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.

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.

Impaired Epstein-Barr Virus-Specific Neutralizing Antibody Response during Acute Infectious Mononucleosis Is Coincident with Global B-Cell Dysfunction.

Here we present evidence for previously unappreciated B-cell immune dysregulation during acute Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). Longitudinal analyses revealed that patients with acute IM have undetectable EBV-specific neutralizing antibodies and gp350-specific B-cell responses, which were associated with a significant reduction in memory B cells and no evidence of circulating antibody-secreting cells. These observations correlate with dysregulation of tumor necrosis factor family members BAFF and APRIL and increased expression of FAS on circulating B cells.

Cytokine-Mediated Loss of Blood Dendritic Cells During Epstein-Barr Virus-Associated Acute Infectious Mononucleosis: Implication for Immune Dysregulation.

Acute infectious mononucleosis (IM) is associated with altered expression of inflammatory cytokines and disturbed T-cell homeostasis, however, the precise mechanism of this immune dysregulation remains unresolved. In the current study we demonstrated a significant loss of circulating myeloid and plasmacytoid dendritic cells (DCs) during acute IM, a loss correlated with the severity of clinical symptoms. In vitro exposure of blood DCs to acute IM plasma resulted in loss of plasmacytoid DCs, and further studies with individual cytokines showed that exposure to interleukin 10 could replicate this effect. Our data provide important mechanistic insight into dysregulated immune homeostasis during acute IM.

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.

Lymph node targeted multi-epitope subunit vaccine promotes effective immunity to EBV in HLA-expressing mice.

The recent emergence of a causal link between Epstein-Barr virus (EBV) and multiple sclerosis has generated considerable interest in the development of an effective vaccine against EBV. Here we describe a vaccine formulation based on a lymph node targeting Amphiphile vaccine adjuvant, Amphiphile-CpG, admixed with EBV gp350 glycoprotein and an engineered EBV polyepitope protein that includes 20 CD8+ T cell epitopes from EBV latent and lytic antigens. Potent gp350-specific IgG responses are induced in mice with titers >100,000 in Amphiphile-CpG vaccinated mice. Immunization including Amphiphile-CpG also induces high frequencies of polyfunctional gp350-specific CD4+ T cells and EBV-specific CD8+ T cells that are 2-fold greater than soluble CpG and are maintained for >7 months post immunization. This combination of broad humoral and cellular immunity against multiple viral determinants is likely to provide better protection against primary infection and control of latently infected B cells leading to protection against the development of EBV-associated diseases.

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.

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.

'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.

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).

Epigenetic programming of T cells impacts immune reconstitution in hematopoietic stem cell transplant recipients.

Immune reconstitution following hematopoietic stem cell transplantation (HSCT) is critical in preventing harmful sequelae in recipients with cytomegalovirus (CMV) infection. To understand the molecular mechanisms underlying immune reconstitution kinetics, we profiled the transcriptome-chromatin accessibility landscape of CMV-specific CD8+ T cells from HCST recipients with different immune reconstitution efficiencies. CMV-specific T cells from HSCT recipients with stable antiviral immunity expressed higher levels of interferon/defense response and cell cycle genes in an interconnected network involving PI3KCG, STAT5B, NFAT, RBPJ, and lower HDAC6, increasing chromatin accessibility at the enhancer regions of immune and T-cell receptor signaling pathway genes. By contrast, the transcriptional and epigenomic signatures of CMV-specific T cells from HSCT recipients with unstable immune reconstitution showed commonalities with T-cell responses in other nonresolving chronic infections. These signatures included higher levels of EGR and KLF factors that, along with lower JARID2 expression, maintained higher accessibility at promoter and CpG-rich regions of genes associated with apoptosis. Furthermore, epigenetic targeting via inhibition of HDAC6 or JARID2 enhanced the transcription of genes associated with differential responses, suggesting that drugs targeting epigenomic modifiers may have therapeutic potential for enhancing immune reconstitution in HSCT recipients. Taken together, these analyses demonstrate that transcription factors and chromatin modulators create different chromatin accessibility landscapes in T cells of HSCT recipients that not only affect immediate gene expression but also differentially prime cells for responses to additional signals. Epigenetic therapy may be a promising strategy to promote immune reconstitution in HSCT recipients.

B cell-derived circulating granzyme B is a feature of acute infectious mononucleosis.

Granzyme B (GzmB) is a serine protease best known for inducing target cell apoptosis when released by cytotoxic T lymphocytes (CTLs) or natural killer cells with pore-forming perforin. As a result, GzmB detected in the serum of virus-infected individuals has typically been attributed to these sources. Here, we show that patients with recently diagnosed infectious mononucleosis caused by Epstein-Barr virus (EBV) have high circulating levels of GzmB that may be derived from infected B cells early in course of disease. We recently reported that human B cells from healthy donors secrete active GzmB when stimulated in vitro through B-cell receptor (BCR) ligation and interleukin (IL)-21. We found that infecting B cells with EBV greatly amplified GzmB secretion in response to the same stimuli, but the expression was terminated once the infection had become latent. Our results represent a rare instance of GzmB expression by non-CTL/natural killer cells in the context of infection with a human pathogen.

Notch signaling controls liver development by regulating biliary differentiation.

In the mammalian liver, bile is transported to the intestine through an intricate network of bile ducts. Notch signaling is required for normal duct formation, but its mode of action has been unclear. Here, we show in mice that bile ducts arise through a novel mechanism of tubulogenesis involving sequential radial differentiation. Notch signaling is activated in a subset of liver progenitor cells fated to become ductal cells, and pathway activation is necessary for biliary fate. Notch signals are also required for bile duct morphogenesis, and activation of Notch signaling in the hepatic lobule promotes ectopic biliary differentiation and tubule formation in a dose-dependent manner. Remarkably, activation of Notch signaling in postnatal hepatocytes causes them to adopt a biliary fate through a process of reprogramming that recapitulates normal bile duct development. These results reconcile previous conflicting reports about the role of Notch during liver development and suggest that Notch acts by coordinating biliary differentiation and morphogenesis.