The study of pathogenic viruses has always posed significant biosafety challenges. In particular, the study of highly pathogenic viruses requires methods with low biological risk but relatively high sensitivity and convenience in detection. In recent years, pseudoviruses, which consist of a backbone of one virus and envelope proteins of another virus, have become one of the most widely used tools for exploring the mechanisms of viruses binding to cells, membrane fusion and viral entry, as well as for screening the libraries of antiviral substances, evaluating the potential of neutralizing monoclonal antibodies, developing neutralization tests, and therapeutic platforms. During the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pseudotyped virus-based assays played a pivotal role in advancing our understanding of virus–cell interactions and the role of its proteins in disease pathogenesis. Such tools facilitated the search for potential therapeutic agents and accelerated epidemiological studies on post-infection and post-vaccination humoral immunity. This review focuses on the use of pseudoviruses as a model for large-scale applications to study enveloped viruses.

Tumor necrosis factor (TNF) is one of many cytokines - protein molecules responsible for communication between the cells of immune system. TNF was discovered and given its grand name because of its striking antitumor effects in experimental systems, but its main physiological functions in the context of whole organism turned out to be completely unrelated to protection against tumors. This short review discusses "man-made" mouse models generated by early genome-editing technologies, which enabled us to establish true functions of TNF in health and certain diseases as well as to unravel potential strategies for improving therapy of TNF-dependent diseases.

Natural killer (NK) cells play a pivotal role in the immune response against viral infections, including SARS-CoV-2. However, our understanding of memory NK cell responses in the context of SARS-CoV-2 remains limited. To address this, we investigated the memory-like response of NK cells to SARS-CoV-2 peptides, presented by autologous cells. Blood samples from 45 donors underwent analysis for SARS-CoV-2 IgG antibodies, categorizing them into four groups based on the antibody kind and level. NK cells from SARS-CoV-2-experienced donors demonstrated enhanced degranulation and activation levels, IFNγ production and proliferative potential in response to SARS-CoV-2 peptides. Investigation of highly proliferating NK cells demonstrated the formation of distinct clusters depending on the SARS-CoV-2 peptide supplementation and the donor group. RNA sequencing revealed differential gene expression patterns, highlighting metabolism, protein transport, and immune response genes. Notably, KIR2DS4 expression correlated with enhanced IFNγ production, degranulation and proliferation levels, suggesting a role in SARS-CoV-2 recognition. Collectively, these findings provide detailed insights into antigen-specific NK cell responses to SARS-CoV-2 peptides, indicating potential mechanisms underlying NK cell activation in antiviral immunity.

The pandemic of coronavirus infection (COVID-19) has stimulated the development, testing and widespread use of preventive vaccines based on various platforms. Our aim was to perform a direct comparison of immunogenicity of various vaccines within a single study in small groups within six months of SARS-CoV-2 vaccination and revaccination. The stdy group included subjects vaccinated with Sputnik V adenovirus vaccine, mRNA vaccines, and CoviVac whole-virion vaccine. Their immune status was assessed by enzyme immunoassay as specific antibody levels. Moreover, the neutralizing ability of detected antibodies was assessed using a cell test system based on pseudoviral technology. All of the mentioned vaccines were shown to elicit an immune response against SARS-CoV-2 RBD antigen, however, appropriate antibody titers and neutralizing capacities differed depending on the type of vaccine. The mRNA vaccines proved to be the most immunogenic, the effectiveness of the immune response to the Sputnik V adenovirus-based vaccine was lower. However, 6 months after vaccination, the effectiveness of virus neutralizing antibodies induced by these vaccines did not differ. The whole-virion CoviVac vaccine with proven efficiency by independent epidemiological studies, induced an antibody response against the RBD protein to a lesser extent. The seropositive participants of the study, both previously exposed to COVID-19 disease or vaccinated, exhibited high-titer production of antibodies already after the first dose of the Sputnik V vaccine, and a significantly higher antibody titer 6 months after the booster immunization as compared with initial level of antibodies, along with direct correlation between the antibody titers and their neutralizing activity.

The binding properties of synthetic and recombinant peptides derived from N-terminal part of ACE2, the main receptor for SARS-CoV-2, were evaluated. Additionally, the ability of these peptides to prevent virus entry in vitro was addressed using both pseudovirus particles decorated with the S protein, as well as through infection of Vero cells with live SARS-CoV-2 virus. Surprisingly, in spite of effective binding to S protein, all linear peptides of various lengths failed to neutralize the viral infection in vitro. However, the P1st peptide that was chemically “stapled” in order to stabilize its alpha-helical structure was able to interfere with virus entry into ACE2-expressing cells. Interestingly, this peptide also neutralized pseudovirus particles decorated with S protein derived from the Omicron BA.1 virus, in spite of variations in key amino acid residues contacting ACE2.

COVID-19 is a systemic inflammatory disease initiated by SARS-CoV-2 virus infection. Multiple vaccines against the Wuhan variant of SARS-CoV-2 have been developed including a whole virion beta-propiolactone-inactivated vaccine based on the B.1.1 strain (CoviVac). Since most of the population has been vaccinated by targeting the original or early variants of SARS-CoV-2, the emergence of novel mutant variants raises concern over possible evasion of vaccine-induced immune responses. Here, we report on the mechanism of protection by CoviVac, a whole virion-based vaccine, against the Omicron variant. CoviVac-immunized K18-hACE2 Tg mice were protected against both prototype B.1.1 and BA.1-like (Omicron) variants. Subsequently, vaccinated K18-hACE2 Tg mice rapidly cleared the infection via cross-reactive T-cell responses and cross-reactive, non-neutralizing antibodies recognizing the Omicron variant Spike protein. Thus, our data indicate that efficient protection from SARS-CoV-2 variants can be achieved by the orchestrated action of cross-reactive T cells and non-neutralizing antibodies.

This short report summarizes the results of recent immunological studies performed at new Sirius University of Science and Technology. The report focuses on studying the features of the immune response to vaccination and revaccination against SARS-CoV-2, as well as on a search of potential agents to prevent infection with this virus.

A highly effective humoral immune response induced by the Sputnik V vaccine was demonstrated in independent studies, as well as in large-scale post-vaccination follow-up studies. However, the shifts in the cell-mediated immunity induced by Sputnik V vaccination are still under investigation. This study was aimed at estimating the impact of Sputnik V on activating and inhibitory receptors, activation and proliferative senescence markers in NK and T lymphocytes. The effects of Sputnik V were evaluated by the comparison of PBMC samples prior to vaccination, and then three days and three weeks following the second (boost) dose. The prime-boost format of Sputnik V vaccination induced a contraction in the T cell fraction of senescent CD57+ cells and a decrease in HLA-DR-expressing T cells. The proportion of NKG2A+ T cells was down-regulated after vaccination, whereas the PD-1 level was not affected significantly. A temporal increase in activation levels of NK cells and NKT-like cells was recorded, dependent on whether the individuals had COVID-19 prior to vaccination. A short-term elevation of the activating NKG2D and CD16 was observed in NK cells. Overall, the findings of the study are in favor of the Sputnik V vaccine not provoking a dramatic phenotypic rearrangement in T and NK cells, although it induces their slight temporal non-specific activation.

Previous studies have shown that polyreactive antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyreactivity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgG and IgE antibodies to acquire polyreactivity following contact with various agents that destabilize protein structure (urea, low pH) or have a pro-oxidative potential (heme, ferrous ions) has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two human monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using immunoblot and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyreactivity.

Peptides are widely used for the diagnostics, prevention, and therapy of certain human diseases. How useful can they be for the disease caused by the SARS-CoV-2 coronavirus? In this review, we discuss the possibility of using synthetic and recombinant peptides and polypeptides for prevention of COVID-19 via blocking the interaction between the virus and its main receptor ACE2, as well as components of antiviral vaccines, in particular, against new emerging virus variants.

SARS-CoV-2 is a new coronavirus that is the cause of COVID-19 pandemic. To enter the cell, the virus interacts via its surface S protein with angiotensin-converting enzyme 2 (ACE2), the main entry receptor on the cell membrane. Most of protective antibodies, including those induced by vaccinations, target the S protein, preventing its interaction with the ACE2 receptor. We have evaluated an alternative strategy for blocking the S-ACE2 interaction using new antipeptide antibodies to the N-terminus of the ACE2 molecule. These antibodies allow detection of human ACE2 in vitro and ex vivo.

Spatial organization and conformational changes of antibodies may significantly affect their biological functions. We assessed the effect of mutual organization of the two VHH domains within bispecific antibodies recognizing human TNF and the surface molecules of murine myeloid cells (F4/80 or CD11b) on TNF retention and inhibition. TNF‐neutralizing properties in vitro and in vivo of MYSTI‐2 and MYSTI‐3 antibodies were compared with new variants with interchanged VHH domains and different linker sequences. The most effective structure of MYSTI‐2 and MYSTI‐3 proteins required the Ser/Gly‐containing ‘superflexible’ linker. The orientation of the modules was crucial for the activity of the proteins, but not for MYSTI‐3 with the Pro/Gln‐containing ‘semi‐rigid’ linker. Our results may contribute toward the development of more effective drug prototypes.

— The aim of this work was to evaluate novel three-domain antibodies consisting of two domains specific for human tumor necrosis factor (hTNF), and of the third domain responsible for binding to myeloid cells. The additional hTNF-binding domain should serve to increase the biological activity of new antibodies. Capacity of these proteins to bind hTNF on the macrophage surface and to neutralize its biological activity in vitro was assessed.

More than 40 years ago ethyl nitrosoеurea was identified as a powerful mutagen for mammalian germ cells resulting in random point mutations in gamete DNA. This feature allowed the use of this mutagen for genetic studies on the mechanisms of various pathological and physiological processes in model organisms. In our study genome-wide mutagenesis in C3H mice by ethyl nitrosourea followed in generation F3 by selection of animals resistant to acute lethal hepatotoxicity caused by a combination of E. coli lipopolysaccharide (LPS) and D-galactosamine (D-gal). Tumor necrosis factor (TNF) is known to be a critical mediator of this pathology. Exposure to D-galactosamine increases sensitivity of hepatocytes to TNF leading to their necrosis and/or apoptosis. After double LPS/D-gal screening in F3 several mice resistant to LPS/D-gal-induced hepatotoxicity were identified, and became the founders of the corresponding “mutant” families. Using outcrossing to C57BL6 background followed by intercrossing, generations F5 and F7 were obtained. Among families of mutant animals only one family showed the resistance to the combination of LPS and D-gal, but sensitivity to TNF-D-galactosamine. This phenotype showed approximately Mendelian inheritance consistent with the recessive mutation hypothesis. This latter fact was confirmed by the sensitivity of mice from “heterozygous generations” (F4 and F6) to lethal LPS/Dgal hepatotoxicity. Primary bone marrow macrophages obtained from half of the mutant mice showed significantly reduced levels of TNF after LPS stimulation in vitro. At the same time, the serum TNF levels 1 hour after the administration of a non-lethal LPS dose did not differ in the mutant family mice and wild-type mice. These results implicate a recessive mutation either in innate TLR4-mediated signaling pathway, including proteins associated with LPS transfer, adapter molecules, components of kinase signaling cascades, transcription factors, or in enzymes involved in regulation of TLR4 cascades, such as components of the ubiquitin cycle, or in genomic regulatory sequences that control the expression of one of these genes, including the tnf gene.

Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF treatment. We generated recombinant fluorescent proteins consisting of the anti-TNF module based on the variable heavy chain (VHH) of camelid antibodies fused with the far-red fluorescent protein Katushka (Kat). Two types of anti-TNF VHH were developed: one (BTN-Kat) that was bound both human or mouse TNF, but did not neutralize their activity, and a second (ITN-Kat) that was binding and neutralizing human TNF. BTN-Kat does not interfere with TNF biological functions and can be used for whole-body imaging. ITN-Kat can be evaluated in humanized mice or in cells isolated from humanized mice. It is able to block human TNF (hTNF) activities both in vitro and in vivo and may be considered as a prototype of a theranostic agent for autoimmune diseases.