Introduction. The SARS-CoV-2 coronavirus pandemic has been a major challenge for all areas of medical science, causing a surge of new developments in various fields ranging from diagnostic techniques to therapeutic and preventive approaches. Intranasal vaccination is an innovative approach to immunization against SARS-CoV-2, which has attracted the attention of many drug developers. Dynamics of blood virus-neutralizing antibodies (VNAs) in recovered COVID-19 patients or vaccinated healthy volunteers is one of the objective parameters for assessing vaccine immunological efficacy, which requires high standards of bioanalytical techniques within the framework of clinical trials. Immunogenicity data on the two-component Salnavak®(intranasal) and Gam-COVID-Vac®(intramuscular) vaccination obtained in randomized double-blind multicenter phase 3 clinical trial interim analysis are presented. The objective of the study was to assess immunogenicity of intranasal and intramuscular vaccination against COVID-19 using a neutralization reaction with pseudoviral particles and HEK293T-hACE2 cell culture.Materials and methods.A total of 137 healthy volunteers with a baseline anti-RBD IgG level not exceeding 100 BAU/ml received immunization by a two-component (Ad26 and Ad5 based) intranasal or intramuscular vaccine administered on day 1 and day 21. Immunogenicity level based on VNA quantitative analysis using a neutralization reaction with pseudoviral particles and a HEK293T-hACE2 cell culture as well as to SARS-CoV-2 S-protein receptor-binding domain (anti-RBD) IgG antibodies on days 21 and 42 after administration of component I was assessed.Results.The geometric mean VNA titer against SARS-CoV-2 on day 42 was 238.34±3.93 and 616.94±3.73 in the Salnavac®and Gam-COVID-Vac®groups, respectively. Trial data shows sufficient immunological efficacy of both intramuscular and intranasal vaccines based on a high protection level at VNA titer of more than 100 while using the pseudoviral neutralization method. The geometric mean of the anti-RBD IgG level by day 42 was 131.22±3.91 and 782.03±3.04 in the Salnavac®and Gam-COVID-Vak®groups, respectively. A direct moderate correlation was shown between VHA and anti-RBD IgG.Conclusion. Neutralization reaction using pseudoviral particles was successfully validated and used to determine the VNA titer during clinical trial. Trial interim data revealed that intranasal vaccine Salnavac®vs intramuscular vaccine Gam-COVID-Vak®resulted in lower but sufficient stringency of humoral immunity.

Using a new method, biolayer interferometry (BLI), it has been shown that the new nasal vaccine Salnavac and Sputnik-V induce the affinity maturation of donor serum antibodies within six months after the vaccination.

With the onset of the COVID-19 pandemic, a number of molecular-based tests have been developed to diagnose SARS-CoV-2 infection. However, numerous available serological tests lack sufficient sensitivity or specificity. They do not detect specific antibodies in a significant proportion of patients with PCR-confirmed COVID-19. There is evidence that some convalescents have a relatively short-lived humoral immunity. In contrast, a number of publications have shown that T-cell response to human coronaviruses, including SARS-CoV-1, MERS, and SARS-CoV-2, can be strong and long-term. Assessment of T-cell immunity to SARS-CoV-2 is important not only for stratification of risks and identification of potentially protected populations with immunity acquired as a result of previous infection, but also for determining immunogenicity and potential efficacy of vaccines under development. The existing methods of quantitative or semi-quantitative assessment of specific T-cell response are mainly used in scientific research and are not standardised. The aim of the study was to develop and verify experimentally a test kit to be used in a standardised procedure for in vitro determination of T-cells specific to SARS-CoV-2 antigens, in human peripheral blood. Materials and methods: the TigraTest® SARS-CoV-2 kit developed by GENERIUM, which determines the number of T-cells secreting interferon gamma in vitro, was tested in the study. Samples of venous blood of volunteers from three different groups were analysed in the study: presumably healthy volunteers; COVID-19 convalescents; individuals vaccinated against SARS-CoV-2. Results: the authors developed the TigraTest® SARS-CoV-2 kit for in vitro determination of T-cells specific to SARS-CoV-2 antigens in human peripheral blood, demonstrated its specificity and performed preliminary assessment of its sensitivity. The study analysed the range and magnitude of the T-cell response in convalescent and vaccinated individuals. A pronounced T-cell response was also shown in some individuals with no symptoms or with unconfirmed diagnosis. It was discovered that the mean T-cell response to peptides of the spike protein (S-protein) was higher in the vaccinated individuals than in the convalescent patients. A correlation was determined between the severity of the disease and the level of T-cell response. Specific contributions of various groups of antigens to the T-cell response after COVID-19 infection were also determined. Conclusions: the TigraTest® SARS-CoV-2 kit is a specific and sensitive tool for the assessment of T-cell immunity to the SARS-CoV-2 virus, which can also be used for vaccinated individuals. The kit may be used in clinical practice for comprehensive assessment of immunity to SARS-CoV-2.

Determination of T-cell immune response to SARS-CoV-2 is important both for diagnosis of the disease in symptomatic patients, and for determination of the total number of people who have had the disease, including its asymptomatic form. In addition, these assays are efficient for assessing the immune response after vaccination, as well as immunity levels in risk groups and in convalescent patients. The most promising method of T-cell immune response assessment is an ELISPOT-based assay measuring the release of interferon-gamma (IGRA) after stimulation with coronavirus-specific antigens. The present review analyses the prospects for using the ELISPOT technological platform in the clinical laboratory setting when dealing with the new coronavirus infection COVID-19, given specific aspects of the immune response. The review summarises data from articles published in peer-reviewed journals, preprints of articles available at arXiv resources, and information that some leading immunologists shared with the authors during private discussions. It has been shown that the introduction of B- and T-cell ELISPOT assays will make it possible to monitor the immunological status of patients, select a treatment strategy, identify the most vulnerable populations, carry out comprehensive assessment of vaccines during the development, clinical trials and implementation stages. The paper discusses the issues of maintaining T-cell immunity in the blood of people who have had HCoV, SARS, MERS, and COVID-19 coronavirus infections. It also discusses the advantages of the T-cell ELISPOT assay over serological tests as regards epidemiological assessment of the prevalence of the new coronavirus infection, and clinical trials of COVID-19 vaccines. Biotechnology companies have a ready-made technological platform for the development and industrial-scale production of ELISPOT kits, and this platform is easily adaptable to specific types of assays and pathogens. The paper supports the need to develop vaccines that would stimulate both cellular and humoral immune responses, and raises the question of the protective potential of cross-immunity acquired before the COVID-19 pandemic.

Therapeutic monoclonal antibodies (mAbs) are within the fastest growing group of pharmaceuticals on the global market. IgG1 subclass is the most potent effector in Fc-related functions. The N-linked glycosylation of mAbs Fc-domain significantly influences its therapeutic activity and the presence of this modification is largely dependent on producer cell and parameters of manufacturing process. Here we examined and characterized cell culture conditions that determine during cultivation selective changes in galactose content of a model therapeutic mAb IgG1, trastuzumab biosimilar. We demonstrated that such in cultivation process shift of galactosylation does not affect binding of the mAb to its antigen yet modifies interaction of the mAb with Fcγ receptors and therefore enhances antibody dependent cellular cytotoxicity (ADCC).

C1 esterase inhibitor (C1INH) is an abundant component of blood plasma (the average concentration is 250 mg/L); it is known to be involved in several biological processes, for instance, in the regulation of the coagulation system, adhesion of leukocytes on endothelial cells, and in the regulation of complement and kallikrein cascades. Lately, the role of C1INH in immunomodulation has gained considerable attention. We used an ex vivo whole blood model to examine the influence of C1INH and its mutated variants on the inflammatory cytokines interleukin (IL)-6, tumor necrosis factor-α (TNFα), and IL-1β. The present study demonstrated for the first time that recombinant C1INH or its Seprin domain can downregulate bacterial endotoxin induced IL-6 release. We also observed that unstructured N-terminal domain of C1INH downregulates the release of IL-1β and TNFα, but not IL-6. Our results suggest that C1INH may have therapeutic potential for treatment of inflammatory conditions.

RNA interference (RNAi) is a powerful tool to study the intracellular membrane transport and membrane organelle behavior in the nematode Caenorhabditis elegans. This model organism has gained popularity in the trafficking field because of its relative simplicity, yet being multicellular. C. elegans is fully sequenced and has an annotated genome, it is easy to maintain, and a growing number of transgenic strains bearing markers for different membrane compartments are available. C. elegans is particularly well suited for protein downregulation by RNAi because of the simple but efficient methods of dsRNA delivery. The phenomenon of systemic RNAi in the worm further facilitates this approach. In this chapter we describe methods and applications of RNAi in the field of membrane traffic. We summarize the fluorescent markers used as a readout for the effects of gene knockdown in different cells and tissues and give details for data acquisition and analysis.

The Wnt/β-catenin signaling pathway is crucial in animal development from sponges to humans. Its activity in the adulthood is less general, with exceptions having huge medical importance. Namely, improper activation of this pathway is carcinogenic in many tissues, most notably in the colon, liver and the breast. On the other hand, the Wnt/β-catenin signaling must be re-activated in cases of tissue damage, and insufficient activation results in regeneration failure and degeneration. These both medically important implications are unified by the emerging importance of this signaling pathway in the control of proliferation of various types of stem cells, crucial for tissue regeneration and, in case of cancer stem cells – cancer progression and relapse. This article aims at briefly reviewing the current state of knowledge in the field of Wnt signaling, followed by a detailed discussion of current medical developments targeting distinct branches of the Wnt pathway for anti-cancer and pro-regeneration therapies.

Fatty liver disease, a type of metabolic disorder, frequently occurs in dairy cows during the parturition period, causing a high culling rate and, therefore, considerable economic losses in the dairy industry owing to the lack of effective diagnostic methods. Here, metabolite biomarkers were identified and validated for the diagnosis of metabolic disorders. A total of 58 participant cows, including severe fatty liver disease and normal control groups, in the discovery set (liver biopsy tested, n = 18), test set (suspected, n = 20) and verification set (liver biopsy tested, n = 20), were strictly recruited and a sample collected for their feces, urine, and serum. Non-targeted GC-MS-based metabolomics methods were used to characterize the metabolite profiles and to screen in the discovery set. Eventually, ten novel biomarkers involved in bile acid, amino acid, and fatty acid were identified and validated in the test set. Each of them had a higher diagnostic ability than the traditional serum biochemical indicators, with an average area under the receiver operating characteristic curve of 0.830 ± 0.0439 (n = 10) versus 0.377 ± 0.182 (n = 9). Especially, combined biomarker panels via different metabolic pipelines had much better diagnostic sensitivity and specificity than every single biomarker, suggesting their powerful utilization potentiality for the early detection of fatty liver disease. Intriguingly, the serum biomarkers were confirmed perfectly in the verification set. Moreover, common biological pathways were found to be underlying the pathogenesis of fatty liver syndrome in cattle via different metabolic pipelines. These newly-discovered and non-invasive metabolic biomarkers are meaningful in reducing the high culling rate of cows and, therefore, benefit the sustainable development of the dairy industry.

The glial cell-derived neurotrophic factor (GDNF) precursor contains several putative sites for prohormone convertase-mediated excision of short peptides. Here, we show that one of the predicted peptides, named BEP (brain excitatory peptide), induces a substantial increase in the synaptic excitability in rat CA1 pyramidal neurons. The excitation is sensitive to N-ethylmaleimide, suggesting involvement of a G-protein-coupled receptor.

The small rab-GTPase RAB-7 acts in endosome and endosome to lysosome traffic. We identified SAND-1 as a protein required for RAB-7 function based on similarities between SAND-1 and RAB-7 RNAi phenotypes. Although the initial uptake of yolk protein in oocytes, or of soluble secreted (ss) GFP in coelomocytes, appeared normal, further transport along the endocytic traffic route was delayed in the absence of SAND- 1 function, and yolk proteins failed to reach yolk granules efficiently. Moreover, in coelomocytes, ssGFP and BSA-Texas-Red were endocytosed but not transported to lysosomes. We show that SAND-1 is essential for RAB-7 function at the transition from early to late endosomes, but not for RAB-7 function at lysosomes.

In many organisms, cortical granules undergo exocytosis following fertilization, releasing cargo proteins that modify the extracellular covering of the zygote. We identified cortical granules in Caenorhabditis elegans and have found that degranulation occurs in a wave that initiates in the vicinity of the meiotic spindle during anaphase I. Previous studies identified genes that confer an embryonic osmotic sensitivity phenotype, thought to result from abnormal eggshell formation. Many of these genes are components of the cell cycle machinery. When we suppressed expression of several of these genes by RNAi, we observed that cortical granule trafficking was disrupted and the eggshell did not form properly. We conclude that osmotic sensitivity phenotypes occur because of defects in trafficking of cortical granules and the subsequent formation of an impermeable eggshell. We identified separase as a key cell cycle component that is required for degranulation. Separase localized to cortically located filamentous structures in prometaphase I upon oocyte maturation. After fertilization, separase disappeared from these structures and appeared on cortical granules by anaphase I. RNAi of sep-1 inhibited degranulation in addition to causing extensive chromosomal segregation failures. Although the temperature-sensitive sep-1(e2406) allele exhibited similar inhibition of degranulation, it had minimal effects on chromosome segregation. These observations lead us to speculate that SEP-1 has two separable yet coordinated functions: to regulate cortical granule exocytosis and to mediate chromosome separation.

Caenorhabditis elegans has recently been used as an attractive model system to gain insight into mechanisms of endocytosis in multicellular organisms. A combination of forward and reverse genetics has identified a number of new membrane trafficking factors. Most of them have mammalian homologues which function in the same transport events. We describe a novel C. elegans gene sand-1, whose loss of function causes profound endocytic defects in many tissues. SAND-1 belongs to a conserved family of proteins present in all eukaryotic species, whose genome is sequenced. However, SAND family has not been previously characterized in metazoa. Our comparison of C. elegans SAND-1 and its yeast homologue, Mon1p, showed a conserved role of the SAND-family proteins in late steps of endocytic transport.

The endoplasmic reticulum (ER) is the major intracellular membrane system. The ER is essential for protein and lipid biosynthesis, transport of proteins along the secretory pathway, and calcium storage. Here, we describe our investigations into the dynamics and regulation of the ER in the early Caenorhabditis elegans embryo. Using a GFP fusion to the ER-resident signal peptidase SP12, we observed the morphological transitions of the ER through fertilization and the early cell-cycles in living embryos. These transitions were tightly coordinated with the division cycle: upon onset of mitosis, the ER formed structured sheets that redispersed at the initiation of cleavage. Although microtubules were not required for the transition of the ER between these different states, the actin cytoskeleton facilitated the dispersal of the ER at the end of mitosis. The ER had an asymmetric distribution in the early embryo, which was dependent on the establishment of polarity by the PAR proteins. The small GTPase ARF-1 played an essential role in the ER dynamics, although this function appeared to be unrelated to the role of ARF-1 in vesicular traffic. In addition, the ER-resident heat shock protein BiP and a homologue of the AAA ATPase Cdc48/p97 were found to be crucial for the ER transitions. Both proteins have been implicated in homotypic ER membrane fusion. We provide evidence that homotypic membrane fusion is required to form the sheet structure in the early embryo.

Glial cell line–derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor α1 (GFRα1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRα1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRα1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRα1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRα1-positive and GFRα1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.

A novel gene named Helix command-specific 2 (HCS2) was shown to be expressed predominantly in four giant parietal interneurons involved in withdrawal behavior of the terrestrial snail Helix lucorum L. and several single neurons in other ganglia. Decrease in spontaneous electrophysiological activity of neurons in the isolated CNS by 24h incubation in saline with elevated Mg(2+) concentration significantly decreased the number of HCS2-expressing neurons. Five short-term serotonin applications (each of 10microM), during a 24h incubation of the nervous system in saline induced expression of the HCS2 gene in many cells in cerebral, parietal, pleural and pedal ganglia. Dopamine applications under similar conditions were not effective. Application of anisomycin or cycloheximide, known to block protein synthesis, did not prevent the induction of HCS2 expression under serotonin influence. Skin injury elicited a significant increase in the number of HCS2-expressing cells 24h later in pleural and cerebral ganglia. Incubation of the isolated nervous system preparations for three days in culture medium elicited close to a maximum increase in number of HCS2-expressing cells. Elevation of the normal Mg(2+) concentration in the culture medium significantly decreased the number of cells demonstrating HCS2 expression. Application of the cAMP activator forskolin (10microM) increased the expression under Mg(2+), indicating that cAMP was involved in the up-regulation of HCS2. Application of thapsigargin (10microM), known to release Ca(2+) from intracellular stores, was also effective in increasing expression, suggesting participation of Ca(2+) in regulation of HCS2 expression. Cellular groups expressing the HCS2 gene under different conditions seem to be functionally related since it was demonstrated earlier that some neurons constituting these clusters are involved in the withdrawal behavior and the response of the organism to stress stimuli. From these results we suggest that the HCS2 pattern of expression can be down-regulated by a decrease in synaptic activity in the nervous system, and up-regulated by external noxious inputs, as well as the application of neurotransmitters and second messengers known to be involved in the withdrawal behavior and maintenance of isolated ganglia in culture medium. When up-regulated, the HCS2 expression appears, at least in part in neurons, to be involved in the withdrawal behavior.

Glial cell line-derived neurotrophic factor (GDNF) family ligands signal through receptor complex consisting of a glycosylphosphatidylinositol-linked GDNF family receptor (GFR) ru subunit and the transmembrane receptor tyrosine kinase RET. The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2), associated with different mutations in RET, is characterized by medullary thyroid carcinoma. GDNF signals via GFR alpha1, neurturin via GFR alpha2, artemin via GFR alpha3, whereas the mammalian GFR alpha: receptor for persephin (PSPN) is unknown. Here we characterize the human GFR alpha4 as the ligand-binding subunit required together with BET for PSPN signaling. Human and mouse GFR alpha4 lack the first Cys-rich domain characteristic of other GFRa: receptors. Unlabeled PSPN displaces I-125-PSPN from GFRA4-transfected cells, which express endogenous Ret. PSPN can be specifically cross-linked to mammalian GFR alpha4 and Ret, and is able to promote autophosphorylation of Ret in GFRA4-transfected cells. PSPN, but not other GDNF family ligands, promotes the survival of cultured sympathetic neurons microinjected with GFRA4. We identified different splice forms of human GFRA4 mRNA encoding for two glycosylphosphatidylinositol-linked and one putative soluble isoform that were predominantly expressed in the thyroid gland, Overlapping expression of RET and GFRA4 but not other GFRA mRNAs in normal and malignant thyroid medullary cells suggests that GFR alpha4 may restrict the MEN2 syndrome to these cells.

The structure and in vivofunctions of the glial cell-derived neurotrophic factor (GDNF) family ligands (GFLs) and their high-affinity receptors are considered. These proteins play an important role in the development of the nervous system, morphogenesis of the kidneys, and regulation of spermatogenesis. Tyrosine kinase Ret is a common receptor component for all GFLs. Its role in multiple endocrine neoplasia type 2 (MEN2) is discussed.