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Journal of Medical Virology
Dose-Dependent Antiviral Activity of
Released-Active Form of Antibodies to
Interferon-Gamma Against Influenza
A/California/07/09(H1N1) in Murine Model
Еlena S. Don,1* Alexandra G. Emelyanova,1 Natalia N. Yakovleva,2 Nataliia V. Petrova,1
Marina V. Nikiforova,2 Evgeniy A. Gorbunov,2 Sergey А. Tarasov,2 Sergey G. Morozov,1
and Оleg I. Epstein1
1
2
The Institute of General Pathology and Pathophysiology, Moscow, Russian Federation
OOO “NPF “MATERIA MEDICA HOLDING”, Moscow, Russian Federation
The assessment of dose–response is an essential part of drug development in terms of
the determination of a drug’s effective dose,
finding the safety endpoint, estimation of the
pharmacokinetic profile, and even validation
of drug activity, especially for therapeutic
agents with a principally novel mechanism
of action. Drugs based on released-active
forms of antibodies are a good example of
such a target. In this study, the efficacy
of the antiviral drug Anaferon for children (released-active form of antibodies to
interferon-gamma) was tested in a dosedependent manner (at doses of 0.13, 0.2, 0.4,
0.8 ml/mouse/day) in a murine model of acute
pneumonia induced by influenza virus pandemic strain A/California/07/09 (H1N1). Administration of the drug at the two highest
doses led to: a reduction in the virus infectious titer in lung tissue up to 4.2 lgEID50/
20 mg of tissue; infected animals’ life prolongation up to 6.7 days; an increase in the
survival rate of up to 40% and a decrease in
morphological signs of inflammation when
compared to the control animals. In this
study, the dose–response effect of Anaferon
for Children was demonstrated on mice for
the first time. This finding is especially important for drugs with a principally novel
mechanism of action like drugs based on
released-active forms of antibodies. J. Med.
Virol.
# 2016 Wiley Periodicals, Inc.
KEY WORDS:
influenza virus; antibodycontaining preparations; antiviral agents; animal models of
infection
C 2016 WILEY PERIODICALS, INC.
INTRODUCTION
There are more than 20 families of human pathogenic viruses varying in their use of RNA or DNA as
genetic material, in genome size, transmission route,
ability to infect diverse tissues, and pathogenicity and,
additionally, RNA viruses are known to evolve extremely rapidly [Sharp, 2002]. One of them, Influenza
A virus, belongs to the Orthomyxoviridae RNA virus
family and is a highly infectious agent causing acute
pulmonary diseases [Lamb and Roberts, 2014].
There are two glycoproteins on the virus surface:
haemagglutinin and neuraminidase. The continuous
proliferation of the influenza virus among humans
relies on antigenic variation of haemagglutinin and
neuraminidase surface proteins, resulting from antigenic drift which leads to recurrent seasonal influenza
epidemics [Krammer and Palese, 2015; Peteranderl
et al., 2016]. The antigenic shift also has the potential
to proliferate globally and cause pandemics, for example, “swine” flu in 2009 [Kong et al., 2015]. Currently,
two classes of influenza antivirals have been approved
for human use in many countries—adamantanes
and neuraminidase inhibitors. Adamantanes inhibit
Abbreviations: AC, Anaferon for children; ANOVA, analysis of
variance; HA, haemagglutinin; IFN-g, interferon-gamma;
MDCK, Madin-Darby canine kidney; MLS, mean life-span; NA,
neuraminidase; p.i., post-infection; PBS, phosphate-buffered
saline; RAF of Abs, Released-active forms of antibodies
Conflicts of interest: The authors declare no conflict of interest.
Four authors have an affiliation to the commercial funders of this
research study (OOO “NPF “MATERIA MEDICA HOLDING”).
Correspondence to: Еlena S. Don, The Institute of General
Pathology and Pathophysiology, 8, Baltiyskaya st., 125315,
Moscow, Russian Federation. E-mail: [email protected]
Accepted 14 October 2016
DOI 10.1002/jmv.24717
Published online in Wiley Online Library
(wileyonlinelibrary.com).
2
influenza A viral replication by blocking the activity of
the M2 ion channel [Moorthy et al., 2014], thus being
ineffective against influenza B viruses [Oh and Hurt,
2014]. The main drawback of adamantane therapy is
the rapid development of drug-resistant viruses [Dong
et al., 2015]. Although neuraminidase inhibitors (e.g.,
oseltamivir) have a broader spectrum of activity
including influenza A and B viruses [NguyenVan-Tam et al., 2015], the recent rapid emergence and
transmission of drug-resistant viruses [Hauge et al.,
2009; Hurt, 2014] demonstrates the necessity to
develop new antiviral drugs against influenza or to
optimize administration regimens of antiviral compounds that are currently in use.
Anaferon for children (AC) is an antiviral drug
containing released-active form of antibodies to
interferon-gamma as its active substance. Antiviral
activity of released-active form of antibodies to
IFN-g against influenza and other respiratory infections has been previously shown in preclinical
and clinical studies [Martyushev-Poklad et al.,
2004; Sergeev et al., 2004; Shishkina et al., 2008;
Vasil’ev et al., 2008; Epstein, 2009; Kudin et al.,
2009; Tarasov et al., 2012; Gavrilova et al., 2014a].
It has been found that the key mechanism of its
pharmacological action is the ability to improve
ligand-receptor interactions of IFN-g and IFN-g
receptor (CD-119) via conformational changes of the
molecule of IFN-g [Zhavbert and Dugina, 2013].
This fact defines the AC’s ability to regulate
functional activity, production of endogenous interferons, and modulate the immune response depending on the initial state of the organism
[Martyushev-Poklad et al., 2004; Sergeev et al.,
2004; Shishkina et al., 2008; Vasil’ev et al., 2008;
Epstein, 2009; Kudin et al., 2009; Tarasov et al.,
2012; Zhavbert and Dugina, 2013].
Historically, studying the relationship between
dose and response has not been a priority during
drug development and, thus, drugs have often been
initially marketed at what were later recognized as
excessive doses, sometimes with adverse consequences [Cottey et al., 2001]. Currently, it is agreed that
the assessment of the dose–response relationship
should be an integral component of drug development
and an inherent part of establishing safety and
efficacy profiles of the drug [ICH, 1994; Reeves et al.,
2015]. During the previous study the effect for dose
of 0AC at 0.4 ml/mouse/day has already been confirmed [Tarasov et al., 2012], but the appropriateness
of this dose had to be examined. Thus, the purpose of
the current study was to test a possible dose–
response of AC antiviral action against influenza A
using a murine model which featured the general
authenticity of the illness presented [Sidwell and
Smee, 2004]. Following an intranasal inoculation of
influenza virus, mice develop a progressive upper and
lower respiratory tract disease with histopathology
virtually identical to that seen in human disease
[Cottey et al., 2001]. Such factors as survival rate,
J. Med. Virol. DOI 10.1002/jmv
Don et al.
weight loss, and virus titer were examined in a dosedependent manner.
MATERIALS AND METHODS
Compounds
AC was supplied as a ready-to-use solution manufactured by OOO “NPF “MATERIA MEDICA HOLDING” (Moscow, Russia). Affinity purified rabbit
polyclonal antibodies to recombinant human IFN-g
were manufactured in accordance with the current
European Union requirements for Good Manufacturing Practice for starting materials by Angel Biotechnology Holdings plc (Edinburg, UK) as a starting
material for commercial production of AC for therapeutic oral application. Released-active forms (RAF)
of rabbit polyclonal antibodies were manufactured
based on a novel patented biotechnological platform
(US Patent 7,572,441 B2, 2009). Briefly, RAF of Abs
were prepared by consecutive reduction of antibodies
to IFN-g (2.5 mg/ml) concentration via their multiple
dilutions under specific conditions in water-ethanol
solutions with either 12, 30, or 50 defined steps of
centesimal dilution. Thus, AC contained RAF of Abs
to IFN-g is a mixture of 12, 30 and 50 centesimal
dilutions of antibodies to IFN-g. Solutions were
prepared avoiding intense direct light in sterile
conditions and were stored at room temperature.
Vehicle (distilled water) was used as a control.
Tamiflu (oseltamivir phosphate, F. Hoffmann–La
Roche Ltd.) (hereinafter “oseltamivir”) was used as
reference drug. All samples (except oseltamivir) were
encoded by the manufacturer and were used blinded
in the study.
Virus and Cells
Influenza virus A/California/07/09 (H1N1)pdm09
was received from the influenza strain depository at
the Influenza Division at the Centers for Disease
Control and Prevention in Atlanta, GA. Prior to the
experiment, the virus was propagated in the allantoic cavity of 10–12-day old chicken embryos for
48 hr. The virus was further adapted to mice by
three serial passages in murine lung tissue as
described previously [Narasaraju et al., 2009]. Lung
homogenate in sterile phosphate-buffered saline
(PBS) was used as the infecting material in further
experiments. Prior to the main study, mouseadapted influenza virus was titrated for lethal effect,
for which purpose anesthetized mice (10 animals
from each experimental group) were intranasally
inoculated with 50 ml of serial decimal dilutions
(101–105) of the mice lung homogenate. The dilution causing death of 50% of the animals within
21 days p.i. (LD50) was calculated as described
previously [Reed, 1938] and was used for subsequent
experiments.
Madin-Darby canine kidney cells were used for the
virus titer determination.
Dose-Related Antiviral Activity of RAF Ab to IFN
Animals
Inbred female BALB/c mice, 16–20 g (5–8 weeks
old), were obtained from the animal breeding facility
of Russian Academy of Medicine “Rappolovo.” Animals were acclimated for at least 7 days after arrival
and prior to any procedures. During the experimental
period, the animals were housed under standard
laboratory conditions (a temperature-controlled
[24˚C] facility, humidity of 50–60%, a 12-hr light/dark
cycle, and appropriate ventilation). Mice were housed
in 590 380 200 mm PC cages (Bioscape) with bedding material Lignocel (J. Rettenmaier & S€ohne
Gmbh þ Co), 8–10 animal per cage. No animals were
numbered within cages. Animals were given access to
balanced food (Laboratorkorm) and water ad libitum.
During housing, animals were monitored daily for
health status. No adverse events were observed
during the therapy. The study in a murine model of
influenza was performed using 225 animals. The
steps to use as little number of animals as possible
were taken from the statistical point of view (see
“Supplementary material”). Animal experiments were
conducted in accordance with the principles of laboratory animals care [Guide for the Care and Use of
Laboratory Animals, 1996] and were approved by the
Institutional Ethical Committee. All sections of this
report adhere to the ARRIVE Guidelines for animal
research reporting [Kilkenny et al., 2010].
General Procedures
In order to evaluate dose-dependence of anti-influenza activity of AC in vivo, mice were infected with
one LD50 or ten LD50 of the previously titrated
virus. Groups of animals (n ¼ 25) were generated
randomly on the bodyweight basis prior to the
treatment initiation. Seven groups were studied: in
the first four groups AC was administered orally via
gavage twice a day at the volumes of 0.065, 0.1, 0.2
and 0.4 ml for 5 days prior to infection and 21 days
p.i. Thus, the doses were 0.13 ml/mouse/day (group
1), 0.2 ml/mouse/day (group 2), 0.4 ml/mouse/day
(group 3), 0.8 ml/mouse/day (group 4), respectively.
The effect for the dose of 0.4 ml/mouse/day had
already been tested previously and it was decided to
try several lower and one higher doses to estimate if
the chosen dose was the most appropriate one
[Tarasov et al., 2012]. Thus, in this study we examined two doses which were two and three times lower
and one dose which was two times higher than the
initial dose (0.4 ml/mouse/day) and at the same time
was the highest possible volume of liquid which can
been injected into a mouse orally via gavage [Waynforth et al., 1998]. Control animals from group 5
received distilled water twice a day in a volume of
0.2 ml/mouse for 5 days prior to infection and 21 days
p.i. The reference drug oseltamivir (final dose 20 mg/
kg body weight) was dissolved in saline and was
administered to the mice in group 6 orally two times
(24 and 1 hr) prior to infection and twice a day for
3
5 days p.i., which was in accordance with the
previously chosen scheme [Tarasov et al., 2012].
Furthermore, the mice in the group of reference drug
were treated with distilled water at the dose of
0.2 ml/mouse twice a day for 5 days prior to infection
and 21 days p.i. Ten uninfected untreated mice were
used as an intact control (group 7). Due to a short
period of time required for oral sample administration, no special efforts to randomize the animals
within groups were made. Moreover, the active
substance of AC was used as drinking water for
groups 1–4, whereas distilled water was used as
drinking water for all other groups.
Five mice from each group were infected with
either 1LD50 or 10LD50 of the virus and were
sacrificed by cervical dislocation by trained personnel
on days 3, 6, and 9 p.i. in order to avoid any
additional influence of anesthetic drugs or gases. The
animals’ chests were opened and the lungs were
isolated. The lungs were used for virus titration
(frozen and stored at 20˚С until the corresponding
experiments). Ten additional mice from each group
underwent identical treatment so their lungs could
undergo histological examination on day 6 p.i. and
further on day 22 p.i. (five animals per group for
each time point).
Survival rate in each group of animals was calculated. Each group was monitored twice a day for
lethal cases for 3 weeks p.i. Based on the data
received, percent of survival rate, protective index
(IP, ratio of mortality percent [which is calculated by
subtraction of survival percent from 100%] in the
control group over the mortality percent in the
experimental group) and mean life-span were calculated. For the lethal dose 10LD50 no humane endpoint was necessary because the time period between
the signs of severe suffering and death was extremely
short. At the dose of 1LD50, the animals were
anesthetized by carbon dioxide (20%/min gradual
displacement up to unconsciousness of animals) and
were sacrificed by cervical dislocation by trained
personnel if during the examination the signs of
suffering were obvious (heavy breathing, coldness,
behavior changing).
For infectious virus titer determinations in lung
tissue, lungs were homogenized in ten volumes of
sterile PBS. Serial dilutions (100–106) were prepared from each homogenate. MDCK cells cultured
in 96-well plates in MEM medium (Biolot, SaintPetersburg, Russia) were inoculated with 0.2 ml of
each dilution and incubated at 36˚C for 48 hr in 5%
CO2. After the incubation, supernatants were harvested and tested for influenza virus by mixing the
fluid in round-bottom wells with an equal volume
of a 1% suspension of chicken erythrocytes in
saline.
The final dilution causing positive haemagglutination reaction in the well was considered as the virus
titer in the lungs which was expressed in decimal
logarithm of 50% infectious dose (lgЕID50) per 20 mg
J. Med. Virol. DOI 10.1002/jmv
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Don et al.
tissue. Activity of the compounds was evaluated by
their ability to decrease infectious titer of the virus in
lung tissue.
Histological Examination
Mice lungs were placed into PBS-formaldehyde,
dehydrated in ethanol, and embedded in paraffin.
Four micrometer slices were cut off and stained with
haemotoxylin–eosin. The relative size of virus-induced foci of inflammation (percent of total lung
surface) were estimated.
Statistical Analysis
All the data were analyzed using the SAS 9.3
statistical software (SAS Institute Inc., Campus Drive
Cary, NC). The statistical analysis included the
comparisons of AC versus control and AC versus
reference drug. Survival analysis was used to compute nonparametric estimates of the survivor functions and to compare survival curves. The Kaplan–
Meier method was used to compute estimates from
survival times which were expressed in the number
of days from inoculation. The log-rank test was used
to compare survival functions. Secondary outcomes
for virus titer in lungs were analyzed by two-way
ANOVA. Foci of pneumonia were analyzed by Kruskal–Wallis one-way analysis.
Animals’ weight was analyzed by Repeated Measures ANOVA, with treatment condition and time
(prior to vs. post-infection) as independent variables.
The statistical significance of changes in weight was
assessed based on P-value of interaction between
time and treatment factors. Each pair of treatment
conditions was compared separately using Tukey’s
adjustment for multiple comparisons. P values of
less than 0.05 were considered as statistically
significant.
RESULTS
Clinical signs typical for severe influenza pneumonia were observed after inoculation of adapted virus
including ataxia, tremor, shortness of breath, as well
as a reduction in water and food consumption (data
not shown) leading to body weight loss. Body weight
reductions started on day 3–4 p.i. reaching its minimal values on days 5–7 (Fig. 1). However, it should
be noted that changes in animal body weight do not
adequately demonstrate the severity of this pathological process, since the drugs ensure the survival of
animals with lower body weight, while in the control
group such animals are the first to expire and by,
thus, artificially increase the mean body weight
values in this group. Therefore, despite the fact that
the drugs showed protective features and that significant differences between the groups were demonstrated by ANOVA with repeated measurement of
group factor and day factor values for 10LD50 dose
(F[33,407] ¼ 1,5770, P ¼ 0.02456), the changes in the
J. Med. Virol. DOI 10.1002/jmv
Fig. 1. Changes in body weight of mice during the pneumonia
induced by influenza virus A/California/07/09(H1N1)v: 1LD50
(a); 10LD50 (b).
body weight of the animals infected with the virus at
both doses were not significantly different from the
values in the control group, AC group (except for
0.4 mg/ml dose) or oseltamivir group.
Administration of AC was shown to increase both
survival rate and MLS in mice infected with Influenza A/California/07/09 (H1N1) virus at the doses
1LD50 and 10LD50.
In the experiment with 1LD50 dose, the survival
rate was significantly higher in the group receiving
AC at 0.4 ml/mouse/day as compared to control group
(x2[1] ¼ 11.0362, ¼ 0.0115); furthermore, a marginally
significant increase in the group receiving AC at
0.8 ml/mouse/day
as
compared
to
control
(x2[1] ¼ 7.5764, ¼ 0.0654) was seen. Additionally, the
differences in survival rate were insignificant between AC (0.8 ml/mouse/day) and oseltamivir group
(x2[1] ¼ 2.2753, ¼ 0.6589).
AC at the doses of 0.4 ml/mouse/day and 0.8 ml/
mouse/day significantly increased the survival rate as
compared to the control by 40% (x2[1] ¼ 15.5964,
¼ 0.0011 and x2[1] ¼ 14.1240, ¼ 0.0024, respectively)
in mice infected with virus at the dose of 10LD50.
Reference drug oseltamivir significantly enhanced the
survival rate by 60% as compared to control group
(x2[1] ¼ 30.1025, < 0.0001) in mice infected with the
same dose of influenza virus.
It was shown that both AC and oseltamivir have
enhanced the specific survival rate and have
Dose-Related Antiviral Activity of RAF Ab to IFN
5
TABLE I. Defensive Activity of AC Against Influenza A(H1N1) Caused Lethal Pneumonia in BALB/c Mice
Virus dose 1 LD50
Treatment
Survive animals (n ¼ 20)
% Survival
Mean life-span, days
Index of protection (%)
13
15
18
16
18
10
65
75
90
80
90
50
16.3
17.6
20.5
19.0
20.6
14.5
30.0
50.0
80.0
60.0
80.0
0
9.4
11.1
15.3
15.0
18.2
8.6
–
5.8
17.6
47.0
47.0
70.5
0
–
AC, 0.13
AC, 0.20
AC, 0.40
AC, 0.80
Oseltamivira, 20
Control, no treatment
AC, 0.13
AC, 0.20
AC, 0.40
AC, 0.80
Oseltamivir, 20
Control, no treatment
Uninfected, no treatment
4
6
11
11
15
3
10 (out of 10)
Virus dose 10 LD50
20
30
55
55
75
15
100
AC, Anaferon for Children, ml/mouse/day.
a
mg/kg/day.
P < 0.05 versus control.
extended MLS (up to 6.7 days in AC group and up to
9.6 days in oseltamivir group) as compared to the
control group values depending on the virus and drug
doses used in the study (Table I).
The virus titer isolated from murine lungs demonstrated that both oseltamivir and AC at almost all
examined doses have reduced the virus titer by 2 or
more lgEID50/20 mg of tissue as compared to control
group (Table SI, “Supplementary material”) on the
day 6 p.i. By day 9 virus titer was rather low in the
group receiving the maximum dose of AC (0.8 ml/
mouse/day): to 0.6 lgEID50/20 mg of tissue (1LD50)
and to 0.8 lgEID50/20 mg of tissue (10LD50). Oseltamivir by this time reduced virus titer to 0.4–0.5
lgEID50/20, while virus titer in the control group for
both doses was 1.7–1.8 lgEID50/20 mg of tissue. For
the virus dose 1LD50 three highest doses of AC as
well as oseltamivir showed significant difference of
virus titer values vs control one ( < 0.05). This is also
true for AC (0.8 ml/mouse/day) and oseltamivir in
case 10LD50 virus dose.
Morphological analysis was conducted to determine
the effects of tested compounds on the lung tissue
structure on day 6 (stage of acute influenza infection)
and on day 22 p.i. (stage of chronic influenza
infection) (Fig. 2).
No macroscopic signs of inflammation were seen in
the intact animals’ lungs on both days 6 and 22
(Fig. 2a). In infected and untreated control groups
morphological changes in lung tissue on day 6 p.i.
were manifested in the form of neutrophil accumulation and cellular detritus in large bronchial lumens
(Fig. 2b). By day 22 of infection cellular exudates
containing neutrophils, lymphocytes, and macrophages replaced serous exudates. The dramatic
changes were observed in lung and bronchial tissue
in untreated surviving animals from the control
group. In the lumens of atelectatic alveoli, intensive
Fig. 2. Morphogenesis of experimental influenza infection. (a)
Intact animals’ lungs; loci of acute influenza pneumonia in mice
on day 6 post-infection with influenza virus A/California/07/09
(H1N1)v, without any treatment (b) or using Ozeltamivir (c) or
Anaferon for Children (d–g); loci of chronic influenza
pneumonia in mice on day 22 post-infection with influenza virus
A/California/07/09(H1N1)v, without any treatment (h) or using
Ozeltamivir (i) or Anaferon for Children (j–m); four micrometer
pieces were cut off and stained with haemotoxylin–eosin (240
for a, j, k and 200 for others).
J. Med. Virol. DOI 10.1002/jmv
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Don et al.
cellular inflammation and substantial volume of
cellular detritus were observed (Fig. 2h).
The principal difference for treated animals as
compared to the untreated group consisted in the
drastic reduction in the intensity of virus specific and
reactive effects of lung tissue at the acute stage of
influenza pneumonia. Thus, on day 6 p.i. bronchial
epithelial cells looked intact, while the control animals had damaged cells with numerous viral inclusions. Inflammatory nidi themselves occupied smaller
areas as compared to those in the control group. AC
in high doses as well as the reference drug oseltavimir normalized lung tissue structure, specifically by
reducing edema and alveolar lesions, decreasing the
count of foreign substances in bronchial lumen and
enhancing the defense of bronchial epithelium
against cell death (Fig. 2c–e, Table SI, “Supplementary material”). The other tested AC doses exerted
less evident effects on lung morphology against
influenza pneumonia (Fig. 2f–g). At the advanced
stages of the pathological process, the trends were
similar to those observed at the stage of acute
pneumonia. Lesion nidi were smaller as compared to
the ones in control animals. Morphological study
revealed moderate epithelium metaplasia and interstitial lung infiltration induced by neutrophils and
globocellular elements. Infiltration was much less
intensive versus control group. The intra-alveolar
septa were distinctive; air cells were well-defined and
contained an increased number of alveolar macrophages. All of the abovementioned observations significantly distinguished these animals from the group
of untreated mice (Fig. 2h–m). Mean size of pneumonia lesion varied significantly between the groups (H
[5, N ¼ 90] ¼ 35.86780 P ¼ 0.0000), while pair-wise
comparisons showed a significant difference for this
parameter in 0.8 AC group versus control (35.9 4.7
vs. 57.0 5.2; P < 0.01) and oseltavimir group
(35.9 4.7 vs. 16.4 2.2; P < 0.001).
been studied [Tarasov et al., 2012], thus for this study
multiple doses were tested in order to determine the
most appropriate dose. This work is especially important because, due to the increasing amount of the
adverse effects’ cases, the pharmaceutical market at
the moment is hardly trying to avoid using excessive
therapeutic doses. The study of survival confirmed
that the dose of 0.4 ml/mouse/day should be used for
further testing and that there is no need to increase
the dose further (Fig. 3a). Interestingly, the dose
increment higher than 0.4 ml/mouse/day was also not
necessary for the low viral dose (1LD50) but it was
important in the case of high viral dose (10LD50)
(Fig. 3b).
To understand the nature of the effect, we considered a number of previous studies which had showed
that released-active form of antibodies to IFN-g significantly augmented production of IFN-g in both humans
and animals [Sherstoboev et al., 2003; Tarasov and
Dugina, 2008; Epstein, 2009; Obraztsova et al., 2009;
Zhavbert and Dugina, 2013]. Generally, IFN-g plays
an important role in the recovery from flu infection by
helping to eliminate the virus [Wiley et al., 2001;
Bruder et al., 2006; Prabhu et al., 2013; Killip et al.,
2015] which means that the regulation of IFN-g level
is among the most limiting factors for antiviral host
response [Khoufache et al., 2009]. It has been established that AC’s influence on the interferon system
involves triggering the mechanisms of innate and
acquired immunity thus providing the antiviral and
immunomodulatory effects, stimulation of the host’s
defense mechanisms against the virus that is indicative of its pharmacological activity [Epstein, 2009;
Tarasov et al., 2012; Epstein, 2013; Zhavbert and
DISCUSSION
Antiviral efficacy of AC was repeatedly demonstrated in several experimental animal models as
shown by the resolution of symptoms and the reduction of viral titer [Sergeev et al., 2004; Susloparov
et al., 2004; Shishkina et al., 2010; Tarasov et al.,
2012]. In accordance with this study, a defensive effect
of oral AC against lethal influenza caused by pandemic
influenza virus A/California/07/09 (H1N1)pdm09 in
mice was shown in a dose-dependent manner. The
infectious virus titer reduction in lung tissue, prolongation of mean life-span, an increased survival rate
among treated animals and normalization of lung
tissue structure were demonstrated as compared to the
control. It is noteworthy that these effects of the higher
doses of AC were not inferior to those of the reference
drug oseltamivir, also the effects were more pronounced at the low inoculating dose (1LD50). The
effect of AC at one dose (0.4 ml/mouse/day) has already
J. Med. Virol. DOI 10.1002/jmv
Fig. 3. Dose–response relationship: in terms of percent of
survival (a) and virus titer (b).
Dose-Related Antiviral Activity of RAF Ab to IFN
Dugina, 2013; Gavrilova and Tarasov, 2014]; which is
important in cases of recurrent influenza infection
[Wiley et al., 2001; Bruder et al., 2006; Prabhu et al.,
2013]. A dose–response relationship was confirmed by
the statistical difference established for the antiviral
activity of AC in comparison to the control group with
the AC dose increment. This manner of action corresponds to the in vitro results obtained for this class of
drugs [Gavrilova et al., 2014b; Pschenitza et al., 2014;
Gorbunov et al., 2015]. We also were able to determine
the most appropriate dose and the direct correlation
between dose of the virus inoculation and the suitable
dose of the drug for treatment. These findings may
confirm the finite specific activity of the biological drug
AC, which is especially important because of the
features of technological treatment of the substance
during preparation of released-active form of antibodies to IFN-g.
Thus, the study confirmed the antiviral activity of
AC shown in previous experimental and clinical
studies, and the effectiveness of its use for the
treatment of influenza infection caused by the pandemic strain of A/California/ 07/09 (H1N1) virus
in a dose–response manner. AC administration
affected the IFN-g system leading to the reduction
of the virus infectious titer in the lung tissue, to
the prolongation of infected animals’ life, and to the
increased survival from influenza as compared to the
control animals.
ACKNOWLEDGMENTS
We would like to express our gratitude to Professor
Svetlana Sergeeva, deceased (2015) for giving us a
good guideline for the study throughout numerous
consultations and for her help in data interpretation.
We would also like to expand our deepest gratitude
to all those who have directly and indirectly guided
us in the writing of this article.
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