Influenza virus infection affects the efficacy of the treatment of dyslipidemia in mice with a polyisoprenoid-based drug

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Introduction

Dyslipidemia, particularly in the context of metabolic syndrome (MS), increases the risk of severe respiratory viral infections, including influenza [1–3]. In turn, the influenza A virus actively alters the host's lipid metabolism, using cholesterol and phospholipids to form lipid rafts and assemble viral particles [4]. This creates direct competition between dyslipidemia therapy and viral replication for shared metabolic resources. In this context, the following question arises: does the lipid-lowering effect of these drugs persist during acute viral infection?

Viral infections, including influenza and COVID-19, can exacerbate the course of cardiovascular diseases by contributing to endothelial damage, systemic inflammation, and platelet activation [5–10]. At the same time, patients with metabolic disorders, such as obesity and dyslipidemia, are particularly vulnerable to severe influenza [11, 12]. Given that the influenza virus actively utilizes host lipids for its own replication [5], it can be assumed that during infection, the efficacy of lipid-lowering therapy may decrease due to the redistribution of lipid resources in favor of the virus. It appears timely to develop drugs that combine effects on the pathogenesis of metabolic syndrome with antiviral, particularly anti-influenza, effects.

To this end, we have developed a drug based on sodium polyprenyl phosphate (PPP) and β-sitosterol (BSS), which is currently undergoing clinical trials. Polyprenyl phosphate, by stimulating the production of type I interferon (IFN), suppresses the activity of the SREBP2 (sterol regulatory element-binding protein 2) transcription factor and, as a result, reduces the synthesis of low-density cholesterol [13]. BSS competitively inhibits cholesterol absorption in the intestine by suppressing its interaction with NPC1L1 receptors (Niemann-Pick C1-like protein 1) [13]. In addition, PPP is the active ingredient in Fortepren, registered as an antiviral agent against herpes, and Fosprenyl, an antiviral drug actively used in veterinary medicine [14–16]. Antiviral activity has also been demonstrated for BSS [17, 18]. In preclinical studies, the drug effectively corrected dyslipidemia in an experimental mouse model [13]; however, its efficacy under conditions of viral infection, which is accompanied by a profound shift in lipid metabolism in favor of pathogen replication, remains unexplored.

The aim of this study was to evaluate the antiviral and antidyslipidemic properties of the drug in mice infected with the influenza virus and exhibiting experimental dyslipidemia induced by repeated administration of poloxamer 407 (Pol 407) [19].

Materials and methods

The drug used was Prenophytol (a product of the Medgamal branch of the N.F. Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of Russia, Roszdravnadzor Authorization No. 362 for the Conduct of Clinical Trials (August 14, 2025)), containing 16 mg of PPF and 20 mg of BSS per tablet.

The influenza A/California/07/2009 (H1N1) virus was used for infection. The influenza A/California/07/2009 (H1N1) virus was propagated in a canine kidney cell line (MDCK) [20]. Infection was performed at a virus-to-cell ratio of 1 : 100 (MOI = 0.01); cultivation was carried out in modified Igla medium (GMEM) with the addition of 2 μg/mL TPCK-trypsin at 35 °C for 48 h. The supernatant was collected, centrifuged at 2000g for 10 min at 4 °C, aliquoted, and stored at –80 °C. Prior to use, the virus titer was determined by the TCID₅₀ method based on cytopathic effect and was 106.5 TCID₅₀/mL [21]. To infect the mice, a suspension was prepared in PBS to a final dose of 1 LD₅₀ (50 μL per animal).

The study used 160 BALB/c mice weighing 20–22 g, obtained from the Central Laboratory Animal Breeding Center of the Russian Academy of Medical Sciences (Andreevka). The animals were housed with free access to water and food. The basic rules for housing and care complied with regulatory documents. Routine animal care procedures were performed in accordance with the Standard Operating Procedures (SOPs) of the N.F. Gamaleya Research Center of Epidemiology and Microbiology (NRCEM) of the Ministry of Health of Russia. Procedures involving the use of laboratory animals were conducted in accordance with the international principles of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1985), as well as the requirements of Order No. 199n of the Ministry of Health of the Russian Federation dated April 1, 2016 "On the Approval of the Rules of Good Laboratory Practice". The study protocol was approved by the Biomedical Ethics Committee of the N.F. Gamaleya National Research Center for Epidemiology and Microbiology (N.F. Gamaleya NRCEM) of the Ministry of Health of Russia (Protocol No. 84 dated December 2, 2024).

Housing. Mice were housed in 26 × 17 × 12 cm³ polycarbonate cages with bedding – 10 animals per cage. The cages were equipped with steel mesh lids with a feeding recess, steel feed dividers, and steel label holders.

Diet. A standard pelleted diet, "PK-120 Compound Feed for Laboratory Rats, Mice, and Hamsters" (Laboratorkorm LLC, Russia), was used.

Water. The animals were provided with water meeting the requirements of GOST "Drinking Water" 28.74–73.

Environmental parameters. Mice were housed under controlled environmental conditions at a temperature of 18–20 °C and relative humidity of 50–60%. Temperature and humidity were continuously monitored.

The quarantine period lasted at least 14 days.

Groups were established to assess the effect of the drug on the condition of mice with artificially induced dyslipidemia using Pol 407 and against the background of a viral infection. Groups: Virus+Pol 407+Drug, Virus+Drug, Virus+Pol 407, Pol 407+Drug, Virus, Control, Intact, Drug. Each group consisted of 20 animals.

A suspension containing the influenza virus was administered intranasally under light ether anesthesia at a volume of 50 μL/mouse at a dose of 1 LD₅₀. The clinical condition of the animals was monitored daily for 14 days after infection. Changes in body weight and mean survival time (MST) served as criteria for evaluating the effectiveness of the infection. MST was calculated as the arithmetic mean of the survival time of all animals in the group until spontaneous death or scheduled euthanasia on day 18 (in cases of survival until the end of observation). Death occurring before day 14 was considered related to the severity of the infection.

Mice in the experimental group were administered Pol 407 intraperitoneally twice a week at a dose of 500 mg/kg [39], while the drug was administered via an intragastric tube five times a week as a suspension containing 0.8 mg of PPP and 1 mg of BSS in 0.5 mL of sodium chloride saline. Administration of both drugs began 7 days prior to influenza virus infection and continued for 3 weeks, ensuring exposure both during the stage of dyslipidemia development and during the acute phase of viral infection. The placebo consisted of 0.5 mL of sodium chloride saline solution.

Blood samples for lipid profile assessment were collected at three time points: 1 day before infection (on the 7th day of therapy), on the 7th day after infection (the peak of the infectious process), and on the 18th day from the start of therapy (corresponding to the 14th day after infection).

The levels of high-density lipoproteins (HDL), low-density lipoproteins (LDL), total cholesterol, and triglycerides in the animals' blood serum were determined using a Beckman Coulter 680 biochemical analyzer.

Statistical analysis. The statistical significance of differences in lipid metabolism parameters was assessed using Student's t-test. Prior to applying Student's t-test, the data distribution was evaluated. Analysis of the coefficient of variation (CV) showed that for most groups it did not exceed 30% (and for key indicators, not even 15%), which indicates a symmetric distribution and allows for the use of a parametric test. Results were considered statistically significant at a p-value < 0.05.

Results

The data obtained from uninfected mice (Table 1, Fig. 1) served as a baseline for assessing the modulatory effect of influenza infection on drug efficacy (Table 2, Figs. 2 and 3). The baseline levels of lipid metabolism parameters in intact animals are presented in Table 1. Administration of Pol 407 to the animals led to a significant increase in blood levels of cholesterol, triglycerides, and LDL. HDL concentration did not change significantly.

 

Table 1. Serum lipid profile in intact and Poloxamer 407 (P-407)-treated BALB/c mice

Таблица 1. Показатели липидного обмена в сыворотке крови интактных и обработанных Pol 407 мышей BALB/c

Parameter Показатель	мМ (М ± m)
	Control Контроль	Pol 407
Cholesterol Холестерин	3.86 ± 0.16	16.20 ± 0.51
Triglycerides Триглицериды	1.54 ± 0.25	18.45 ± 2.78
HDL ЛПВП	2.55 ± 0.05	2.90 ± 0.42
LDL ЛПНП	0.75 ± 0.10	2.46 ± 0.7

 

Fig. 1. Serum lipid parameters in dyslipidemic mice infected with influenza virus.

* – р < 0.05, ** – р < 0.001 versus baseline (day 0) within the respective group. In uninfected mice, the drug induced a significant reduction in cholesterol, triglycerides, and LDL, along with an increase in HDL by day 18, indicating a pronounced hypolipidemic effect.

Рис. 1. Показатели липидного обмена у дислипидемичных мышей после введения ЛС.

* – р < 0,05, ** – р < 0,001 по сравнению с точкой «0» в соответствующей группе. У незараженных мышей ЛС вызывало достоверное снижение холестерина, триглицеридов и ЛПНП, а также повышение ЛПВП к 18-м суткам, что свидетельствует о выраженном гиполипидемическом эффекте.

 

Table 2. Dynamics of the lipid profile in mice with Poloxamer 407 (P-407)-induced dyslipidemia receiving drug therapy with and without concurrent influenza virus infection (mmol/L)

Таблица 2. Динамика липидного профиля у мышей с дислипидемией, вызванной Pol 407, на фоне терапии ЛС при вирусной инфекции и без нее (ммоль/л)

Group Группа	Parameter Показатель	Pre-infection (day 0) До заражения (0 сут)	Infection peak (day 7) Пик инфекции (7 сут)	Recovery (day 18) Восстановление (18 сут)
P-407 + drug Pol 407 + ЛС	TC ХС	16.20	15.09	10.27
	TG ТГ	18.45	14.86	12.87
	LDL-C ЛПНП	2.46	0.75	1.68
	HDL-C ЛПВП	2.90	6.32	3.53
P-407 + Virus + drug Pol 407 + Вирус + ЛС	TC ХС	16.20	13.36	9.44
	TG ТГ	18.45	15.08	18.15
	LDL-C ЛПНП	2.46	1.06	2.02
	HDL-C ЛПВП	2.90	4.69	2.26

Note. TC – cholesterol; TG – triglycerides; LDL-C – low-density lipoproteins; HDL-C – high-density lipoproteins. Bold values indicate statistically significant differences from baseline (p < 0.05). Data are presented as mean ± SD (n = 6–9 per group).

Примечание. ХС – холестерин; ТГ – триглицериды; ЛПНП – липопротеины низкой плотности; ЛПВП – липопротеины высокой плотности. Жирным выделены значения, достоверно отличающиеся от исходного уровня (p < 0,05). Данные представлены как среднее значение (n = 6–9).

 

Fig. 2. Profile in dyslipidemic mice infected with influenza virus following drug administration.

* – р < 0.05 versus baseline (day 0) in the respective group. In infected mice, spontaneous reduction in cholesterol and LDL was observed alongside increased HDL, reflecting the impact of viral infection itself on lipid metabolism.

Рис. 2. Показатели липидного обмена у дислипидемичных мышей, зараженных вирусом гриппа.

* – р < 0,05, по сравнению с точкой «0» в соответствующей группе. У зараженных мышей наблюдали спонтанное снижение холестерина и ЛПНП на фоне роста ЛПВП, что отражает влияние самой вирусной инфекции на липидный обмен.

 

Fig. 3. Serum lipid profile in dyslipidemic mice infected with influenza virus following drug administration.

** – р < 0.001 versus baseline (day 0) within the respective group. In the presence of viral infection, the drug significantly reduced only total cholesterol levels, whereas its effects on the remaining lipid fractions (TG, HDL-C, LDL-C) were not statistically significant, indicating attenuated therapeutic efficacy.

Рис. 3. Показатели липидного обмена у зараженных вирусом гриппа дислипидемичных мышей после введения ЛС.

** – р < 0,001 по сравнению с точкой «0» в соответствующей группе. На фоне вирусной инфекции ЛС достоверно снижало только уровень холестерина, тогда как воздействие на остальные липидные фракции было статистически незначимым, что указывает на ослабление терапевтической эффективности.

 

Administration of the drug to mice with dyslipidemia resulted in a significant reduction in cholesterol and triglyceride levels within 18 days of the start of treatment (Fig. 1). LDL levels decreased significantly by day 7. At the same time, HDL levels increased.

A somewhat unexpected result was observed in a group of dyslipidemic mice infected with the influenza virus (Fig. 2). Although triglyceride levels remained unchanged, blood cholesterol levels in the experimental animals decreased as the infection progressed. At the same time, on the 7th day after infection, HDL levels increased with a parallel decrease in LDL levels. To a certain extent, the influenza virus had the same effect as the drug.

A different picture was observed in the treatment of dyslipidemic animals infected with the influenza virus. The drug had a significant effect only on cholesterol levels, causing them to decrease (Fig. 3). Although the other parameters tended toward normalization, as after drug administration to dyslipidemic uninfected animals, they did not differ significantly from baseline levels (point "0").

It was shown that the drug exerted an antiviral effect, increasing the average lifespan of the animals (Fig. 4b, Vir + Drug group). However, in the group of mice with experimental dyslipidemia, this effect disappeared. Moreover, administration of Pol 407 led to an exacerbation of the viral infection.

 

Fig. 4. Body weight changes (a) and mean survival time (MST) (b) over the 18-day observation period in experimental mouse groups.

Virus, influenza A virus; P-407, Poloxamer 407; drug, test compound; MST, mean survival time. Experimental groups: Virus – influenza A virus-infected mice; Virus + drug – infected mice treated with the drug; Virus + P-407 – infected mice with P-407 – induced dyslipidemia; Virus + P-407 + drug – infected, dyslipidemic mice treated with the drug. Body weight was recorded daily throughout the 18-day observation period. In mice treated with P-407 + Virus, weight loss began on days 3–4 post-infection, peaked on days 7–10 (mean loss: 20–25% of baseline body weight), and was accompanied by high mortality. Animals additionally receiving the drug exhibited attenuated weight loss (maximum 15–18% on days 9–11) and faster body weight recovery after day 12. Control groups (drug alone, Virus + drug) displayed only minor physiological fluctuations in body weight (≤ 5–7%).

Рис. 4. Изменение массы тела (а) и средняя продолжительность жизни (б) за период наблюдения (18 сут) у животных разных групп.

Вирус – мыши, зараженные вирусом гриппа; Вирус + ЛС – зараженные животные, которым вводили ЛС; Вирус + Pol 407 – зараженные и обработанные полоксамером животные; Вирус + Pol 407 + ЛС – зараженные и обработанные полоксамером животные, которым вводили ЛС; СПЖ – средняя продолжительность жизни. Массу тела животных регистрировали ежедневно в течение всего периода наблюдения (18 сут). У мышей, получавших Pol 407 + Вирус, снижение массы начиналось с 3–4-х суток после заражения, достигало максимума на 7–10-е сутки (средняя потеря – 20–25% от исходной массы) и сопровождалось высокой смертностью. У животных, дополнительно получавших ЛС, потеря массы была менее выраженной (максимум – 15–18% на 9–11-е сутки) и сопровождалась более быстрым восстановлением массы тела после 12 сут. У контрольных групп (ЛС, Вирус + ЛС) колебания массы не превышали 5–7% и носили физиологический характер.

 

A similar conclusion can be drawn from the analysis of changes in body weight in experimental animals from different groups (Fig. 4a).

Discussion

The aim of this study was to evaluate the antiviral and anti-dyslipidemic properties of the drug in mice infected with the influenza virus and suffering from experimental dyslipidemia induced by repeated administration of Pol 407. Statins are traditionally prescribed for the treatment of dyslipidemia, but their use may be accompanied by serious side effects [37, 40]. In this regard, there is a pressing need to develop new, effective, and safe agents for the prevention and treatment of metabolic syndrome, one of the manifestations of which is dyslipidemia.

To this end, we applied a new complex drug based on PPP and BSS. The first component of this drug – PPP – has long been successfully used as an antiviral agent, including for infections caused by the influenza virus [14]. PPP activates TLR2/4 [22], and consequently, IRF3 (interferon-regulatory factor 3), and increases the production of type I IFN – all of which leads to the suppression of SREBP2 (sterol regulatory element-binding protein 2) activity and, as a result, reduces the synthesis of cholesterol and products of the mevalonate pathway [48]. Activation of the TLR2/4 → IRF3 pathway in response to PPP exposure not only suppresses SREBP2 but also limits the prenylation of viral proteins by inhibiting the mevalonate pathway, which may weaken influenza replication [13]. Prenylation is a process of post-translational modification of proteins in which a lipophilic isoprenyl group binds to a viral protein synthesized de novo. Prenylated proteins are involved in virtually all stages of the viral life cycle — during binding to the cell, entry into the cell and the nucleus, as well as during viral genome replication [23]. Inhibition of prenylation disrupts the assembly and production of viral particles, which can lead to the formation of defective viral particles. The latter has been repeatedly confirmed upon exposure of PPP to viruses belonging to various taxonomic groups [14]. A number of studies have demonstrated the antiviral activity of PPP against influenza infection. In in vitro experiments, PPP suppressed the replication of the influenza A virus strain WSN of serotype H1N1 [24] as well as highly pathogenic strains of avian influenza A virus (H5N1) [25]. In vivo experiments demonstrated the therapeutic and prophylactic effects of PPF against infection caused by the WSN strain of influenza A virus (H1N1), as well as the AICHI 6/68 strain of influenza A virus (H3N2) [26].

The second component of the complex drug — BSS — competitively blocks the NPC1L1 receptor in the intestine, inhibiting cholesterol absorption [13, 27, 41]. This dual mechanism of action — suppression of synthesis and absorption — makes the PPP + BSS combination particularly promising for the treatment of dyslipidemia [13, 41, 42, 44]. Furthermore, BSS stimulates the T-cell response [43] and the functional activity of the NK cells in viral infections, autoimmune diseases, and other pathological processes [28]. The immunostimulatory activity of BSS has been confirmed in numerous studies [29, 30]. The role of BSS as a potential agent against pathogens that use cholesterol-dependent toxins for infection has also been noted [31]. Furthermore, BSS exhibits antiviral activity in vitro and in vivo against infections caused by the influenza virus strains Puerto Rico/8/34 (H1N1) and A/FM/1/47(H1N1) [17]. It has also been shown that BSS can suppress the entry of coronaviruses into host cells via angiotensin-converting enzyme-2 (ACE-2) by inhibiting its interaction with the receptor-binding domain of the SARS-CoV-2 spike glycoprotein [18].

Both components of the drug exhibit anti-inflammatory activity. However, while the anti-inflammatory effect of PPP is primarily associated with the inhibition of lipoxygenase-5 and -15 [32], the anti-inflammatory properties of BSS are partly due to its ability to modulate signaling via RIG-I (retinoic acid-inducible gene 1), which prevents excessive activation of STAT1 and the overproduction of pro-inflammatory cytokines in response to type I IFN [17]. This effect does not suppress but rather balances the interferon response, which is particularly important in acute viral infections accompanied by a cytokine storm, and, consequently, leads to a reduction in pro-inflammatory reactions induced by ISGF3 (interferon-stimulated growth factor 3) complexes in interferon-sensitive cells [33].

Furthermore, both PPP and BSS exhibit antioxidant activity [34, 35, 45].

Thus, the drug potentially possesses a comprehensive therapeutic effect, which includes correction of dyslipidemia, counteraction of viral infection, and anti-inflammatory and antioxidant activity.

In preclinical studies, the drug effectively corrected dyslipidemia in mouse experiments [13]. To model dyslipidemia in this study, Pol 407 was used — a nonionic surfactant that inhibits lipoprotein lipase and 7-α-hydroxylase, leading to a sustained increase in cholesterol and triglycerides [19, 36, 38, 39]. This model allows for the reproduction of key features of metabolic disorders and the evaluation of the efficacy of lipid-lowering agents.

The data obtained in this study confirmed that in an experimental model using Pol 407, the drug complex exerts a pronounced effect, leading to a reduction in cholesterol, triglyceride, and LDL levels with concomitant increase in HDL levels.

However, it remained unclear whether the drug's lipid-lowering efficacy persists under conditions of acute viral infection, when lipid resources are reallocated in favor of pathogen replication [46]. It is known that the influenza A virus actively utilizes host cholesterol and phospholipids to form lipid rafts and assemble viral particles, leading to a reorganization of lipid metabolism in infected cells [5]. This leads to a certain degree of competition between the virus and lipid-lowering therapy for shared metabolic resources.

As expected, the drug exhibited antiviral activity, increasing the MST of animals (Fig. 4). It was also found that, under conditions of influenza infection, the drug's antiviral effect persists, while its therapeutic effect on dyslipidemia becomes weaker. This paradoxical observation can be explained by the fact that Pol 407 itself causes severe metabolic disturbances: it inhibits lipoprotein lipase and 7-α-hydroxylase, leading to a sharp increase in cholesterol and triglyceride levels [19, 36]. This condition creates a background "metabolic burden" that depletes the body's resources and reduces its resistance to infection. Under these conditions, even an effective antiviral drug cannot exert its full activity.

Conclusions

We conclude that Pol 407 does not directly block the effect of the drug, but rather creates conditions under which the antiviral effect of the drug becomes insufficient to counteract the severity of the infection. This confirms that the metabolic state of the host critically influences the outcome of a viral infection.

The results obtained raise the issue of optimizing the treatment of atherosclerosis in the presence of concomitant acute and chronic viral infections. The polyisoprenoid-based drug under study demonstrated precisely the following combination: it significantly reduces cholesterol, triglyceride, and LDL levels in uninfected animals, and in the context of influenza infection, it retains its antiviral effect while partially mitigating the course of the disease.

The study experimentally substantiates a personalized approach to the treatment of dyslipidemia in patients with acute respiratory infections and confirms the feasibility of developing drugs with dual hypolipidemic and antiviral effects for comorbid patients.

About the authors

Evgeny A. Makiev

National Research Centre for Epidemiology and Microbiology named after the Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Author for correspondence.
Email: makiev31121994@yandex.ru
ORCID iD: 0000-0002-7062-958X

PhD Student, Junior Researcher, Laboratory of Cellular Immunity

Russian Federation, Moscow

Tatyana N. Kozhevnikova

National Research Centre for Epidemiology and Microbiology named after the Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: tatiana140663@gmail.com
ORCID iD: 0000-0003-0507-1935

PhD, Researcher, Laboratory of Cellular Immunity

Russian Federation, Moscow

Alexander V. Pronin

National Research Centre for Epidemiology and Microbiology named after the Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: proninalexander@yandex.ru
ORCID iD: 0000-0001-5266-9783

DSc, Professor, Deputy Director for Research

Russian Federation, Moscow

Alexander N. Narovlyansky

National Research Centre for Epidemiology and Microbiology named after the Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: narovl@yandex.ru
ORCID iD: 0000-0003-0601-7148

DSc, Professor, Chief Researcher, Laboratory of Cytokines

Russian Federation, Moscow

Alexander V. Sanin

National Research Centre for Epidemiology and Microbiology named after the Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: saninalex@inbox.ru
ORCID iD: 0000-0003-3091-5802

DSc, Professor, Head of Laboratory of Cellular Immunity

Russian Federation, Moscow

Irina V. Ganshina

National Research Centre for Epidemiology and Microbiology named after the Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: ganshina.iren@yandex.ru
ORCID iD: 0000-0003-4049-7907

Researcher, Laboratory of Cellular Immunity

Russian Federation, Moscow

Maxim M. Shmarov

National Research Centre for Epidemiology and Microbiology named after the Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation

Email: mmshmarov@gmail.com
ORCID iD: 0000-0002-5268-1296

DSc, Head of Laboratory of Molecular Biotechnology

Russian Federation, Moscow

References

Supplementary files