Bioengineered small extracellular vesicles deliver multiple SARS-CoV-2 antigenic fragments and drive a broad immunological response.

The COVID-19 pandemic highlighted the clear risk that zoonotic viruses pose to global health and economies. The scientific community responded by developing several efficacious vaccines which were expedited by the global need for vaccines. The emergence of SARS-CoV-2 breakthrough infections highlights the need for additional vaccine modalities to provide stronger, long-lived protective immunity. Here we report the design and preclinical testing of small extracellular vesicles (sEVs) as a multi-subunit vaccine. Cell lines were engineered to produce sEVs containing either the SARS-CoV-2 Spike receptor-binding domain, or an antigenic region from SARS-CoV-2 Nucleocapsid, or both in combination, and we tested their ability to evoke immune responses in vitro and in vivo. B cells incubated with bioengineered sEVs were potent activators of antigen-specific T cell clones. Mice immunised with sEVs containing both sRBD and Nucleocapsid antigens generated sRBD-specific IgGs, nucleocapsid-specific IgGs, which neutralised SARS-CoV-2 infection. sEV-based vaccines allow multiple antigens to be delivered simultaneously resulting in potent, broad immunity, and provide a quick, cheap, and reliable method to test vaccine candidates.

Immunology of THymectomy And childhood CArdiac transplant (ITHACA): protocol for a UK-wide prospective observational cohort study to identify immunological risk factors of post-transplant lymphoproliferative disease (PTLD) in thymectomised children.

INTRODUCTION: Paediatric heart transplant patients are disproportionately affected by Epstein-Barr virus (EBV)-related post-transplant lymphoproliferative disease (PTLD) compared with other childhood solid organ recipients. The drivers for this disparity remain poorly understood. A potential risk factor within this cohort is the routine surgical removal of the thymus-a gland critical for the normal development of T-lymphocyte-mediated antiviral immunity-in early life, which does not occur in other solid organ transplant recipients. Our study aims to describe the key immunological differences associated with early thymectomy, its impact on the temporal immune response to EBV infection and subsequent risk of PTLD. METHODS AND ANALYSIS: Prospective and sequential immune monitoring will be performed for 34 heart transplant recipients and 6 renal transplant patients (aged 0-18 years), stratified into early (<1 year), late (>1 year) and non-thymectomy groups. Peripheral blood samples and clinical data will be taken before transplant and at 3, 6, 12 and 24 months post-transplant. Single cell analysis of circulating immune cells and enumeration of EBV-specific T-lymphocytes will be performed using high-dimensional spectral flow cytometry with peptide-Major Histocompatibilty Complex (pMHC) I/II tetramer assay, respectively. The functional status of EBV-specific T-lymphocytes, along with EBV antibodies and viral load will be monitored at each of the predefined study time points. ETHICS AND DISSEMINATION: Ethical approval for this study has been obtained from the North of Scotland Research Ethics Committee. The results will be disseminated through publications in peer-reviewed journals, presentations at scientific conferences and patient-centred forums, including social media. TRIAL REGISTRATION NUMBER: ISRCTN10096625.

Structural definition of HLA class II-presented SARS-CoV-2 epitopes reveals a mechanism to escape pre-existing CD4+ T cell immunity.

CD4+ T cells recognize a broad range of peptide epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which contribute to immune memory and limit COVID-19 disease. We demonstrate that the immunogenicity of SARS-CoV-2 peptides, in the context of the model allotype HLA-DR1, does not correlate with their binding affinity to the HLA heterodimer. Analyzing six epitopes, some with very low binding affinity, we solve X-ray crystallographic structures of each bound to HLA-DR1. Further structural definitions reveal the precise molecular impact of viral variant mutations on epitope presentation. Omicron escaped ancestral SARS-CoV-2 immunity to two epitopes through two distinct mechanisms: (1) mutations to TCR-facing epitope positions and (2) a mechanism whereby a single amino acid substitution caused a register shift within the HLA binding groove, completely altering the peptide-HLA structure. This HLA-II-specific paradigm of immune escape highlights how CD4+ T cell memory is finely poised at the level of peptide-HLA-II presentation.

Tspan6 stimulates the chemoattractive potential of breast cancer cells for B cells in an EV- and LXR-dependent manner.

The immune microenvironment in breast cancer (BCa) is controlled by a complex network of communication between various cell types. Here, we find that recruitment of B lymphocytes to BCa tissues is controlled via mechanisms associated with cancer cell-derived extracellular vesicles (CCD-EVs). Gene expression profiling identifies the Liver X receptor (LXR)-dependent transcriptional network as a key pathway that controls both CCD-EVs-induced migration of B cells and accumulation of B cells in BCa tissues. The increased accumulation oxysterol ligands for LXR (i.e., 25-hydroxycholesterol and 27-hydroxycholesterol) in CCD-EVs is regulated by the tetraspanin 6 (Tspan6). Tspan6 stimulates the chemoattractive potential of BCa cells for B cells in an EV- and LXR-dependent manner. These results demonstrate that tetraspanins control intercellular trafficking of oxysterols via CCD-EVs. Furthermore, tetraspanin-dependent changes in the oxysterol composition of CCD-EVs and the LXR signaling axis play a key role in specific changes in the tumor immune microenvironment.

Mutations in SARS-CoV-2 spike protein impair epitope-specific CD4+ T cell recognition.

CD4+ T cells are essential for protection against viruses, including SARS-CoV-2. The sensitivity of CD4+ T cells to mutations in SARS-CoV-2 variants of concern (VOCs) is poorly understood. Here, we isolated 159 SARS-CoV-2-specific CD4+ T cell clones from healthcare workers previously infected with wild-type SARS-CoV-2 (D614G) and defined 21 epitopes in spike, membrane and nucleoprotein. Lack of CD4+ T cell cross-reactivity between SARS-CoV-2 and endemic beta-coronaviruses suggested these responses arose from naïve rather than pre-existing cross-reactive coronavirus-specific T cells. Of the 17 epitopes located in the spike protein, 10 were mutated in VOCs and CD4+ T cell clone recognition of 7 of them was impaired, including 3 of the 4 epitopes mutated in omicron. Our results indicated that broad targeting of epitopes by CD4+ T cells likely limits evasion by current VOCs. However, continued genomic surveillance is vital to identify new mutations able to evade CD4+ T cell immunity.

Cytotoxic CD4+ T-cells specific for EBV capsid antigen BORF1 are maintained in long-term latently infected healthy donors.

Epstein Barr Virus (EBV) infects more than 95% of the population whereupon it establishes a latent infection of B-cells that persists for life under immune control. Primary EBV infection can cause infectious mononucleosis (IM) and long-term viral carriage is associated with several malignancies and certain autoimmune diseases. Current efforts developing EBV prophylactic vaccination have focussed on neutralising antibodies. An alternative strategy, that could enhance the efficacy of such vaccines or be used alone, is to generate T-cell responses capable of recognising and eliminating newly EBV-infected cells before the virus initiates its growth transformation program. T-cell responses against the EBV structural proteins, brought into the newly infected cell by the incoming virion, are prime candidates for such responses. Here we show the structural EBV capsid proteins BcLF1, BDLF1 and BORF1 are frequent targets of T-cell responses in EBV infected people, identify new CD8+ and CD4+ T-cell epitopes and map their HLA restricting alleles. Using T-cell clones we demonstrate that CD4+ but not CD8+ T-cell clones specific for the capsid proteins can recognise newly EBV-infected B-cells and control B-cell outgrowth via cytotoxicity. Using MHC-II tetramers we show a CD4+ T-cell response to an epitope within the BORF1 capsid protein epitope is present during acute EBV infection and in long-term viral carriage. In common with other EBV-specific CD4+ T-cell responses the BORF1-specific CD4+ T-cells in IM patients expressed perforin and granzyme-B. Unexpectedly, perforin and granzyme-B expression was sustained over time even when the donor had entered the long-term infected state. These data further our understanding of EBV structural proteins as targets of T-cell responses and how CD4+ T-cell responses to EBV change from acute disease into convalescence. They also identify new targets for prophylactic EBV vaccine development.

Robust SARS-CoV-2-specific T cell immunity is maintained at 6 months following primary infection.

The immune response to SARS-CoV-2 is critical in controlling disease, but there is concern that waning immunity may predispose to reinfection. We analyzed the magnitude and phenotype of the SARS-CoV-2-specific T cell response in 100 donors at 6 months following infection. T cell responses were present by ELISPOT and/or intracellular cytokine staining analysis in all donors and characterized by predominant CD4+ T cell responses with strong interleukin (IL)-2 cytokine expression. Median T cell responses were 50% higher in donors who had experienced a symptomatic infection, indicating that the severity of primary infection establishes a 'set point' for cellular immunity. T cell responses to spike and nucleoprotein/membrane proteins were correlated with peak antibody levels. Furthermore, higher levels of nucleoprotein-specific T cells were associated with preservation of nucleoprotein-specific antibody level although no such correlation was observed in relation to spike-specific responses. In conclusion, our data are reassuring that functional SARS-CoV-2-specific T cell responses are retained at 6 months following infection.

BCL-XL inhibition by BH3-mimetic drugs induces apoptosis in models of Epstein-Barr virus-associated T/NK-cell lymphoma.

Epstein-Barr virus (EBV)-associated T- and natural killer (NK)-cell malignancies, such as extranodal NK-/T-cell lymphoma (ENKTL), exhibit high chemoresistance and, accordingly, such patients have a poor prognosis. The rare nature of such cancers and nonmalignant T/NK lymphoproliferative disorders, such as chronic active EBV (CAEBV), has limited our understanding of the pathogenesis of these diseases. Here, we characterize a panel of ENKTL- and CAEBV-derived cell lines that had been established from human tumors to be used as preclinical models of these diseases. These cell lines were interleukin-2 dependent and found to carry EBV in a latency II gene-expression pattern. All cell lines demonstrated resistance to cell death induction by DNA damage-inducing agents, the current standard of care for patients with these malignancies. This resistance was not correlated with the function of the multidrug efflux pump, P-glycoprotein. However, apoptotic cell death could be consistently induced following treatment with A-1331852, a BH3-mimetic drug that specifically inhibits the prosurvival protein BCL-XL. A-1331852-induced apoptosis was most efficacious when prosurvival MCL-1 was additionally targeted, either by BH3-mimetics or genetic deletion. Xenograft models established from the ENKTL cell line SNK6 provided evidence that A-1331852 treatment could be therapeutically beneficial in vivo. The data here suggest that therapeutic targeting of BCL-XL would be effective for patients with EBV-driven T/NK proliferative diseases, however, MCL-1 could be a potential resistance factor.

Tracking immunodynamics by identification of S-G2/M-phase T cells in human peripheral blood.

The ready availability of human blood makes it the first choice for immuno-monitoring. However, this has been largely confined to static metrics, particularly resting T cell phenotypes. Conversely, dynamic assessments have mostly relied on cell stimulation in vitro which is subject to multiple variables. Here, immunodynamic insights from the peripheral blood are shown to be obtainable by applying a revised approach to cell-cycle analysis. Specifically, refined flow cytometric protocols were employed, assuring the reliable quantification of T cells in the S-G2/M phases of the cell-cycle (collectively termed "T Double S" for T cells in S-phase in Sanguine: in short "TDS" cells). Without protocol refinement, TDS could be either missed, as most of them layed out of the conventional lymphocyte gates, or confused with cell doublets artefactually displaying high DNA-content. To illustrate the nature of TDS cells, and their relationship to different immunodynamic scenarios, we examined them in healthy donors (HD); infectious mononucleosis (IM) patients versus asymptomatic EBV+ carriers; and recently-diagnosed T1D patients. TDS were reproducibly more abundant among CD8+ T cells and a defined subset of T-regulatory CD4+ T cells, and were substantially increased in IM and a subset of T1D patients. Of note, islet antigen-reactive TDS cell frequencies were associated with an aggressive T cell effector phenotype, suggesting that peripheral blood can reflect immune events within tissues in T1D, and possibly in other organ-specific autoimmune diseases. Our results suggest that tracking TDS cells may provide a widely applicable means of gaining insight into ongoing immune response dynamics in a variety of settings, including tissue immunopathologies where the peripheral blood has often not been considered insightful.

The CD151-midkine pathway regulates the immune microenvironment in inflammatory breast cancer.

The immune microenvironment in inflammatory breast cancer (IBC) is poorly characterised, and molecular and cellular pathways that control accumulation of various immune cells in IBC tissues remain largely unknown. Here, we discovered a novel pathway linking the expression of the tetraspanin protein CD151 in tumour cells with increased accumulation of macrophages in cancerous tissues. It is notable that elevated expression of CD151 and a higher number of tumour-infiltrating macrophages correlated with better patient responses to chemotherapy. Accordingly, CD151-expressing IBC xenografts were characterised by the increased infiltration of macrophages. In vitro migration experiments demonstrated that CD151 stimulates the chemoattractive potential of IBC cells for monocytes via mechanisms involving midkine (a heparin-binding growth factor), integrin α6ß1, and production of extracellular vesicles (EVs). Profiling of chemokines secreted by IBC cells demonstrated that CD151 increases production of midkine. Purified midkine specifically stimulated migration of monocytes, but not other immune cells. Further experiments demonstrated that the chemoattractive potential of IBC-derived EVs is blocked by anti-midkine antibodies. These results demonstrate for the first time that changes in the expression of a tetraspanin protein by tumour cells can affect the formation of the immune microenvironment by modulating recruitment of effector cells to cancerous tissues. Therefore, a CD151-midkine pathway can be considered as a novel target for controlled changes of the immune landscape in IBC. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Non-uniform in vivo Expansion of Epstein-Barr Virus-Specific T-Cells Following Donor Lymphocyte Infusion for Post-transplant Lymphoproliferative Disease.

Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) is a life-threatening complication of T-lymphocyte deplete allogeneic hematopoietic stem cell transplantation (allo-HSCT). For patients with PTLD refractory to Rituximab, donor lymphocyte infusion (DLI) is established as a successful option for salvage therapy. However, although in vivo lymphocyte expansion has been correlated with good clinical outcome following DLI, the specificity and functional characteristics of EBV-specific T-cell responses remain poorly characterized. Here we describe two patients with Rituximab-refractory PTLD complicating T-cell deplete allo-HSCT, both of whom were successfully rescued with 1 × 106/Kg unselected stem cell donor-derived DLI. Prospective analyses revealed that complete clinical and radiological responses were associated with in vivo expansion of T and NK cells. Furthermore, EBV MHC tetramer, and interferon gamma analyses revealed a marked increase in EBV-specific T-cell frequency from 4 weeks after DLI. Reactivity was demonstrated against a range of EBV latent and lytic antigens, including those detected in tumor biopsy material. The immunodominant EBV-specific T cell response expanding in vivo following infusion matched the dominant response present in the DLI preparations prior to administration. Furthermore, differences in the repertoire of subdominant antigen-specific T-cells were also detected, suggesting that antigen-encounter in vivo can shape the immune response. These results demonstrate the value of prospectively studying in vivo T-cell responses, by facilitating the identification of important specificities required for clinical efficacy. Applying this approach on a larger scale promises to yield data which may be essential for the optimization of future adoptive immunotherapeutic strategies for PTLD.

The T-cell Response to Epstein-Barr Virus-New Tricks From an Old Dog.

Epstein-Barr virus (EBV) infects most people and establishes life-long infection controlled by the host's immune system. The genetic stability of the virus, deep understanding of the viral antigens and immune epitopes recognized by the host's T-cell system and the fact that recent infection can be identified by the development of symptomatic infectious mononucleosis makes EBV a powerful system in which to study human immunology. The association between EBV and multiple cancers also means that the lessons learned have strong translational potential. Increasing evidence of a role for resident memory T-cells and non-conventional γδ T-cells in controlling EBV infection suggests new opportunities for research and means the virus will continue to provide exciting new insights into human biology and immunology into the future.

Primary EBV Infection Induces an Acute Wave of Activated Antigen-Specific Cytotoxic CD4+ T Cells.

CD4+ T cells are essential for immune protection against viruses, yet their multiple roles remain ill-defined at the single-cell level in humans. Using HLA class II tetramers, we studied the functional properties and clonotypic architecture of EBV-specific CD4+ T cells in patients with infectious mononucleosis, a symptomatic manifestation of primary EBV infection, and in long-term healthy carriers of EBV. We found that primary infection elicited oligoclonal expansions of TH1-like EBV-specific CD4+ T cells armed with cytotoxic proteins that responded immediately ex vivo to challenge with EBV-infected B cells. Importantly, these acutely generated cytotoxic CD4+ T cells were highly activated and transcriptionally distinct from classically described cytotoxic CD4+ memory T cells that accumulate during other persistent viral infections, including CMV and HIV. In contrast, EBV-specific memory CD4+ T cells displayed increased cytokine polyfunctionality but lacked cytotoxic activity. These findings suggested an important effector role for acutely generated cytotoxic CD4+ T cells that could potentially be harnessed to improve the efficacy of vaccines against EBV.

Isolation of T cell receptors targeting recurrent neoantigens in hematological malignancies.

Mutation-derived neoantigens represent an important class of tumour-specific, tumour rejection antigens, and are attractive targets for TCR gene therapy of cancer. The majority of such mutations are patient-specific and targeting these requires a fully personalized approach. However, some mutations are found recurrently among cancer patients, and represent potential targets for neoantigen-specific TCR gene therapy that is more widely applicable. Therefore, we have investigated if some cancer mutations found recurrently in hematological malignancies encode immunogenic neoantigens presented by common European Caucasoid HLA class I alleles and can form targets for TCR gene therapy. We initially focused on identifying HLA class I neoepitopes derived from calreticulin (CALR) exon 9 mutations, found in ~ 80% of JAK2wt myeloproliferative neoplasms (MPN). Based on MHC class I peptide predictions, a number of peptides derived from mutant CALR (mCALR) were predicted to bind to HLA-A*03:01 and HLA-B*07:02. However, using mass spectrometry and ex vivo pMHC multimer staining of PBMC from MPN patients with CALR exon 9 mutations, we found no evidence that these peptides were naturally processed and presented on the surface of mCALR-expressing target cells. We next developed a protocol utilizing pMHC multimers to isolate CD8+ T cells from healthy human donor PBMC that are specific for mCALR and additional putative neoepitopes found recurrently in hematological malignancies. Using this approach, CD8+ T cells specific for HLA-A*03:01- and HLA-B*07:02-presented mCALR peptides and an HLA-A*11:01-presented mutant FBXW7 (mFBXW7) peptide were successfully isolated. TCRs isolated from mCALR-specific CD8+ T cell populations were not able to recognize target cells engineered to express mCALR. In contrast, mFBXW7-specific CD8+ T cells were able to recognize target cells engineered to express mFBXW7. In conclusion, while we found no evidence for mCALR derived neoepitope presentation in the context of the HLA class I alleles studied, our data suggests that the recurrent pR465H mutation in FBXW7 may encode an HLA-A*11:01 presented neoepitope, and warrants further investigation as a target for T cell based immunotherapy of cancer.

Asymptomatic Primary Infection with Epstein-Barr Virus: Observations on Young Adult Cases.

Epstein-Barr virus (EBV) is typically acquired asymptomatically in childhood. In contrast, infection later in life often leads to infectious mononucleosis (IM), a febrile illness characterized by anti-EBV IgM antibody positivity, high loads of circulating latently infected B cells, and a marked lymphocytosis caused by hyperexpansion of EBV-specific CD8+ T cells plus a milder expansion of CD56dim NKG2A+ KIR- natural killer (NK) cells. How the two situations compare is unclear due to the paucity of studies on clinically silent infection. Here we describe five prospectively studied patients with asymptomatic infections identified in a seroepidemiologic survey of university entrants. In each case, the key blood sample had high cell-associated viral loads without a marked CD8 lymphocytosis or NK cell disturbance like those seen in patients during the acute phase of IM. Two of the cases with the highest viral loads showed a coincident expansion of activated EBV-specific CD8+ T cells, but overall CD8+ T cell numbers were either unaffected or only mildly increased. Two cases with slightly lower loads, in whom serology suggests the infection may have been caught earlier in the course of infection, also showed no T or NK cell expansion at the time. Interestingly, in another case with a higher viral load, in which T and NK cell responses were undetectable in the primary blood sample in which infection was detected, EBV-specific T cell responses did not appear until several months later, by which time the viral loads in the blood had already fallen. Thus, some patients with asymptomatic primary infections have very high circulating viral loads similar to those in patients during the acute phase of IM and a cell-mediated immune response that is qualitatively similar to that in IM patients but of a lower magnitude. However, other patients may have quite different immune responses that ultimately could reveal novel mechanisms of host control.IMPORTANCE Epstein-Barr virus (EBV) is transmitted orally, replicates in the throat, and then invades the B lymphocyte pool through a growth-transforming latent infection. While primary infection in childhood is usually asymptomatic, delayed infection is associated with infectious mononucleosis (IM), a febrile illness in which patients have high circulating viral loads and an exaggerated virus-induced immune response involving both CD8+ T cells and natural killer (NK) cells. Here we show that in five cases of asymptomatic infection, viral loads in the blood were as high as those in patients during the acute phase of IM, whereas the cell-mediated responses, even when they resembled those in patients during the acute phase of IM in timing and quality, were never as exaggerated. We infer that IM symptoms arise as a consequence not of the virus infection per se but of the hyperactivated immune response. Interestingly, there were idiosyncratic differences among asymptomatic cases in the relationship between the viral load and the response kinetics, emphasizing how much there is still to learn about primary EBV infection.

CXCR5(+) follicular cytotoxic T cells control viral infection in B cell follicles.

During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (TFH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (TC cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected TFH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (TFC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided TFC cell development. The identification of TFC cells has far-reaching implications for the development of strategies to control infections that target B cells and TFH cells and to treat B cell-derived malignancies.

Early T Cell Recognition of B Cells following Epstein-Barr Virus Infection: Identifying Potential Targets for Prophylactic Vaccination.

Epstein-Barr virus, a B-lymphotropic herpesvirus, is the cause of infectious mononucleosis, has strong aetiologic links with several malignancies and has been implicated in certain autoimmune diseases. Efforts to develop a prophylactic vaccine to prevent or reduce EBV-associated disease have, to date, focused on the induction of neutralising antibody responses. However, such vaccines might be further improved by inducing T cell responses capable of recognising and killing recently-infected B cells. In that context, EBNA2, EBNA-LP and BHRF1 are the first viral antigens expressed during the initial stage of B cell growth transformation, yet have been poorly characterised as CD8+ T cell targets. Here we describe CD8+ T cell responses against each of these three "first wave" proteins, identifying target epitopes and HLA restricting alleles. While EBNA-LP and BHRF1 each contained one strong CD8 epitope, epitopes within EBNA2 induced immunodominant responses through several less common HLA class I alleles (e.g. B*3801 and B*5501), as well as subdominant responses through common class I alleles (e.g. B7 and C*0304). Importantly, such EBNA2-specific CD8+ T cells recognised B cells within the first day post-infection, prior to CD8+ T cells against well-characterised latent target antigens such as EBNA3B or LMP2, and effectively inhibited outgrowth of EBV-transformed B cell lines. We infer that "first wave" antigens of the growth-transforming infection, especially EBNA2, constitute potential CD8+ T cell immunogens for inclusion in prophylactic EBV vaccine design.