Protective activity of CR9114 universal antibody isotypes against influenza A virus in vivo

Full Text

Introduction

Seasonal influenza is an acute respiratory infection caused by influenza viruses that circulate throughout the year around the world. Influenza can cause illness of varying severity, sometimes leading to hospitalization or death¹. The most promising strategies for reducing morbidity and preventing the risks of severe consequences of infection are the development of a universal vaccine or broad-spectrum antibodies (Abs).

It is known that the antiviral action of Abs is due to their functional structure: a unique Fab (fragment antigen binding) fragment formed by variable regions of Abs provides highly specific binding of viral antigens, while the constant region of Abs (Fc) is responsible for effector functions [1, 2]. Although antigen recognition by the Fab fragment is crucial for pathogen neutralization in vitro, it is becoming increasingly clear that Fc-mediated effector function plays an equally important role in providing protection in vivo [3]. The interaction of Ab Fc fragments with immune cell receptors (FcR) initiates a series of pro-inflammatory, anti-inflammatory, and adaptive immune responses in the host, leading to protection or, in some cases, worsening the course of the disease [4]. The effector function of Abs depends on its isotype (IgG, IgA, IgM or IgD), i.e., on the ability of Abs to interact with its corresponding FcR on the surface of immune cells.

All currently clinically approved full-size Abs are of the IgG isotype due to their longer half-life; in addition, large-scale production and purification technologies have been developed specifically for IgG [5]. However, over the past 20 years, various Fc-mediated functions have been characterized in detail not only for IgG antibodies, but also for IgA isotypes; the unique effector properties of IgA have been demonstrated, potentially allowing the development of more effective broad-spectrum therapeutic Abs based on this Ab isotype [6].

IgA, the most common class of antibodies found on the surface of mucous membranes, plays an important role in the immune system. IgA is capable of interacting with various classes of immune cells expressing the FcαRI (CD89) receptor, such as polymorphonuclear cells, monocytes, macrophages, and Kupffer cells [7]. Furthermore, IgA can activate dendritic or T cells by binding to alternative cell receptors such as DC-SIGN, transferrin receptor, or FcRL4 [8].

To date, about a dozen broad-spectrum Abs have been isolated and characterized, most of which target conserved regions in the stem of the surface protein hemagglutinin, as well as several Abs that specifically bind and block the catalytic centers of influenza virus neuraminidase [9–12].

This study focuses on the broad-spectrum antiviral CR9114, which specifically binds to a highly conserved epitope in the stem domain of hemagglutinin and exhibits cross-reactivity against influenza A and B viruses [12]. In 2024, it was shown that CR9114 provides effective protection in mice not only against infections caused by seasonal strains, but also against lethal infection caused by the H5N1 strain with high pandemic potential [13]. Based on the known variable fragments of CR9114, we obtained three recombinant human antibodies (rAbs) of the IgG1, IgA1, and IgA2 isotypes.

The aim of this study was to evaluate the protective activity of CR9114 rAbs of IgG and IgA isotypes when administered systemically and locally against experimental influenza infection in mice.

Materials and methods

Obtaining and purifying rAbs

Based on the sequences of variable domains of AT CR9114 (GenBank JX213639.1 and JX213640.1 for heavy and light chains, respectively), human rAb isotypes IgG1 and IgG2 were designed, differing in the structure of the constant domains of the heavy chain. The sequences of the constant regions of the heavy chains were identical to the sequences P01857 (IgG1), P01876 (IgA1), and P01877 (IgA2) presented in the UniProt database [14]. All types of rAbs obtained contained the same kappa isotype light chain (constant region P01834). Antibodies were assembled using a biplasmid system for the expression of the corresponding heavy and light (kappa) Ab chains in eukaryotic cells. A modified pcDNA3.4 plasmid (Thermo Fisher Scientific) containing the constant regions of Ab chains and universal signal peptide sequences was used as the starting vector.

The production of experimental samples of IgA1 and IgA2 isotypes, as well as control Ab of the IgG1 isotype, was carried out in CHO cells in transient expression mode.

To obtain Ab drugs, the culture medium was clarified by centrifugation and the proteins were purified by affinity chromatography using the ÄKTA pure system (Cytiva) on a KappaSelect column (1 mL; Cytiva). The principle of the protein purification method is based on the fact that Ab specific to kappa chains of immunoglobulins immobilized on the carrier are strongly affinity-bound to synthesized rAb isotypes IgG1, IgA1 and IgA2. Protein elution from the chromatographic column was performed at low pH values, at which the affinity interactions between the sorbent and recombinant immunoglobulins are disrupted.

High-resolution exclusion chromatography

The Ab samples were analyzed using high-performance liquid chromatography in accordance with the requirements of the State Pharmacopoeia. The study used a Waters Breeze chromatography system (Waters) with Empower 3 control software; The analysis was performed on a Bio-Silect SEC 250-5 column (Bio-Rad) measuring 7.8 mm × 30 cm. A single phosphate-salt buffer was used as the mobile phase. The samples were diluted with the mobile phase to a final concentration of 100 μg/ml. Next, 50 µL of the solution was injected into the chromatograph using an autosampler and eluted with 25 mL of mobile phase at a flow rate of 1 mL/min. Detection was performed at a wavelength of 280 nm in the range of 4–15 min. The chromatograms were analyzed using Empower 3.

Laboratory animals

The study used female BALB/c mice weighing 19–22 g (Scientific Center for Biomedical Technologies of the Federal Medical Biological Agency, Stolbovaya branch). The mice were kept under standard conditions in accordance with Directive 2010/63/EU and sanitary rules and standards SanPiN 3.3686-21 “Sanitary and epidemiological requirements for the prevention of infectious diseases.” All experiments involving laboratory animals were approved by the Bioethics Committee of the A.A. Smorodintsev Research Institute of Influenza (protocol No. 6 dated 03.04.2025).

Immunization and infection of mice, collection of sera and organs

Intranasal administration was performed using a dispenser under inhalation anesthesia, administering 50 μL of the drug. Intraperitoneal administration of the drugs was performed in a volume of 250 μL using sterile disposable syringes with a 30G needle.

For the therapeutic and prophylactic study regimen, rAb drugs (CR9114_G1 and CR9114_A1) were administered to mice at a dose of 150 μg 4 hours before infection. As a control for possible nonspecific effects of rAb-based drugs, the commercial drug Synagis (100 mg/mL; AstraZeneca) was used as a control for possible nonspecific effects of rAT-based drugs. Synagis is a humanized monoclonal IgG1 antibody with specific activity against respiratory syncytial virus but not influenza virus [15]. The mice in the placebo group received an equivalent volume of phosphate-saline buffer. Four hours after administration of the drugs, the mice were infected with influenza A/California/07/09 (H1N1)pdm09 virus at a dose of 5 MLD₅₀. Twenty-four hours after infection, the drugs were re-administered to the mice. On the fourth day of the study, blood samples were collected from the mice to assess the level of rAb remaining since administration, and organs were collected to determine the viral load in the tissues of the lower and upper respiratory tract (lungs and nasal passages), as well as to assess the level of rAb in lung homogenates.

For the preventive study regimen, rAb drugs (CR9114_G1, CR9114_A1, and CR9114_A2) were administered to the mice at a dose of 100 or 20 μg 24 hours prior to infection with influenza A/California/07/09 (H1N1)pdm09 virus at a dose of 10 MLD₅₀.

For the therapeutic study regimen, rAb drugs (CR9114_G1, CR9114_A1, and CR9114_A2) were administered to mice at a dose of 100 μg 24 hours after infection with influenza A/California/07/09 (H1N1)pdm09 virus at a dose of 10 MLD₅₀.

Blood samples for serum collection were taken from the mandibular vein. The serum was stored at –20°C until use. Tissue and organ samples (lungs and nasal passages) were collected from the mice after euthanasia by cervical dislocation. Ten percent tissue homogenates were prepared in phosphate-saline buffer using a TissueLyser II homogenizer (Qiagen), clarified by centrifugation, and stored at –80°C.

Experimental infection was performed using a dispenser with a suspension of influenza A/California/07/09 (H1N1)pdm09 (collection of the A.A. Smorodintsev Research Institute of Influenza), capable of causing lethal infection in mice, at a dose of 5 MLD₅₀ (50% mouse lethal dose), under ether anesthesia, in a volume of 50 μL. Within 14 days after infection, the mice were evaluated for body weight dynamics and mortality was recorded.

Assessment of viral load in organ homogenates

The viral load in homogenates of laboratory animal organs was assessed by titration on MDCK cell culture in a 96-well plate format on AlphaMEM medium (Biolot) medium with the addition of 1% antibiotic-antimycotic (Gibco) and TPCK trypsin at a final concentration of 1 μg/mL. The infectious titer of the virus was calculated using the Reed and Munchau method² and expressed as log10 TCID₅₀/mL (50% tissue infectious dose).

Assessment of rAb levels in blood serum and tissue homogenates

The level of rAbs in blood serum and tissue homogenates was assessed using an enzyme-linked immunosorbent assay with 96-well Microlon High Binding immunological plates (Greiner Bio-One). Inactivated influenza A/California/07/09 (H1N1)pdm09 virus at a concentration of 1 μg/mL was used as the antigen. The analysis was performed for two dilutions of sera and lung homogenates (1:50, 1:100, and 1:200, 1:500, respectively). Goat Anti-Human IgG (H+L) conjugate labeled with horseradish peroxidase (Bio-Rad) was used to detect IgG. Goat Anti-Mouse IgA (α-chain) conjugate labeled with horseradish peroxidase (Sigma-Aldrich) was used to detect IgA. The conjugates were used at a dilution of 1:1000. Tetramethylbenzidine (Hema) was used as the substrate, and the color reaction was stopped with monobasic sulfuric acid (Vecton). The optical density (OD) was measured using a Multiskan SkyHigh spectrophotometer (Thermo Fisher Scientific). The OD corresponding to the absorption of tetramethylbenzidine was calculated as the difference between OD_{450 nm} and OD_{620 nm}.

Microneutralization reaction, determination of the half-maximal inhibitory concentration

The virus-neutralizing activity of rAbs was evaluated in a monolayer culture of MDCK cells using the method described earlier [16]. A series of threefold dilutions of rAb drugs were mixed with an equivalent volume of growth medium containing 100 TCID₅₀ of influenza virus, and after 1 hour of incubation at room temperature, the resulting dilutions were transferred to plates with a daily monolayer of MDCK cells. The plates were incubated for 3 days at 37°C until the development of a characteristic cytopathic effect, which was confirmed by the hemagglutination reaction method. The neutralizing titer was considered to be the highest Ab dilution at which complete inhibition of the cytopathic effect of the virus was observed. The obtained values were converted into the percentage of inhibition of the cytopathic effect of the virus at a certain Ab concentration. The half-maximal inhibitory concentration (IC₅₀) was calculated based on the results of constructing a four-parameter dose-response curve using GraphPad Prism v. 9.5.1 software based on three independent replicates.

Primary data and statistical processing

Statistical analysis of primary data was performed using Microsoft Office Excel 2010 and GraphPad Prism v. 9.5.1 software packages. The following statistical indicators were used to present the data: arithmetic mean, standard deviation, standard error of the mean. To determine the significance of differences between group means, one-way ANOVA was used for group comparison or its nonparametric analogue, the Kruskal–Wallis test, then, if the null hypothesis was rejected, Tukey's test or Dunn's nonparametric test for posterior pairwise comparisons with the placebo group. The nonparametric log-rank test was used to compare survival curves. The a priori significance level was set at α = 0.05. Differences were considered significant at a significance level of p < α.

Results

Obtaining CR9114 rAbs of various isotypes

Based on variable sequences of CR9114 rAbs [12], genetic constructs were created for the accumulation of human rAbs of IgG and IgA isotypes in eukaryotic cell lines.

Experimental samples of IgG1, IgA1 and IgA2 rAb isotypes were obtained by transient expression. Analysis of the samples by exclusion chromatography showed that the retention time of the main substance in the IgA antibody samples was 7.5–7.8 min, and in the IgG1 samples, it was 8.3 min (Fig. 1, a). According to the column calibration curve, these values correspond to the monomeric forms of IgA (~160 kDa) and IgG (~150 kDa). According to the results obtained, the rAb drugs contained almost no undesirable aggregates and met the required purity criteria.

 

Fig. 1. Analysis of CR9114 rAb drugs of IgG1, IgA1, and IgA2 isotypes.

a — gel filtration chromatograms of purified CR9114 of different isotypes (the x-axis represents time in minutes; the images show elution peaks); b — neutralizing activity of CR9114 rAbs against influenza virus A/California/07/09 (H1N1)pdm09 in vitro. The neutralizing activity was determined in 3 independent replicates; for each point on the graph, the average normalized percentage of inhibition ± standard deviation is shown.

 

To confirm the antiviral activity of the engineered CR9114 rAbs in the IgG1 format (CR9114_G1) and in the IgA1 format (CR9114_A1), their neutralizing activity against the influenza A/California/07/09 (H1N1)pdm09. The IC₅₀ for CR9114_G1 Abs was 0.06152 μg/mL, and for CR9114_A1 Abs it was 0.3133 μg/mL (Fig. 2, a). The specific activity indices of rAbs of different isotypes were comparable, and at the next stage, the obtained experimental Ab drugs were used to evaluate the protective efficacy against influenza infection in mice.

 

Fig. 2. Specific activity of CR9114 rAbs of IgG1 and IgA1 isotypes.

a — viral load in the respiratory tract of mice (lungs and nasal passages) infected with influenza A/California/07/09 (H1N1)pdm09 virus, which received rAbs according to a therapeutic and prophylactic regimen. Individual symbols show the values of individual virus titers for each mouse, the column shows the average log10 value for the group ± standard deviation (number of mice in the group n = 5).

b — quantitative determination of rAbs in the blood serum and lungs of infected mice. Individual symbols show individual values of rAbs for each mouse, the column and the numerical value above it show the average value for the group ± standard deviation.

 

Effect of the method of administration on the specific activity of CR9114 rAbs against influenza A virus

In the first stage, to study the specific activity of rAbs, the protective efficacy of experimental drugs against influenza infection in mice was evaluated when administered according to a therapeutic and prophylactic regimen. To do this, 4 hours before infection with the influenza A/California/07/09 (H1N1)pdm09 virus, the mice were administered CR9114 rAb drugs in the IgG1 (CR9114_G1) or IgA1 (CR9114_A1) format at a dose of 150 μg. Twenty-four hours after infection, the drugs were administered to the mice again. Two methods of rAb administration were tested in the study: intranasal and parenteral (intraperitoneal).

The data obtained showed that intranasal administration of rAbs of both isotypes leads to a significant reduction in viral load in the tissues of the upper and lower respiratory tract (Fig. 2, a). Compared to the control group, the infectious virus titer decreased by more than 5 log10, up to complete elimination of the pathogen (p < 0.0001). At the same time, intraperitoneal administration of rAbs, regardless of isotype, did not lead to a significant decrease in the infectious virus titer in the respiratory tract of infected mice (p > 0.5). No non-specific protective effect of Synagis against influenza infection was demonstrated.

Assessment of the level of rAbs in blood serum showed that rAbs of the IgG1 isotype penetrates into the systemic bloodstream regardless of the method of administration and remains at a high level (3.4– 48.0 μg/mL) for up to 4 days. With intraperitoneal administration, compared with intranasal administration, the level of residual rAbs in the blood serum of mice was significantly higher (p < 0.05), while in lung tissue, the level of rAbs was significantly higher with intranasal administration (Fig. 2, b).

It was not possible to detect IgA1 isotype rAbs in the blood serum of laboratory animals on the 4^th day after administration using any of the methods of administration of experimental drugs (data not shown). In the tissues of the respiratory tract (in the lungs), IgA1 antibodies were detected only with intranasal administration, their concentration was 2.7 μg/mL.

Intranasal administration of CR9114 rAbs protects mice from lethal influenza infection

The efficacy of intranasal administration of rAbs for passive immunotherapy of influenza infection in mice, confirmed in the previous section, determined the choice of this route of administration for subsequent experiments to evaluate protective activity.

In the next stage, the protective efficacy of rAb drugs of IgG1 (CR9114_G1), IgA (IgA1 (CR9114_A1) and IgA2 (CR9114_A2) isotypes was evaluated in a single-dose regimen for prophylactic or therapeutic use with dose escalation. The experimental rAb drugs were administered to the mice 24 hours before infection or 24 hours after infection. Lethal infection was modeled using influenza virus A/California/07/09 (H1N1)pdm09 at a dose of 10 MLD₅₀. For 14 days after infection, body weight dynamics were assessed in mice and mortality was recorded.

It has been shown that a single prophylactic administration of CR9114_G1 and CR9114_A1 rAbs at a dose of 100 μg provides complete protection against lethal infection, with 100% survival in these groups of mice and body weight dynamics not differing from those of the intact group. Protection indicators in the group of mice receiving CR9114_A2 rAbs at a dose of 100 μg were lower: survival was 80%, and body weight was significantly lower than in intact mice, starting from the 7th day of observation (p < 0.05). When using a low dose of rAbs (20 μg), a decrease in the protective effect was shown for all 3 drugs tested. Thus, survival in the group of mice receiving CR9114_G1 rAbs was 80%, in the group receiving CR9114_A1 — 70%, and in the group receiving CR9114_A2 — 60% (Fig. 3, a).

 

Fig. 3. Kaplan–Meier survival curves and body weight dynamics of mice receiving a single dose of CR9114 prophylactically (a) or therapeutically (b).

Experimental infection with influenza virus A/California/07/09 (H1N1)pdm09 (10 MLD₅₀), 10 mice per group. Survival curves and body weight dynamics relative to day 0 are shown for groups of mice receiving CR9114 rAbs of the IgG isotype (CR9114_G1), IgA1 isotype (CR9114_A1), and IgA2 isotype (CR9114_A2) 1 day before infection at doses of 100 and 20 μg or 1 day after infection at a dose of 100 μg.

 

A single therapeutic dose of all three rAb drugs at a dose of 100 μg did not provide complete protection against lethal infection. All mice in these groups showed a decrease in body weight compared to uninfected mice. Survival in groups of mice that received rAbs only after infection was 50% for CR9114_G1 and CR9114_A2 and 20% for CR9114_A1 (Fig. 3, b).

Thus, rAbs of the IgG1 and IgA1 isotypes demonstrated comparable protective efficacy. At the same time, the efficacy of rAbs of the IgA2 isotype was significantly lower than that of IgA1.

Discussion

As previously demonstrated, CR9114 binds to the stem domain of hemagglutinin and exhibits cross-protection against infection caused by influenza A and B viruses [12, 13]. Thus, the CR9114 antibody has high therapeutic potential for protection not only against seasonal influenza viruses, but also against emerging zoonotic strains [17].

Since the upper respiratory tract is the entry point for the influenza virus, the immune system of the mucous membranes plays a central role in antiviral protection. The distribution of immunoglobulins in the mucous membrane and in the blood serum differs. On the mucous membrane, the predominant class of Abs is IgA (~74% of all mucosal immunoglobulins), followed by IgG (~25%) and IgM (~2%) [18]. In blood serum, the predominant isotype is IgG (75–80% of serum immunoglobulins), followed by IgA (15%) and IgM (10%) [8]. The total production of IgA (40–60 mg/kg per day) is higher than that of all other Ab isotypes combined [8]. Thus, a comparative analysis of the efficacy of CR9114 Abs of different isotypes as a means of passive immunotherapy is logical and justified.

It has previously been established that, depending on the method of administration, CR9114 Abs can provide an Fc-dependent or Fc-independent mechanism of protection [19]. In particular, A.L. Beukenhorst et al. compared the protective efficacy of Ab CR9114 in the full-length IgG1 format, IgG1 with Fc-deficient function, and in the F(abʹ)₂ format [19].

In this study, there was particular focus on studying the protective activity of Ab CR9114 depending on various effector functions mediated by Fc regions of different isotypes. Three human rAbs were developed and obtained based on the variable regions of Ab CR9114 [12]: one of the IgG1 isotype and two of the IgA isotype (IgA1 and IgA2, in monomeric form). The effector role of IgG is carried out by binding to FcγR (I, II, III) receptors, and that of IgA by binding to FcαRI/CD89 [20]. The structural difference between the IgA1 and IgA2 subclasses is the length of the flexible hinge region that separates the antigen-binding and effector parts of the antibody. IgA1 has a hinge region consisting of 22 amino acids, which contains up to 5 O-linked glycans on serine and threonine residues. In contrast, IgA2 has a hinge of 9 amino acids and lacks O-linked glycans [21].

At the first stage of the study, the high degree of purity and integrity of the target Abs in all drugs was confirmed, which allowed us to proceed to experiments on laboratory animals. The IC₅₀ was determined for the obtained Ab drugs on a cellular model of influenza infection. It is worth noting that the IC₅₀ value for CR9114 rAbs of the IgG1 isotype was approximately 5 times lower than that for the IgA isotype. One possible explanation for this may be the difference in the segmental flexibility of the isotypes, which affects the accessibility of antigen-binding sites.

The protective efficacy of rAb drugs was evaluated in a mouse model of influenza infection following parenteral and intranasal administration. It was shown that intranasal administration of rAbs of both isotypes reduces the viral load in the respiratory tract tissues of infected mice and completely suppresses pathogen replication. At the same time, parenteral administration of IgG1 (but not IgA1) also reduced the virus titer in the nasal passages (but not in the lungs) of mice.

In preclinical studies, Abs with species-specific constant regions is sometimes used to prevent immunogenicity, or experiments are conducted on humanized mice with a human Fc receptor. Human IgG1 has a high functional similarity to mouse IgG2α in terms of pharmacokinetics and Fc-mediated effector function [22]. In contrast, according to current experimental data, mice do not have homologues of human IgA1 and FcαRI [4].

Depending on the isotype (IgG1 or IgA1), our study showed different distributions of rAbs in the respiratory tract and systemic blood flow of laboratory mice. The differences in the distribution of rAbs in serum and in their antiviral activity are likely related to the degree of homology of human Abs (and their receptors) with mouse analogues.

In mouse models, passive immunization against influenza is usually performed by peripheral systemic injection [9, 22]. Our study demonstrates the advantage of local intranasal administration over systemic parenteral administration, probably due to the creation of a therapeutic concentration of Abs directly on the respiratory tract mucosa — the primary site of infection. Less invasiveness and targeted delivery confirm the promise of developing intranasal drugs for the prevention and treatment of influenza.

Thus, only intranasal administration was used to evaluate the dose-dependent antiviral activity of rAbs. Our results generally confirm the conclusions of a recently published study [19] that intranasal administration of CR9114 Abs is more effective if it contains an Fc domain, although the relative contribution of the Fc fragment is lower with intranasal administration than with parenteral administration.

This study shows that treatment with CR9114 rAbs in the IgG1, IgA1 or IgA2 format did not result in effective protection of mice from lethal infection. Prophylactic administration of IgG1 or IgA1 at a dose of 100 μg ensured 100% survival of mice with lethal infection with influenza A/California/07/09 (H1N1)pdm09 virus.

Conclusion

The study demonstrates that intranasal prophylactic administration of human neutralizing CR9114 antibodies of the IgG1 or IgA1 isotypes provides 100% antiviral protection against lethal influenza virus infection in mice. At the same time, Fc fragments of immunoglobulins of different isotypes, which are responsible for effector functions, appear to influence the degree of antiviral protection.

 

¹ World Health Organization. Global Influenza Programme.

URL: https://www.who.int/teams/global-influenza-programme

² Reed L.J., Muench H. A simple method of estimating fifty percent endpoints. Am. J. Trop. Med. Hygiene. 1938. 27(20):493–497.

About the authors

Ekaterina A. Romanovskaya-Romanko

Smorodintsev Research Institute of Influenza

Email: romromka@yandex.ru
ORCID iD: 0000-0001-7560-398X

Cand. Sci. (Biol.), leading researcher, Laboratory of Vector Vaccines

Russian Federation, St. Petersburg

Marina A. Plotnikova

Smorodintsev Research Institute of Influenza

Email: biomalinka@mail.ru
ORCID iD: 0000-0001-8196-3156

Cand. Sci. (Biol.), seniour researcher, Laboratory of Vector Vaccines

Russian Federation, St. Petersburg

Veronika A. Oleynik

Smorodintsev Research Institute of Influenza

Email: working.lyutik@gmail.com
ORCID iD: 0009-0005-3081-0463

laboratory researcher, Laboratory of immunobiological technologies

Russian Federation, St. Petersburg

Aram A. Shaldzhyan

Smorodintsev Research Institute of Influenza

Email: shaldzhyan@yandex.ru
ORCID iD: 0000-0002-8646-6252

laboratory researcher, Laboratory of genetic engineering and recombinant protein expression

Russian Federation, St. Petersburg

Varvara S. Monakhova

Smorodintsev Research Institute of Influenza

Email: varvara.bio@gmail.com
ORCID iD: 0009-0002-9519-5316

Cand. Sci. (Biol.), researcher, Laboratory of immunobiological technologies

Russian Federation, St. Petersburg

Dmitry S. Balabashin

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: dbalabashin@mail.ru
ORCID iD: 0000-0002-7627-0600

Cand. Sci. (Biol.), junior researcher, Laboratory of protein engineering

Russian Federation, Moscow

Viktoriya A. Toporova

Smorodintsev Research Institute of Influenza; Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: toporova.viktorija@gmail.com
ORCID iD: 0000-0002-7450-7096

laboratory researcher, Laboratory of immunobiological technologies, Smorodintsev Research Institute of Influenza; researcher, Laboratory of protein engineering, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry

Russian Federation, St. Petersburg; Moscow

Teymur K. Aliev

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Lomonosov Moscow State University

Email: ta12345@list.ru
ORCID iD: 0000-0002-1753-9614

Cand. Sci. (Chem.), Deputy Head, Competence Center of the National Technological Initiative, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry; researcher, Department of chemical enzymology, Lomonosov Moscow State University

Russian Federation, Moscow; Moscow

Sergey A. Klotchenko

Smorodintsev Research Institute of Influenza

Author for correspondence.
Email: fosfatik@mail.ru
ORCID iD: 0000-0003-0289-6560

Cand. Sci. (Biol.), Head, Laboratory of immunobiological technologies

Russian Federation, St. Petersburg

References

Supplementary files