Breakthrough SARS-COV-2 infection induces broad anti-viral T cell immunity.

Vaccines have curtailed the devastation wrought by COVID-19. Nevertheless, emerging variants result in a high incidence of breakthrough infections. Here we assess the impact of vaccination and breakthrough infection on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T cell immunity. We demonstrate that COVID-19 vaccination induces robust spike-specific T cell responses that, within the CD4+ compartment, display comparable IFN-γ responses to SARS-CoV-2 infection without vaccination. Vaccine-induced CD8+ IFN-γ responses however, were significantly greater than those primed by SARS-CoV-2 infection alone. This increased responsiveness is associated with induction of novel HLA-restricted CD8+ T cell epitopes not primed by infection alone (without vaccination). Despite these augmented responses, breakthrough infection still induced de novo T cell responses against additional SARS-CoV-2 CD8+ epitopes that display HLA-associated immunodominance hierarchies consistent with those in unvaccinated COVID-19 convalescent individuals. This study demonstrates the unique modulation of anti-viral T cell responses against multiple viral antigens following consecutive yet distinct priming events in COVID-19 vaccination and breakthrough infection.

Ablation of CD8+ T cell recognition of an immunodominant epitope in SARS-CoV-2 Omicron variants BA.1, BA.2 and BA.3.

The emergence of the SARS-CoV-2 Omicron variant has raised concerns of escape from vaccine-induced immunity. A number of studies have demonstrated a reduction in antibody-mediated neutralization of the Omicron variant in vaccinated individuals. Preliminary observations have suggested that T cells are less likely to be affected by changes in Omicron. However, the complexity of human leukocyte antigen genetics and its impact upon immunodominant T cell epitope selection suggests that the maintenance of T cell immunity may not be universal. In this study, we describe the impact that changes in Omicron BA.1, BA.2 and BA.3 have on recognition by spike-specific T cells. These T cells constitute the immunodominant CD8+ T cell response in HLA-A*29:02+ COVID-19 convalescent and vaccinated individuals; however, they fail to recognize the Omicron-encoded sequence. These observations demonstrate that in addition to evasion of antibody-mediated immunity, changes in Omicron variants can also lead to evasion of recognition by immunodominant T cell responses.

Limited Recognition of Highly Conserved Regions of SARS-CoV-2.

Understanding the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) is critical to overcome the current coronavirus disease (COVID-19) pandemic. Efforts are being made to understand the potential cross-protective immunity of memory T cells, induced by prior encounters with seasonal coronaviruses, in providing protection against severe COVID-19. In this study we assessed T-cell responses directed against highly conserved regions of SARS-CoV-2. Epitope mapping revealed 16 CD8+ T-cell epitopes across the nucleocapsid (N), spike (S), and open reading frame (ORF)3a proteins of SARS-CoV-2 and five CD8+ T-cell epitopes encoded within the highly conserved regions of the ORF1ab polyprotein of SARS-CoV-2. Comparative sequence analysis showed high conservation of SARS-CoV-2 ORF1ab T-cell epitopes in seasonal coronaviruses. Paradoxically, the immune responses directed against the conserved ORF1ab epitopes were infrequent and subdominant in both convalescent and unexposed participants. This subdominant immune response was consistent with a low abundance of ORF1ab encoded proteins in SARS-CoV-2 infected cells. Overall, these observations suggest that while cross-reactive CD8+ T cells likely exist in unexposed individuals, they are not common and therefore are unlikely to play a significant role in providing broad preexisting immunity in the community. IMPORTANCE T cells play a critical role in protection against SARS-CoV-2. Despite being highly topical, the protective role of preexisting memory CD8+ T cells, induced by prior exposure to circulating common coronavirus strains, remains less clear. In this study, we established a robust approach to specifically assess T cell responses to highly conserved regions within SARS-CoV-2. Consistent with recent observations we demonstrate that recognition of these highly conserved regions is associated with an increased likelihood of milder disease. However, extending these observations we observed that recognition of these conserved regions is rare in both exposed and unexposed volunteers, which we believe is associated with the low abundance of these proteins in SARS-CoV-2 infected cells. These observations have important implications for the likely role preexisting immunity plays in controlling severe disease, further emphasizing the importance of vaccination to generate the immunodominant T cells required for immune protection.

SARS-CoV-2-specific T cells generated for adoptive immunotherapy are capable of recognizing multiple SARS-CoV-2 variants.

Adoptive T-cell immunotherapy has provided promising results in the treatment of viral complications in humans, particularly in the context of immunocompromised patients who have exhausted all other clinical options. The capacity to expand T cells from healthy immune individuals is providing a new approach to anti-viral immunotherapy, offering rapid off-the-shelf treatment with tailor-made human leukocyte antigen (HLA)-matched T cells. While most of this research has focused on the treatment of latent viral infections, emerging evidence that SARS-CoV-2-specific T cells play an important role in protection against COVID-19 suggests that the transfer of HLA-matched allogeneic off-the-shelf virus-specific T cells could provide a treatment option for patients with active COVID-19 or at risk of developing COVID-19. We initially screened 60 convalescent individuals and based on HLA typing and T-cell response profile, 12 individuals were selected for the development of a SARS-CoV-2-specific T-cell bank. We demonstrate that these T cells are specific for up to four SARS-CoV-2 antigens presented by a broad range of both HLA class I and class II alleles. These T cells show consistent functional and phenotypic properties, display cytotoxic potential against HLA-matched targets and can recognize HLA-matched cells infected with different SARS-CoV-2 variants. These observations demonstrate a robust approach for the production of SARS-CoV-2-specific T cells and provide the impetus for the development of a T-cell repository for clinical assessment.

Rapid whole-blood assay to detect SARS-CoV-2-specific memory T-cell immunity following a single dose of AstraZeneca ChAdOx1-S COVID-19 vaccine.

OBJECTIVES: With the ongoing emergence of SARS-CoV-2 variants and potential to evade vaccine-induced neutralisation, understanding the magnitude and breadth of vaccine-induced T-cell immunity will be critical for the ongoing optimisation of vaccine approaches. Strategies that provide a rapid and easily translatable means of assessing virus-specific T-cell responses provide an opportunity to monitor the impact of vaccine rollouts in the community. In this study, we assessed whether our recently developed SARS-CoV-2 whole-blood assay could be used effectively to analyse T-cell responses following vaccination. METHODS: Following a median of 15 days after the first dose of the ChAdOx1-S (AstraZeneca®) vaccine, peripheral blood was isolated from 58 participants. Blood was incubated overnight with an overlapping set of spike protein peptides and assessed for cytokine production using a cytometric bead array. RESULTS: The majority of vaccine recipients (51/58) generated a T helper 1 response (IFN-γ and/or IL-2) following a single dose of ChAdOx1-S. The magnitude of the IFN-γ and IL-2 response strongly correlated in vaccine recipients. While the production of other cytokines was evident in individuals who did not generate IFN-γ and IL-2, they showed no correlation in magnitude, nor did we see a correlation between sex or age and the magnitude of the response. CONCLUSIONS: The whole-blood cytokine assay provides a rapid approach to assessing T-cell immunity against SARS-CoV-2 in vaccine recipients. While the majority of participants generated a robust SARS-CoV-2-specific T-cell response following their first dose, some did not, demonstrating the likely importance of the booster dose in improving T-cell immunity.

CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope cross-react with selective seasonal coronaviruses.

Efforts are being made worldwide to understand the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic, including the impact of T cell immunity and cross-recognition with seasonal coronaviruses. Screening of SARS-CoV-2 peptide pools revealed that the nucleocapsid (N) protein induced an immunodominant response in HLA-B7+ COVID-19-recovered individuals that was also detectable in unexposed donors. A single N-encoded epitope that was highly conserved across circulating coronaviruses drove this immunodominant response. In vitro peptide stimulation and crystal structure analyses revealed T cell-mediated cross-reactivity toward circulating OC43 and HKU-1 betacoronaviruses but not 229E or NL63 alphacoronaviruses because of different peptide conformations. T cell receptor (TCR) sequencing indicated that cross-reactivity was driven by private TCR repertoires with a bias for TRBV27 and a long CDR3ß loop. Our findings demonstrate the basis of selective T cell cross-reactivity for an immunodominant SARS-CoV-2 epitope and its homologs from seasonal coronaviruses, suggesting long-lasting protective immunity.

Complete response to PD-1 blockade following EBV-specific T-cell therapy in metastatic nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated heterogeneous disease and is characterized by peritumoral immune infiltrate. Adoptive T-cell therapy (ACT) has emerged as a potential therapeutic strategy for NPC. However, the tumor microenvironment remains a major roadblock for the successful implementation of ACT in clinical settings. Expression of checkpoint molecules by malignant cells can inhibit the effector function of adoptively transferred EBV-specific T cells. Here we present a novel case report of a patient with metastatic NPC who was successfully treated with a combination of EBV-specific ACT and programmed cell death-1 blockade therapy. Following combination immunotherapy, the patient showed complete resolution of metastatic disease with no evidence of disease relapse for 22 months. Follow-up immunological analysis revealed dramatic restructuring of the global T-cell repertoire that was coincident with the clinical response. This case report provides an important platform for translating these findings to a larger cohort of NPC patients.

'Off-the-shelf' allogeneic antigen-specific adoptive T-cell therapy for the treatment of multiple EBV-associated malignancies.

BACKGROUND: Epstein-Barr virus (EBV), an oncogenic human gammaherpesvirus, is associated with a wide range of human malignancies of epithelial and B-cell origin. Recent studies have demonstrated promising safety and clinical efficacy of allogeneic 'off-the-shelf' virus-specific T-cell therapies for post-transplant viral complications. METHODS: Taking a clue from these studies, we developed a highly efficient EBV-specific T-cell expansion process using a replication-deficient AdE1-LMPpoly vector that specifically targets EBV-encoded nuclear antigen 1 (EBNA1) and latent membrane proteins 1 and 2 (LMP1 and LMP2), expressed in latency II malignancies. RESULTS: These allogeneic EBV-specific T cells efficiently recognized human leukocyte antigen (HLA)-matched EBNA1-expressing and/or LMP1 and LMP2-expressing malignant cells and demonstrated therapeutic potential in a number of in vivo models, including EBV lymphomas that emerged spontaneously in humanized mice following EBV infection. Interestingly, we were able to override resistance to T-cell therapy in vivo using a 'restriction-switching' approach, through sequential infusion of two different allogeneic T-cell therapies restricted through different HLA alleles. Furthermore, we have shown that inhibition of the programmed cell death protein-1/programmed death-ligand 1 axis in combination with EBV-specific T-cell therapy significantly improved overall survival of tumor-bearing mice when compared with monotherapy. CONCLUSION: These findings suggest that restriction switching by sequential infusion of allogeneic T-cell therapies that target EBV through distinct HLA alleles may improve clinical response.

Pretransplant Cytomegalovirus-Specific Cellular Immunity and Risk of Viral Reactivation Following Lung Transplantation: A Prospective Cohort Study.

Cytomegalovirus (CMV) remains a significant burden in lung transplant recipients. Deficiencies in T-cell immunity posttransplant increase the risk of CMV-associated complications. However, it is not clear if underlying poor pretransplant immunity increases risk. To assess this, we recruited 39 prospective lung transplant patients and performed QuantiFERON-CMV on their peripheral blood. More than a third of prospective CMV-seropositive transplant recipients were CMV non-immune reactive (CMV-NIR) pretransplant. CMV-NIR status was associated with a significantly higher incidence of CMV reactivation posttransplant, demonstrating that dysfunctional CMV immunity in prospective lung transplant recipients is associated with an increased risk of viral reactivation posttransplant.

Rapid detection of SARS-CoV-2-specific memory T-cell immunity in recovered COVID-19 cases.

OBJECTIVES: There is emerging evidence that SARS-CoV-2-specific memory T-cell responses are likely to provide critical long-term protection against COVID-19. Strategies to rapidly assess T-cell responses are therefore likely to be important for assessing immunity in the global population. METHODS: Here, we have developed a rapid immune-monitoring strategy to assess virus-specific memory T-cell responses in the peripheral blood of COVID-19 convalescent individuals. We validated SARS-CoV-2-specific memory T-cell responses detected in whole blood using in vitro expansion with SARS-CoV-2 proteins. RESULTS: T-cell immunity characterised by the production of IFN-γ and IL-2 could be consistently detected in the whole blood of recovered participants. T cells predominantly recognised structural SARS-CoV-2 proteins. In vitro expansion demonstrated that while CD8+ T cells recognised nucleocapsid protein, spike protein and ORF3a, CD4+ T cells more broadly targeted multiple SARS-CoV-2 proteins. CONCLUSION: These observations provide a timely monitoring approach for identifying SARS-CoV-2 cellular immunity and may serve as a diagnostic for the stratification of risk in immunocompromised and other at-risk individuals.

Autologous CMV-specific T cells are a safe adjuvant immunotherapy for primary glioblastoma multiforme.

BACKGROUNDThe recent failure of checkpoint-blockade therapies for glioblastoma multiforme (GBM) in late-phase clinical trials has directed interest toward adoptive cellular therapies (ACTs). In this open-label, first-in-human trial, we have assessed the safety and therapeutic potential of cytomegalovirus-specific (CMV-specific) ACT in an adjuvant setting for patients with primary GBM, with an ultimate goal to prevent or delay recurrence and prolong overall survival.METHODSTwenty-eight patients with primary GBM were recruited to this prospective study, 25 of whom were treated with in vitro-expanded autologous CMV-specific T cells. Participants were monitored for safety, progression-free survival, overall survival (OS), and immune reconstitution.RESULTSNo participants showed evidence of ACT-related toxicities. Of 25 evaluable participants, 10 were alive at the completion of follow-up, while 5 were disease free. Reconstitution of CMV-specific T cell immunity was evident and CMV-specific ACT may trigger a bystander effect leading to additional T cell responses to nonviral tumor-associated antigens through epitope spreading. Long-term follow-up of participants treated before recurrence showed significantly improved OS when compared with those who progressed before ACT (median 23 months, range 7-65 vs. median 14 months, range 5-19; P = 0.018). Gene expression analysis of the ACT products indicated that a favorable T cell gene signature was associated with improved long-term survival.CONCLUSIONData presented in this study demonstrate that CMV-specific ACT can be safely used as an adjuvant therapy for primary GBM and, if offered before recurrence, this therapy may improve OS of GBM patients.TRIAL REGISTRATIONanzctr.org.au: ACTRN12615000656538.FUNDINGPhilanthropic funding and the National Health and Medical Research Council (Australia).

Profiling HPV-16-specific T cell responses reveals broad antigen reactivities in oropharyngeal cancer patients.

Cellular immunotherapeutics targeting the human papillomavirus (HPV)-16 E6 and E7 proteins have achieved limited success in HPV-positive oropharyngeal cancer (OPC). Here we have conducted proteome-wide profiling of HPV-16-specific T cell responses in a cohort of 66 patients with HPV-associated OPC and 22 healthy individuals. Unexpectedly, HPV-specific T cell responses from OPC patients were not constrained to the E6 and E7 antigens; they also recognized E1, E2, E4, E5, and L1 proteins as dominant targets for virus-specific CD8+ and CD4+ T cells. Multivariate analysis incorporating tumor staging, treatment status, and smoking history revealed that treatment status had the most significant impact on HPV-specific CD8+ and CD4+ T cell immunity. Specifically, the breadth and overall strength of HPV-specific T cell responses were significantly higher before the commencement of curative therapy than after therapy. These data provide the first glimpse of the overall human T cell response to HPV in a clinical setting and offer groundbreaking insight into future development of cellular immunotherapies for HPV-associated OPC patients.

T-cell adoptive immunotherapy for BK nephropathy in renal transplantation.

INTRODUCTION: BK virus (BKPyV) nephropathy occurs in 1%-10% of kidney transplant recipients, with suboptimal therapeutic options. CASE: A 54-year-old woman received a transplant in March 2017. BKPyV was detected at 1.5 × 102  copies/mL within a month, necessitating halving of mycophenolate and addition of leflunomide. Allograft histology in December showed polyomavirus nephropathy treated with intravenous immunoglobulin and cessation of mycophenolate. In February 2018, cidofovir and ciprofloxacin were commenced. In April, tacrolimus was reduced while introducing everolimus. A second graft biopsy in August showed increasing polyoma virus infection and a subsequent biopsy in September for worsening renal function showed 30% of tubular reactivity for simian virus 40 (SV40). Allogeneic BKPyV-reactive T cells were generated from the patient's daughter and infused over 10 sessions starting late September. The fourth allograft biopsy in November 2018 demonstrated involvement of BKPyV in 50% of tubules. Allograft function continued to decline, requiring hemodialysis from December 2018. Allograft nephrectomy after 6 months showed <1% SV40 in preserved tubules and 80% interstitial fibrosis. DISCUSSION: We conclude that the T-cell adoptive immunotherapy reduced BKPyV load significantly despite extensive infection, but attendant fibrosis and tubular atrophy led to graft failure. Early intervention with T-cell therapy may prove efficacious in BKPyV nephropathy.

T cell repertoire remodeling following post-transplant T cell therapy coincides with clinical response.

BACKGROUNDImpaired T cell immunity in transplant recipients is associated with infection-related morbidity and mortality. We recently reported the successful use of adoptive T cell therapy (ACT) against drug-resistant/recurrent cytomegalovirus in solid-organ transplant recipients.METHODSIn the present study, we used high-throughput T cell receptor Vß sequencing and T cell functional profiling to delineate the impact of ACT on T cell repertoire remodeling in the context of pretherapy immunity and ACT products.RESULTSThese analyses indicated that a clinical response was coincident with significant changes in the T cell receptor Vß landscape after therapy. This restructuring was associated with the emergence of effector memory T cells in responding patients, while nonresponders displayed dramatic pretherapy T cell expansions with minimal change following ACT. Furthermore, immune reconstitution included both adoptively transferred clonotypes and endogenous clonotypes not detected in the ACT products.CONCLUSIONThese observations demonstrate that immune control following ACT requires significant repertoire remodeling, which may be impaired in nonresponders because of the preexisting immune environment. Immunological interventions that can modulate this environment may improve clinical outcomes.TRIAL REGISTRATIONAustralian New Zealand Clinical Trial Registry, ACTRN12613000981729.FUNDINGThis study was supported by funding from the National Health and Medical Research Council, Australia (APP1132519 and APP1062074).

Prophylactic and therapeutic strategies for Epstein-Barr virus-associated diseases: emerging strategies for clinical development.

INTRODUCTION: Epstein-Barr virus (EBV) infects more than 95% of the world's population and is associated with infectious mononucleosis as well as a number of cancers in various geographical locations. Despite its significant health burden, no licenced prophylactic or therapeutic vaccines are available. Areas covered: Over the last two decades, our understanding of the role of EBV infection in the pathogenesis and immune regulation of EBV-associated diseases has provided new lines of research to conceptualize various novel prophylactic and therapeutic approaches to control EBV-associated disease. In this review, we evaluate the prophylactic and therapeutic vaccine approaches against EBV and various immunotherapeutic strategies against a number of EBV-associated malignancies. This review also describes the existing and future prospects of improved EBV-targeted therapeutic strategies. Expert opinion: It is anticipated that these emerging strategies will provide answers for the major challenges in EBV vaccine development and help improve the efficacy of novel therapeutic strategies.

Autologous Adoptive T-cell Therapy for Recurrent or Drug-resistant Cytomegalovirus Complications in Solid Organ Transplant Recipients: A Single-arm Open-label Phase I Clinical Trial.

Background: Opportunistic infections including cytomegalovirus (CMV) are a major cause of morbidity and mortality in solid organ transplant (SOT) recipients. The recurrent and protracted use of antiviral drugs with eventual emergence of drug resistance represents a significant constraint to therapy. Although adoptive T-cell therapy has been successfully used in hematopoietic stem cell transplant recipients, its extension to the SOT setting poses a considerable challenge because of the inhibitory effects of immunosuppressive drugs on the virus-specific T-cell response in vivo and the perceived risk of graft rejection. Methods: In this prospective study, 22 SOT recipients (13 renal and 8 lung and 1 heart transplants) with recurrent or ganciclovir-resistant CMV infection were recruited, and 13 of them were treated with in vitro-expanded autologous CMV-specific T cells. These patients were monitored for safety, clinical symptoms, and immune reconstitution. Results: Autologous CMV-specific T-cell manufacture was attempted for 21 patients, and was successful in 20. The use of this adoptive immunotherapy was associated with no therapy-related serious adverse events. Eleven (84%) of the 13 treated patients showed improvement in symptoms, including complete resolution or reduction in DNAemia and CMV-associated end-organ disease and/or the cessation or reduced use of antiviral drugs. Furthermore, four of these patients showed coincident increased frequency of CMV-specific T cells in peripheral blood after completion of T-cell therapy. Conclusions: The data presented here demonstrate for the first time the clinical safety of CMV-specific adoptive T-cell therapy and its potential therapeutic benefit for SOT recipients with recurrent and/or drug-resistant CMV infection or disease. Clinical Trials Registration: ACTRN12613000981729.

Impact of pre-therapy glioblastoma multiforme microenvironment on clinical response to autologous CMV-specific T-cell therapy.

OBJECTIVES: Clinical response to antibody-based immunotherapies targeting checkpoint inhibitors is critically dependent on the tumor immune microenvironment (TIME). However, the precise impact of the TIME on adoptive cellular immunotherapy remains unexplored. Here we have conducted a long-term follow-up analysis of patients with recurrent glioblastoma multiforme (GBM) who were treated with autologous CMV-specific T-cell therapy to delineate the potential impact of the TIME on their clinical response. METHODS: Multiplexed immunohistochemical analysis of CD3, PD-L1 and Sox-2 in GBM tissue biopsies obtained before autologous T-cell therapy was carried out and correlated with long-term survival of GBM patients adoptively treated with T-cell therapy. RESULTS: Tumor microenvironment analyses revealed that the pre-treatment cellular composition of the tumor tissue may influence the subsequent response to adoptive T-cell therapy. GBM patients who showed prolonged overall survival following T-cell therapy had a significantly lower number of tumor-infiltrating CD3+ T cells in recurrent tumors than that in patients with short-term survival. Furthermore, long-term surviving patients showed low or undetectable PD-L1 expression in tumor cells in recurrent GBM biopsies. CONCLUSION: We hypothesise that lack of PD-L1-mediated immunosuppression in the TIME may allow efficient immune control following adoptive T-cell therapy. Future studies combining anti-PD-L1 or genetically modified T cells with PD-1 receptor knockdown could be considered to improve clinical responses in patients who have high PD-L1 expression in their tumors.