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.

An Intensive Approach to Improving Diversity, Equity, and Inclusion in an Academic Emergency Department.

A healthcare workforce that demonstrates cultural competence and humility while reflecting the diversity of the surrounding community has the potential to significantly benefit the patient population it serves. In this context and given numerous societal influences and the events of 2020, the leadership of the Department of Emergency Medicine at Albany Medical Center recognized the need to promote diversity, equity, and inclusion (DEI) in multiple areas. These included premedical education, medical education, postgraduate medical education, faculty development, staff satisfaction, and patient care. The department formed a DEI taskforce that developed an ongoing, multipronged, interdisciplinary approach to address these important aspects of our work and clinical environment with the goals of improving staff wellbeing, reducing burnout, and promoting the health of our community. Our experience is shared here to illustrate how a small, dedicated team can implement a variety of DEI initiatives quickly and with relatively little cost at a large academic medical center.

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.

Evaluating and evolving a screening library in academia: the St Jude approach.

The quality of lead compounds is a key factor for determining the success of chemical probe and drug discovery programs. Given that high-throughput screening (HTS) continues to be a dominant lead generation paradigm, access to high-quality screening libraries is crucial for such efforts in both industry and academia. Here, we discuss the strategy implemented a decade ago to build from scratch one of the largest compound collections in academia, containing ∼575 000 carefully annotated small molecules, and a recent multidisciplinary effort designed to further enhance the collection to meet our research demands for the next decade.

Characterizing EBV-associated lymphoproliferative diseases and the role of myeloid-derived suppressor cells.

Chronic active Epstein-Barr virus (CAEBV) typically presents as persistent infectious mononucleosis-like disease and/or hemophagocytic lymphohistocytosis (HLH), reflecting ectopic Epstein-Barr virus (EBV) infection and lymphoproliferation of T and/or NK cells. Clinical behavior ranges from indolent, stable disease through to rapidly progressive, life-threatening disease. Although it is thought the chronicity and/or progression reflect an escape from immune control, very little is known about the phenotype and function of the infected cells vs coresident noninfected population, nor about the mechanisms that could underpin their evasion of host immune surveillance. To investigate these questions, we developed a multicolor flow cytometry technique combining phenotypic and functional marker staining with in situ hybridization for the EBV-encoded RNAs (EBERs) expressed in every infected cell. This allows the identification, phenotyping, and functional comparison of infected (EBERPOS) and noninfected (EBERNEG) lymphocyte subset(s) in patients' blood samples ex vivo. We have characterized CAEBV and HLH cases with monoclonal populations of discrete EBV-activated T-cell subsets, in some cases accompanied by EBV-activated NK-cell subsets, with longitudinal data on the infected cells' progression despite standard steroid-based therapy. Given that cytotoxic CD8+ T cells with relevant EBV antigen specificity were detectable in the blood of the best studied patient, we searched for means whereby host surveillance might be impaired. This revealed a unique feature in almost every patient with CAEBV studied: the presence of large numbers of myeloid-derived suppressor cells that exhibited robust inhibition of T-cell growth. We suggest that their influence is likely to explain the host's failure to contain EBV-positive T/NK-cell proliferation.

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.

Quality Assessment of Expired Naloxone Products from First-Responders' Supplies.

Objective: Naloxone is an opioid receptor antagonist that reverses life-threatening effects of opioid overdose. Since the 1970s, naloxone products have been developed as injectable solutions, and more recently as nasal sprays. Naloxone products have saved many lives in emergency settings. These products are routinely carried by public safety first-responders including fire fighters (FF), law enforcement officers (LEO), and emergency medical services (EMS). Now, they are also distributed through community access programs to the public. While public safety medications are monitored, those publically distributed are not, so expired products can be possibly found on-hand in an emergency. This study analyzed the quality and stability of expired Naloxone HCl Solutions for Injection, to assess their remaining efficacies and potential risks. Methods: The samples were collected from EMS or law enforcement training supplies and expired returns, with expiration dates ranging from 1990 to 2018. Using standardized techniques, the remaining naloxone was quantified, and the main degradation products, nornaloxone (also known as noroxymorphone) and other possible species, were monitored and quantified systematically. Results: Most tested samples were found containing more than 90% of labeled naloxone, including those stored for nearly 30 years. The naloxone degradation was slow, but generally correlated with storage time length. There was no significant amount of degradation products detected across all samples. Nornaloxone was detected from some older samples, but all less than 1%. Therefore, although it is an opioid agonist, the risk caused by nornaloxone should be low. Conclusion: This quality assessment demonstrates that expired naloxone products may still meet USP standards, even after many years. Further pharmaceutical, clinical, and regulatory investigation should be conducted to confirm our findings, especially for new naloxone products with different formulations and routes of administration. Extending the shelf-life of naloxone products may have important financial and public health consequences in addressing future drug shortages and meeting the needs for this critical drug.

Long-Term Ibrutinib Therapy Reverses CD8+ T Cell Exhaustion in B Cell Chronic Lymphocytic Leukaemia.

Chronic Lymphocytic Leukaemia (CLL) is associated with immune suppression and susceptibility to infection. CD8+ T cell numbers are increased and demonstrate elevated expression of PD-1 and impaired function. The mechanisms driving these features of exhaustion are uncertain but are likely to include chronic immune recognition of tumor and/or infectious agents. We investigated the number, phenotype and function of total and virus-specific CD8+ T cells in 65 patients with CLL and 14 patients undergoing long-term ibrutinib therapy (median 21 months). Ibrutinib substantially reduced the number of both CD3+ T cells and CD8+ T cells. Importantly, this was associated with a reduction in PD-1 expression on CD8+ T cells (median 28 vs. 24%; p = 0.042) and 3.5 fold increase in cytokine production following mitogen stimulation. The influence of ibrutinib on antigen-specific CD8+ T cell function was assessed by HLA-peptide tetramers and revealed increased IFNγ and TNFα cytokine responses following stimulation with CMV or EBV peptides together with a 55% reduction in the frequency of "inflated" virus-specific CD8+ T cells. These findings reveal that long-term ibrutinib therapy is associated with substantial reversal of T cell exhaustion in B-CLL and is likely to contribute to the reduced infection risk seen in association with this agent.