130 Immunogenic potential of chimeric antigen receptor (CAR)-engineered T cells expressing inducible nuclease-deactivated SpCas9 (dCas9)

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A143-A143
Author(s):  
Dharmeshkumar Patel ◽  
Dharmeshkumar Patel ◽  
Angshumala Goswami ◽  
Vitaly Balan ◽  
Zhifen Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Peptide Sequence ◽  
T Cell Epitopes ◽  
Cell Responses ◽  
Car T Cells ◽  
Car T

BackgroundThe application of CRISPR-Cas9 for personalized medicine is potentially revolutionary for the treatment of several diseases including cancer. However, the bacterial origin of the Cas9 protein raises concerns about immunogenicity. Recent ELISA-based assays detected antibodies against Cas9 from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in 5–10% of sera from 343 normal healthy individuals.1,2 SpCas9-specific memory CD8 T cell responses were not demonstrated in those individuals. To date, there are no conclusive studies assessing whether CRISPR-Cas9-modified CAR-T could raise CD8 T cell-mediated immunogenicity in humans. Refuge CAR-T cell platform employs an inducible, non-gene editing, nuclease deactivated Cas9 (dCas9) to modulate gene expression in response to external stimuli such as antigen-dependent CAR signaling to suppress PD-1 expression.MethodsIn the present study, we analyzed two putative HLA-A*02:01 and two HLA-B*07:02-associated SpCas9 T cell epitopes. The candidate epitopes were derived from a prediction algorithm that incorporates T cell receptor contact residue hydrophobicity and HLA binding affinity. We engaged in-vitro sensitization (IVS) assay to identify immunogenic potential of dCas9 peptides.ResultsAutologous IVS assay of T cells in two healthy donor PBMCs identified CD8-T cell responses after two rounds of stimulation against only one HLA-A*02:01-associated Cas9 peptide (sequence NLIALSLGL) P1– while the other candidate epitopes did not elicit any response. Dextramer analysis demonstrated that 15% of CD8+ T cells were specific for P1 and ~11% of CD8+ cells produced INFG upon challenge with P1-loaded T2 cells.ConclusionsOur in-vitro sensitization assay was able to demonstrate that dCas9 epitope P1 is immunogenic and may elicit adaptive immune response against gene edited CAR-T cells. Endogenous processing and presentation of P1 and other putative epitopes by Refuge CAR-T cells are currently being analyzed.AcknowledgementsRefuge Biotechnologies Inc. Menlo Park, California, 94025Trial RegistrationN/AEthics ApprovalN/AConsentN/AReferencesSimhadri VL, McGill J, McMahon S, Wang J, Jiang H, Sauna ZE. Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population. Mol Ther Methods Clin Dev 2018;10:105–112. Published 2018 Jun 15. doi:10.1016/j.omtm.2018.06.006Ferdosi SR, Ewaisha R, Moghadam F, et al. Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes. Nat Commun2019;10(1):1842. Published 2019 Apr 23. doi:10.1038/s41467-019-09693-x

scholarly journals Protection against Vaccinia Virus Challenge by CD8 Memory T Cells Resolved by Molecular Mimicry

2006 ◽  
Vol 81 (2) ◽  
pp. 934-944 ◽  
Author(s):  
Markus Cornberg ◽  
Brian S. Sheridan ◽  
Frances M. Saccoccio ◽  
Michael A. Brehm ◽  
Liisa K. Selin
Keyword(s):  
T Cells ◽  
T Cell ◽  
Vaccinia Virus ◽  
Cd8 T Cells ◽  
Protective Immunity ◽  
Molecular Mimicry ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
T Cell Epitopes ◽  
Cell Responses

ABSTRACT Live vaccinia virus (VV) vaccination has been highly successful in eradicating smallpox. However, the mechanisms of immunity involved in mediating this protective effect are still poorly understood, and the roles of CD8 T-cell responses in primary and secondary VV infections are not clearly identified. By applying the concept of molecular mimicry to identify potential CD8 T-cell epitopes that stimulate cross-reactive T cells specific to lymphocytic choriomeningitis virus (LCMV) and VV, we identified after screening only 115 peptides two VV-specific immunogenic epitopes that mediated protective immunity against VV. An immunodominant epitope, VV-e7r130, did not generate cross-reactive T-cell responses to LCMV, and a subdominant epitope, VV-a11r198, did generate cross-reactive responses to LCMV. Infection with VV induced strong epitope-specific responses which were stable into long-term memory and peaked at the time virus was cleared, consistent with CD8 T cells assisting in the control of VV. Two different approaches, direct adoptive transfer of VV-e7r-specific CD8 T cells and prior immunization with a VV-e7r-expressing ubiquitinated minigene, demonstrated that memory CD8 T cells alone could play a significant role in protective immunity against VV. These studies suggest that exploiting cross-reactive responses between viruses may be a useful tool to complement existing technology in predicting immunogenic epitopes to large viruses, such as VV, leading to a better understanding of the role CD8 T cells play during these viral infections.

BK Polyomavirus–Specific CD8 T-Cell Expansion In Vitro Using 27mer Peptide Antigens for Developing Adoptive T-Cell Transfer and Vaccination

2020 ◽  
Author(s):  
Maud Wilhelm ◽  
Amandeep Kaur ◽  
Marion Wernli ◽  
Hans H Hirsch
Keyword(s):  
T Cells ◽  
T Cell ◽  
Kidney Transplant ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Bk Polyomavirus

Abstract Background BK polyomavirus (BKPyV) remains a significant cause of premature kidney transplant failure. In the absence of effective antivirals, current treatments rely on reducing immunosuppression to regain immune control over BKPyV replication. Increasing BKPyV-specific CD8 T cells correlate with clearance of BKPyV DNAemia in kidney transplant patients. We characterized a novel approach for expanding BKPyV-specific CD8 T cells in vitro using 27mer-long synthetic BKPyV peptides, different types of antigen-presenting cells, and CD4 T cells. Methods Langerhans cells and immature or mature monocyte-derived dendritic cells (Mo-DCs) were generated from peripheral blood mononuclear cells of healthy blood donors, pulsed with synthetic peptide pools consisting of 36 overlapping 27mers (27mP) or 180 15mers (15mP). BKPyV-specific CD8 T-cell responses were assessed by cytokine release assays using 15mP or immunodominant 9mers. Results BKPyV-specific CD8 T cells expanded using 27mP and required mature Mo-DCs (P = .0312) and CD4 T cells (P = .0156) for highest responses. The resulting BKPyV-specific CD8 T cells proliferated, secreted multiple cytokines including interferon γ and tumor necrosis factor α, and were functional (CD107a+/PD1–) and cytotoxic. Conclusions Synthetic 27mP permit expanding BKPyV-specific CD8 T-cell responses when pulsing mature Mo-DCs in presence of CD4 T cells, suggesting novel and safe approaches to vaccination and adoptive T-cell therapies for patients before and after kidney transplantation.

scholarly journals Increasing the potency of dendritic cell based vaccines for the treatment of cancer

2021 ◽  
Author(s):  
◽  
Dianne Sika-Paotonu
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Vaccination Strategies ◽  
Monophosphoryl Lipid A ◽  
Tlr Agonist

<p>Tumours can be eradicated by T cells that recognise unique tumour-associated antigens. These T cells are initially stimulated by dendritic cells (DCs) that have acquired antigens from tumour tissue. Vaccination strategies that increase the frequencies of tumour-specific T cells by enhancing the activity of DCs are being evaluated in the clinic as novel cancer therapies. Our hypothesis is that existing DC-based vaccination strategies can be improved by stimulating toll-like receptor (TLR) signalling in the DCs, and also by encouraging interactions with iNKT cells, as these two activities are known to enhance DC function. It was also hypothesised that superior T cell responses could be induced by combining these two activities together. We used the TLR 4 agonist monophosphoryl lipid A (MPL) alone and in combination with other TLR agonists to achieve effective activation of bone marrow-derived DCs (BM-DCs) cultured in-vitro, which was characterised by upregulated expression of maturation markers on the cell surface, and enhanced release of pro-inflammatory cytokines. Some TLR agonist combinations provided significantly enhanced activities in this regard, notably the combination of MPL with either the TLR 2 agonist Pam3Cys, or the TLR 7/8 agonist Resiquimod. Although in-vitro activated BM-DCs were unable to induce stronger antigen-specific CD8+ T cell responses after intravenous injection when compared to BMDCs without TLR stimulation, enhanced CD8+ T cell responses were achieved in-vivo with the co-administration of TLR ligands, implying that TLR stimulation needed to act on cells of the host. Further studies identified the langerin-expressing CD8ɑ+ splenic DC subset in the spleen as recipients of antigen that was transferred from injected cells, and that these recipients were participants in the cross-presentation and T cell priming activities driving the CD8+ T cell response after vaccination. Antigen-loaded BM-DCs carrying the NKT cell ligand ɑ-galactosylceramide (ɑ-GalCer) were found to consistently increase antigen-specific CD8+ T cell responses in-vivo, and also cytotoxic responses as seen in cytotoxic killing assays. Again, langerin-expressing CD8ɑ+ splenic DCs were shown to be involved in this response by acquiring antigen and ɑ-GalCer from the injected vaccine BM-DCs. Finally, it was possible to achieve even greater CD8+ T cell responses in-vivo by injecting BM-DCs carrying antigen and ɑ-GalCer, together with timely co-administration of the TLR agonist. These results suggest a reassessment of the activities of DC-based vaccines to include the important role of “courier” to DCs already resident in the host that can be exploited to improve vaccination outcomes.</p>

scholarly journals Enhanced Anti-HIV Functional Activity Associated with Gag-Specific CD8 T-Cell Responses

2010 ◽  
Vol 84 (11) ◽  
pp. 5540-5549 ◽  
Author(s):  
B. Julg ◽  
K. L. Williams ◽  
S. Reddy ◽  
K. Bishop ◽  
Y. Qi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Virus Replication ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Cell Counts ◽  
Inhibit Virus Replication

ABSTRACT Effective HIV-specific T-cell immunity requires the ability to inhibit virus replication in the infected host, but the functional characteristics of cells able to mediate this effect are not well defined. Since Gag-specific CD8 T cells have repeatedly been associated with lower viremia, we examined the influence of Gag specificity on the ability of unstimulated CD8 T cells from chronically infected persons to inhibit virus replication in autologous CD4 T cells. Persons with broad (≥6; n = 13) or narrow (≤1; n = 13) Gag-specific responses, as assessed by gamma interferon enzyme-linked immunospot assay, were selected from 288 highly active antiretroviral therapy (HAART)-naive HIV-1 clade C-infected South Africans, matching groups for total magnitude of HIV-specific CD8 T-cell responses and CD4 T-cell counts. CD8 T cells from high Gag responders suppressed in vitro replication of a heterologous HIV strain in autologous CD4 cells more potently than did those from low Gag responders (P < 0.003) and were associated with lower viral loads in vivo (P < 0.002). As previously shown in subjects with low viremia, CD8 T cells from high Gag responders exhibited a more polyfunctional cytokine profile and a stronger ability to proliferate in response to HIV stimulation than did low Gag responders, which mainly exhibited monofunctional CD8 T-cell responses. Furthermore, increased polyfunctionality was significantly correlated with greater inhibition of viral replication in vitro. These data indicate that enhanced suppression of HIV replication is associated with broader targeting of Gag. We conclude that it is not the overall magnitude but rather the breadth, magnitude, and functional capacity of CD8 T-cell responses to certain conserved proteins, like Gag, which predict effective antiviral HIV-specific CD8 T-cell function.

scholarly journals Bone marrow MSC from pediatric patients with B-ALL highly immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity

2020 ◽  
Vol 8 (2) ◽  
pp. e001419
Author(s):  
Samanta Romina Zanetti ◽  
Paola Alejandra Romecin ◽  
Meritxell Vinyoles ◽  
Manel Juan ◽  
José Luis Fuster ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Activity ◽  
Cell Responses ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Severe Colitis

BackgroundAlthough adoptive transfer of CD19-directed chimeric antigen receptor (CAR) T-cells (CD19-CAR T-cells) achieves high rates of complete response in patients with B-cell acute lymphoblastic leukemia (B-ALL), relapse is common. Bone marrow (BM) mesenchymal stem/stromal cells (BM-MSC) are key components of the hematopoietic niche and are implicated in B-ALL pathogenesis and therapy resistance. MSC exert an immunosuppressive effect on T-cells; however, their impact on CD19-CAR T-cell activity is understudied.MethodsWe performed a detailed characterization of BM-MSC from pediatric patients with B-ALL (B-ALL BM-MSC), evaluated their immunomodulatory properties and their impact on CD19-CAR T-cell activity in vitro using microscopy, qRT-PCR, ELISA, flow cytometry analysis and in vivo using a preclinical model of severe colitis and a B-ALL xenograft model.ResultsWhile B-ALL BM-MSC were less proliferative than those from age-matched healthy donors (HD), the morphology, immunophenotype, differentiation potential and chemoprotection was very similar. Likewise, both BM-MSC populations were equally immunosuppressive in vitro and anti-inflammatory in an in vivo model of severe colitis. Interestingly, BM-MSC failed to impair CD19-CAR T-cell cytotoxicity or cytokine production in vitro using B-ALL cell lines and primary B-ALL cells. Finally, the growth of NALM6 cells was controlled in vivo by CD19-CAR T-cells irrespective of the absence/presence of BM-MSC.ConclusionsCollectively, our data demonstrate that pediatric B-ALL and HD BM-MSC equally immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity.

scholarly journals Increasing the potency of dendritic cell based vaccines for the treatment of cancer

2021 ◽  
Author(s):  
◽  
Dianne Sika-Paotonu
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Vaccination Strategies ◽  
Monophosphoryl Lipid A ◽  
Tlr Agonist

<p>Tumours can be eradicated by T cells that recognise unique tumour-associated antigens. These T cells are initially stimulated by dendritic cells (DCs) that have acquired antigens from tumour tissue. Vaccination strategies that increase the frequencies of tumour-specific T cells by enhancing the activity of DCs are being evaluated in the clinic as novel cancer therapies. Our hypothesis is that existing DC-based vaccination strategies can be improved by stimulating toll-like receptor (TLR) signalling in the DCs, and also by encouraging interactions with iNKT cells, as these two activities are known to enhance DC function. It was also hypothesised that superior T cell responses could be induced by combining these two activities together. We used the TLR 4 agonist monophosphoryl lipid A (MPL) alone and in combination with other TLR agonists to achieve effective activation of bone marrow-derived DCs (BM-DCs) cultured in-vitro, which was characterised by upregulated expression of maturation markers on the cell surface, and enhanced release of pro-inflammatory cytokines. Some TLR agonist combinations provided significantly enhanced activities in this regard, notably the combination of MPL with either the TLR 2 agonist Pam3Cys, or the TLR 7/8 agonist Resiquimod. Although in-vitro activated BM-DCs were unable to induce stronger antigen-specific CD8+ T cell responses after intravenous injection when compared to BMDCs without TLR stimulation, enhanced CD8+ T cell responses were achieved in-vivo with the co-administration of TLR ligands, implying that TLR stimulation needed to act on cells of the host. Further studies identified the langerin-expressing CD8ɑ+ splenic DC subset in the spleen as recipients of antigen that was transferred from injected cells, and that these recipients were participants in the cross-presentation and T cell priming activities driving the CD8+ T cell response after vaccination. Antigen-loaded BM-DCs carrying the NKT cell ligand ɑ-galactosylceramide (ɑ-GalCer) were found to consistently increase antigen-specific CD8+ T cell responses in-vivo, and also cytotoxic responses as seen in cytotoxic killing assays. Again, langerin-expressing CD8ɑ+ splenic DCs were shown to be involved in this response by acquiring antigen and ɑ-GalCer from the injected vaccine BM-DCs. Finally, it was possible to achieve even greater CD8+ T cell responses in-vivo by injecting BM-DCs carrying antigen and ɑ-GalCer, together with timely co-administration of the TLR agonist. These results suggest a reassessment of the activities of DC-based vaccines to include the important role of “courier” to DCs already resident in the host that can be exploited to improve vaccination outcomes.</p>

New SARS-CoV-2 lineages could evade CD8+ T-cells response

2021 ◽  
Author(s):  
Marco Antonio M. Pretti ◽  
Rômulo G. Galvani ◽  
Alessandro S Farias ◽  
Mariana Boroni
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Reference Sequence ◽  
T Cell Epitopes ◽  
Antibody Recognition ◽  
Cell Responses ◽  
The Uk

AbstractBackgroundMany SARS-CoV-2 variants of concern have emerged since the Covid-19 outburst, notably the lineages detected in the UK, South Africa, and Brazil. Their increased transmissibility and higher viral load put them in the spotlight. Much has been investigated on the ability of those new variants to evade antibody recognition. However, not enough attention has been given to pre-existing and induced SARS-CoV-2-specific CD8+ T cell responses during the natural course of infection by new lineages.MethodsIn this work, we investigated the SARS-CoV-2-specific CD8+ T cell epitopes from the main variants of concern and the potential of associated mutations to trigger or hinder CD8+ T-cells response. We also estimated the population’s coverage of these different lineages, considering peptide binding predictions to class I HLA alleles from 29 countries to investigate differences in the fraction of individuals expected to respond to a given epitope set from new and previous lineages.ResultsWe observed a lower populational coverage for 20B/S.484K (P2 lineage) in contrast to an increased coverage found for 20H/501Y.V2 (B.1.351 Lineage) and 20J/501Y.V3 (P1 lineage) compared to a reference lineage. Moreover, mutations such as Spike N501Y and Nucleocapsid T205I were predicted to have an overall higher affinity through HLA-I than the reference sequence.ConclusionsIn summary, the data in this work provided evidence for the existence of potentially immunogenic and conserved epitopes across new SARS-CoV-2 variants, but also highlights the reduced populational’s coverage for the Brazilian lineage P.1, suggesting its potential to evade from CD8+ T-cell responses. Our results also may guide efforts to characterize and validate relevant peptides to trigger CD8+ T-cell responses, and design new universal T-cell-inducing vaccine candidates that minimize detrimental effects of viral diversification and at the same time induce responses to a broad human population.

Peptide-Induced Antigen-Specific CD4+CD25+FoxP3+ T Cells Suppress Cytotoxicity T Cell Responses Directed Against the AAV Capsid.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3769-3769 ◽  
Author(s):  
Daniel J. Hui ◽  
Etiena Basner-Tschakarjan ◽  
Gary C. Pien ◽  
William D. Martin ◽  
Annie S. DeGroot ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Equity Ownership

Abstract Abstract 3769 Recent advances in adeno-associated viral (AAV) vector-mediated gene transfer continue to offer hope for the correction of monogenic disorders such as hemophilia B. However, unanticipated T cell responses directed against viral capsid epitopes may limit the efficacy of AAV gene transfer. A phase I clinical study in which an AAV2 vector expressing human factor IX (FIX) was delivered systemically provided the first evidence that AAV vector administration at high doses may trigger the expansion of memory CD8+ T cells directed against AAV capsid epitopes. This response was associated with the loss of FIX transgene expression and a transient increase in liver enzymes. Additional studies in human subjects undergoing AAV gene transfer suggest that the capsid antigen load is an important determinant of capsid-specific T cell activation. Thus, strategies for the modulation of capsid T cell responses could contribute to achieving sustained transgene expression following high dose delivery of AAV. MHC class II peptide ligands identified within the human IgG Fc fragment (Tregitopes, Blood 2008;112:3303) have been shown to expand regulatory T cells (Tregs). Restimulation of human peripheral blood mononuclear cells (PBMC) in vitro with AAV capsid antigen in the presence of Tregitopes resulted in the suppression of capsid-specific CD8+ T cells and in the expansion of CD4+CD25+FoxP3+ Tregs. To better define the nature of Tregitope-induced Tregs, we depleted CD25+ cells from PBMC prior to in vitro restimulation. This completely prevented capsid-specific CTL suppression and the expansion of Tregs, suggesting that Tregitopes act by expanding natural Tregs. Cytokine ELISA on conditioned media from PBMC co-cultured with AAV antigen and Tregitopes showed a 50% decrease in IL-2 levels and a >500-fold increase in IL-10 levels. These results suggest that the effect of Tregitopes may be cytokine mediated. To test this hypothesis, we used a transwell system in which the CD4+ T cell fraction of Tregitope-restimulated PBMC was co-cultured with the capsid-specific CD8+ T cells. Without cell contact, a nearly 50% suppression of anti-capsid CD8+ T cell responses was observed. Further evidence supporting the role of cytokine-mediated suppression came from the observation that Tregitope-treated capsid-specific CD8+ T cells appeared to be anergic, and depletion of CD4+ T cells (Tregs) followed by a 24-hour incubation of CD8+CD4− T cells with IL-2 restored >80% of CTL activity. Finally, antigen specificity of Tregitope-induced Tregs was tested by expanding PBMC in vitro with HLA-B*0702-restricted epitopes from either the AAV capsid or the Epstein-Barr Virus (EBV). After in vitro restimulation, negatively-isolated CD4+ T cells expanded in the presence of EBV antigen and Tregitopes were co-incubated with either CD8+ T cells expanded against the AAV capsid or against EBV. Suppression of CTL activity was observed only when EBV Tregs were co-incubated with EBV CD8+ T cells. Similarly, Tregs isolated from AAV and Tregitope cultures suppressed AAV-specific CD8+ T cells but not EBV-specific CD8+T cells. These results suggest that inhibition of CD8+ T cell responses is antigen-specific. We conclude that Tregitopes induce the expansion of Tregs, which can mediate a potent antigen-specific inhibition of CD8+ T cell responses directed to the AAV capsid. Disclosures: Hui: Children's Hospital of Philadelphia: Patents & Royalties. Martin:EpiVax: Employment, Equity Ownership, Patents & Royalties. DeGroot:EpiVax: Employment, Equity Ownership. High:Children's Hospital of Philadelphia: Patents & Royalties. Mingozzi:Children's Hospital of Philadelphia: Patents & Royalties.

scholarly journals SARS-CoV-2–specific T cells are rapidly expanded for therapeutic use and target conserved regions of the membrane protein

Blood ◽  
2020 ◽  
Vol 136 (25) ◽  
pp. 2905-2917 ◽  
Author(s):  
Michael D. Keller ◽  
Katherine M. Harris ◽  
Mariah A. Jensen-Wachspress ◽  
Vaishnavi V. Kankate ◽  
Haili Lang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Membrane Protein ◽  
T Cell Responses ◽  
Effective Vaccine ◽  
T Cell Epitopes ◽  
T Cell Therapy ◽  
Cell Responses ◽  
Conserved Regions

Abstract T-cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals. In this report, we demonstrate that SARS-CoV-2–specific T cells can be expanded from convalescent donors and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleocapsid. Following in vitro expansion using a good manufacturing practice-compliant methodology (designed to allow the rapid translation of this novel SARS-CoV-2 T-cell therapy to the clinic), membrane, spike, and nucleocapsid peptides elicited interferon-γ production, in 27 (59%), 12 (26%), and 10 (22%) convalescent donors (respectively), as well as in 2 of 15 unexposed controls. We identified multiple polyfunctional CD4-restricted T-cell epitopes within a highly conserved region of membrane protein, which induced polyfunctional T-cell responses, which may be critical for the development of effective vaccine and T-cell therapies. Hence, our study shows that SARS-CoV-2 directed T-cell immunotherapy targeting structural proteins, most importantly membrane protein, should be feasible for the prevention or early treatment of SARS-CoV-2 infection in immunocompromised patients with blood disorders or after bone marrow transplantation to achieve antiviral control while mitigating uncontrolled inflammation.

scholarly journals Identification of Immunogenic Epitopes That Permit the Detection of Antigen-Specific T Cell Reponses to Coxsackievirus B3

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Troy Wesson ◽  
Adeeba Dhalech ◽  
Christopher M. Robinson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Viral Myocarditis ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
The United States ◽  
T Cell Epitopes ◽  
Cell Responses ◽  
Cvb3 Infection

Background and Hypothesis:Coxsackievirus B3 (CVB3) is a non-enveloped RNA virus from the Picornaviridae family and is a primary cause of viral myocarditis in the United States. Approximately 5% of all symptomatic CVB3 infections are fatal. Therefore, there is a need to identify the mechanism(s) that regulate a protective immune response to CVB3. However, viral epitopes that stimulate T cell responses to CVB3 remain poorly characterized. To this end, we used a mouse model of CVB3 infection to identify the viral immunogenic CD8 T cell epitopes. We hypothesized that isolated antigen-experienced CD8 T cells from infected mice would be stimulated in the presence of predicted viral epitopes, confirming CVB3-specific T cells. Experimental Design: To identify novel CD8 T cell epitopes, predicted 9-mer MHC binding peptides from the CVB3-Nancy polyprotein were identified using the Immune Epitope Database (IEDB) analysis resource consensus tool. The top ten predicted peptides were synthesized for our assays. Splenocytes from CVB3-infected male and female IFNAR -/- mice were stimulated with each peptide in the presence of brefeldin A for 6 hours at 37˚C. Following stimulation, cells were surfaced stained with antibodies specific for antigen-experienced CD8 T cells. Next, we performed intracellular staining for IFN-gamma. Cells were analyzed using flow cytometry. Candidate epitopes were identified as having results ≥2 standard deviations over the control. Results: Thus far, our analysis has revealed responses to three novel CD8 T cell epitopes within the peptide library, including the viral epitopes within VP1 protein and the RNA-dependent RNA polymerase. Conclusion and Potential ImpactOverall, these data provide an advancement in CVB3 immunology. Further, these data generate new tools like MHC-tetramers to track endogenous T cell responses to CVB3 infection.

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