Transcript Synergistic Inhibition of Avian Influenza (H5N1) by Poly I

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
Background of Ampligen®
Synergy Studies
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 welltolerated, synergistic drug combinations active against
(H5N1) influenza. Ampligen®, poly I: poly C12U, is a bifunctional, 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.
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.
Results of Ampligen®
Synergy Studies
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.
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
Oseltamivir Carboxylate (µg/ml)
0.1
0.032
0.01
0.0032
Ampligen
(µg/ml)
0.32
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
0
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
Percent Cell Destruction
Oseltamivir Carboxylate (µg/ml)
0.1
0.032
0.01
0.0032
Ampligen
(µg/ml)
0.32
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
0
Synergy of
Ampligen and Oseltamivir
†
Combination Index (CI) †
Ampligen:
Oseltamivir
Ratio
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
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 Cytopathic Effect
Zanamivir (µg/ml)
0.032
0.01
Ampligen
(µg/ml)
0.32
0.1
100
0
0
0
32
0
0
10
0
3.2
0.0032
0
0
0
0
0
0
0
0
0
0
0
0
0
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 Neutral Red Uptake Assay
Percent Cell Destruction
Zanamivir (µg/ml)
0.032
0.01
Ampligen
(µg/ml)
0.32
0.1
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
0.0032
0
Synergy of
Ampligen and Zanamivir
†
Combination Index (CI) †
Ampligen:
Zanamivir
Ratio
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
Recommended Symbols and Interpretation of
Combination Index (CI) for Describing Synergism
or Antagonism in Drug Combination Studiesa
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
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 crossprotective 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 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.
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.
3
4
Activity of Ampligen as a Vaccine Adjuvant:
Vaccination with A/Vietnam (H5N1) and
Challenge with A/Vietnam (H5N1)
* : p < 0.01
< 1*
400 0 1 2 3 4 5 6
0
1
2
A/VN virus titer
in nasal wash
( PFU / ml ; 10n )
Anti-A/VN IgG
in serum
( U/ml )
0
200
Anti-A/VN IgA
in nasal wash
( U/ml )
Vaccination
Route
IN
IN
IN
SC
SC
AMP (μg)
10
-
10
10
-
A/VN (μg)
1
1
-
1
1
1
0
Anti-A/VN IgA
in nasal wash
( U/ml )
200
Anti-A/VN IgG
in serum
( U/ml )
400 0 1 2 3 4 5 6
0
A/HK virus titer
in nasal wash
( PFU / ml ; 10n )
2
3
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
-
10
+
+
+
Structure of Ampligen® and TLR 3
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.
CONCLUSIONS
• Cell culture studies show that Ampligen combined
with either oseltamivir or zanamivir, synergistically
inhibit CPE of avian influenza (H5N1) infection of
MDCK cells.
• Studies in mice show that Ampligen given intranasally
with A/Vietnam (H5N1) influenza vaccine increase
mucosal IgA anti-influenza antibodies.
• Virus challenge with influenza A/Vietnam (H5N1) of
mice vaccinated with influenza A/Vietnam show
reduced virus titers in nasal wash when Ampligen is
included as an intranasal adjuvant.
• Virus challenge with A/Hong Kong (H5N1) of mice
vaccinated with influenza A/Vietnam (H5N1) show
heterologous cross-protection with reduced virus
titers when Ampligen is used as an intranasal
adjuvant.
• Ampligen has been generally well-tolerated in prior
clinical testing for anti-viral/cancer indications utilizing
over 75,000 administered doses.
• These studies suggest a new and potentially pivotal
role of dsRNA therapeutics in improving the efficacy of
the present standards of care for influenza
prevention/treatment.