Efficacy of the interferon inducer tilorone and its combination with antibacterial drugs in a murine model of secondary bacterial pneumonia caused by  S. aureus  after influenza infection

Irina N. Falynskova; Фалынскова Ирина Николаевна; Artem A. Poromov; Поромов Артем Андреевич; Natalia A. Mikhailova; Михайлова Наталья Александровна; Nadezhda P. Kartashova; Карташова Надежда Павловна; Ekaterina A. Glubokova; Глубокова Екатерина Андреевна; Anna V. Ivanina; Иванина Анна Валерьевна; Natalia V. Gudova; Гудова Наталия Владимировна; Irina A. Leneva; Ленева Ирина Анатольевна

doi:10.36233/0507-4088-363

Efficacy of the interferon inducer tilorone and its combination with antibacterial drugs in a murine model of secondary bacterial pneumonia caused by S. aureus after influenza infection

Authors: Falynskova I.N.¹, Poromov A.A.¹, Mikhailova N.A.¹, Kartashova N.P.¹, Glubokova E.A.¹, Ivanina A.V.¹, Gudova N.V.¹, Leneva I.A.¹
Affiliations:
1. Mechnikov Research Institute of Vaccines and Sera
Issue: Vol 71, No 1 (2026)
Pages: 73-82
Section: ORIGINAL RESEARCHES
URL: https://virusjour.crie.ru/jour/article/view/16845
DOI: https://doi.org/10.36233/0507-4088-363
EDN: https://elibrary.ru/gxgumt
ID: 16845

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Abstract

Introduction. Secondary bacterial pneumonias are complications responsible for most fatalities following influenza infection, and antiviral drugs are used to prevent their development.

The aim of the study is to evaluate the efficacy of tilorone and its combination with antibacterial agents in a murine model of secondary bacterial pneumonia caused by Staphylococcus aureus after influenza infection.

Materials and methods. BALB/c mice were infected with influenza virus A/California/04/2009 (pdm H1N1 2009) virus, followed by S. aureus infection four days later. Treatments included the interferon inducer tilorone, comparator oseltamivir, antibacterial agents cefuroxime and amoxicillin, or combinations of antivirals with antibiotics. Efficacy was assessed by increased survival, reduced weight loss, and inhibition of pathogen proliferation in the lungs.

Results. The efficacy of tilorone, as indicated by increased survival, reduced weight loss, and decreased pathogen load in the lungs, in a mouse model of secondary bacterial pneumonia following influenza infection, increased with reducing viral dose, and intraperitoneal administration of the drug was more effective than oral administration. The combination of tilorone with the antibiotic cefuroxime, to which S. aureus was sensitive, was more effective than either agent alone and completely protected animals from death and from viral and bacterial proliferation in the lungs. In contrast, the combination with amoxicillin, to which S. aureus was resistant, had no effect on disease outcome.

Conclusion. The efficacy of tilorone in this model depended on viral dose and drug administration route. Combining tilorone with cefuroxime was more effective than monotherapy.

Keywords

influenza virus, Staphylococcus aureus, secondary bacterial pneumonia following influenza infection, tiloron

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Introduction

Influenza is a respiratory disease whose annual epidemics, according to the World Health Organization, cause about 1 billion cases of infection worldwide [1]. Bacterial complications, primarily pneumonia, are the main cause of death after influenza [2]. The incidence of bacterial complications of influenza can reach 20% [1]; according to Russian epidemiological studies, it is 18% among outpatients from risk groups [3]. According to a meta-analysis of 135 studies (2010–2020), bacterial co-infection is diagnosed in an average of 11% of hospitalized patients with influenza, significantly worsening the prognosis and increasing the risk of death by 3.4 times [4]. Staphylococcus aureus is one of the most common causative agents of secondary pneumonia [5, 6].

The mortality rate for secondary bacterial pneumonia associated with influenza infection remains high despite the use of antibacterial drugs [7], so limiting primary influenza virus infection is necessary to prevent secondary complications. In the clinical guidelines “Influenza in Adults,” approved by the Russian Ministry of Health on October 21, 2025, patients are recommended to use antiviral drugs, including drugs with immunomodulatory properties, and in the development of viral-bacterial pneumonia, to supplement therapy with antibacterial drugs [8].

In Russia, interferon and its inducers are used for their antiviral and immunomodulatory effects. Interferon inducers cause the synthesis of endogenous interferon and ensure the prolonged circulation of interferons, whereas achieving similar concentrations with exogenous interferons requires multiple administrations of significant doses of the drugs [9].

Tilorone is a low-molecular-weight synthetic interferon inducer that stimulates the formation of alpha, beta, gamma, and lambda interferons in the body. The main structures that produce interferon in response to tilorone administration are intestinal epithelial cells, hepatocytes, T lymphocytes, neutrophils, and granulocytes. After oral administration of the drug, maximum interferon production is determined in the “intestine-liver-blood” sequence after 4-24 hours [10]. IFN-λ is the first line of defense of the respiratory epithelium of both the upper and lower respiratory tract against viral infections [11, 12]. Tilorone has an affinity for the corresponding receptors on alveolar macrophages and induces the formation of interferons in the lungs [13, 14]. The mechanism of antiviral action is associated with the inhibition of the translation of virus-specific proteins in infected cells^¹.

We previously developed a model of secondary viral-bacterial pneumonia in mice [15] to evaluate the effectiveness of therapy with drugs that have different mechanisms of action.

The aim of the study was to investigate the efficacy of the interferon inducer tilorone and a combination of tilorone with the antibacterial drugs cefuroxime and amoxicillin in a model of secondary bacterial pneumonia in mice after influenza infection.

Materials and methods

Pathogens. The influenza A/California/04/2009 (H1N1 2009) virus strain was obtained from the National Influenza Center at the A.A. Smorodintsev Research Institute of Influenza; strain S. aureus No. 1986 was obtained from the Microorganism Collection of the I.I. Mechnikov Research Institute of Virology and Immunology.

Laboratory animals. BALB/c female mice weighing 12–14 g were purchased from the Andreevka Branch of the National Center for Biomedical Technologies (NCBMT) of the Federal Medical Biological Agency (FMBA) of Russia and randomly divided into groups so that the individual body weight did not deviate from the average by more than ± 10%. Each group contained 9 to 15 animals. The study was approved by the Local Ethics Committee of the Mechnikov Research Institute of Vaccines and Sera (meeting protocol No. 15/2025 dated October 24, 2025).

Drugs. The drugs were purchased from a pharmacy chain. Tilorone was crushed and dissolved in distilled water. The control drug oseltamivir (Tamiflu capsules containing 75 mg of oseltamivir; manufacturer Hoffmann-La Roche, Switzerland) was dissolved in distilled water. Cefuroxime (powder for preparation of a solution for intravenous and intramuscular administration Cefuroxime, containing 750 mg of cefuroxime; manufacturer PJSC Kraspharma, Russia) was dissolved in distilled water. Amoxicillin (Flemoxin Solutab dispersible tablets containing 125 mg of amoxicillin; manufacturer: Haupt Pharma Latina, Italy) was dissolved in distilled water.

Determination of the effectiveness of tilorone and its combination with antibacterial drugs in a model of secondary bacterial pneumonia in mice after influenza infection. Mice were infected intranasally under general gas anesthesia with isoflurane with influenza virus at different doses (from 10³ to 10^4.2 TCID₅₀/0.1 mL), and after 4 days – with S. aureus at a dose of 2 × 10⁹ CFU/mL. Treatment of animals with tilorone at a dose of 30 mg/kg orally or 10 mg/kg intraperitoneally was started 24 and 4 hours before virus infection, then the drug was administered 24 hours after infection, with a total treatment duration of 3 days. Therapy with oseltamivir was started 4 hours before viral infection, then the drug was administered 4 hours after viral infection, then twice a day, with a total treatment duration of 5 days. Treatment with antibacterial drugs was started 3 hours after infection with S. aureus and continued for 3 days, once a day. The drugs were administered in a volume of 200 μL, and their doses were calculated at 1 mg/kg of body weight per day. The mice were observed daily for 16 days (Figs. 1, 2). The control group of animals infected with influenza virus and bacteria received 200 μL of distilled water as a placebo. Changes in body weight were recorded as described in the study [15]. Six days after infection with the influenza virus (two days after bacterial infection), three animals in each group were euthanized and their lungs were removed to determine the bacterial content using the Petri dish culture method with nutrient medium No. 10 (manufactured by the State Research Center for Applied Biotechnology and Microbiology) to identify S. aureus and the virus in MDCK cell culture, as described earlier [15].

Fig. 1. Study design for evaluating the efficacy of tilorone at different infectious doses in a secondary bacterial pneumonia model following influenza infection.

Рис. 1. Схема эксперимента по изучению эффективности тилорона при различных заражающих дозах на модели вторичной бактериальной пневмонии после гриппозной инфекции.

Fig. 2. Study design for evaluating the efficacy of tilorone and its combination with antibacterial agents in a secondary bacterial pneumonia model following influenza infection.

Рис. 2. Схема эксперимента по изучению эффективности тилорона и его комбинации с антибактериальными препаратами на модели вторичной бактериальной пневмонии после гриппозной инфекции.

The spectrum of sensitivity of the S. aureus 1986 strain to antibiotics was determined by the disc diffusion method in accordance with MG 4.2.1890-04 “Determination of the sensitivity of microorganisms to antibacterial drugs.”

Performance evaluation criteria: clinical signs (survival rate and weight loss); viral characteristics (virus titer in the lungs); bacterial characteristics (bacterial content in the lungs).

Statistical data processing was performed using Statistica 10 software. For intergroup comparisons of quantitative indicators presented as mean values with standard deviation, Dunn’s nonparametric test with Bonferroni correction was used, and preliminary analysis was performed using the Kruskal–Wallis criterion. Nonlinear four-parameter logistic regression was used to compare changes in body weight and survival between groups. Survival curves were constructed using the Kaplan–Meier method. Differences were considered statistically significant at p ≤ 0.05. The virus titer in the lung homogenate was calculated using the Reed and Mench limit serial dilution method [16], expressed in lg TCID₅₀/mL, and a difference of more than 2 log10 was considered significant [17].

Results

Study of the effectiveness of tilorone at different infectious doses of the influenza virus

The efficacy of tilorone was studied in a mouse model of secondary bacterial pneumonia in three experiments (Fig. 1) with different infectious doses of the virus: high (10^4.2 TCID₅₀/0.1 mL), medium (10⁴ TCID₅₀/0.1 mL), and low (10³ TCID₅₀/0.1 mL). The infecting dose of S. aureus for all experiments was 2 × 10⁹ CFU/mL.

At a high infecting dose (10^4.2 TCID₅₀/0.1 mL), combined sequential infection with pathogens resulted in the death of 93% of animals by the end of the experiment; body weight loss reached 15–18% by 9–11 days. Treatment with tilorone, both orally and intraperitoneally, as well as with oseltamivir orally, was ineffective; animal mortality and body weight loss were comparable to those in the control group.

Given the lack of effect of the drugs, in the next experiment, the virus infection dose was reduced (10⁴ TCID₅₀/0.1 mL). In the control group, 90% of the animals died on the 8th day after viral infection. The effectiveness of treatment with all drugs was higher when the infectious dose was reduced: oral administration of tilorone protected half of the animals in the group from death, and intraperitoneal administration completely protected all animals from death. Oral administration of oseltamivir increased survival to 80%. The results of the lung study corresponded to the survival data: Tilorone therapy reduced the virus titer by 3–4 log10 TCID₅₀/0.1 mL and the bacterial density in the lung homogenate by 2 log10 CFU/mL with intraperitoneal administration compared to untreated control animals (Table 1, Fig. 3).

Table 1. Efficacy of tilorone in different routes of administration and different infection doses of influenza virus A/H1N1/California/04/2009 (2009 H1N1 pandemic strain) in a secondary bacterial pneumonia murine model

Таблица 1. Эффективность тилорона при разных способах введения и заражающих дозах вируса гриппа А/Калифорния/04/2009 (пндм H1N1 2009) на модели вторичной бактериальной пневмонии у мышей

Group Группа	Drug Препарат	Survival rate Выживаемость		Viral titer, log10 TCID₅₀/0.1 mL Титр вируса, lg ТЦИД₅₀/0,1 мл	Bacterial count, log10 CFU/mL Содержание бактерий, lg КОЕ/мл
Group Группа	Drug Препарат	surviv./all жив./общ.	%
High dose (10^4.2TCID₅₀/0.1 mL) Высокая доза (10^4,2 ТЦИД₅₀/0,1 мл)	Tiloron, oral, 30 mg/kg Тилорон, перорально, 30 мг/кг	2/15 (р = 0.55)	13.3	6.7 ± 0.6	5.5 ± 0.1
	Tiloron, intraperitoneal, 10 mg/kg Тилорон, внутрибрюшинно, 10 мг/кг	4/15 (р = 0.15)	26.7	6.3 ± 0.6*	5.29 ± 0.2*
	Oseltanivir, oral, 20 mg/kg Осельтамивир, перорально, 20 мг/кг	5/15 (р = 0.07)	33.3	6.3 ± 0.6	5.51 ± 0.6
	Control A/California/04/2009 + S. aureus Контроль А/Калифорния/04/2009 + S. aureus	1/15	6.7	7 ± 0	5.6 ± 0.1
Medium dose (10⁴ TCID₅₀/0.1 mL) Средняя доза (10⁴ ТЦИД₅₀/0,1 мл)	Tiloron, oral, 30 mg/kg Тилорон, перорально, 30 мг/кг	5/10 (р = 0.05)	50.0	4.0 ± 0.5**	5.15 ± 0.1*
	Tiloron, intraperitoneal, 10 mg/kg Тилорон, внутрибрюшинно, 10 мг/кг	10/10 (р = 0.001)	100.0	3.2 ± 0.3**	3.35 ± 0.04**
	Oseltanivir, oral, 20 mg/kg Осельтамивир, перорально, 20 мг/кг	8/10 (р = 0.001)	80.0	3.2 ± 0**	3.49 ± 0.01**
	Control A/California/04/2009 + S. aureus Контроль А/Калифорния/04/2009 + S. aureus	1/10	10.0	6.7 ± 0.6	5.68 ± 0.05
Low dose (10³ TCID₅₀/0.1 mL) Низкая доза (10³ ТЦИД₅₀/0,1 мл)	Tiloron, oral, 30 mg/kg Тилорон, перорально, 30 мг/кг	8/9 (р = 0.01)	89.0	Не выявлено Not detected	Не выявлено Not detected
	Tiloron, intraperitoneal, 10 mg/kg Тилорон, внутрибрюшинно, 10 мг/кг	9/9 (р = 0.001)	100.0	Не выявлено Not detected	Не выявлено Not detected
	Oseltanivir, oral, 20mg/kg Осельтамивир, перорально, 20 мг/кг	10/10 (р = 0.001)	100	Не выявлено Not detected	Не выявлено Not detected
	Control A/California/04/2009 + S. aureus Контроль А/Калифорния/04/2009 + S. aureus	3/9	33	5.8 ± 0.3	4.5 ± 0.1

Note. Comparison of treatment groups with control, using the Mann–Whitney test with Bonferroni correction: * – p < 0.05; ** – p < 0.01.

Примечание. Сравнение групп терапии с контролем, критерий Манна–Уитни с поправкой Бонферрони: * – р < 0,05; ** – р < 0,01.

Fig. 3. Survival rate (a) and weight change (b) during tilorone treatment at high (10^4,2TCID₅₀/0.1 mL), medium (10⁴ TCID₅₀/0.1 mL), and low (10⁴TCID₅₀/0.1 mL) infectious doses of Influenza A/California/04/2009 (pdm H1N1 2009) in a secondary bacterial pneumonia model following influenza infection.

Рис. 3. Выживаемость мышей (а) и изменение массы тела (б) при лечении тилороном при высокой (10^4,2 ТЦИД₅₀/0,1 мл), средней (10⁴ ТЦИД₅₀/0,1 мл) и низкой (10³ ТЦИД₅₀/0,1 мл) заражающей дозе вируса гриппа А/Калифорния/04/2009 (пндм H1N1 2009) на модели вторичной бактериальной пневмонии после гриппозной инфекции.

With a further reduction in the infectious dose to 10³ TCID₅₀/0.1 mL, 67% of animals in the control group died. Oral administration of tilorone protected 89% of animals from death, while intraperitoneal administration of tilorone and oral administration of oseltamivir completely prevented the death of the study animals and the reproduction of pathogens in the lungs.

Thus, the interferon inducer tilorone was effective in a model of secondary pneumonia caused by infection with A/California/04/2009 followed by infection with S. aureus, both when administered intraperitoneally and orally. The effectiveness of treatment increased with a decrease in the infecting dose of the virus. Tilorone therapy prevented the death of mice, reduced their weight loss, and also reduced viral replication in the lungs.

Study of the effectiveness of combined use of tilorone and antibacterial drugs in a model of secondary pneumonia in mice after influenza infection

To study the combined effect of tilorone with antibacterial drugs, an average infecting dose of 10⁴ TCID₅₀/0.1 mL was selected, since in a previous experiment, oral administration of tilorone at this dose was effective in killing 90% of the animals in the control group. Oseltamivir was administered at a dose of 10 mg/kg, and the dose of tilorone was reduced to 15 mg/kg.

One of the problems in combating bacterial complications is the increase in bacterial resistance to antibacterial drugs. In this regard, information about the sensitivity of the pathogen to different types of antibacterial agents is necessary to select the appropriate drug. Studies of the sensitivity spectrum of the S. aureus 1986 strain to antibacterial drugs have revealed the resistance of bacteria to amoxicillin and azithromycin.

Thus, two antibacterial drugs were selected for the study: cefuroxime at a dose of 20 mg/kg as the antibiotic to which the studied S. aureus strain was found to be sensitive; as the antibiotic to which resistance was detected, amoxicillin at a dose of 20 mg/kg.

Complete death of animals in the control group was observed on the 9th day after viral infection (Table 2). The use of both antiviral and antibacterial agents increased survival by no more than 20%, did not prevent weight loss in animals, and did not reduce viral replication in the lungs. Antiviral drugs alone, as well as amoxicillin, to which S. aureus was found to be resistant, had no effect on the bacteria in the lungs. Furthermore, the combined use of tilorone and oseltamivir with amoxicillin did not affect the survival of mice or the replication of the virus in the lungs of mice.

Table 2. Effect of combined antiviral and antibacterial therapy on survival, viral and bacterial titers in the lungs during treatment of secondary bacterial pneumonia caused by S. aureus following influenza infection

Таблица 2. Влияние комбинации противовирусных и антибактериальных препаратов на выживаемость, титры вирусов и бактерий в легких при лечении вторичной бактериальной пневмонии, вызванной S. aureus после гриппозной инфекции

Group Группа	Survival Выживаемость		Viral titer, log10 EID₅₀/0.1 mL Титр вируса, lg ТЦИД₅₀/0,1 мл	Bacterial count, log10 CFU/mL Содержание бактерий, lg КОЕ/мл
Group Группа	жив./общ. surviv./all	%
Amoxicillin, oral, 20 mg/kg Амоксициллин, перорально, 20 мг/кг	0/10	0	6.5 ± 0.5	5.54 ± 0.06
Cefuroxime, oral, 20 mg/kg Цефуроксим, перорально, 20 мг/кг	2/10 (р = 0,150)	20	5.0 ± 0.5	3.37 ± 0.18*
Oseltamivir, oral, 10 mg/kg Осельтамивир, перорально, 10 мг/кг	2/10 (р = 0,150)	20	5.7 ± 0.3	5.7 ± 0.14
Tiloron, oral, 15 mg/kg Тилорон, пеорально, 15 мг/кг	2/10 (р = 0,150)	10	6.3 ± 0.6	5.52 ± 0.11
Oseltamivir, oral, 10 mg/kg + Cefuroxime, oral, 10 mg/kg Осельтамивир, перорально, 10 мг/кг + цефуроксим, перорально, 10 мг/кг	10/10	100	Не выявлено Not detected	Не выявлено Not detected
Oseltamivir, oral, 10 mg/kg + Amoxicillin, oral, 10 mg/kg Осельтамивир, перорально, 10 мг/кг + амоксициллин, перорально, 10 мг/кг	2/10 (р = 0,150)	20	5.8 ± 0.3	5.62 ± 0.05
Tiloron, oral, 15 mg/kg + Cefuroxime, oral, 20 mg/kg Тилорон, перорально, 15 мг/кг + цефуроксим, перорально, 20 мг/кг	8/10 (р = 0,001)	80	Не выявлено Not detected	Не выявлено Not detected
Tiloron, oral, 15 mg/kg + Amoxicillin, oral, 20 mg/kg Тилорон, перорально, 15 мг/кг + амоксициллин, перорально, 20 мг/кг	2/10 (р = 0,150)	20	6.2 ± 0.3	5.48 ± 0.11
Control A/California/04/2009 + S. aureus Контроль А/Калифорния/04/2009 + S. aureus	0/10	0	6.7 ± 0.6	5.43 ± 0.1

Note. Comparison of treatment groups with control, using the Mann–Whitney test with Bonferroni correction: * – p < 0.05.

Примечание. Сравнение групп терапии с контролем, критерий Манна–Уитни с поправкой Бонферрони: * – р < 0,05.

The use of cefuroxime reduced the number of bacteria by 100 times, and its combination with tilorone or oseltamivir increased survival to 80–100%, completely prevented weight loss in animals, and completely suppressed the reproduction of the virus and bacteria in the lungs, indicating the additive nature of both combinations (Table 2, Fig. 4).

Fig. 4. Survival rate (a) and weight change (b) during treatment with tilorone and oseltamivir at different infectious doses in a secondary bacterial pneumonia model following influenza infection in mice.

Рис. 4. Выживаемость мышей (а) и изменение массы тела (б) при лечении тилороном и осельтамивиром при различных заражающих дозах на модели вторичной бактериальной пневмонии после гриппозной инфекции у мышей.

Thus, combined treatment with tilorone and the antibacterial drug cefuroxime showed greater efficacy than treatment with each drug separately.

Discussion

in a murine model of secondary bacterial pneumonia following influenza infection, it was shown that the efficacy of the interferon inducer tilorone, expressed as increased survival, reduced weight loss, and reduced pathogen content in the lungs, increased with reducing virus dose and intraperitoneal administration was more effective than oral administration.

The combination of tilorone with the antibacterial drug cefuroxime, to which the studied strain of S. aureus was sensitive, resulted in complete protection of animals from death, complete suppression of virus replication, and elimination of bacterial colonization of the lungs. In contrast, the combination of tilorone with amoxicillin, to which the pathogen was resistant, had no therapeutic effect. This highlights the importance of selecting an appropriate antibacterial agent based on the sensitivity of the pathogen and the promise of a strategy combining antibiotics and antiviral drugs for post-influenza bacterial complications. A similar study previously showed that combination therapy with oseltamivir and ampicillin resulted in 100% survival in mice sequentially infected with S. pneumoniae after influenza A/Puerto Rico/8/34 (H1N1) virus [18]. Experimental results in a mouse model of secondary bacterial pneumonia were consistent with clinical observations: combination therapy of severe influenza with oseltamivir and antibiotics suppressed the inflammatory response, reduced the length of hospital stay, and decreased the frequency of admissions to the intensive care unit [1].

Tilorone stimulates the production of endogenous interferons of all major types (α, β, γ, and λ) and activates macrophages, NK cells, and T lymphocytes, which may explain its contribution to the increased effectiveness of combination therapy. Of particular importance is the induction of IFN-λ in lung tissue, which has a pronounced antiviral and anti-inflammatory effect at the level of the respiratory epithelium [9, 11]. In the context of secondary viral-bacterial infection, such activation of immune mechanisms contributes to limiting virus replication, restoring the barrier function of the mucosa, and increasing the effectiveness of antibacterial therapy. Previously, in a similar model, it was shown that intranasal administration of IFN-λ was associated with a decrease in the bacterial load in the lungs and a reduction in the weight loss of animals, which corresponds to our data [11].

The experimental data obtained can serve as a basis for developing combined therapy regimens for influenza and its bacterial complications, aimed at improving treatment efficacy and reducing the risk of developing antibiotic resistance.

¹ Instructions for medical use of the medicinal product Amixin® (tilorone, LP No. (004817) (RG RU)) [Electronic resource]. Moscow: Ministry of Health of the Russian Federation, State Register of Medicines / JSC Otisifarm Pro. Available at: https://grls.pharm-portal.ru/grls/8a267191-4cf2-4d87-af75-64c2b971c787 (date of access: 16.01.2026).