhibition of Avian Influenza (H5N1) by Poly I: Poly C12U Combined

with Oseltamivir or Zanamivir

D. Strayer1, W. Carter1, W. Mitchell2, D. Smee3, H. Hasegawa4;

1Hemispherx Biopharma, Inc., Philadelphia, PA, 2Vanderbilt University, Nashville, TN,

3Utah State University, Logan, UT,4National Institute of Infectious Diseases,

Tokyo, Japan

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

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.

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

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 13 29 100 71 100

1.0 13 13 50 100 100 100

0.32 13 13 50 100 100 100

0 25 54 100 100 100 100

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 95 100 100 100

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

Ampligen: Oseltamivir

Ratio

Combination Index (CI) †

ED50 ED75 ED90

32:1 0.31 0.21 0.16

100:1 0.31 0.18 0.12

320:1 0.33 0.21 .015

1000:1 0.26 0.21 .018

3200:1 0.32 0.26 0.22

† Combination Indices < 0.9 Indicate Synergism

Synergy of Ampligen and Oseltamivir

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 88 100 100

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

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

Ampligen: Zanamivir

Ratio

Combination Index (CI) †

ED50 ED75 ED90

3:1 <0.01 <0.01 0.14

10:1 0.02 0.08 0.32

32:1 0.33 0.34 .035

100:1 0.11 0.17 .028

320:1 0.08 0.16 0.31

† Combination Indices < 0.9 Indicate Synergism

Synergy of Ampligen and Zanamivir

Range of CI Symbol Description

< 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 Studiesa

a The combination index method is based on that described by Chou an Talalay (Adv. Enz. Regul.; 22: 27-55 (1984)).

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.

Materials and Methods for In Vivo Study of Ampligen®

as a Vaccine AdjuvantImmunization 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.

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.

* : p < 0.01

Anti-A/VN IgAin nasal wash

( U/ml )

Anti-A/VN IgGin serum( U/ml )

020

040

00

32

45

61

A/VN virus titerin nasal wash

( PFU / ml ; 10n )

03

21

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 IN IN SC SC

10 - 10 10 -

1 1 - 1 1

Anti-A/VN IgAin nasal wash

( U/ml )

Anti-A/VN IgGin serum( U/ml )

200

400

03

24

56

1

A/HK virus titerin nasal wash

( PFU / ml ; 10n )

03

21

04

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 - 10

+ + +

Figure 1 Figure 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

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