Heterologous prime-boost viral vector vaccines for the treatment of a P1A-expressing BGL-1 glioblastoma model

Glioblastoma is an extremely aggressive and difficult cancer to treat, which may partly be due to its limited ability to induce T-cell responses. However, combining viral vector vaccines with other therapies to generate tumor-specific T cells may provide a meaningful benefit to patients. Here, we investigated whether heterologous prime-boost vaccination with chimpanzee-derived adenoviral vector ChAdOx1 and modified vaccinia Ankara (MVA) vaccines could generate therapeutically effective CD8+ T-cell responses against a model antigen P1A, a mouse homolog of human tumorassociated Melanoma Antigen GenE (MAGE)-type antigens, expressed by a BGL-1 mouse glioblastoma cell line. We demonstrated that heterologous prime-boost vaccination with ChAdOx1/MVA vaccines targeting P1A generated a high magnitude of CD8+ T cells specific for the P1A35-43 epitope presented by the MHC class I molecule H-2Ld . Prophylactic vaccination with ChAdOx1/MVA-P1A significantly prolonged the survival of syngeneic mice subcutaneously challenged with P1A-expressing BGL-1 tumors. Furthermore, different vaccination schedules significantly impact the magnitude of antigen-specific CD8+ T-cell responses and may impact protective efficacy. However, the substantial induction of myeloid-derived suppressor cells (MDSCs) by this tumor model presents a significant challenge in the therapeutic setting. Future work will investigate the efficacy of this vaccination strategy on intracranial P1A-expressing BGL-1 models.

T-cell mediated response to emerging COVID19 strains in patients with cancer studied via deep learning

Cancer patients, particularly those receiving B cell-depleting therapy for lymphoid malignancies, are at risk of prolonged SARS-CoV-2 infection, poorer clinical outcomes, and delayed initiation or disruption of cancer-directed therapy (Lee at al., 2022, Clark et al., 2021). We first studied T-cell mediated response to the Wuhan strain of SARS-CoV-2 in a cohort of 69 patients with hematologic and solid cancers, including 18 patients who received prior B-cell depleting therapy. Patients with prolonged COVID-19 clearance, defined by a positive PCR test for longer than 30 days, had a broad but poorly converged CD8+ dominant response and a lacking CD4+ response. To conduct this analysis, we performed bulk T-cell receptor (TCR) sequencing of 121 blood samples and tracked over time TCR repertoire statistics such as clonality, convergence, breadth, and depth of COVID-19-associated TCRs during the active and convalescent periods of COVID-19 infection. These SARS-CoV-2-associated TCRs were identified leveraging immunoSEQ T-MAP database (Snyder et al., 2020), a set of TCR sequences derived from COVID-19 patients and experimentally identified as responsive to MHC Class I and II epitopes from the Wuhan SARS-CoV-2 strain using the multiplex identification of TCR antigen assay (Klinger et al., 2015). To extend our TCR repertoire analysis to other SARS-CoV-2 variants, including Omicron, we developed a deep learning (DL) method to predict TCR specificities for new SARS-CoV-2 epitopes. This DL approach also permits the identification of SARS-CoV2-responsive TCRs private to an individual. Combining this DL approach with our TCR statistics methodology, we studied the dynamics of T-cell response to COVID-19 vaccinations in a cohort of 50 patients with cancer and analyzed TCR repertoire characteristics associated with different degrees of COVID-19 severity in a cohort of 42 cancer patients who contracted the Omicron. Understanding cellular response to novel infections is critical for patient care in the context of cancer, and our novel DL-based approach can leverage existing datasets to analyze and track response to emerging viral strains.

Covid-19 Vaccine-Induced Myositis

Background/Aims Through the COVID pandemic there have emerged reports of autoimmunity or new rheumatic diseases presenting in patients after they had COVID-19. This is thought to be caused by cross-reactivity of the COVID-19 spike protein to human antigens. Given the use of mRNA COVID-19 vaccinations which express the spike protein we might expect to see presentation of new rheumatic diseases following their use. We discuss a case where this appears to have occurred. Methods Our patient is a 24-year-old male with mixed phenotype acute leukaemia who had been treated with allogenic stem cell transplant and was currently in remission. He presented with fevers, palpitations, myalgia and bilateral arm and leg swelling. Symptoms began the day after receiving the first dose of an mRNA COVID-19 vaccination (Pfizer/BioNTech.) There were no other symptoms or recent change in medications. Physical examination revealed tender oedema in his forearms, biceps and thighs bilaterally with sparring of the hands. He had reduced power with shoulder (MRC 3/5), elbow (4), wrist (4+) and hip (4) movements. Observations revealed tachycardia and fevers up to 40C. Results Laboratory studies showed markedly elevated C-reactive protein (202), creatinine kinase (6697) and troponin (593) whilst investigations for infection were negative. An autoimmune panel was positive for anti- PM-SCL-75-Ab. An electrocardiogram showed sinus tachycardia. Echocardiogram was normal. Bilateral upper limb dopplers revealed no deep vein thrombus. An MRI of his thighs showed diffuse symmetrical oedema within the muscles, in keeping with an inflammatory myositis. A quadricep muscle biopsy showed evidence of MHC class 1 up-regulation, suggesting an inflammatory process. In addition, there were numerous macrophages evident in the endomysium. While this can be seen in graft-versus-host disease (GVHD), they would usually be found in the perimysium. After discussion between haematology, rheumatology and neurology, this was felt to be a case of vaccine induced myositis and myocarditis. Autoimmune myositis was thought to be less likely due to the relative sparing of the hands and the absence of Raynaud's phenomenon. 1 gram of intravenous methylprednisolone was then given for 3 days. The patient had a marked response with defervescence, improving laboratory markers, improved myalgia and decreased limb swelling. The patient was stepped down to a reducing regime of prednisolone and discharged. Due to relapse whilst weaning he has started on mycophenalate mofetil and rituximab and now continues to improve. Conclusion There are case reports of myositis following COVID-19 vaccination but our patient's case is complicated by the differential diagnosis of GVHD and concurrent myocarditis. Ongoing work is needed to clarify the exact link between vaccination and the presentation of a new inflammatory myositis, but it is important to recognise and start treatment early in order to preserve muscle bulk and ensure recovery.

Sars-Cov-2 Associated Myopathy

An association between SARS-CoV-2 infection and myopathy was suspected early in the pandemic: patients with severe COVID-19 showed increased levels of creatine kinase that could not be solely explained by cardiac affection. On the other hand, myalgia and muscle weakness are frequent symptoms in patients with mild or moderate COVID-19 - as with many other viral infections -and subsets of infected patients report persistent muscular weakness and fatigue even months after the initial infection. We performed a case-control autopsy comparing patients with severe COVID-19 to patients with other critical illnesses and assessed inflammation of skeletal muscle tissue by quantification of immune cell infiltrates, expression of major histocompatibility complex (MHC) class I and class II antigens on the sarcolemma. Relevant expression of MHC class I antigens on the sarcolemma was present in 23 of 42 specimens from patients with COVID-19 (55%) and upregulation of MHC class II antigens in 7 of 42 specimens from patients with COVID-19 (17%), but neither were found in any of the controls. In a subset of patients, MHC class I and MHC class II expression showed a clear perifascicular pattern. Signs of degenerating and necrotic fibers could also be found, however there was no statistically significant difference in the frequency of occurrence when compared to non-COVID-19 critically ill patients. We interpreted this as non-specific signs of muscular damage in critically ill patients. Numbers of macrophages, lymphocytes and natural killer cells were found to be increased in muscles from patients with COVID-19. Interestingly, no relevant expression of MxA on myofibers could be found by immunohistochemistry, but in some cases, expression of MxA was found on capillaries. Ultrastructural analysis of selected muscles with perifascicular MHC-expression did not show tubuloreticular inclusions. However, capillaries of the analyzed samples showed basement membrane alterations and signs of ongoing regenerative processes. In addition, we evaluated inflammation of cardiac muscles by quantification of immune cell infiltrates in the same patients, and found that skeletal muscles showed more inflammatory features than cardiac muscles. Moreover, inflammation was most pronounced in patients with COVID-19 with chronic courses. In some muscle specimens, SARS-CoV-2 RNA was detected by reverse transcription-polymerase chain reaction, but no evidence for a direct viral infection of myofibers was found by immunohistochemistry or electron microscopy. This suggests that SARS-CoV-2 may be associated with a postinfectious, immune-mediated myopathy.

ROLE OF IFNgamma IN PATHOGENESIS OF SARS-CoV-2 INFECTION.

To date, there is no consensus explaining the relationship between varying concentrations of IFNgamma and the severity of infection caused by SARS-CoV-2. The aim of this article was to analyze and formulate conclusions from the selected studies and publications, which, in sum, provide a potentially reasonable view on the role of IFNgamma in COVID-19 pathogenesis. This article highlights current data on the immunological role of IFNgamma which affects differentiation of naive T helper cells, acting as a polarizing factor. It activates the major histocompatibility complex (MHC) class I and II, by increasing the expression of MHC I/II subunits, inhibiting replication of the viral particles by initiating activation of interferon-stimulated genes followed by subsequent synthesis of antiviral proteins. Moreover, IFNgamma activates the production of cytokines by T cells, enhancing cytotoxic activity of the T killers. IFNgamma exerts immunostimulatory and immunomodulatory effects via STAT1, SOCS1 and PIAS genes, thus regulating activation of the JAK-STAT signaling pathway. A number of studies were considered where the patterns of changes in serum IFNgamma concentration were examined in viral infections and SARS-CoV-2. We performed a systemic analysis of the results of studies that showed a relationship between high concentrations of IFNgamma and COVID-19 severity. In a number of studies, the significantly high levels of IFNgamma in COVID-19 patients were often associated with a poor outcome of the disease. The median values of the IFNgamma concentration in severe COVID-19 were found to be significantly higher compared to the results obtained in the cases of moderate severity. It shows an increase, in parallel with viral load in the nasopharyngeal samples upon worsening of the clinical condition. Based on the data on the decreased IFNgamma concentrations in convalescent patients, the mechanism of antagonism between IFNgamma and IL-4 is considered, where the decreases serum concentrations of IFNgamma along with increasing level of IL-4 may be an indirect proof of normal adaptive immune response with subsequent development of antibodies to SARS-CoV-2 and gradual elimination of the virus from the body. Moreover, the evidence is discussed that the patients harboring some parasitic infections (Toxoplasma gondii, Cryptosporidium, Blastocystis hominis, Giardia duodenalis, Entamoeba histolytica) with persistently elevated level of IFNgamma are at reduced risk for severe course of COVID-19. Copyright © 2022, SPb RAACI.

Inverse correlation between central hypogonadism and circulating IL-10 levels in males with severe COVID-19

Introduction & Objectives: After the early and dramatic induction of inflammatory cytokines, IL-6 emerged to be associated with severe outcomes in patients with COVID-19. Likewise, high IL-10 plasma levels have been reported, and central hypogonadism has been recently observed in male patients with severe clinical outcomes (i.e., Intensive Care Unit (ICU) admission or death) of COVID-19. We aimed to investigate the role of IL-10 over the pathophysiology of COVID-19 and its relationship with hypogonadism in males. Materials & Methods: Plasma from 281 voluntary healthy males (HC) and 258 laboratory-confirmed COVID-19 males (i.e., asymptomatic (n=24);symptomatic (n=155);ICU patients (n=48);and, deceased (n=31)) was collected to measure levels of total testosterone (TT), IL-10 and the nonclassical MHC class I HLA-G (HLA-G) molecule - associated to IL-10 and involved in immune escape after viral infection - by specific enzyme-linked immunosorbent assay. Results: An inverse correlation between TT and IL-10 levels was identified, with TT levels progressively decreasing from HC (median (IQR) 10.4 (8.1-13.4) nmol/L) to asymptomatic COVID-19 (3.9 (3.1-5.3) nmol/L), to symptomatic COVID-19 (3.0 (1.8-5.7) nmol/L), ICU (1.0 (0.5-1.8) nml/L) and deceased (0.7 (0.3-2.3) nmol/L) patients, respectively (p<0.0001). Conversely, IL-10 levels progressively decreased from deceased COVID-19 patients (11.3 (4.5-37.7) pg/ml), to ICU (8.0 (2.6-16.7) pg/mL), symptomatic (6.0 (3.0-10.9) pg/mL), asymptomatic COVID-19 patients (6.0 (1.6-6.0) pg/mL), and HC (3.0 (1.3-3.0) pg/mL), respectively (p<0.0001). Similarly, HLA-G levels, progressively increased from HC to COVID-19 patients with most severe clinical outcomes. Conclusions: These data indicate that circulating TT is inversely associated to both IL-10 and HLA-G levels in men with COVID-19, where lower TT and higher IL-10 levels are associated with the most severe clinical outcomes. Further investigations are required to better define whether TT and IL-10 might be early effective biomarkers of clinical severity in males with COVID-19 and to exploit if TT is involved in promoting IL-10 and HLA-G induction.

Immune-mediated Hepatitis associated with SARS-CoV-2 mRNA vaccination

In a fraction of SARS-CoV-2 vaccinees, hepatitis compatible with features of autoimmune hepatitis (AIH) have been observed. However, it remains unclear whether the association is coincidental, reflects drug-induced liver injury, or involves vaccine-induced antigen-specific immune activation. Here, we report a case of a 52-year-old male developing a transient hepatitis after the first mRNA vaccination and severe AIH-compatible hepatitis after the second. The intrahepatic immune cell infiltrate was analysed by highly multiplexed imaging mass cytometry broadly covering key immune cell populations. Liver and longitudinal blood samples were analysed for the presence and phenotype of SARS-CoV-2 Spike-specific CD8 T cells using MHC class I tetramer technology. Additionally, Serum titers against SARS-Cov2-Spike antibodies were assessed. We identified a panlobular CD8 T cell dominant immune cell infiltrate in the liver without significant plasma cell components. Spike-specific CD8 T cells were highly enriched within the intrahepatic CD8 T cell population expressing activation markers and a tissue-resident phenotype. The activation phenotype correlated with the circulating Spike-specific CD8 T cell profile and longitudinal analysis revealed a rapid decline of T cell activation after the initiation of budesonid therapy. However, the patient experienced a mild relapse under therapy that was paralleled by the peripheral activation of Spike-specific CD8 T cells and was controlled under systemic steroid therapy. Collectively, our results indicate that an immune-mediated hepatitis after COVID19 vaccination can present with typical clinical features of an AIH but can be pathophysiologically separated from a classical AIH. Whether a long-term immunosuppressive regimen will be required remains to be determined.

Manufacture and Immunological Characterization of GMP-Compliant Functional Sars-COV-2 Cytotoxic T Lymphocytes (CTLs) Utilizing the Clinimacs ® Cytokine Capture System

Background: The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pandemic that has taken millions of lives around the globe. Treatment of patients with moderate and severe COVID-19 disease has included dexamethasone, tocilizumab, Remdesivir, convalescent plasma, and targeted antibodies, however, currently, there are no FDA approved targeted cellular therapies in the treatment of mild or moderate SARS-CoV-2 disease. Virus-specific cytotoxic T cell lymphocytes (vCTLs) have shown therapeutic efficacy in immunocompromised patients with viral infections. We developed a multicenter and multidisciplinary Viral Cytotoxic T-Cell Consortium (VIRCTLC) to investigate the use of vCTLs manufactured by direct enrichment using the Cytokine Capture System (CCS) on the CliniMACS® Prodigy device. SARS-CoV-2 specific PepTivator Peptides consist of overlapping peptides that span the entire sequence of the protein (Protein N and M), or the length of its immunodominant domain (Protein S). The peptides can bind to either MHC class I or MHC class II molecules and are therefore able to target both CD4 and CD8 T cells. Objective: To screen, manufacture, and characterize SARS-CoV-2 vCTLs generated from convalescent COVID-19 donors using the CliniMACS® Cytokine Capture System on the CliniMACS® Prodigy device. Methods: Donor screening was done utilizing PBMNCs from 15 convalescent COVID-19 donors after informed consent. PBMNCs were stimulated with a mix of PepTivator peptides (Miltenyi Biotech®) contained in the S, M and N proteins. IFN-γ levels were examined in CD3, CD4, and CD8 T cells by flow cytometry analysis. After informed consent, PBMNCs from three convalescent COVID-19 donors who screened positively to the PepTivator® peptide pools of SARS-CoV-2 Proteins M, N and S were collected by apheresis using the SPECTRA Optia® apheresis instrument. PBMNCs were incubated with the PepTivator® peptide pools for 4 hours. After incubation, the SARS-CoV-2 vCTLs were enriched using the CliniMACS Cytokine Capture System as we have previously described (Flower/Cairo, et al, ASTCT, 2020). Samples were taken from the enriched vCTLs and tested in gram stains, sterility cultures, cell counts, viability and IFN-γ cytokine staining (CD3/CD4/CD8/IFN-γ marker panel) by flow cytometry. Amplification and sequencing of TCRβ CDR3 regions of pre-stimulated PBMNC, stimulated PBMNCs samples taken from the QC bag (QC samples) and the enriched SARS-CoV-2 vCTLs were performed on the ImmunoSEQ platform using ImmunoSEQ® TCRB Assay kit (Adaptive Biotechnologies, Seattle, WA, USA). Characterization of immune subsets was done by mass cytometry analysis with 41 Immunophenotypic markers. Transcriptome of the immune landscape of QC samples, and enriched vCTLs was compared with the pre samples using the human nCounter PanCancer Immune Profiling Panel on the nCounter system. Results: We demonstrate that 93.3% of convalescent donor blood samples passed the screening criteria for clinical manufacture. Three validation runs resulted in enriched T cells that consisted of 79% + 21% (mean + SEM) IFNγ + T cells (Fig.1). TCRβ sequencing showed that convalescent COVID-19 donors have a highly diverse TCR repertoire and we identified TCRβ CDR3 clones that are known to be associated with SARS-CoV-2 T cell responses. Immunophenotyping analysis demonstrated more CD4 T cells than CD8 T cells in the SARS CoV-2 vCTLs, an increase in memory CD8 and CD4 cells, especially CD8 T EM, CD4 T cm and CD4 T EMRA cells (Fig.2) and an increase DC cells in the SARS CoV-2 vCTL products as compared to pre-stimulated PBMNCs. Expression of the exhaustion markers was not enhanced in the SARS CoV-2 vCTLs as compared to pre-stimulated PBMNCs. Transcriptome analysis showed increased gene expression in T-cell function, interleukin, pathogen defense, and TNF superfamily pathway genes in the SARS CoV-2 vCTLs as compared to pre-stimulated PBMNCs. Conclusion: Our study demonstrates that highly functional SARS-CoV-2 vCTLs can be rapidly generat d by direct cytokine enrichment from convalescent donor peripheral blood mononuclear cells. These data serve as pre-clinical validation for an ongoing clinical trial utilizing related HLA-matched and haplo-identical SARS CoV-2 vCTLs for the treatment of patients with mild and moderate SARS-CoV-2 disease (IND #27260, NCT# 04896606). [Formula presented] Disclosures: Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding;Courier Therapeutics: Current holder of individual stocks in a privately-held company. Johnson: Miltenyi Biotec: Research Funding. Cairo: Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Amgen: Speakers Bureau;Sanofi: Speakers Bureau;Servier: Speakers Bureau;Sobi: Speakers Bureau;Omeros: Membership on an entity's Board of Directors or advisory committees;Nektar: Membership on an entity's Board of Directors or advisory committees.