Search

Cellular immunity plays an important role in the pathogenesis and formation of protective immune defense against the SARS‑CoV‑2 virus.The aim of the work was to study the cellular immunity of rhesus monkeys applying flow cytometry after experimental infection with the SARS‑CoV‑2 virus.Materials and methods. Male rhesus monkeys were intranasally inoculated with the SARS‑CoV‑2 virus, Isolate B strain and hCoV-19/Russia/SP48-1226/2020 strain (abbreviated name U-2), at a dose of 5.0 lg PFU. Using flow cytometry, the levels of 21 populations/subpopulations of mononuclear cells in the peripheral blood of animals were determined before experimental infection with the pathogen and on day 14 after infection. SARS‑CoV‑2 coronavirus RNA was assessed using real-time polymerase chain reaction. Determination of the titer of virus-neutralizing antibodies to the SARS‑CoV‑2 virus in the blood sera of animals was conducted through neutralization test evaluating the ability to suppress negative colonies.Results and discussion. Infection with Isolate B strain culture has led to an increase in the relative content of total T-lymphocytes (p˂0.2), cytotoxic T-lymphocytes (p˂0.1), as well as monocytes expressing the early activation marker CD25 (p˂0.2). The decrease in levels has been observed for total B-lymphocytes (p˂0.2) and T-helper cells (p˂0.1). Infection with the U-2 strain culture revealed an increase in the relative content of monocytes expressing the early activation marker CD25 (p˂0.2). Thus, for the first time in the Russian Federation, flow cytometry was used to study the cellular immunity of rhesus monkeys before and after experimental infection with the SARS‑CoV‑2 virus. The obtained information can be used for studying the pathogenesis of SARS‑CoV‑2 infection, course, and outcome of the disease, and developing strategies for vaccination and treatment.

Although successes in antiretroviral therapy (ART) turned AIDS from lethal illness into sluggishly progressing disease, its prevention and treatment remain one of the most socially significant concerns. The increase in the number of patients infected with human immunodeficiency virus (HIV), especially in the USA, South America and Europe, determines the need in creating a vaccine against this disease. Existing vaccination practice has demonstrated efficiency of priming/boosting scheme for the development of immune responses. As anti-vector immunity of priming vector can constrain the response to boosting immunization with the same vaccine, heterologous priming/boosting vector constructs are used. An ideal AIDS vaccine would prevent virus dissemination and control viral replication, but it also must be safe for HIV-infected contingent. The vaccination of HIV-infected individuals is used for enhancing immune-mediated elimination of persistently HIV-infected CD4+ Т-cells during long-term ART in order to purge the latently infected viral reservoirs. The paper considers the results of clinical trials of DNA-anti-HIV/AIDS vaccines and recombinant MVA strain of vaccinia virus, expressing different combination of HIV genes, which demonstrated the safety and tolerability both, in HIV-infected and non-HIV-infected volunteers. All implemented schedules of vaccination induced cell-mediated and humoral immune responses in all volunteers. And though there are no data on acquiring AIDS by HIV-uninfected volunteers from groups at low risk of HIV-infection, there are no grounds to conclude the sufficiency of induced protection for the prevention of possible HIV infection.

Objective. Selection of indicators of lymphocyte populations in rhesus macaques determined by flow cytometry to evaluate variations of cellular constituent of their immune status.Materials and methods. Blood of 11 healthy rhesus macaque males, 2,0–2,5 years old, weighing 2,5–3,0 kg, was used. Monkeys were examined simultaneously in each of 7 months of observation (since May till November). Immunophenotyping was conducted by FC500 cytofluorimeter using Affymetrix eBioscience monoclonal antibodies. The following cellular constituent indicators of immune status were differentiated: total T lymphocytes (phenotype CD2+CD20-); total B lymphocytes (phenotype CD2-CD20+); natural killer cells (phenotype CD2+CD56+); T helper cells (phenotype CD2+CD4+); cytotoxic T lymphocytes (phenotype CD2+CD8+); double-positive T lymphocytes (phenotype CD4+CD8+) and T lymphocytes positive by antigens CD2 and CD20 (phenotype CD2+CD20+).Results and discussion. Statistical analysis of the obtained results revealed the absence of the effect of research time factor on the stated indicators. To assess changes in the cellular constituent of immune status of rhesus macaques, it is possible to use indicators that are less variable: total T and B lymphocytes, T helper cells, cytotoxic T lymphocytes and T lymphocytes with phenotype (CD2+CD20+). The use of CD56 as a marker of natural killer cells of rhesus macaques is impractical due to its low expression and a small size of the population bearing this marker. The research results may form the basis of the normative indicators of the subpopulation cell composition of immune system in rhesus macaques, which will allow the study of infected animals when assessing the quality of medical products in relation to dangerous and particularly dangerous infections.

Chikungunya virus belongs to Alphavirus genus of the Togaviridae family. It is a member of Semliki Forest virus antigenic complex that includes antigenic related Semliki Forest, Chikungunya, O’ Nyong-nyong, Ross River viruses. Chikungunya virus is the causative agent of acute febrile illness with myalgia and arthralgia in humans. Since its discovery in 1952, Chikungunya virus caused sporadic and infrequent outbreaks. Since 2004, global Chikungunya outbreaks have occurred. Now Chikungunya is viewed as a global public health issue in many countries, where Aedes mosquito vectors are widespread. Currently, four genotypes of Chikungunya virus (West African, South African, Asian and Indian Ocean) are distinguished. Appearance of different genotypes is associated with adaptive mutations in peplomers of E1 and E2 glycoproteins. It is shown, that a single mutation in E1 glycoprotein (alanin for valin substitution in 226 position) leads to increasing virus virulence (50-100 times). This mutation is instrumental for epidemic potential increase. For virus variants with this mutation, secondary substitutions enhancing viral virulence are described too. Аedes aegypti mosquitoes are common vector for all genotypes of Chikungunya virus, Аedes albopictus mosquitoes are vector, mainly, for South African and Asian genotypes. They play the leading role in epidemic potential increase over the last decade. The effectiveness of Chikungunya virus transmission by Аedes аegypti mosquitoes is 83.3 %, by Аedes albopictus mosquitoes - 96.7 %. The Аedes albopictus are more widely disseminated than Аedes аegypti (about 40 percent of all land territory). Demonstrated is the possibility of transcontinental spread of Аedes albopictus mosquitoes by aviation and naval transport. This review highlights the most recent advances in our knowledge of the ecology, epidemiology and molecular biology of Chikungunya virus. These data play an important role in the development of preventive, treatment and vaccination strategies of Chikungunya fever.

The review presents the results of preclinical use of vector vaccines against human immunodeficiency virus (HIV) disease and simian immunodeficiency virus (SIV) disease. Application of antiretroviral therapy exclusively is insufficient for elimination of HIV from patient’s body. This dictates the need for an effective vaccine which will reduce the number of new cases of the disease and reduce the risk of virus transmission. Current practice of medicinal product development showed the effectiveness of heterologous prime-boost regimens for the induction of expressed immune response in laboratory animals. Various vector constructs were used as priming vaccines: DNA vaccines, Bacille Calmette-Guerin vaccine, chimpanzee adenovirus, vesicular stomatitis virus, alphavirus repli-clone. Booster vaccine was represented by recombinant MVA strain. In all vector vaccines, different genes of immunodominant antigens of HIV and SIV agents were inserted. On rhesus macaques, murine, rabbit models, it was demonstrated that deployed vaccination schemes were safe and induced immune response. Because membrane HIV protein is highly variable, strongly glycoziled and subjected to structural changes during receptor binding, it cannot be viewed as a target for induction of virus neutralized antibodies. Therefore, we mainly studied the cell immune response that was presented by poly-functional CD8+ T-cells. However, some recent researches are aimed at such modification of envelope HIV immunogene that would provide for virus neutralizing antibody induction. The study of protective efficiency of the induced immunity in rhesus macaques, immunized with recombinant vectors expressing SIV’ s immunodominant antigens, in case of subsequent inoculation with virulent SIV strain has revealed that all monkeys developed illness. Assuming that the constructions with SIV’ s immunodominant antigens under protective efficiency testing on rhesus macaques imitate AIDS in humans, it seems that vaccines, developed up-to-date, will not be effective for collective immunity formation against AIDS. Therefore, the search for novel combinations of expressed immunodominant antigens for the inclusion into the composition of priming and booster vaccines remains a priority area at present time.

Presently, the Arenavirus genus (Arenaviridae family) includes 26 individual species of viruses. It is divided into two main groups – Old World arenaviruses and New World arenaviruses. The New World arenaviruses comprise four clades: А, B, C, D; pathogenic for humans New World arenaviruses are attributed to clade B. Lassa, Lujo, Machupo, Junin, Guanarito and Sabia viruses are the agents of extremely hazardous hemorrhagic fevers (Lassa hemorrhagic fever, Lujo hemorrhagic fever, Bolivian hemorrhagic fever, Argentinean hemorrhagic fever, Venezuelan hemorrhagic fever, Brazilian hemorrhagic fever, accordingly). These arenaviruses pose a potential threat to national public health due to the possibility of their accidental importation into the territory of the Russian Federation. The vaccination of risk group is the most effective and money-saving means of protection against epidemic spread. Objective of this review is to analyze the specific preparations for arenaviral hemorrhagic fever prevention that are currently under development. Production of live vaccines based on attenuated strains of the agents, the DNA vaccines, vector recombinant vaccines and vaccines on the basis of RNA-replicons is viewed as the main trends in the area. Тhe most important results in the development of effective prophylactic means against arenaviral hemorrhagic fevers are discussed in this paper.

Nodular dermatitis, (dermatitis nodularis), is an exotic for the Russian Federation poxviral disease. Some epizootical characteristics of it, problems of laboratory diagnostics, and possible reasons of extension of agent spread areal are discussed in this review. Phylogenetic analysis of P32 gene nucleotide sequences of dermatitis nodularis virus has also been conducted. It is noted that taking into account the economic loss caused by the disease and possibility of its import into non-enzootic regions, it is necessary to use existing effective means of specific prophylacsis extensively. The swift outspread of the disease in the Russian Federation should be considered as the risk factor for biological safety of our country.

The Arenaviridae family consists of a large group of single strand ambisense RNA viruses that are separated phylogenetically, serologically and geographically into Old World and New World viruses. Recent studies indicate that cellular entry of arenaviruses requires a series of cellular protein interaction and molecular mechanisms. The arenaviruses entry into cells is initiated by the interaction of viral glycoprotein with one or more receptors on the surface of host cells. The main host cell factors that are involved in filovirus entry are attachment factors (α-dystroglycan for Old World and human transferrin receptor 1 for New World viruses), endolisosomal host cell factors (cathepsins B and L and Niemann-Pick C1 protein). The review presents the modern knowledge about the role of structural proteins of arenaviruses and some cell factors in pathogenesis of the diseases, caused by arenaviruses.

In 2009, a novel virus, named severe fever with thrombocytopenia syndrome (SFTS) virus, was isolated from a patient in China. The illness caused by this novel virus is characterized by a sudden onset of fever and respiratory or gastrointestinal disorders, followed by progressive thrombocytopenia and leucocytopenia, the case-fatality rate amounting to 6–30 %. Genomic sequencing of the isolated agent indicated that the SFTS virus constituted a new (third) group of Phlebovirus genus, Bunyaviridae family. Presently, different means for specific diagnostics of SFTS (real-time reverse transcription polymerase chain reaction, indirect fluorescent antibodies method, enzyme-linked immune-sorbent assay) are developed. Constructing of diagnostic kits, basic characteristics of methods for determination of causative agent of infection or specific antibodies against it are considered in this review.

Nowadays vaccination of the population living in the endemic regions and widespread implementation of the potent therapies for the emergency prophylaxis and treatment into the clinical practice are regarded as the basic efficient and cost-effective measures for Ebola epidemic spread control. Objective of the review is to analyze current state of the development of aids for the immediate prophylaxis and treatment of Ebola fever. Focus area of the activities is the construction of drugs on the basis of virus-specific anti-bodies (including monoclonal), small interfering RNA, and anti-sense phosphordiamidate morpholine oligomers and interferons. The paper discusses the most significant achievements in this sphere.

Ebola virus, representative of the Ebolavirus genus, Filoviridae family, causes severe hemorrhagic fever in humans, with lethality rates amounting up to 90 %. The members of Ebolavirus genus infect a broad range of mammalian cells. Recent studies indicate that entry of Ebola virus into cells requires a series of cellular protein interactions and molecular mechanisms, some of which are unique to filoviruses, while others are commonly used by all viral glycoproteins. The cellular factors deployed by filoviruses for their entry into permissive cells are defined incompletely. The aim of this review is to analyze peculiarity of the Ebola virus penetration into permissive cells at molecular level. The Ebola virus entry into cells is initiated by the interaction of viral glycoprotein with one or more receptors on the surface of host-cell. The main host-cell factors, involved in filovirus entry, are: attachment factors (cell lectins and human T-cell mucin 1 (TIM-1)), signaling factors (tyrosinkinase receptors and α5β1-integrin), and endolisosomal host-cell factors (cathepsins B and L and Niemann-Pick C1 protein. The study of the complex set of virus entry events provides potential avenues for the development of antiviral therapies against Ebola fever.

Representative of Ebolavirus gender, Filoviridae family, Ebola virus is an etiological agent of particularly dangerous viral fever, the lethality of which comes up to 88 %. According to the leading specialists and experts in the sphere, vaccination is the most effective and cost-efficient method for the protection from epidemic spread. Objective of the review is to analyze current state of the development of next generation vaccines against Ebola fever. It is established that focus areas of the activities are the construction of vaccines on the basis of alpha-virus replicons, virus-like particles, and the development of DNA-vaccines and vector recombinant vaccines. The paper discusses the most significant achievements in the sphere of obtainment of potent therapies for prophylaxis as regards Ebola fever. To date manufactured, using various approaches, have been the next-generation vaccine preparations, for a number of which high protective capacity is demonstrated in the course of experiments on the nonhuman primates. The most advanced and prospective prototype is the vector recombinant vesicular stomatitis virus-based vaccine.

The molecular genetic peculiarities of genomic structure of the Ebolavirus genus members are viewed in the review. The Ebola virus disease outbreaks in West African countries constitute a threat not only for Africa, but for the whole world in view of possible introduction of the agent in non-endemic regions. The members of the Ebolavirus genus have different pathogenicity for humans, thus differ severity and mortality of the disease they cause. There is a significant genetic divergence among members of the Ebolavirus genus. The differences of pathogenic potential of members of the Ebolavirus genus may be explained as the result of mutations in the genes of virus structural proteins. It is possible, that some of these mutations may affect virulence of strains within one virus species. So far as most effective modern medicines for specific prophylaxis and treatment of Ebola fever are target-oriented, genotyping of the agent will promote elaboration of strategy of such preparations development.

Coronaviruses are enveloped viruses with a single-strand “+” RNA, its genome size varying from 25 to 32 thousands of nucleotides. They cause respiratory and intestinal diseases in animals and humans. The review contains the data on human infection cases induced by a new coronavirus (NCoV), as well as the information about probable natural agent reservoirs, mechanisms of transmission, some characteristic features of the etiological agent, methods of diagnostics and identification, complete genome sequence, and NCoV relation to the established coronaviruses.

Middle-East respiratory syndrome is a human disease caused by a new coronavirus. In December, 2012 WHO published draft regulatory document on diagnostics of the virus. It was recommended to use two methods of disease diagnostics - two-phase reverse-transcription real-time PCR and enzyme immunoassay. The first phase of the PCR-diagnostics should include reverse-transcription real-time PCR targeted on the genome fragment upwards of upE. The second (control) PCR-test may be alternatively targeted within the bonds of the genome, its target being non-crisscross with upE gene. It should include sequencing of, at least, a segment of one of the viral genomes and comparative analysis of the obtained sequence along with the like ones deposited in the GenBank. Enzyme immunoassay is retrospectively used for virus-specific antibody detection in convalescents’ blood sera. Examined are the key specifications of the methods for the detection of ethiological agent or specific antibodies to it, and WHO methodological recommendations in case of Middle-East respiratory syndrome diagnostics.

The members of the Filoviridae (Marburg and Ebola viruses) and Arenaviridae (Lassa, Lujo, Machupo, Junin, Guanarito, Sabia viruses) families are the etiological agents of particularly dangerous viral hemorrhagic fevers. These agents pose a potential threat to public health care in view of the possibility of their unintended import into the non-endemic regions, and thus construction of specific medical protectors as regards induced by them diseases is a pressing issue. According to leading experts, vaccination of the cohorts that fall in the risk groups is the most effective and least expensive method to prevent the development of epidemics. The review contains information on a new prospective line of protective preparations development as regards particularly dangerous viral infections - construction of alphavirus-replicon-based vaccine. Elaboration of recombinant replicons does not require cultivation of pathogenic microorganisms. RNA-replicons are distinguished by their incapacity to produce infective progeny, which is of a great importance for the development of vaccines against particularly dangerous viral hemorrhagic fevers. Advantages of alphaviral replicons over other RNA-replicons are as follows: high levels of heterologous gene expression and resistance to anti-vector immunity. RNA-replicons of alphaviruses combine the safety of inactivated, and immunogenicity of live attenuated vaccines. Alphaviruses-based replicons are suitable for express vaccine development with the purpose of specific prophylaxis of viral infectious diseases.

Reviewed are some peculiarities of circulation of Venezuelan equine encephalomyelitis virus (VEE), the enzootic strains of which play an increasingly important part as etiological agents of human infections nowadays. The majority of VEE cases among the residents of the endemic regions within the inter-epidemic period are caused by the ID subtype of VEE that includes 6 genotypes. Isolated from humans have been the genotypes Columbia/Venezuela, Panama/Peru, and Peru/Bolivia, ID subtype of VEE. Possibility of emergence of new VEE outbreaks is associated with ID subtype mutation into the IC subtype, which is assumed to be a potential threat as etiological agent of the disease at present.