Omicron (B.1.1.529) BA.1 or BA.2-related effects on immune responses in previously naïve versus imprinted individuals: immune imprinting as an advantage in the humoral immune response against novel variants.

Background: Immune imprinting is a phenomenon in which a person's immune system develops a specific immunological memory of the pathogen or vaccine due to a previous exposure. This memory basically leads to a faster and stronger immune response in a subsequent contact to the same pathogen or vaccine. However, what happens if the pathogen has changed considerably in the meantime due to mutations in the main target region of antibodies, as in the evolution of SARS-CoV-2 from the ancestral strain to B.1.1.529 (Omicron)? In this case, does immune imprinting also confer an advantage in repeated contact and does it lead to a stronger immune response? Methods: To clarify these questions, we investigated the effects of immune imprinting in the context of SARS-CoV-2 by comparing a group of previously infection-naïve versus imprinted study participants and determined differences in humoral and cellular immune responses during and after infection with strain SARS-CoV-2 B.1.1.529 BA.1 and BA.2, respectively. We used a commercial CLIA, immunoblots, IFN-γ ELISpots and a plaque-reduction neutralization test to generate a clear and comparable picture of the humoral and cellular immune response in the two study groups. Results: Imprinted participants developed significantly higher antibody titers and showed significantly stronger neutralization capacity against the ancestral strain, BA.1 and BA.5. The immune response of naïve study participants was narrower and related mainly to the receptor-binding domain, which resulted in a lower neutralization capacity against other strains including BA.5. Naïve study participants showed a significantly higher cellular immune response than the imprinted study group, indicating a higher antigenic challenge. The cellular immune response was directed against general structures of SARS-CoV-2 and not specifically against the receptor-binding domain. Conclusion: Viral variant infection elicits variant-specific antibodies and prior mRNA vaccination or infection with a previous SARS-CoV-2 variant imprints serological responses toward the ancestral strain rather than variant antigens. On the other hand, our study shows that the initially higher specific antibody titers due to former imprinting via vaccination or prior infection significantly increased the humoral immune response, and therefore outperformed the humoral immune response of naïve study participants.

MERS-CoV‒Specific T-Cell Responses in Camels after Single MVA-MERS-S Vaccination.

We developed an ELISPOT assay for evaluating Middle East respiratory syndrome coronavirus (MERS-CoV)‒specific T-cell responses in dromedary camels. After single modified vaccinia virus Ankara-MERS-S vaccination, seropositive camels showed increased levels of MERS-CoV‒specific T cells and antibodies, indicating suitability of camel vaccinations in disease-endemic areas as a promising approach to control infection.

CELL-MEDIATED AND HUMORAL IMMUNITY DURING COVID-19 IN THE REPUBLIC OF CRIMEA

The COVID-19 (coronavirus disease 2019) pandemic has spurred the development ofhighly effective quantitative methods for assessing the adaptive immune response to the SARS-CoV-2 (severe acute respiratory syndrome-related coronavirus 2) virus. In order to assess the humoralcomponentofthe immune response, various methods for detecting immunoglobulins A, M, Gare widely used. ELISPOTseems to be the most accessible and effective method to assess the level of T cells that specifically respond to the SARS-CoV-2 virus antigens. The aim. To assess cell-mediated and humoral immunity in COVID-19 in residents of the Republic of Crimea. Methods. The study was performed on 24 volunteers: the presence of coronavirus antibodies was determinedby ELISAmethod, andthe presence ofcontact withcoronavirus proteins - by the ELISPOT "TigraTest® SARS-CoV-2” method (Generium, Russia). For retrospective study of humoral immunity in the population, we assessed 10 000 ELISA tests (ECOlab IgM and IgG, Russia) performed in our laboratory for the period from July 2020 to January 2022. Results. The results show the effectiveness of using the ELISPOT method to detect latent forms of coronavirus infection. It is important to note that there is statistically significant relationship between the timing of the disease and the number of spots in both antigen panels. After vaccination against SARS-CoV-2, cell-mediated immunity lasts up to 6 months or more. Conclusions. As a result of the study, it was found that during 2021, the level of immunization of the population of the Republic of Crimea against COVID-19 has significantly increased;the proportion of residents who have positive IgG test has increasedfrom 27%to 87%. The results ofELISPOTstudies using a setofreagents for in vitro detection ofbloodT-lymphocytes thatspecifically respondto SARS-COV-2 virus antigens ("TigraTest® SARS-CoV-2”) showed that this method is more sensitive than ELISA in detecting latent diseases. © 2023 Voprosy Literatury. All rights reserved.

Impaired SARS-CoV-2 specific T-cell response in patients with severe COVID-19.

Cellular immune responses are of pivotal importance to understand SARS-CoV-2 pathogenicity. Using an enzyme-linked immunosorbent spot (ELISpot) interferon-γ release assay with wild-type spike, membrane and nucleocapsid peptide pools, we longitudinally characterized functional SARS-CoV-2 specific T-cell responses in a cohort of patients with mild, moderate and severe COVID-19. All patients were included before emergence of the Omicron (B.1.1.529) variant. Our most important finding was an impaired development of early IFN-γ-secreting virus-specific T-cells in severe patients compared to patients with moderate disease, indicating that absence of virus-specific cellular responses in the acute phase may act as a prognostic factor for severe disease. Remarkably, in addition to reactivity against the spike protein, a substantial proportion of the SARS-CoV-2 specific T-cell response was directed against the conserved membrane protein. This may be relevant for diagnostics and vaccine design, especially considering new variants with heavily mutated spike proteins. Our data further strengthen the hypothesis that dysregulated adaptive immunity plays a central role in COVID-19 immunopathogenesis.

INTRA-LABORATORY VALIDATED "TIGRATEST (R) SARS-CoV-2" - TEST ASSESSING RELEASE OF INTERFERON GAMMA IN VITRO TO IDENTIFY PERIPHERAL BLOOD T-LYMPHOCYTES SPECIFICALLY RESPONDING AGAINST SARS-CoV-2 VIRUS ANTIGENS

SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), also known as 2019-nCoV (2019-Novel Coronavirus) is a strain of coronavirus from the genus Betacoronavirus, discovered in China at the end of 2019 in patients with pneumonia. "Coronavirus infection COVID-19" (COrona VIrus Disease 2019) caused by coronavirus 2019 (COVID-19) has spread around the world at a very fast pace, with death toll exceeding more than 5.2 million people worldwide. Limited success in developing new drugs as well as use of existing drugs for the treatment of COVID-19 resulted in situation when the main prevention measures for a long time were based on testing and isolation of sick subjects, which started to reverse due to vaccination. Monitoring the formation of humoral and T-cell population immunity against the SARS-CoV-2 virus during the COVID-19 pandemic is a necessary element for epidemiological surveillance. ELISA-based methods are widely used to assess humoral immunity, and various test systems including ELISPOT (Enzyme-Linked ImmunoSpot) are used to analyze cellular immunity. The ELISPOT assay is a highly sensitive and specific method for quantifying individual cytokine-secreting T cells after being stimulated with a specific antigen. "TigraTest (R) SARS-CoV-2" Test assessing release of interferon gamma in vitro to detect peripheral blood T-lymphocytes that specifically respond to the SARS-CoV-2 virus antigens manufactured by GENERIUM JSC, is created on the ELISPOT platform. This study describes the procedure for laboratory validation of this test system to analyze the following parameters: specificity of antibody pair, effect of interfering substances, sensitivity and specificity, precision, stability of blood samples till isolation of target cells. The developed test system showed high diagnostic sensitivity and specificity. The specificity of TigraTest (R) SARS-CoV-2 was 100%, the sensitivity for subjects immunized with the Gam-COVID-Vac vaccine (Sputnik V) was 91.67%, and the sensitivity in convalescent COVID-19 patients was 95.45%. At the same time, the data variability both during within and between series comparison did not exceed 25%, whereas 24-hour storage of peripheral blood samples at (18-25)degrees C after blood collection followed by isolation of target cells did not affect the test results.

Factors associated with vaccine-induced T cell immune responses against SARS-CoV-2 in kidney transplant recipients.

Vaccination against SARS-CoV-2 is an important prophylactic measure in kidney transplant recipients (KTRs), however, the immune response is often impaired. Here, we examined the T cell immune response against SARS-CoV-2 in 148 KTRs after three or four vaccine doses including 35 KTRs with subsequent SARS-CoV-2 infection. The frequency of spike-specific T cells was lower in KTRs compared to immunocompetent controls and correlated with the level of spike-specific antibodies. Positive predictors for detection of vaccine-induced T cells were detection of spike-specific antibodies, heterologous immunization with mRNA and a vector vaccine and longer time past transplant. In vaccinated KTRs with subsequent SARS-CoV-2 infection, the T-cell response was greatly enhanced and was significantly higher than in vaccinated KTRs without SARS-CoV-2 infection. Overall, the data show a correlation between impaired humoral and T-cell immunity to SARS-CoV-2 vaccination and provide evidence for greater robustness of hybrid immunity in KTRs.

Assessment of specific T-cell immunity in patients who have been ill and vaccinated against COVID-19.

Introduction. In the context of a pandemic of a new coronavirus infection (COVID-19), research on the peculiarities of the formation of an immune response to SARS-CoV-2 in patients who have been ill and vaccinated is of particular relevance. However, most studies are currently devoted to evaluating only the humoral link of immunity, and its cellular component remains insufficiently studied. The aim of the study was to evaluate the features of the formation and changes of the T-cell link of immunity in patients with a new coronavirus infection and vaccinated against this disease. Materials and methods. The study was performed on the basis of the clinical and diagnostic laboratory of the European Medical Center "UMMC-Health "LLC. Specific T-cell immunity was evaluated using ELISPOT technology. In the course of the study, 72 blood samples of employees of medical organizations were analyzed, including 26 from those who had a new coronavirus infection, 23 from persons who were intact according to COVID-19 before vaccination and 23 from the same employees after vaccination (<<Gam-Covid-Vac>>). In addition, each of the study participants was examined to determine specific class G antibodies (IgG) by solid-phase enzyme immunoassay using SARS-CoV-2-IgG-ELISA-BEST test systems (manufactured by VECTOR-BEST JSC). Results and discussion. In the group of patients (26 people), T-lymphocytes capable of specifically reacting to SARSCoV-2 antigens were detected in 100% of cases, even in individuals with IgG elimination. It should be noted that the response was more pronounced when meeting with M-and N-pepdids, compared with S-protein. 22 out of 23 COVID-19 intact individuals had no T-cell immunity to coronavirus infection before vaccination, but one employee had a response to 3 proteins-M, N, S, which indicates that he had previously encountered the SARS-CoV-2 virus. After vaccination with the drug "Gam-Covid-Vac", 22 (95.6%) employees revealed a T-cell response, while 21-only to S-protein, and an employee with a previously detected immune response-after vaccination, the response to M -, N-proteins remained almost at the same level, and the cellular response to S-peptide doubled. Conclusion. Thus, based on the results of the study, important materials were obtained on the peculiarities of the formation of a specific T-cell immune response to a new coronavirus infection. The obtained data provide a broader understanding of the immune response in new coronavirus infection in patients who have been ill and vaccinated and can be used in the future when planning preventive and anti-epidemic measures.Copyright © 2022 Interregional public organization Association of infectious disease specialists of Saint-Petersburg and Leningrad region (IPO AIDSSPbR). All rights reserved.

Assessment of specific T-cell immunity in patients who have been ill and vaccinated against COVID-19.

Introduction. In the context of a pandemic of a new coronavirus infection (COVID-19), research on the peculiarities of the formation of an immune response to SARS-CoV-2 in patients who have been ill and vaccinated is of particular relevance. However, most studies are currently devoted to evaluating only the humoral link of immunity, and its cellular component remains insufficiently studied. The aim of the study was to evaluate the features of the formation and changes of the T-cell link of immunity in patients with a new coronavirus infection and vaccinated against this disease. Materials and methods. The study was performed on the basis of the clinical and diagnostic laboratory of the European Medical Center "UMMC-Health "LLC. Specific T-cell immunity was evaluated using ELISPOT technology. In the course of the study, 72 blood samples of employees of medical organizations were analyzed, including 26 from those who had a new coronavirus infection, 23 from persons who were intact according to COVID-19 before vaccination and 23 from the same employees after vaccination (<<Gam-Covid-Vac>>). In addition, each of the study participants was examined to determine specific class G antibodies (IgG) by solid-phase enzyme immunoassay using SARS-CoV-2-IgG-ELISA-BEST test systems (manufactured by VECTOR-BEST JSC). Results and discussion. In the group of patients (26 people), T-lymphocytes capable of specifically reacting to SARSCoV-2 antigens were detected in 100% of cases, even in individuals with IgG elimination. It should be noted that the response was more pronounced when meeting with M-and N-pepdids, compared with S-protein. 22 out of 23 COVID-19 intact individuals had no T-cell immunity to coronavirus infection before vaccination, but one employee had a response to 3 proteins-M, N, S, which indicates that he had previously encountered the SARS-CoV-2 virus. After vaccination with the drug "Gam-Covid-Vac", 22 (95.6%) employees revealed a T-cell response, while 21-only to S-protein, and an employee with a previously detected immune response-after vaccination, the response to M -, N-proteins remained almost at the same level, and the cellular response to S-peptide doubled. Conclusion. Thus, based on the results of the study, important materials were obtained on the peculiarities of the formation of a specific T-cell immune response to a new coronavirus infection. The obtained data provide a broader understanding of the immune response in new coronavirus infection in patients who have been ill and vaccinated and can be used in the future when planning preventive and anti-epidemic measures.Copyright © 2022 Interregional public organization Association of infectious disease specialists of Saint-Petersburg and Leningrad region (IPO AIDSSPbR). All rights reserved.

Evaluation of QuantiFERON SARS-CoV-2 interferon-γ release assay following SARS-CoV-2 infection and vaccination.

T cells are important in preventing severe disease from SARS-CoV-2, but scalable and field-adaptable alternatives to expert T-cell assays are needed. The interferon-gamma release assay QuantiFERON platform was developed to detect T-cell responses to SARS-CoV-2 from whole blood with relatively basic equipment and flexibility of processing timelines. Forty-eight participants with different infection and vaccination backgrounds were recruited. Whole blood samples were analysed using the QuantiFERON SARS-CoV-2 assay in parallel with the well-established 'Protective Immunity from T Cells in Healthcare workers' (PITCH) ELISpot, which can evaluate spike-specific T-cell responses. The primary aims of this cross-sectional observational cohort study were to establish if the QuantiFERON SARS-Co-V-2 assay could discern differences between specified groups and to assess the sensitivity of the assay compared with the PITCH ELISpot. The QuantiFERON SARS-CoV-2 distinguished acutely infected individuals (12-21 days post positive PCR) from naïve individuals (P < 0.0001) with 100% sensitivity and specificity for SARS-CoV-2 T cells, whilst the PITCH ELISpot had reduced sensitivity (62.5%) for the acute infection group. Sensitivity with QuantiFERON for previous infection was 12.5% (172-444 days post positive test) and was inferior to the PITCH ELISpot (75%). Although the QuantiFERON assay could discern differences between unvaccinated and vaccinated individuals (55-166 days since second vaccination), the latter also had reduced sensitivity (44.4%) compared to the PITCH ELISpot (66.6%). The QuantiFERON SARS-CoV-2 assay showed potential as a T- cell evaluation tool soon after SARS-CoV-2 infection but has lower sensitivity for use in reliable evaluation of vaccination or more distant infection.

Anti-dsDNA B-Cell ELISpot as a Monitoring and Flare Prediction Tool in SLE Patients.

Anti-dsDNA autoantibodies quantification and complement levels are widely used to monitor disease activity in systemic lupus erythematosus (SLE). However, better biomarkers are still needed. We hypothesised whether the dsDNA antibody-secreting B-cells could be a complementary biomarker in disease activity and prognosis of SLE patients. Fifty-two SLE patients were enrolled and followed for up to 12 months. Additionally, 39 controls were included. An activity cut-off (comparing active and non-active patients according to clinical SLEDAI-2K) was established for SLE-ELISpot, chemiluminescence and Crithidia luciliae indirect immunofluorescence tests (≥11.24, ≥374.1 and ≥1, respectively). Assays performances together with complement status were compared regarding major organ involvement at the inclusion and flare-up risk prediction after follow-up. SLE-ELISpot showed the best performance in identifying active patients. High SLE-ELISpot results were associated with haematological involvement and, after follow-up, with an increased hazard ratio for disease flare-up (3.4) and especially renal flare (6.5). Additionally, the combination of hypocomplementemia and high SLE-ELISpot results increased those risks up to 5.2 and 32.9, respectively. SLE-ELISpot offers complementary information to anti-dsDNA autoantibodies to evaluate the risk of a flare-up in the following year. In some cases, adding SLE-ELISpot to the current follow-up protocol for SLE patients can improve clinicians' personalised care decisions.

Persistent memory despite rapid contraction of circulating T Cell responses to SARS-CoV-2 mRNA vaccination.

Introduction: While antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined. Methods: Interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD). Results: In two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination. Discussion: Overall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

T-Cell Immunity in COVID-19-Recovered Individuals and Individuals Vaccinated with the Combined Vector Vaccine Gam-COVID-Vac.

The COVID-19 pandemic has required extensive research on the new coronavirus SARS-CoV-2 and the creation of new highly effective vaccines. The presence of T-cells in the body that respond to virus antigens suggests adequate antiviral immunity. We investigated T-cell immunity in individuals who recovered from mild and moderate COVID-19 and in individuals vaccinated with the Gam-COVID-Vac combined vector vaccine. The ELISPOT method was used to determine the number of T-cells responding with IFN-γ synthesis to stimulation by peptides containing epitopes of the S-protein or N-, M-, ORF3, and ORF7 proteins, using peripheral blood mononuclear cells (PBMCs). At the same time, the multiplex method was used to determine the accumulation of IFN-γ and other cytokines in the culture medium. According to the data obtained, the proportion of positive conclusions about the T-cell immune response to SARS-CoV-2 antigens in control, recovered, and vaccinated individuals was 12%, 70%, and 52%, respectively. At the same time, more than half of the vaccinated individuals with a T-cell response were sensitized to the antigens of N-, M-, ORF3, and ORF7 proteins not produced by Gam-COVID-Vac, indicating a high likelihood of asymptomatic SARS-CoV-2 infection. Increased IFN-γ release by single sensitized T-cells in response to specific stimulation in recovered and vaccinated individuals did not result in the accumulation of this and other cytokines in the culture medium. These findings suggest a balance between cytokine production and utilization by immunocompetent cells as a prerequisite for providing a controlled cytokine signal and avoiding a "cytokine storm".

Coronavirus-Specific Antibody and T Cell Responses Developed after Sputnik V Vaccination in Patients with Chronic Lymphocytic Leukemia.

The clinical course of the new coronavirus disease 2019 (COVID-19) has shown that patients with chronic lymphocytic leukemia (CLL) are characterized by a high mortality rate, poor response to standard treatment, and low virus-specific antibody response after recovery and/or vaccination. To date, there are no data on the safety and efficacy of the combined vector vaccine Sputnik V in patients with CLL. Here, we analyzed and compared the magnitudes of the antibody and T cell responses after vaccination with the Sputnik V vaccine among healthy donors and individuals with CLL with different statuses of preexposure to coronavirus. We found that vaccination of the COVID-19-recovered individuals resulted in the boosting of pre-existing immune responses in both healthy donors and CLL patients. However, the COVID-19-naïve CLL patients demonstrated a considerably lower antibody response than the healthy donors, although they developed a robust T cell response. Regardless of the previous infection, the individuals over 70 years old demonstrated a decreased response to vaccination, as did those receiving anti-CD20 therapy. In summary, we showed that Sputnik V, like other vaccines, did not induce a robust antibody response in individuals with CLL; however, it provided for the development of a significant anti-COVID-19 T cell response.

ELISPOT ASSAY OF THE SARS-COV-2 SPECIFIC T CELLS IMMUNE RESPONSE

The COVID-19 pandemichas stimulate dinterestin the developmen to fbiotechnology, as well as in the search for new solutions in the diagnostics of immune processes. The response of immunoglobulins A, Mand Ghada significantrole in the assessment of virus-specific immune responses. Later, it was understood that for a comprehensive assessment of adaptive immunity processes, it is reasonable to study its cellular component. One of the most affordable methods for assessing T cell immunity, which has proven itself in the diagnosis of other infectious diseases, such as latent tuberculosis infection, is IGRA ELISPOT. The aim of the study. To determine SARS-СoV-2 specific immune response ofT lymphocytes in vitro in the peripheral blood of volunteers from various groups using IGRA ELISPOT method. We evaluated the applicability of the method to assess T cell immune response to infection andvaccination. In addition, we determinedthe duration of the maintenance period of the SARS-CoV-2 specific T cells immune response induced by vaccination. Materials andmethods. The study was carriedout on venous bloodsamples ofvolunteers from three groups: 1) hospital patients withCOVID-19;2) COVID-19convalescents;3) vaccinatedagainst COVID-19. The T cellimmune response was assessed using the TigraTest® SARS-CoV-2 test system, which determines in vitro the number of T cells secreting interferon-gamma in response to stimulation with SARS-СoV-2 peptides in two antigens panels: 1) peptides of the spike protein (S);2) peptides of N, M, Orf3a and Orf7a proteins. Conclusion. The IGRA ELISPOT assay is a specific and sensitive tool in the assessment of T cell immunity to the SARS-CoV-2 virus. The method makes it possible to assess SARS-CoV-2 specific T cell responses induced both by natural encounter with the pathogen and by vaccination. It is advisable to use the method in routine practice for comprehensive assessment of immunity to SARS-CoV-2. © 2022 Acta Biomedica Scientifica. All rights reserved.

HLA-A*01:01 allele diminishing in COVID-19 patients population associated with non-structural epitope abundance in CD8+ T-cell repertoire.

In mid-2021, the SARS-CoV-2 Delta variant caused the third wave of the COVID-19 pandemic in several countries worldwide. The pivotal studies were aimed at studying changes in the efficiency of neutralizing antibodies to the spike protein. However, much less attention was paid to the T-cell response and the presentation of virus peptides by MHC-I molecules. In this study, we compared the features of the HLA-I genotype in symptomatic patients with COVID-19 in the first and third waves of the pandemic. As a result, we could identify the diminishing of carriers of the HLA-A*01:01 allele in the third wave and demonstrate the unique properties of this allele. Thus, HLA-A*01:01-binding immunoprevalent epitopes are mostly derived from ORF1ab. A set of epitopes from ORF1ab was tested, and their high immunogenicity was confirmed. Moreover, analysis of the results of single-cell phenotyping of T-cells in recovered patients showed that the predominant phenotype in HLA-A*01:01 carriers is central memory T-cells. The predominance of T-lymphocytes of this phenotype may contribute to forming long-term T-cell immunity in carriers of this allele. Our results can be the basis for highly effective vaccines based on ORF1ab peptides.

Profiling the B cell immune response elicited by vaccination against the respiratory virus SARS-CoV-2.

B cells play a fundamental role in host defenses against viral infections. Profiling the B cell response elicited by SARS-CoV-2 vaccination, including the generation and persistence of antigen-specific memory B cells, is essential for improving the knowledge of vaccine immune responsiveness, beyond the antibody response. mRNA-based vaccines have shown to induce a robust class-switched memory B cell response that persists overtime and is boosted by further vaccine administration, suggesting that memory B cells are critical in driving a recall response upon re-exposure to SARS-CoV-2 antigens. Here, we focus on the role of the B cell response in the context of SARS-CoV-2 vaccination, offering an overview of the different technologies that can be used to identify spike-specific B cells, characterize their phenotype using machine learning approaches, measure their capacity to reactivate following antigen encounter, and tracking the maturation of the B cell receptor antigenic affinity.

Specific Antibody and the T-Cell Response Elicited by BNT162b2 Boosting After Two ChAdOx1 nCoV-19 in Common Variable Immunodeficiency.

Common variable immunodeficiency (CVID) patients have markedly decreased immune response to vaccinations. In this study we evaluated humoral and T cell-mediated responses against severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2) with additional flow cytometric changes in CVID patients receiving booster vaccination with BNT162b2 after two ChAdOx1 nCoV-19. The BNT162b2 vaccine raised the anti-spike protein S immunoglobulin G over the cut-off value from 70% to 83% in CVID, anti-neutralizing antibody had been raised over a cut-off value from 70% to 80% but levels after boosting were significantly less in both tests than in healthy controls (*p=0.02; **p=0.009 respectively). Anti-SARS-CoV-2 immunoglobulin A became less positive in CVID after boosting, but the difference was not significant. The cumulative interferon-γ positive T cell response by ELISpot was over the cut-off value in 53% of the tested individuals and raised to 83% after boosting. This and flow cytometric control of cumulative CD4+ and CD8+ virus-specific T cell absolute counts in CVID were also statistically not different from healthy individuals after boosting. Additional flow cytometric measures for CD45+ lymphocytes, CD3+, and CD19+ cells have not shown significant differences from controls except for lower CD4+T cell counts at both time points (**p=0.003; **p=0.002), in parallel CD4+ virus-specific T-cell ratio was significantly lower in CVID patients at the first time point (*p: 0.03). After boosting, in more than 33% of both CVID patients and also in their healthy controls we detected a decrease in absolute CD45+, CD3+, CD3+CD4+, and CD3+CD8+, CD19+, and CD16+56+ cell counts. CD16+CD56+ cell counts were significantly lower compared to controls before and after boosting (*p=0.02, *p=0.02). CVID patients receiving immunosuppressive therapy throughout the previous year or autologous stem cell transplantation two years before vaccination had worse responses in anti-spike, anti-neutralizing antibody, CD3+CD4+T, CD19+ B, and natural killer cell counts than the whole CVID group. Vaccinations had few side effects. Based on these data, CVID patients receiving booster vaccination with BNT162b2 after two ChadOx1 can effectively elevate the levels of protection against COVID-19 infection, but the duration of the immune response together with COVID-19 morbidity data needs further investigation among these patients.

Antibody Levels Poorly Reflect on the Frequency of Memory B Cells Generated following SARS-CoV-2, Seasonal Influenza, or EBV Infection.

The scope of immune monitoring is to define the existence, magnitude, and quality of immune mechanisms operational in a host. In clinical trials and praxis, the assessment of humoral immunity is commonly confined to measurements of serum antibody reactivity without accounting for the memory B cell potential. Relying on fundamentally different mechanisms, however, passive immunity conveyed by pre-existing antibodies needs to be distinguished from active B cell memory. Here, we tested whether, in healthy human individuals, the antibody titers to SARS-CoV-2, seasonal influenza, or Epstein-Barr virus antigens correlated with the frequency of recirculating memory B cells reactive with the respective antigens. Weak correlations were found. The data suggest that the assessment of humoral immunity by measurement of antibody levels does not reflect on memory B cell frequencies and thus an individual's potential to engage in an anamnestic antibody response against the same or an antigenically related virus. Direct monitoring of the antigen-reactive memory B cell compartment is both required and feasible towards that goal.

Divergent SARS-CoV-2-specific T cell responses in intensive care unit workers following mRNA COVID-19 vaccination.

The cellular immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in response to full mRNA COVID-19 vaccination could be variable among healthy individuals. Studies based only in specific antibody levels could show an erroneous immune protection at long times. For that, we analyze the antibody levels specific to the S protein and the presence of SARS-CoV-2-specific T cells by ELISpot and AIM assays in intensive care unit (ICU) workers with no antecedents of COVID-19 and vaccinated with two doses of mRNA COVID-19 vaccines. All individuals were seronegative for the SARS-CoV-2 protein S before vaccination (Pre-v), but 34.1% (14/41) of them showed pre-existing T lymphocytes specific for some viral proteins (S, M and N). One month after receiving two doses of COVID-19 mRNA vaccine (Post-v1), all cases showed seroconversion with high levels of total and neutralizing antibodies to the spike protein, but six of them (14.6%) had no T cells reactive to the S protein. Specifically, they lack of specific CD8+ T cells, but maintain the contribution of CD4+ T cells. Analysis of the immune response against SARS-CoV-2 at 10 months after full vaccination (Post-v10), exhibited a significant reduction in the antibody levels (p<0.0001) and protein S-reactive T cells (p=0.0073) in all analyzed individuals, although none of the individuals become seronegative and 77% of them maintained a competent immune response. Thus, we can suggest that the immune response to SARS-CoV-2 elicited by the mRNA vaccines was highly variable among ICU workers. A non-negligible proportion of individuals did not develop a specific T cell response mediated by CD8+ T cells after vaccination, that may condition the susceptibility to further viral infections with SARS-CoV-2. By contrast, around 77% of individuals developed strong humoral and cellular immune responses to SARS-CoV-2 that persisted even after 10 months. Analysis of the cellular immune response is highly recommended for providing exact information about immune protection against SARS-CoV-2.

Time-dependent contraction of the SARS-CoV-2-specific T-cell responses in convalescent individuals.

Background: Adaptive immunity in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is decisive for disease control. Delayed activation of T cells is associated with a worse outcome in coronavirus disease 2019 (COVID-19). Although convalescent individuals exhibit solid T-cell immunity, to date, long-term immunity to SARS-CoV-2 is still under investigation. Objectives: We aimed to characterize the specific T-cell response on the basis of the in vitro recall of IFN-γ-producing cells to in silico-predicted peptides in samples from SARS-CoV-2 convalescent individuals. Methods: The sequence of the SARS-CoV-2 genome was screened, leading to the identification of specific and promiscuous peptides predicted to be recognized by CD4+ and CD8+ T cells. Next, we performed an in vitro recall of specific T cells from PBMC samples from the participants. The results were analyzed according to clinical features of the cohort and HLA diversity. Results: Our results indicated heterogeneous T-cell responsiveness among the participants. Compared with patients who exhibited mild symptoms, hospitalized patients had a significantly higher magnitude of response. In addition, male and older patients showed a lower number of IFN-γ-producing cells. Analysis of samples collected after 180 days revealed a reduction in the number of specific circulating IFN-γ-producing T cells, suggesting decreased immunity against viral peptides. Conclusion: Our data are evidence that in silico-predicted peptides are highly recognized by T cells from convalescent individuals, suggesting a possible application for vaccine design. However, the number of specific T cells decreases 180 days after infection, which might be associated with reduced protection against reinfection over time.