Safety and immunogenicity of live intranasal pertussis vaccine GamLVP in the experimental infant hamadryas baboon model

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INTRODUCTION

Pertussis, also known as whooping cough, is a highly contagious infection transmitted via airborne droplets and caused by the bacteria Bordetella pertussis. Despite successful preventive vaccination programs, pertussis remains a serious public health problem in all countries worldwide. The whole-cell pertussis vaccines (wPVs) developed and introduced in public health practices in the second half of the 20^th century reduced the incidence to a few cases per 100,000 population. The vaccination was so epidemiologically effective that pertussis was classified as a vaccine-preventable disease, and it was commonly believed that the disease was effectively controlled through vaccination programs. In the meantime, although being successful in bringing the incidence rates down, the years-long mass immunization with wPVs demonstrated adverse side effects and postvaccinal complications. In the 1990s, a number of countries discontinued vaccination against pertussis, thus triggering a sharp resurgence of the disease, including severe cases with a fatal outcome. In the economically developed countries, wPVs have been replaced with less reactogenic acellular pertussis vaccines (aPVs) [1].

Nevertheless, the incidence of pertussis is steadily rising, and has increased almost ten-fold over the last 20 years [1]. Around 1 million deaths associated with pertussis are reported each year, ranking this disease sixth among infections causing infant mortality. The reported pertussis incidence is also increasing in countries with high vaccination coverage of the target population, including economically developed countries [1]. In Russia, the increasing pertussis incidence is characterized by local outbreaks and clusters of different intensity in school settings. Based on the data from the Federal Service for Surveillance on Consumer Rights Protection and Wellbeing (Rospotrebnadzor), in 2019, the number of pertussis cases increased almost by 40%¹ or 2.7 times compared to 2018.

The immunity induced by modern wPVs and aPVs turned out to be of a shorter term than expected, thus, apparently, failing to prevent the increasing number of teenagers and adults susceptible to the pathogen. The increasing atypical cases make it difficult to diagnose pertussis. Adults are a reservoir of B. pertussis bacteria for infants and older children, while children can transmit the disease to adults. Uncontrolled sources of pertussis infection, especially in families with new-born babies and in childcare settings, make early immunization and revaccination of all age groups of population highly important. However, currently, wPVs or aPVs are administered for vaccination against pertussis not earlier than at 2–3 months of age. The immunization consists of three doses of the vaccine administered intramuscularly at 1.5-month intervals, followed by revaccination at the age of 18 months. Currently approved vaccines are not recommended for newborns and infants younger than two months, though at this age, babies are most susceptible to pertussis infection and are more likely to have severe disease.

Currently, the percentage of circulating B. pertussis bacteria with mutations in genes responsible for production of protective antigens encoding proteins included in aPVs is steadily increasing. Teenagers and adults are revaccinated only with a DTaP vaccine (containing an acellular pertussis component). In the meantime, the recent studies have demonstrated that booster vaccination is more effective when infants of the first year of life are administered priming immunization with DTP containing wPVs rather than aPVs. Nevertheless, with both vaccines, antibodies lasted for only 1–3 years, and revaccination did not provide antibacterial protection. As presently believed, the pertussis pathogen can be eradicated when there is not only a humoral immune response, but also a cellular response mediated by T helper Th1 and Th17 cells [1]. The experiments on primates showed that protection against virulent bacteria B. pertussis was established after recovery from the pertussis infection; it was less pronounced after immunization with wPVs; no antibacterial immunity was found after the monkeys were vaccinated with aPVs [2][3].

At present, the GamLPV (live pertussis) vaccine developed at the Gamaleya National Center of Epidemiology and Microbiology is an alternative to intranasal wPVs and aPVs based on recombinant attenuated Bordetella pertussis bacteria [4]. The similar live recombinant pertussis vaccine was also developed in France [5]. Currently, both live recombinant pertussis vaccines go through different stages of clinical trials involving adult volunteers. After the clinical trials are completed and approval documents are received, the GamLPV vaccine is expected to be used for revaccination of adolescents and adults. In the meantime, the increasing pertussis incidence necessitates more intensive research so that the safe, easy-to-use intranasal vaccine, inducing antibacterial immunity would be available for vaccination of the most vulnerable category of the population — newborns and infants of the early months of life. Preclinical studies in experimental models of infant laboratory animals are of high importance, considering the requirements for therapeutic agents and vaccines administered for treatment and prevention of diseases of infants and the approval of clinical trials of the GamLPV vaccine for infants.

We have demonstrated the safety of the GamLPV vaccine in the experiments in suckling mice and rats [6]. The second experimental model, most similar to a human, are primates [7][8]. Since we have developed an experimental model of pertussis infection using rhesus monkeys (Macaca mulatta) and hamadryas baboons (Papio hamadryas) and have proved the safety, immunogenicity, and antibacterial activity of the GamLPV vaccine administered to adult species [8–10], we believe that preclinical studies of the GamLPV vaccine can and should be performed in infant monkeys.

The aim of the study was to assess safety and immunogenicity of the GamLPV vaccine administered intranasally for single dose, two-dose, and three-dose immunization of P. hamadryas infants.

MATERIALS AND METHODS

The health of the experimental monkeys was monitored through veterinary checkups. The examination of mothers and infants included complete blood count, sera tests for antibodies to bacteria B. pertussis, physical examination of the oropharyngeal cavity and weight measuring; oral and nasopharyngeal swab specimen collection for further tests and assessment; the infants’ weight check, their physical examination by the veterinarian, general health status evaluation, mouth and throat assessment, evaluation of behavior and reactions to external factors.

The experimental work in animals was performed in compliance with GOST 33218-2014 (Russia) and the rules of work with laboratory animals [12].

The immunization, blood drawing, and nasopharyngeal sampling in the infants were performed without anesthesia. The mothers were put into a medication-induced sleep by injecting intramuscularly 0.03–0.04 ml of zoletil (Virbac) at a concentration of 100 mg/ml (including premedication with xylazine hydrochloride, 20 mg/ml).

Immediately prior to the intranasal application, 0.70 ml of sterile 0.85% NaCl solution was added to the vial with the GamLPV vaccine. After lyophilizate was dissolved within 1 minute, the vial was gently swirled; the suspension was delivered intranasally, 0.3 ml into each nostril, with a 2ml nasal spray syringe (Shenzhen Bona Pharma Technology).

The infants were restrained manually, placed on their backs and kept in this position until the liquid reached the nasal cavities. The mothers were inoculated with virulent bacteria using anesthesia and pressure cages [8].

In the infants, blood for the blood test was collected from the femoral vein. Nasopharyngeal and oropharyngeal aspirate specimens were collected with nasopharyngeal probes and oropharyngeal tampons. Blood and aspirate specimens from the mothers were collected following the previously described procedure, using anesthesia and pressure cages [8].

The blood samples were tested on the automated Micros ES 60 hematology analyzer (Horibo ABX). The glucose content was measured with the Accu-Chek blood glucose meter (Roche).

The serum samples were tested for specific immunoglobulins using the enzyme immunoassay (EIA) and the reagent kit for detection of IgG antibodies to B. pertussis (RIDASCREEN Bordetella IgG). For this purpose, the Sero G HD rabbit anti-human IgG antibody conjugate was replaced with the rabbit anti-monkey IgG antibody conjugate (Abcam). The adapted testing system, most likely, cannot be used for a quantitative analysis of IgG antibodies, though it can be used for assessment of changes in the antibody levels. The direct agglutination test (DAT) was performed using the Pertussis Diagnosticum Liquid kit (Ecolab).

For the molecular and biological analysis, we used B. pertussis DNA isolated from washing samples of posterior pharyngeal and nasopharyngeal swabs. Their pellets were centrifuged and treated with guanidine thiocyanate solution, followed by DNA adsorption onto magnetic sorbent (Promega) [13][14]. To measure the quantity of B. pertussis DNA genome equivalents in the aspirate samples, we used our own designed and validated assay based on the real-time polymerase chain reaction (real-time PCR) [13][14].

Study design

The biomaterial was collected following the schedule below:

• before immunization (background values) — control time point (c.t.p.) 1.0;
• 1 hour after immunization (swab only);
• on the 3^rd day after immunization;
• 1 week after immunization — c.t.p. 1.1;
• 2 weeks after immunization — c.t.p. 1.2;
• 1 month after immunization — c.t.p. 1.3;
• 5 months after immunization — c.t.p. 1.4;
• 2 months after immunization — c.t.p. 1.5;
• before first repeat immunization — c.t.p. 2.0;
• 1 hour after first repeat immunization (swab);
• 3 days after first repeat immunization;
• 1 week after first repeat immunization — c.t.p. 2.1;
• 2 weeks after first repeat immunization — c.t.p. 2.2;
• 1 month after first repeat immunization — c.t.p. 2.3;
• 5 months after first repeat immunization — c.t.p. 2.4;
• 3–4 months after first repeat immunization — c.t.p. 2.5;
• before second repeat immunization — c.t.p. 3.0;
• 1 hour after second repeat immunization (swab);
• 1 week after second repeat immunization — c.t.p. 3.1;
• 2 weeks after second repeat immunization — c.t.p. 3.2;
• 1 month after second repeat immunization — c.t.p. 3.3;
• 5 months after second repeat immunization — c.t.p. 3.4;
• 3 months after second repeat immunization — c.t.p. 3.5.

Biomaterial was collected from the mothers, following the same schedule, except the time point “one hour after the application of the vaccine”. 12 months after the second re-immunization, the animals were experimentally infected with virulent isogenic bacteria B. pertussis 475. The material for the further analysis was collected, following the same schedule that was used after the immunization.

RESULTS

Development of the experimental model of pertussis infection in infant monkeys

The results of experiments involving infant monkeys do not have sufficient coverage in scientific literature. Therefore, during the first stage, the priority should be given to assessment of the possibility and selection of methods of working with infant monkeys (1–2-month-olds). Addressing this problem and being aware of social behavioral patterns typical of animals with infants as well as keeping the existing negative experience in mind regarding the separation of infants from mothers, we decided to separate mothers together with their infants from the rest of the family. Considering the scope of tests, especially blood tests required for assessment of vaccine safety and analysis of pertussis infection development, we opted for P. hamadryas baboons that were bigger than other monkeys and more available for experiments as a model.

By the beginning of the experiment, all the monkeys were clinically and somatically healthy; all of them were seronegative to B. pertussis antigens. The females were monitored during their pregnancy and after the childbirth. The infants’ dates of birth were different; therefore, the immunization and examination of the monkeys were performed at different time.

The experiment included 3 P. hamadryas females, each with its infant. Numbers were assigned to the onemonth- old monkeys as it was impossible to take the infants away from their mothers earlier. The documents included the registration number of the mother and father, the location of the family and the number assigned to the infant. When the infants turned one month old, the mothers, together with their infants, were placed in a pressure cage. The mother was put into a medication-induced sleep; the infants were brought to the manipulation room for performing all the required procedures. All the manipulations with the infants were performed on the disposable underpad that had been prepared earlier. The relocation of the infants and all the manipulations were supervised by the veterinarian. The manipulations with the sleeping mothers (swab specimen and blood collection, physical examination) were performed in a separate room. The infants were returned to their mothers before the latter woke up. The mothers and infants participating in the experiment stayed in a common area (an enclosure), separately from the other members of the family, during the entire period of monitoring.

Safety of intranasal administration of the GamLPV vaccine to infant monkeys

The infants had their first immunization at the age of 1 month; when they became older, they had 2 re-immunizations at 3.0–3.5-month intervals. 12 months after the second re-immunization, the infants were experimentally infected with isogenic virulent pertussis bacteria.

The assessment criteria applicable both to safety of the GamLPV vaccine and to inoculation with virulent bacteria B. pertussis 475 were the absence of any deviations from the blood test normal values, the good health status of the monkeys — absence of local reactions, weight loss, and behavioral changes. The infants’ and mothers’ behavior was monitored during the entire experiment.

During the monitoring period, no adverse changes in the health and development of the infants were detected; the infants did not have any cough, changes in the temperature, and interruptions in the weight gain, any local reactions to each of three intranasal immunizations with the GamLPV vaccine and inoculations with experimental infection. The results of the complete blood count during the first 2 weeks of monitoring — the timeframe identified as the most important based on the results of the previous studies in adult animals — are presented in the Table. No significant changes in ESR, general somatic status, and blood test values were detected in the mothers (only the results after the experimental infection are presented), who were in constant contact with their immunized infants.

 

Immunogenicity of the GamLPV vaccine after intranasal administration to infant monkeys and their experimental infection with B. pertussis

In all the previously mentioned studies, the monkeys’ sera were tested for the presence of specific immunoglobulins using reagent kits (RIDASCREEN Bordetella IgG) designed for human sera EIA [6][8–10]. To adapt the testing system to immunoglobulins, we replaced the rabbit anti-human IgG antibody conjugate in the kit with the rabbit anti-monkey IgG antibody conjugate. The required dilutions of the conjugate and the tested sera were selected through control EIA tests using plates with specific antigens — pertussis toxin (PT) and filamentous hemagglutinin (FHA) — and the rabbit anti-monkey IgG antibody conjugate. The previously selected and described sera samples from the control monkeys and the monkeys experimentally infected with virulent bacteria B. pertussis were used as the reference standard. The working dilution of the conjugate was 1 : 40,000 and that of the serum was 1 : 20 to be further used for the EIA test with the modified kit.

Fig. 1 presents the measurement results for the optical density (OD) in the EIA plate wells containing diluted sera from immunized monkeys, capturing changes after the first intranasal application of the GamLPV vaccine and the two subsequent re-immunizations. The OD values were used to estimate levels of IgG antibodies in the tested sample. After the first immunization, only one infant demonstrated some background IgG antibodies with the levels being highest on the 28^th–43^rd day. After the 2 subsequent immunizations, a significant increase in specific IgG antibodies was recorded 1 week later and reached the peak 14–30 days after the first re-immunization and 7–14 days after the second re-immunization.

Using DAT, we measured titers of anti-pertussis antibodies resulting after the intranasal immunization by the GamLPV vaccine. To perform the test, we used the suspension of bacteria B. pertussis and different dilutions of sera from the immunized monkeys. After the first immunization, serum-induced agglutination was hardly detectable, while after two re-immunizations, it increased significantly and reached the highest level in 14–30 days (Fig. 2).

12 months after the second re-immunization, the animals were infected experimentally with virulent isogenic bacteria B. pertussis 475. After the experimental infection, the levels of specific IgGs (PT + FHA) and the agglutination titer in the mothers and infants reached the peak on the 14^th day and remained high till the end of the monitoring (Fig. 3, 4).

DISCUSSION

Our previous preclinical studies showed that multiple- dose intranasal immunization of adult monkeys with the GamLPV vaccine did not cause any deviations from the normal status of somatic health and any changes in the behavior. The subsequent experimental infection of immunized monkeys with virulent bacteria B. pertussis did not cause any clinical symptoms or positive laboratory test results indicative of pertussis infection [8–10]. Our findings confirmed safety, high tolerability, and immunogenicity of the GamLPV vaccine. The results of 2 stages of the GamLPV vaccine clinical trial involving healthy volunteers supported our conclusion. The experimental infection of native (control, non-immunized) monkeys was accompanied by a number of clinical symptoms and positive laboratory test results typical of pertussis infection and immune response development observed in people [8–10]. The monitoring of the P. hamadryas infants and mothers also did not reveal any deviations from the norm or any changes in the measured parameters, including increased lymphocytes counts and decreased glucose levels typical of pertussis infection in monkeys and humans. As the amount of collectible blood was limited to 1 ml, we were able only to measure the complete blood count and the glucose levels with a glucose meter. The presented results confirm the safety and high tolerability of the GamLPV vaccine — absence of local and systemic reactions, stability of all measured variables: complete blood count and biochemical profile test after the three-dose intranasal administration to P. hamadryas infants.

The specifics of the work involving an experimental model implies limiting requirements for blood amounts that can be collected from one-month-olds as well as the physiological and psychological attachment between mothers and infants. The above implications were taken into consideration and applied to selection of parameters for the analysis, especially after the first immunization. The parameters shown in this article were selected as most significant for assessment of safety and immunogenicity. The next stage of the study will include the assessment of the safety parameters (alanine- and aspartate aminotransferases), which were not included in this study.

The previous stage of the study involving adult monkeys showed that after the immunization with the GamLPV vaccine, the experimental infection with virulent bacteria B. pertussis triggers defense responses of the host body, namely a pronounced increase in specific IgG levels, which reach their peak 7–14 days after the second immunization or repeat infection, as well as rapid removal of bacteria B. pertussis from the mouth, nose, and throat [8]. The similar picture was observed after the re-immunization of the monkeys with the GamLPV vaccine [8–10]. The increasing immune response was also observed after the repeat vaccination of the volunteers during the clinical trials of the Gam- LPV vaccine [15].

To assess the immune response in the infant monkeys, we used the RIDASCREEN Bordetella IgG test system designed for the EIA testing on the human serum [6][8–10]. However, the test systems of another lot, which were purchased from the same manufacturer, did not detect IgG antibodies in seropositive sera from adult P. hamadryas and M. mulatta monkeys. After the consultation with the manufacturer, we assumed that the negative result could be caused by the absence of binding of human-specific conjugate from the new kit with monkey immunoglobulins. Therefore, the human conjugate was replaced with the M. mulatta monkey conjugate. Fig. 1 shows the results of measurement of the levels of specific pertussis IgG antibodies using the modified test system. The data presented in Fig. 1 are not significantly different from the results demonstrating changes in the increasing levels of IgG antibodies after the immunization of adult monkeys and volunteers [6][8–10]. Similar to the first case, after the first immunization, only some of the monkeys demonstrated certain levels of background IgG antibodies, which significantly increased after the re-immunizations.

Another currently popular method of assessment of the immunogenicity of pertussis vaccines is measuring titers of agglutination with suspension of bacteria B. pertussis and dilutions of sera from immunized people and animals. The 1 : 160 ratio is included in the national standard of Russia as the reference value for the assessment of protective potency of wPVs used in full-scale programs of primary vaccination of children². The results presented in Fig. 2 demonstrate that the changes in the agglutination titers do not differ significantly from the changes in the levels of IgG antibodies in the monkeys’ sera. After the first immunization, 2 out of 3 infants did not demonstrate any increase in the titers in DAT and in the levels of serum-specific pertussis antibodies of class IgG. In 1.5 month, infant No. 32317 demonstrated a four-fold increase in the levels of IgG antibodies compared to the initial levels and an increase in DAT to the 1 : 40 value. Three months later, the IgG levels and the agglutination titer decreased to the initial level.

The repeat administration of the vaccine (re-immunization) to all the 3 monkey infants resulted in a significantly faster increase in the levels of IgG antibodies in sera. All the infants demonstrated a significant increase in the IgG levels one week after the immunization, with the levels reaching their peak during the 2^nd–4^th week. Six months after the first immunization, only one monkey had the IgG levels significantly higher than the initial levels. The results of the third vaccine administration (second re-immunization) did not differ significantly from the results observed after the second administration, similar to the results observed after the experimental infection, demonstrating the similar patterns with the adult monkeys. After the first immunization, in the infants, the titers of IgG antibodies reached their peak a little later (in 14-28 days) than in the adult monkeys (7–14 days). After the second re-immunization, IgG antibodies in the infants reached their peak levels already in 7–14 days. Note that the re-immunizations in 3.0–3.5 months were followed by a pronounced booster effect and were performed when the IgG levels were low and close to the initial levels. After the repeat administration of the GamLPV vaccine to the infants, the mothers who had close contact with them did not show any significant increase in the IgG levels and in the agglutination titer in DAT.

Thus, the intranasal immunization of P. hamadryas infants with the GamLPV vaccine resulted in development of an anti-pertussis immune response, demonstrating increasing levels of specific IgGs (PT + FHA) and titers of antibodies in DAT. The booster effect of the repeat immunization supported the usefulness of re-immunization aimed to achieve the highest protective effect. The obtained results were consistent with the results we had described previously for adult monkeys [8–10] and lead to the assumption that although the average levels of IgG antibodies (PT + FHA) in EIA and the levels of common anti-pertussis antibodies in DAT are relatively low, the primary immunization of monkey infants with the GamLPV vaccine can induce a protective immune response during the repeat exposure to pertussis infection. The efficacy of single or double-dose primary immunization with the GamLPV vaccine for development of intense and longer lasting anti-bacterial immunity will be studied further in experimental monkey models and clinical trials involving healthy volunteers.

12 months after the second re-immunization, the infant monkeys were infected experimentally with virulent isogenic bacteria B. pertussis 475. The changes in the levels of IgG antibodies (PT + FHA) in the infants were consistent with the changes in adult monkeys, which we described previously [9]. Special attention should be given to the rapid and significant increase in the levels of IgG antibodies in sera from the mothers, while they were almost undetectable during the entire period after the immunization of the infants. The increase in the antibody titers in the mothers’ sera was detected approximately at the same time when it was detected in the infants, i.e. on the 14^th, 28^th days after the infection (Fig. 4); however, it was less pronounced and the titers quickly dropped down to the initial level. The rapid increase in the levels of specific antibodies in the mothers’ sera after the experimental infection suggests the existence of passive immunization of the mothers who had close contact with the immunized infants. This assumption is supported by the absence of positive laboratory test results and clinical symptoms of pertussis infection in the mothers experimentally infected with virulent bacteria.

The intensity and the time needed for development of protective immunity against pertussis after the vaccination and/or the infection are measured by the humoral and cellular antibacterial response. In the latter case, when it comes to anti-pertussis immunity, the priority in assessment is given to production of interferon and interleukin-17 induced by polymorphonuclear blood cells [1]. We performed the related studies previously in preclinical experiments with adult monkeys and are continuing them within the framework of clinical trials in healthy volunteers [9][15]. However, the most adequate assessment of the defense potential of the Gam- LPV vaccine, in our opinion, can be offered by the comparative data showing the time needed for elimination of bacteria B. pertussis (virulent and/or attenuated) in immunized and native animals.

In future, we are planning to delve into the structure of the population of attenuated bacteria B. pertussis and their persistence in the host body. The rate of elimination of bacteria B. pertussis and changes in their DNA levels in nasopharyngeal and oropharyngeal aspirates will be studied using our real-time PCR assay.

CONCLUSION

The absence of local and systemic reactions in P. hamadryas infants as well as the consistency of the results of clinical blood tests and the pronounced booster effect after the re-immunization and experimental infection can be seen as a reliable proof of existing defensive mechanisms in the infants immunized with the GamLPV vaccine against pertussis infection and as a proof of the safety of this vaccine administered intranasally. The fact that the non-immunized mothers, who were in close contact with their immunized infants, demonstrated a booster antibody response after the experimental infection proves that the mothers successfully developed an antibacterial immune response.

The attenuated bacteria B. pertussis-based Gam- LPV vaccine that we developed offers most favorable prospects for developing herd immunity against pertussis and for the so-called family cocooning.

About the authors

A. A. Djidaryan

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia

Email: fake@neicon.ru
ORCID iD: 0000-0002-8792-5289

Anush A. Djidaryan — senior laboratory assistant with higher
education, Laboratory of virology and immunology

Sukhum

A. Z. Matua

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia

Email: fake@neicon.ru
ORCID iD: 0000-0003-3275-0941

Alisa Z. Matua — Cand. Sci. (Biol.), Deputy Director of science,
Head, Laboratory of immunology and virology

Sukhum

A. Yu. Medkova

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya

Email: fake@neicon.ru
ORCID iD: 0000-0002-1509-0622

Alisa Yu. Medkova — senior researcher of l, Laboratory of bacteria
genetics

Moscow

Россия

E. G. Semin

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya

Email: fake@neicon.ru
ORCID iD: 0000-0001-6696-8362

Evgeniy G. Semin — researcher, Laboratory of bacteria genetics

Moscow

Россия

L. N. Sinyashina

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya

Email: fake@neicon.ru
ORCID iD: 0000-0003-1708-5453

Lyudmila N. Sinyashina — D. Sci. (Med.), leading researcher, Laboratory of bacteria genetics

Moscow

Россия

I. N. Dyakov

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya; I. Mechnikov Research Institute for Vaccines and Sera

Email: fake@neicon.ru
ORCID iD: 0000-0001-5384-9866

lya N. Dyakov — Cand. Sci. (Biol.), leading researcher, Head

Moscow

Россия

I. N. Chernyshova

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya; I. Mechnikov Research Institute for Vaccines and Sera

Email: fake@neicon.ru
ORCID iD: 0000-0001-5053-2433

Irina N. Chernyshova — Cand. Sci. (Med.), senior researcher

Moscow

Россия

D. T. Kubrava

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia

Email: fake@neicon.ru
ORCID iD: 0000-0001-9104-4014

Dzhenni T. Kubrava — junior researcher, Laboratory of virology and
immunology

Sukhum

A. A. Amichba

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia

Email: fake@neicon.ru
ORCID iD: 0000-0002-4986-1392

Astanda A. Amichba — junior researcher, Laboratory of virology and immunology

Sukhum

I. G. Kondzariya

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia

Email: fake@neicon.ru
ORCID iD: 0000-0003-3707-4874

Irina G. Kondzariya — Cand. Sci. (Biol.), senior researcher, Laboratory of virology and immunology

Sukhum

Z. Ya. Mikvabiya

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia

Email: fake@neicon.ru
ORCID iD: 0000-0002-0729-6516

Zurab Yа. Mikvabiya — D. Sci. (Med.), Professor, Director

Sukhum

G. I. Karataev

National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya

Author for correspondence.
Email: karataevgi@rambler.ru
ORCID iD: 0000-0001-8771-6092

Gennadiy I. Karataev — D. Sci. (Biol.), Head, Laboratory of bacteria
genetics

Moscow

Россия

References

Supplementary files