Assessment Of Cell-mediated Immune Responses Research Articles

Assessment of cell-mediated immune responses to SARS-CoV-2 in Syrian hamsters

BACKGROUND: The pandemic caused by the SARS-CoV-2 virus at the end of 2019 remains to be a serious healthcare problem. Constant antigenic drift of the pathogen led to a decrease of licensed COVID-19 vaccines effectiveness. And the development of broad-spectrum vaccines with high effectiveness rate against evolutionarily divergent SARS-CoV-2 variants remains an urgent issue. Unlike virus-specific antibodies with limited spectrum of action, T-cell immunity has a wider cross-protective potential. Syrian hamsters are the most appropriate model for preclinical evaluation of new vaccine candidates, since these animals are susceptible to SARS-CoV-2 infection and show clinical symptoms of the disease. However, study of T-cell vaccine response in hamsters is complicated by the lack of available reagents and test systems for adequate assessment of the virus-specific cellular immunity levels after vaccination.
 AIM: In this work, we report an optimized protocol of stimulation of Syrian hamsters immune cells with a live SARS-CoV-2 virus to assess virus-specific T-cell responses.
 MATERIALS AND METHODS: Intranasal infection of animals with SARS-CoV-2 virus followed by stimulation of immune cells with different doses of whole live coronavirus and counting of IFN-producing cells by ELISpot method.
 RESULTS: Stimulation of spleen and lung cells with SARS-CoV-2 at a dose 0.1 TCID50/cell is the most optimal viral concentration for detecting maximum of cytokine-producing cells in SARS-CoV-2-infected animals. Stimulation of cells with whole virus revealed greater number of virus-specific cells compared to a stimulation with pools of SARS-CoV-2 lyophilized peptides (S and N proteins).
 CONCLUSIONS: Overall, the new methodology allows assessment of the immunogenicity of COVID-19 T-cell vaccines more accurately in preclinical studies using the Syrian hamsters model.

Identification of immunodominant CD8 epitope in the stalk domain of influenza B viral hemagglutinin

Human infections by type B influenza virus constitute about 25% of all influenza cases. The viral hemagglutinin is comprised of two subunits, HA1 and HA2. While HA1 is constantly evolving in an unpredictable fashion, the HA2 subunit is highly conserved, making it a potential candidate for a universal vaccine. However, immunodominant epitopes in the HA2 subunit remain largely unknown. To delineate MHC Class I epitopes, we first identified 9-mer H-2Kd-restricted CD8 T cell epitopes in the HA2 domain by in silico analyses, followed by evaluating the immunodominance of these peptides in mice challenged with the virus. Of three peptides selected through in silico analysis, the universally conserved peptide, YYSTAASSL (B/HA2-190), possessed the highest predicted binding affinity to MHC Class I and was most effective in inducing IL-2 and TNF-α in mouse splenocytes. Importantly, the peptide demonstrated best capability of stimulating peptide-specific ex-vivo cytotoxicity against target cells. Taken together, this finding would be of value for assessment of cell-mediated immune responses elicited by vaccines based on the highly conserved HA2 stalk domain.

METHODS OF SPECIFIC DIAGNOSIS OF TUBERCULOSIS: A MODERN VIEW ON THE PROBLEM

This review presents modern views on the problems of immunodiagnosis of tuberculosis. We have examined the advisability of the use of immunological methods for diagnosis of active tuberculosis and detection of latent tuberculous infection. It is shown that nowadays the use of serological diagnostic methods is not recommended because of contradictory information about their accuracy and limited diagnostic significance. Advantages and disadvantages of current methods of assessment of cell-mediated immune response that are used as screening tests for diagnosis of latent tuberculosis have been analyzed. We point to advisability of combination of different methods that are available nowadays with the use of skin tests as the first step of diagnosis, and improvement of techniques of antigens injections that are used for them.

Assessment of Cell-Mediated Immune Response in Goats Immunized with Lyophilized SaponifiedMycoplasmaCapriVaccine

Assessment of Cell-Mediated Immune Response in Goats Immunized with Lyophilized Saponified<i>Mycoplasma</i><i>Capri</i>Vaccine

Intracellular ATP Production in CD4+ T Cells as a Predictor for Infection and Allograft Rejection in Trough-Level Guided Pediatric Liver Transplant Recipients Under Calcineurin-Inhibitor Therapy

The assessment of cell-mediated immune responses through the measurement of intracellular adenosine-tri-phosphate (iATP) production (Cylex ImmuKnow) as a pharmacodynamic biomarker of immune function represents a potential tool to optimize individual immunosuppressive therapy independent of drug dosage or trough levels. This study aims to investigate the correlations between iATP and adverse events, immunosuppression, calcineurin-inhibitor-trough levels, and age. In this prospective trial, 31 nontransplant pediatric subjects and 50 consecutive children were included after they underwent liver transplantation (LTX). During the study period, 4 allograft rejections and 3 acute infections occurred. The patients were treated with cyclosporine, tacrolimus, mycophenolate mofetil, and everolimus either as monotherapy or in combinations. The reactivity of the immune system was measured as iATP concentration in CD4+ T-cells after in vitro stimulation by phytohemagglutinin. The iATP concentrations in patients with intercurrent, clinically significant infections were in the low immune response range (median iATP 181 versus 251 ng/mL, P = 0.308), whereas the patients with incidental allograft rejection had significantly higher iATP concentrations as compared with the event-free group (median iATP 444 versus 251 ng/mL, P = 0.017). However, there was a wide range of iATP concentrations in both nontransplant and LTX patient groups, and no clear iATP cut-off values for an increased risk of infection or rejection could be defined. Post LTX, stable-phase patients showed a significantly lower iATP compared with respective controls (median iATP 297 versus 384 ng/mL, P = 0.013). No significant correlation between calcineurin-inhibitor-trough concentrations and iATP was found. iATP was not correlated with age, but was inversely correlated with time after transplantation. The observed correlation between clinical events and iATP concentrations is similar to the findings previously reported in adult patients who underwent transplantation. The lack of correlation of iATP with trough drug concentrations suggests that the ImmuKnow assay provides independent information that may be useful to guide immunosuppressive therapy in pediatric (liver) transplant patients. However, the wide range of iATP levels in event-free patients suggests that serial iATP measurements will be necessary to assess and guide the individual immunosuppressive therapy. Further investigations are needed to evaluate and extend these findings.

Optimal Blood Mononuclear Cell Isolation Procedures for Gamma Interferon Enzyme-Linked Immunospot Testing of Healthy Swedish and Tanzanian Subjects

Determination of antigen-specific T-cell responses is an important part of vaccine assessment. High levels of recovery, viability, and functionality of peripheral blood mononuclear cells (PBMCs) are essential for reliable assessment of cell-mediated immune responses. Here, we sought to find the cell preparation technique best suited for two clinical vaccine trial sites: Stockholm, Sweden, and Dar es Salaam, Tanzania. Standard Ficoll-Paque gradient centrifugation, BD Vacutainer cell preparation tube (CPT), and Greiner Bio-One LeucoSep tube techniques were tested. Cell yield and viability were recorded. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) testing was used to assess cell functionality. No differences in mean recovery or mean viability of fresh PBMCs were observed between Ficoll-Paque gradient centrifugation and CPT techniques as used in Stockholm. In Dar es Salaam, recovery of PBMCs isolated by use of the Ficoll-Paque gradient technique was higher than that seen with CPT (1.58 +/- 0.6 versus 1.34 +/- 0.4 million cells/ml of blood [P = 0.0469]), and the viability of PBMCs processed by Ficoll-Paque gradient was higher than that seen with CPT-purified cells (95.8% +/- 2.3% versus 92.6% +/- 4.8% [P = 0.0081]). Furthermore, LeucoSep cell separation gave higher levels of yield (1.10 +/- 0.3 versus 0.92 +/- 0.3 million cells/ml of blood [P = 0.0022]) and viability (95.7% +/- 2.0% versus 93.4% +/- 3.2% [P = 0.0012]) than Ficoll-Paque cell separation. The cells purified by the different techniques at the two sites performed equally well in IFN-gamma ELISPOT assays. Both techniques generated cell preparations with excellent yield, viability, and functionality in Stockholm. In Dar es Salaam, CPT did not perform as well as Ficoll-Paque separation. In a subsequent comparison, LeucoSep performed better than Ficoll-Paque separation. Our findings emphasize the need for on-site assessment of PBMC purification techniques for optimal evaluation of cell-mediated immune responses.

Characterization of primary cell cultures as potential target cells for analysis of bovine cytotoxic T lymphocytes

In domestic animal species, assessment of cell-mediated immune responses to virus infection is hampered by the requirement for class I MHC compatibility between target and effector cells. Additional complicating factors can include an inability to infect target cells in vitro, or virus-induced lysis of infected target cells. One way to circumvent these problems is to use virus-mediated gene transfer to deliver individual viral genes to autologous primary target cells. Several primary bovine cell cultures were assessed as potential target cells for cytotoxic T lymphocyte (CTL) assays by measuring their levels of class I MHC expression and susceptibilities to retroviral gene delivery. High levels in both class I MHC expression and susceptibility to gene delivery were seen in adherent cell cultures isolated from peripheral blood (PBAC). PBAC, which arose as an outgrowth of adherent peripheral blood mononuclear cell cultures, had morphology, protein expression patterns, and response to functional assays characteristic of high endothelial cells. Expression of viral vector-delivered genes in PBAC cells was confirmed with a recombinant retrovirus carrying the green fluorescent protein (GFP) gene. The use of vector-mediated delivery of viral genes to bovine high endothelial cells is a promising method for assessment of cell-mediated immunity in cattle.