Adenovirus vaccines, particularly the COVID-19 Ad5-nCoV adenovirus vaccine, have emerged as promising tools in the fight against infectious diseases. In this study, we investigated the structure of the T cell response to the Spike protein of the SARS-CoV-2 virus used in the COVID-19 Ad5-nCoV adenoviral vaccine in a phase 3 clinical trial (NCT04540419). In 69 participants, we collected peripheral blood samples at four time points after vaccination or placebo injection. Sequencing of T cell receptor repertoires from Spike-stimulated T cell cultures at day 14 from 17 vaccinated revealed a more diverse CD4+ T cell repertoire compared to CD8+. Nevertheless, CD8+ clonotypes accounted for more than half of the Spike-specific repertoire. Our longitudinal analysis showed a peak T cell response at day 14, followed by a decline until month 6. Remarkably, multiple T cell clonotypes persisted for at least 6 months after vaccination, as demonstrated by ex vivo stimulation. Examination of CDR3 regions revealed homologous sequences in both CD4+ and CD8+ clonotypes, with major CD8+ clonotypes sharing high similarity with annotated sequences specific for the NYNYLYRLF peptide, suggesting potential immunodominance. In conclusion, our study demonstrates the immunogenicity of the Ad5-nCoV adenoviral vaccine and highlights its ability to induce robust and durable T cell responses. These findings provide valuable insight into the efficacy of the vaccine against COVID-19 and provide critical information for ongoing efforts to control infectious diseases.

T cells play a crucial role in combatting SARS-CoV-2 and forming long-term memory responses to this coronavirus. The emergence of SARS-CoV-2 variants that can evade T cell im-munity has raised concerns about vaccine efficacy and the risk of reinfection. Some SARS-CoV-2 T cell epitopes elicit clonally restricted CD8+ T cell responses characterized by T cell re-ceptors (TCRs) that lack structural diversity. Mutations in such epitopes can lead to loss of recognition by most T cells specific for that epitope, facilitating viral escape. Here, we studied an HLA-A2-restricted spike protein epitope (RLQ) that elicits CD8+ T cell responses in COVID-19 convalescent patients characterized by highly diverse TCRs. We previously reported the structure of an RLQ-specific TCR (RLQ3) with greatly reduced recognition of the most common natural variant of the RLQ epitope (T1006I). Opposite to RLQ3, TCR RLQ7 recog-nizes T1006I with even higher functional avidity than the WT epitope. To explain the ability of RLQ7, but not RLQ3, to tolerate the T1006I mutation, we determined structures of RLQ7 bound to RLQ-HLA-A2 and T1006I-HLA-A2. These complexes show that there are multiple structural solutions to recognizing RLQ and thereby generating a clonally diverse T cell response to this epitope that assures protection against viral escape and T cell clonal loss.

Background To determine the immunogenicity, efficacy, reactogenicity, and safety of a single dose of recombinant adenovirus type-5 vectored COVID-19 vaccine (Ad5-nCoV, 5 × 1010 viral particles per 0.5 mL dose), we conducted a single-dose, randomised, double-blind, placebo-controlled, parallel group (3:1 Ad5-nCoV:placebo), phase 3 trial (Prometheus). Methods From 11-September-2020 to 05-May-2021, across six sites in the Russian Federation, 496 participants were injected with either placebo or Ad5-nCoV expressing the full-length spike (S) protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Results Seroconversion (the primary endpoint) rates of 78.5% (95% CI: 73.9; 82.6) against receptor binding domain (RBD), 90.6% (95% CI: 87.2; 93.4) against S protein and 59.0% (95% CI: 53.3; 64.6) seroconversion of neutralising antibodies against SARS-CoV-2 at 28 days post-vaccination were observed. Geometric mean titres (GMTs) were also elevated for antibodies against the RBD (405 [95% CI: 366; 449]) and S protein (677 [95% CI: 608; 753]) compared to the GMT of neutralising antibodies against SARS-CoV-2 (16.7 [95% CI: 15.3; 18.3]). Using an IFN-γ ELISpot assay after stimulating the cells with recombinant S protein ectodomain we showed that the Ad5-nCoV vaccine induced the most robust cellular immune response on Days 14 and 28. Up to Day 28, the primary and all secondary endpoints of the Ad5-nCoV vaccine were statistically significant compared with the placebo (р<0.001). Systemic reactions were reported in 113 of 496 (22.8%) participants (Ad5-nCoV, 26.9%; Placebo, 10.5%), and local reactions were reported in 108 (21.8%) participants (Ad5-nCoV, 28.5%; Placebo, 1.6%). These were generally mild and resolved within 7 days after vaccination. Of the six serious adverse events reported, none of the events were vaccine related. There were no deaths or premature withdrawals. Conclusion A single-dose of Ad5-nCoV vaccine induced a marked specific humoral and cellular immune response with a favourable safety profile. Trial registration Trial registration: ClinicalTrials.gov: NCT04540419.

Topic: 22. Stem cell transplantation - Clinical Background: A large number of reviews is devoted to affection of different types of graft-versus-host disease (GvHD) prophylaxis regimens on the reconstitution of the T-cell immunity after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Association of the posttransplant cyclophosphamide (PT-CY) together with TCR αβ+/CD19+ cell depletion is associated with the deeper T cell deficiency after allo-HSCT. Cytomegalovirus (CMV) specific T lymphocytes transfusion as adoptive T cell therapy (ACT) can boost reconstitution of the CMV-specific immunity and provide long-term protection against CMV-infection. Aims: To evaluate the impact of ACT with using CMV-specific T cells on the reconstitution of CMV-specific T-cell immunity after allo-HSCT. Methods: Total 64 recipients of peripheral blood stem cells (PBSC) as the graft source after TCR αβ+/CD19+ cells depletion and PT-CY as GvHD prophylaxis were included in the analysis. Seventeen (26.5%) patients received ACT at the +30 day after the engraftment. All patients reached engraftment after allo-HSCT. Absolute count of CMV-specific T cells at +30, +90, +180 day after allo-HSCT in peripheral blood samples was estimated via flow cytometry analysis. The detection of CMV-specific T cells was performed by using tetramers formed from MHC class I monomers loaded with immunodominant epitopes of cytomegalovirus pp65 protein (HLA-A*02/NLV, HLA-B*07/TPR, HLA-B*07/RPH) and Streptavidin, R-Phycoerythrin conjugate (SAPE). We formed two groups due to the presence of the ACT after engraftment. These groups were balanced by two main factors affecting CMV-specific immunity: GvHD prophylaxis regimen and CMV-serologic status of Donor-Recipient pairs (D/ R). Results: As shown in figure 1 the absolute number of CMV-specific T cells had not significantly differed between groups before ACT (on day +30). After that on day +90 in the group with CMV-specific T cell transfusion the number of CMV-specific T cells was significantly higher compared to the group without ACT (p=0.013).Figure 1. The comparison of the absolute count of CMV-specific T cells (cell/µl) in peripheral blood samples at +30, +90, +180 day after allo-HSCT depending on the use of adoptive T cell therapy. Summary/Conclusion: Transfusion of donor CMV-specific T-lymphocytes after the engraftment from +30 day after allo-HSCT effectively boost the reconstruction of CMV-specific immunity in patients with highly aggressive GvHD prophylaxis regimens, including PT-CY and TCR αβ+/CD19+ cells depletion. Thus, tge presence of the ACT in prophylaxis protocols for management of CMV infection after allo-HSCT can accelerate virus‐specific immune reconstitution. Keywords: CMV infection, Adoptive immunotherapy, Stem cell transplant, T cell reconstitution

A significant share of allogeneic hematopoietic stem cell transplantations (allo-HSCT) results in the relapse of malignant disease. The T cell immune response to minor histocompatibility antigens (MiHAs) promotes a favorable graft-versus-leukemia response. The immunogenic MiHA HA-1 is a promising target for leukemia immunotherapy, as it is predominantly expressed in hematopoietic tissues and presented by the common HLA A*02:01 allele. Adoptive transfer of HA-1-specific modified CD8+ T cells could complement allo-HSCT from HA-1- donors to HA-1+ recipients. Using bioinformatic analysis and a reporter T cell line, we discovered 13 T cell receptors (TCRs) specific for HA-1. Their affinities were measured by the response of the TCR-transduced reporter cell lines to HA-1+ cells. The studied TCRs showed no cross-reactivity to the panel of donor peripheral mononuclear blood cells with 28 common HLA alleles. CD8+ T cells after endogenous TCR knock out and introduction of transgenic HA-1-specific TCR were able to lyse hematopoietic cells from HA-1+ patients with acute myeloid, T-, and B-cell lymphocytic leukemia (n = 15). No cytotoxic effect was observed on cells from HA-1- or HLA-A*02-negative donors (n = 10). The results support the use of HA-1 as a target for post-transplant T cell therapy.

Minor histocompatibility antigens (MiHAs) are polymorphic peptides on the cell surface derived from self-proteins that are capable to induce an immune response during allogeneic hematopoietic stem cells transplantation. Their presentation occurs in the context of the certain major histocompatibility complex (HLA – human leucocyte antigen) alleles. One of the most common HLA alleles is HLA-A*02:01. Accordingly, for a significant number of donors and recipients pairs, it is possible to use the MiHAs presented in the HLA-A*02:01 as a target for relapsed leukemia therapy. This review discusses the main known MiHAs presented in the context of HLA-A*02:01, their characteristics and approaches used for identification. The described approaches may be used to search for new MiHAs for immunotherapy.

T cells play a pivotal role in reducing disease severity during SARS-CoV-2 infection and formation of long-term immune memory. We studied 50 COVID-19 convalescent patients and found that T cell response was induced more frequently and persisted longer than circulating antibodies. We identified 756 clonotypes specific to nine CD8+ T cell epitopes. Some epitopes were recognized by highly similar public clonotypes. Receptors for other epitopes were extremely diverse, suggesting alternative modes of recognition. We tracked persistence of epitope-specific response and individual clonotypes for a median of eight months after infection. The number of recognized epitopes per patient and quantity of epitope-specific clonotypes decreased over time, but the studied epitopes were characterized by uneven decline in the number of specific T cells. Epitopes with more clonally diverse TCR repertoires induced more pronounced and durable responses. In contrast, the abundance of specific clonotypes in peripheral circulation had no influence on their persistence.

T cells play a vital role in combatting SARS-CoV-2 and forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T-cell receptors (TCRs) bound to their peptide-MHC targets is lacking. Here we determine the structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARSCoV-2 spike protein epitopes (YLQ and RLQ). The structures reveal the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of the TCRs to recognize natural variants of RLQ but not YLQ. Neither TCR recognizes homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit pan-coronavirus immunity.

The development of effective vaccines against SARS-CoV-2 remains a global health priority. Despite extensive use, the effects of Sputnik V on B cell immunity need to be explored in detail. We performed comprehensive profiling of humoral and B cell responses in a cohort of vaccinated subjects (n = 22), and demonstrate that Sputnik vaccination results in robust B cell immunity. We show that B memory cell (MBC) and antibody responses to Sputnik V were heavily dependent on whether the vaccinee had a history of SARS-CoV-2 infection or not. 85 days after the first dose of the vaccine, ex vivo stimulated MBCs from the vast majority of Sputnik V vaccinees produced antibodies that robustly neutralized the Wuhan Spike-pseudotyped lentivirus. MBC-derived antibodies from all previously infected and some of the naïve vaccine recipients could also cross-neutralize Beta (B.1.351) variant of SARS-CoV-2. Virus-neutralizing activity of MBC-derived antibodies correlated well with that of the serum antibodies, suggesting the interplay between the MBC and long-lived plasma cell responses. Thus, our in-depth analysis of MBC responses in Sputnik V vaccinees complements traditional serological approaches and may provide important outlook into future B cell responses upon re-encounter with the emerging variants of SARS-CoV-2.

In a prospective study involving 5340 individuals, humoral and cellular responses revealed magnitude-dependent protection from COVID-19. Antibodies alone significantly decreased infection rates; isolated cellular response provided an intermediate level of protection. The lowest COVID-19 incidence was in the double-positive group. Background During the ongoing coronavirus disease 2019 (COVID-19) pandemic, many individuals were infected with and have cleared the virus, developing virus-specific antibodies and effector/memory T cells. An important unanswered question is what levels of T-cell and antibody responses are sufficient to protect from the infection. Methods In 5340 Moscow residents, we evaluated anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin M (IgM)/immunoglobulin G (IgG) titers and frequencies of the T cells specific to the membrane, nucleocapsid, and spike proteins of SARS-CoV-2, using interferon gamma (IFN-gamma) enzyme-linked immunosorbent spot (ELISpot) assay. Additionally, we evaluated the fractions of virus-specific CD4(+) and CD8(+) T cells using intracellular staining of IFN-gamma and interleukin 2 followed by flow cytometry. We analyzed the COVID-19 rates as a function of the assessed antibody and T-cell responses, using the Kaplan-Meier estimator method, for up to 300 days postinclusion. Results We showed that T-cell and antibody responses are closely interconnected and are commonly induced concurrently. Magnitudes of both responses inversely correlated with infection probability. Individuals positive for both responses demonstrated the highest levels of protectivity against the SARS-CoV-2 infection. A comparable level of protection was found in individuals with antibody response only, whereas the T-cell response by itself granted only intermediate protection. Conclusions We found that the contribution of the virus-specific antibodies to protection against SARS-CoV-2 infection is more pronounced than that of the T cells. The data on the virus-specific IgG titers may be instructive for making decisions in personalized healthcare and public anti-COVID-19 policies.

Despite measures taken world-wide, the coronavirus disease 2019 (COVID-19) pandemic continues. Because efficient antiviral drugs are not yet widely available, vaccination is the best option to control the infection rate. Although this option is obvious in the case of COVID-19-naive individuals, it is still unclear when individuals who have recovered from a previous SARS-CoV-2 infection should be vaccinated and whether the vaccination raises immune responses against the coronavirus and its novel variants. In this study, we collected peripheral blood from 84 healthy human donors of different COVID-19 status who were vaccinated with the Sputnik Light vaccine and measured the dynamics of the Ab and T cell responses, as well as the virus-neutralizing activity (VNA) in serum, against two SARS-CoV-2 variants, B.1.1.1 and B.1.617.2. We showed that vaccination of individuals previously exposed to the virus considerably boosts the existing immune response. In these individuals, receptor-binding domain (RBD)-specific IgG titers and VNA in serum were already elevated on the 7th day after vaccination, whereas COVID-19-naive individuals developed the Ab response and VNA mainly 21 d postvaccination. Additionally, we found a strong correlation between RBD-specific IgG titers and VNA in serum, and according to these data vaccination may be recommended when the RBD-specific IgG titers drop to 142.7 binding Ab units/ml or below. In summary, the results of the study demonstrate that vaccination is beneficial for both COVID-19-naive and recovered individuals, especially since it raises serum VNA against the B.1.617.2 variant, one of the five SARS-CoV-2 variants of concern.

Background: Background: Cytomegalovirus infection (CMV infection) is the most frequent and life-threatening complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The development of CMV infection is controlled by T-cell immunity. The type of the immunosuppressive regimen is the most important reason of decreased CMV-specific CD8+ immune response following allo-HSCT. Aims: Aims: In our study we try to assess the influence of different types of immunosuppressive regimens on recovery of the CMV-specific CD8+ T cells in patients after allo-HSCT. Methods: Methods: Analysis of peripheral blood CMV-specific CD8+ T cells from 108 patients performed by flow cytometry at + 30, +90, +180 days after allo-HSCT. We used fluorochrome-labeled monoclonal antibodies against CD3, CD8, CD45 molecules, viability reagent and tetramers which were formed from MHC class I monomers loaded with immunodominant epitopes of cytomegalovirus pp65 protein (HLA-A*0201/NLV, HLA-B*0702/TPR, Summary/Conclusion: Summary/Conclusion: Our results show that patients who received PT-Cy as GVHD prophylaxis and T-cell depletion-grafts have highest risk for developing CMV infection after allo-HSCT. The management of CMV infection by monitoring of CMV by polymerase chain reaction, use of antiviral prophylaxis and prompt use of pre-emptive therapy are very important for such patients’ groups.

Abstract As a part of phase 3 clinical trial of a single-dose adenoviral COVID-19 vaccine, we evaluated the dynamics and clonal structure of T cell response to the SARS-CoV-2 Spike protein. T cell response measured in 50 donors by IFNg ELISpot after Spike stimulation peaked on the 14th day (d14) after vaccination. Flow cytometry demonstrated that the response was skewed to CD4+ cells. To isolate Spike-specific T cells we performed short-term T cell expansion of d14 samples (n=17 donors) with the recombinant Spike, restimulated and FACS sorted IFNg+ cells. T cell receptor (TCR) beta chain sequencing identified from 36 to 629 Spike-specific clones (median 186) per individual. CD4+ response was more clonal than CD8+ (median 116 and 17 respectively) but CD8+ occupied a larger share of the total TCR repertoire (median 0.25% for CD4+ and 0.52% for CD8+) as a result of the larger average clone size at d14 (3.4×10−5 for CD8+ vs. 6.6×10−6 for CD4+). Both the number and total share of Spike-specific clones in the TCR repertoire decreased at day 28 and further at day 180. At 6 months a median of 1 (0 to 18) of the initial Spike-specific clones were detectable in the peripheral blood. This number increased to 39 (9 to 105) as a result of T cell expansion. For 14 donors we also performed rapid expansion of T cells sampled at d14 with 11 Spike-epitopes (9 MHC I and 2 MHC II) followed by sorting of activated cells and TCR sequencing. We identified on average 41 unique epitope-specific clones (2 to 141). They were mostly undetectable in the peripheral blood at 6 months (0 to 8 clones), but their numbers increased to the median of 7 clones (0 to 35) after cultivation with Spike. Overall this demonstrates the induction of polyclonal and stable T cell response by a single injection of adenoviral vaccine. The work was supported by the Russian Science Foundation grant 20-15-00395.

Memory T cells play a crucial role in acceleration of immune response during reinfection with the same pathogen. Since the beginning of the COVID-19 pandemic, attention has been focused on determining the epitopes of SARS-CoV-2 that give rise to long-lived memory T cells. We examined the immune response to 12 immunogenic CD8+ epitopes in 26 COVID-19 convalescent patients. Paired blood samples were collected shortly after infection and approximately 8 months after. Using ex vivo T cell expansions with a subsequent MHC-tetramer+ cell-sorting and T cell receptor beta-chain (TCRβ) sequencing we studied the epitope-specific CD8+ response on the clonal level. In total in both time points we identified 756 epitope-specific TCRβ sequences. Nine epitopes were immunodominant while five immunogenic epitopes (YLQ, KCY, LLY, KTF and ALW) yielded response in 100% of tested individuals. This coincided with a high number of clones per individual specific to these epitopes (median = 16.5). Despite the very low frequency of specific cells in the total repertoire most (75.4%) of responses were retained at 8-month post-infection. The median number of clones for the top five most immunogenic epitopes dropped to 6. The TCRβ repertoires of T cells specific to the studied epitopes were characterized by different degrees of CDR3 homology. LLY and YLQ-specific TCRβ had the highest degree of mutual similarity while KCY and KTF-specific TCRβ were the most diverse. Our data demonstrates that despite the overall decrease of T cell reactivity and clonality most epitopes are still recognized due to the ability of memory SARS-CoV-2-specific CD8+ cells to survive over prolonged periods of time. The work was supported by the Russian Science Foundation grant 20-15-00395.

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.

The ongoing COVID-19 pandemic calls for more effective diagnostic tools. T cell response assessment serves as an independent indicator of prior COVID-19 exposure while also contributing to a more comprehensive characterization of SARS-CoV-2 immunity. In this study, we systematically assessed the immunogenicity of 118 epitopes with immune cells collected from multiple cohorts of vaccinated, convalescent, healthy unexposed, and SARS-CoV-2???exposed donors. We identified 75 immunogenic epitopes, 24 of which were immunodominant. We further confirmed HLA restriction for 49 epitopes and described association with more than 1 HLA allele for 14 of these. Exclusion of 2 cross-reactive epitopes that generated a response in prepandemic samples left us with a 73-epitope set that offered excellent diagnostic specificity without losing sensitivity compared with full-length antigens, and this evoked a robust cross-reactive response. We subsequently incorporated this set of epitopes into an in vitro diagnostic Corona-T-test, which achieved a diagnostic accuracy of 95% in a clinical trial. In a cohort of asymptomatic seronegative individuals with a history of prolonged SARS-CoV-2 exposure, we observed a complete absence of T cell response to our epitope panel. In combination with strong reactivity to full-length antigens, this suggests that a cross-reactive response might protect these individuals.

Since multiple different T-cell receptor (TCR) sequences can bind to the same peptide-MHC combination and the number of TCR-sequences that can theoretically be generated even exceeds the number of T cells in a human body, the likelihood that many public identical (PUB-I) TCR-sequences frequently contribute to immune responses has been estimated to be low. Here, we quantitatively analyzed the TCR-repertoires of 190 purified virus-specific memory T-cell populations, directed against 21 epitopes of Cytomegalovirus, Epstein-Barr virus and Adenovirus isolated from 29 healthy individuals, and determined the magnitude, defined as prevalence within the population and frequencies within individuals, of PUB-I TCR and of TCR-sequences that are highly-similar (PUB-HS) to these PUB-I TCR-sequences. We found that almost one third of all TCR nucleotide-sequences represented PUB-I TCR amino-acid (AA) sequences and found an additional 12% of PUB-HS TCRs differing by maximally 3 AAs. We illustrate that these PUB-I and PUB-HS TCRs were structurally related and contained shared core-sequences in their TCR-sequences. We found a prevalence of PUB-I and PUB-HS TCRs of up to 50% among individuals and showed frequencies of virus-specific PUB-I and PUB-HS TCRs making up more than 10% of each virus-specific T-cell population. These findings were confirmed by using an independent TCR-database of virus-specific TCRs. We therefore conclude that the magnitude of the contribution of PUB-I and PUB-HS TCRs to these virus-specific T-cell responses is high. Because the T cells from these virus-specific memory TCR-repertoires were the result of successful control of the virus in these healthy individuals, these PUB-HS TCRs and PUB-I TCRs may be attractive candidates for immunotherapy in immunocompromised patients that lack virus-specific T cells to control viral reactivation.

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.