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

4

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

6

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. REFERENCES Bruder D, Srikiatkhachorn A, Enelow RI. 2006. Cellular immunity and lung injury in respiratory virus infection. Viral Immunol 19:147–155. Cottey R, Rowe CA, Bender BS. 2001. Influenza virus. Curr Protoc Immunol 42:19.11.1–19.11.32. Dong G, Peng C, Luo J, Wang C, Han L, Wu B, Ji G, He H. 2015. Adamantane-resistant influenza A viruses in the world (1902– 2013): Frequency and distribution of M2 gene mutations. PLoS ONE 10:e0119115. Epstein OI. 2009. Ultralow doses (history of one research). Moscow: RAMS Publishing House, p 336. Epstein OI. 2013. The phenomenon of release activity and the hypothesis of “spatial” homeostasis. Usp Fiziol Nauk 44: 54–76. Gavrilova E, Gorbunov E, Borshcheva A, Guryanova N, Tarasov S. 2014a. Application of drugs based on release-active antibodies as immunotherapy agents. J Clin Cell Immunol 5:62. Gavrilova ES, Bobrovnik SA, Sherriff G, Myslivets AA, Tarasov SA, Epstein OI. 2014b. Novel approach to activity evaluation for release-active forms of anti-interferon-gamma antibodies based on enzyme-linked immunoassay. PLoS ONE 9:e97017. Gavrilova ES, Tarasov SA. 2014. Modern approaches for the investigation of natural autoantibodies to cytokine by various immunoassays. Immunologiya 35:107–112.

7 Gorbunov EA, Ertuzun IA, Kachaeva EV, Tarasov SA, Epstein OI. 2015. In vitro screening of major neurotransmitter systems possibly involved in the mechanism of action of antibodies to S100 protein in released-active form. Neuropsychiatr Dis Treat 11:2837–2846. Guide for the Care and Use of Laboratory Animals. 1996. 7th edition. Washington D.C.: National Academy Press. p 125. Hauge SH, Dudman S, Borgen K, Lackenby A, Hungnes O. 2009. Oseltamivir-resistant influenza viruses A (H1N1), Norway, 2007–08. Emerg Infect Dis 15:155–162. Hurt AC. 2014. The epidemiology and spread of drug resistant human influenza viruses. Curr Opin Virol 8:22–29. ICH E.W.G. 1994. Guideline for industry. Dose-response information to support drug registration. ICH-E41994.1994. Rockville: CDER. p 15. Khoufache K, LeBouder F, Morello E, Laurent F, Riffault S, Andrade-Gordon P, Boullier S, Rousset P, Vergnolle N, Riteau B. 2009. Protective role for protease-activated receptor-2 against influenza virus pathogenesis via an IFN-gamma-dependent pathway. J Immunol 182:7795–7802. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. 2010. Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. PLoS Biol 8:e1000412. Killip MJ, Fodor E, Randall RE. 2015. Influenza virus activation of the interferon system. Virus Res 209:11–22. Kong W, Wang F, Dong B, Ou C, Meng D, Liu J, Fan ZC. 2015. Novel reassortant influenza viruses between pandemic (H1N1) 2009 and other influenza viruses pose a risk to public health. Microb Pathog 89:62–72. Krammer F, Palese P. 2015. Advances in the development of influenza virus vaccines. Nat Rev Drug Discov 14:167–182. Kudin MV, Tarasov SA, Kachanova MV, Skripkin AV, Fedorov YN. 2009. Anaferon (pediatric formulation) in prophylactics of acute respiratory viral infection in children. Bull Exp Biol Med 148:279–282. Lamb RA, Roberts KL. 2014. In: Influenza (MS 654). Chapter 02606, Caplan MJ, editor. Reference Module in Biomedical Research. Amsterdam: Elsevier Inc. Martyushev-Poklad AV, Kotelnikova MP, Uchaikin VF, Dugina JL, Epstein OI, Sergeeva SA. 2004. A novel antiviral based on oral antibodies: Clinical benefits in paediatric upper respiratory infections. Clin Microbiol Infect 10:125–126. Moorthy NS, Poongavanam V, Pratheepa V. 2014. Viral M2 ion channel protein: A promising target for anti-influenza drug discovery. Mini Rev Med Chem 14:819–830. Narasaraju T, Sim MK, Ng HH, Phoon MC, Shanker N, Lal SK, Chow VT. 2009. Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: Insights into viral virulence, tissue tropism and host pathogenesis. Microbes Infect 11:2–11. Nguyen-Van-Tam JS, Venkatesan S, Muthuri SG, Myles PR. 2015. Neuraminidase inhibitors: Who, when, where? Clin Microbiol Infect 21:222–225. Obraztsova EV, Osidak LV, Golovacheva EG, Afanas’eva OI, Mil’kint KK, Koroleva EG, Drinevskii VP, Vasil’eva IA. 2009. Interferon status in children during acute respiratory infections. Therapy with interferon. Bull Exp Biol Med 148:275–278. Oh DY, Hurt AC. 2014. A review of the antiviral susceptibility of human and avian influenza viruses over the last decade. Scientifica (Cairo) 2014:430629. Peteranderl C, Herold S, Schmoldt C. 2016. Human influenza virus infections. Semin Respir Crit Care Med 37:487–500. Prabhu N, Ho AW, Wong KH, Hutchinson PE, Chua YL, Kandasamy M, Lee DC, Sivasankar B, Kemeny DM. 2013. Gamma interferon regulates contraction of the influenza virusspecific CD8 T cell response and limits the size of the memory population. J Virol 87:12510–12522. Pschenitza M, Gavrilova ES, Tarasov SA, Knopp D, Niessner R, Epstein OI. 2014. Application of a heterogeneous immunoassay for the quality control testing of release-active forms of diclofenac. Int Immunopharmacol 21:225–230. Reed LJ. 1938. A simple method of estimating fifty percent endpoints. Am J Hyg 27:493–497. PT, Roesch C, Raghnaill MN. 2015. In: Drug action and pharmacodynamics. Reeves PT, Roesch C, Raghnaill MN, editors. Merck Manual. Vol. 2015. Merck, http://www.merckvetmanual.com/ mvm/pharmacology/pharmacology_introduction/drug_action_ and_ pharmacodynamics.html

J. Med. Virol. DOI 10.1002/jmv

8 Sergeev AN, P’Iankov OV, Shishkina LN, Duben LG, Petrishchenko VA, Zhukov VA, P’Iankova OG, Sviatchenko LI, Sherstoboev E, Karimova TV, Martiushev-Poklad AV, Sergeeva SA, Epshtein OI, Glotov AG, Glotova TI. 2004. Antiviral activity of oral ultralow doses of antibodies to gammainterferon: Experimental study of influenza infection in mice. Antibiot Khimioter 49:7–11. Sharp PM. 2002. Origins of human virus diversity. Cell 108:305– 312. Sherstoboev EY, Masnaya NM, Churin AA, Borsuk OS, Martyushev AV, Sergeeva SA, Epstein OI, Dygai AM, Goldberg ED. 2003. Immunotropic effects of potentiated antibodies to human interferon-gamma. Bull Exp Biol Med 135:70–72. Shishkina LN, Sergeev AN, Kabanov AS, Skarnovich MO, Evtin NK, Mazurkova NA, Sergeev AA, Belopolskaya MV, Kheyfets IA, Dugina JL, Tarasov SA, Sergeeva SA, Epstein OI. 2008. Study of efficiency of therapeutic and preventive anaferon (pediatric formulation) in mice with influenza infection. Bull Exp Biol Med 146:763–765. Shishkina LN, Skarnovich MO, Kabanov AS, Sergeev AA, Olkin SE, Tarasov SA, Belopolskaya MV, Sergeeva SA, Epstein OI, Malkova EM, Stavsky EA, Drozdov IG. 2010. Antiviral activity of Anaferon (pediatric formulation) in mice infected with pandemic influenza virus A(H1N1/09). Bull Exp Biol Med 149:612–614. Sidwell RW, Smee DF. 2004. Experimental disease models of influenza virus infections: Recent developments. Drug Discov Today Dis Models 1:57–63. Susloparov MA, Makhova NM, Noskova NV, Pliasunov IV, Susloparov IM, Sergeev AN, Sherstoboev E, MartiushevPoklad AV, Sergeeva SA, Epshtein OI, Glotov AG, Glotova TI. 2004. Efficacy of therapeutic and prophylactic actions of ultralow doses of antibodies to gamma-interferon in experimental murine model of herpes virus. Antibiot Khimioter 49:3–6.

J. Med. Virol. DOI 10.1002/jmv

Don et al. Tarasov SA, Dugina JL. 2008. Oral antibody to interferon gamma in ultra low doses: Clinical efficacy and interferon stimulation in patients. 5th Congress of European Pharmacology Society. Vol. 22. Manchester, UK: Fundamental & Clinical Pharmacology, p 37. Tarasov SA, Zarubaev VV, Gorbunov EA, Sergeeva SA, Epstein OI. 2012. Activity of ultra-low doses of antibodies to gammainterferon against lethal influenza A(H1N1)2009 virus infection in mice. Antiviral Res 93:219–224. Vasil’ev AN, Sergeeva SA, Kachanova MV, Tarasov SA, Elfimova UV, Dugina JL, Epshtein OI. 2008. Use of ultralow doses of antibodies to gamma-interferon in the treatment and prophylaxis of viral infections. Antibiot Khimioter 53:32–35. Waynforth DHB, Brain PP, Sharpe MT, Stewart MDF, Applebee MKA, Darke DPGG. 1998. Good Practice Guidelines. Administration of Substances (Rat, Mouse, Guinea Pig, Rabbit). In: Association, L.A.S. (Ed.), issue 1, PO Box 3993, Tamworth, Staffordshire, B78 3QU, p 4. Wiley JA, Cerwenka A, Harkema JR, Dutton RW, Harmsen AG. 2001. Production of interferon-gamma by influenza hemagglutinin-specific CD8 effector T cells influences the development of pulmonary immunopathology. Am J Pathol 158:119–130. Zhavbert ES, IuL Dugina, Epshtein OI. 2013. Immunotropic properties of anaferon and anaferon pediatric. Antibiot Khimioter 58:17–23.

SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s web-site.