An mRNA-based T-cell-inducing antigen strengthens COVID-19 vaccine against SARS-CoV-2 variants.

Herd immunity achieved through mass vaccination is an effective approach to prevent contagious diseases. Nonetheless, emerging SARS-CoV-2 variants with frequent mutations largely evaded humoral immunity induced by Spike-based COVID-19 vaccines. Herein, we develop a lipid nanoparticle (LNP)-formulated mRNA-based T-cell-inducing antigen, which targeted three SARS-CoV-2 proteome regions that enriched human HLA-I epitopes (HLA-EPs). Immunization of HLA-EPs induces potent cellular responses to prevent SARS-CoV-2 infection in humanized HLA-A*02:01/DR1 and HLA-A*11:01/DR1 transgenic mice. Of note, the sequences of HLA-EPs are highly conserved among SARS-CoV-2 variants of concern. In humanized HLA-transgenic mice and female rhesus macaques, dual immunization with the LNP-formulated mRNAs encoding HLA-EPs and the receptor-binding domain of the SARS-CoV-2 B.1.351 variant (RBDbeta) is more efficacious in preventing infection of SARS-CoV-2 Beta and Omicron BA.1 variants than single immunization of LNP-RBDbeta. This study demonstrates the necessity to strengthen the vaccine effectiveness by comprehensively stimulating both humoral and cellular responses, thereby offering insight for optimizing the design of COVID-19 vaccines.

Potentiating the Cross-Reactive IFN-γ T Cell and Polyfunctional T Cell Responses by Heterologous GX-19N DNA Booster in Mice Primed with Either a COVID-19 mRNA Vaccine or Inactivated Vaccine.

Waning vaccine-induced immunity, coupled with the emergence of SARS-CoV-2 variants, has inspired the widespread implementation of COVID-19 booster vaccinations. Here, we evaluated the potential of the GX-19N DNA vaccine as a heterologous booster to enhance the protective immune response to SARS-CoV-2 in mice primed with either an inactivated virus particle (VP) or an mRNA vaccine. We found that in the VP-primed condition, GX-19N enhanced the response of both vaccine-specific antibodies and cross-reactive T Cells to the SARS-CoV-2 variant of concern (VOC), compared to the homologous VP vaccine prime-boost. Under the mRNA-primed condition, GX-19N induced higher vaccine-induced T Cell responses but lower antibody responses than the homologous mRNA vaccine prime-boost. Furthermore, the heterologous GX-19N boost induced higher S-specific polyfunctional CD4+ and CD8+ T cell responses than the homologous VP or mRNA prime-boost vaccinations. Our results provide new insights into booster vaccination strategies for the management of novel COVID-19 variants.

Intradermal skin test with mRNA vaccines as a surrogate marker of T cell immunity in immunocompromised patients.

OBJECTIVES: Intradermal skin test (IDT) with mRNA vaccines may represent a simple, reliable, and affordable tool to measure T cell response in immunocompromised patients who failed to mount serological responses following vaccination with mRNA covid-19 vaccines. METHODS: We compared anti-SARS-CoV-2 antibodies and cellular responses in vaccinated immunocompromised patients (n = 58), healthy seronegative naive controls (NC, n = 8), and healthy seropositive vaccinated controls (VC, n = 32) by Luminex, spike-induced IFN-γ Elispot and an IDT. A skin biopsy 24 h after IDT and single-cell RNAseq was performed in three vaccinated volunteers. RESULTS: Twenty-five percent of seronegative NC had a positive Elispot (2/8) and IDT (1/4), compared to 95% (20/21) and 93% (28/30) in seropositive VC, respectively. Single-cell RNAseq data in the skin of VC showed a predominant mixed population of effector helper and cytotoxic T cells. The TCR repertoire revealed 18/1064 clonotypes with known specificities against SARS-CoV-2, among which six were spike-specific. Seronegative immunocompromised patients with positive Elispot and IDT were in 83% (5/6) treated with B cell-depleting reagents, while those with negative IDT were all transplant recipients. CONCLUSIONS: Our results indicate that delayed local reaction to IDT reflects vaccine-induced T-cell immunity opening new perspectives to monitor seronegative patients and elderly populations with waning immunity.

Dynamic Evolution of Humoral and T-Cell Specific Immune Response to COVID-19 mRNA Vaccine in Patients with Multiple Sclerosis Followed until the Booster Dose.

This study characterizes antibody and T-cell immune responses over time until the booster dose of COronaVIrus Disease 2019 (COVID-19) vaccines in patients with multiple sclerosis (PwMS) undergoing different disease-modifying treatments (DMTs). We prospectively enrolled 134 PwMS and 99 health care workers (HCWs) having completed the two-dose schedule of a COVID-19 mRNA vaccine within the last 2-4 weeks (T0) and followed them 24 weeks after the first dose (T1) and 4-6 weeks after the booster (T2). PwMS presented a significant reduction in the seroconversion rate and anti-receptor-binding domain (RBD)-Immunoglobulin (IgG) titers from T0 to T1 (p < 0.0001) and a significant increase from T1 to T2 (p < 0.0001). The booster dose in PwMS showed a good improvement in the serologic response, even greater than HCWs, as it promoted a significant five-fold increase of anti-RBD-IgG titers compared with T0 (p < 0.0001). Similarly, the T-cell response showed a significant 1.5- and 3.8-fold increase in PwMS at T2 compared with T0 (p = 0.013) and T1 (p < 0.0001), respectively, without significant modulation in the number of responders. Regardless of the time elapsed since vaccination, most ocrelizumab- (77.3%) and fingolimod-treated patients (93.3%) showed only a T-cell-specific or humoral-specific response, respectively. The booster dose reinforces humoral- and cell-mediated-specific immune responses and highlights specific DMT-induced immune frailties, suggesting the need for specifically tailored strategies for immune-compromised patients to provide primary prophylaxis, early SARS-CoV-2 detection and the timely management of COVID-19 antiviral treatments.

Dual targeting of CD19 and CD22 with bicistronic CAR-T cells in patients with relapsed/refractory large B-cell lymphoma.

Relapse after CD19-directed chimeric antigen receptor T-cell (CAR-T) therapy for large B-cell lymphoma (LBCL) is commonly ascribed to antigen loss or CAR-T exhaustion. Multiantigen targeting and programmed cell death protein-1 blockade are rational approaches to prevent relapse. Here, we test CD19/22 dual-targeting CAR-T (AUTO3) plus pembrolizumab in relapsed/refractory LBCL (NCT03289455). End points include toxicity (primary) and response rates (secondary). Fifty-two patients received AUTO3 and 48/52 received pembrolizumab. Median age was 59 years (range, 27-83), 46/52 had stage III/ IV disease and median follow-up was 21.6 months. AUTO3 was safe; grade 1-2 and grade 3 cytokine release syndrome affected 18/52 (34.6%) and 1/52 (1.9%) patients, neurotoxicity arose in 4 patients (2/4, grade 3-4), and hemophagocytic lymphohistiocytosis affected 2 patients. Outpatient administration was tested in 20 patients, saving a median of 14 hospital days per patient. Overall response rates were 66% (48.9%, complete response [CR]; 17%, partial response). Median duration of remission (DOR) for CR patients was not reached and for all responding patients was 8.3 months (95% confidence interval [CI]: 3.0-not evaluable). 54.4% (CI: 32.8-71.7) of CR patients and 42.6% of all responding patients were projected to remain progression-free at ≥12 months. AUTO3 ± pembrolizumab for relapsed/refractory LBCL was safe and delivered durable remissions in 54.4% of complete responders, associated with robust CAR-T expansion. Neither dual-targeting CAR-T nor pembrolizumab prevented relapse in a significant proportion of patients, and future developments include next-generation-AUTO3, engineered for superior expansion in vivo, and selection of CAR binders active at low antigen densities.

GRT-R910: a self-amplifying mRNA SARS-CoV-2 vaccine boosts immunity for ≥6 months in previously-vaccinated older adults.

SARS-CoV-2 has resulted in high levels of morbidity and mortality world-wide, and severe complications can occur in older populations. Humoral immunity induced by authorized vaccines wanes within 6 months, and frequent boosts may only offer transient protection. GRT-R910 is an investigational self-amplifying mRNA (samRNA)-based SARS-CoV-2 vaccine delivering full-length Spike and selected conserved non-Spike T cell epitopes. This study reports interim analyses for a phase I open-label dose-escalation trial evaluating GRT-R910 in previously vaccinated healthy older adults (NCT05148962). Primary endpoints of safety and tolerability were assessed. Most solicited local and systemic adverse events (AEs) following GRT-R910 dosing were mild to moderate and transient, and no treatment-related serious AEs were observed. The secondary endpoint of immunogenicity was assessed via IgG binding assays, neutralization assays, interferon-gamma ELISpot, and intracellular cytokine staining. Neutralizing antibody titers against ancestral Spike and variants of concern were boosted or induced by GRT-R910 and, contrasting to authorized vaccines, persisted through at least 6 months after the booster dose. GRT-R910 increased and/or broadened functional Spike-specific T cell responses and primed functional T cell responses to conserved non-Spike epitopes. This study is limited due to small sample size, and additional data from ongoing studies will be required to corroborate these interim findings.

Idiotope-Driven T-Cell/B-Cell Collaboration-Based T-Cell Epitope Prediction Using B-Cell Receptor Repertoire Sequences in Infectious Diseases.

T-cell recognition of antigen epitopes is a crucial step for the induction of adaptive immune responses, and the identification of such T-cell epitopes is, therefore, important for understanding diverse immune responses and controlling T-cell immunity. A number of bioinformatic tools exist that predict T-cell epitopes; however, many of these methods highly rely on evaluating conventional peptide presentation by major histocompatibility complex (MHC) molecules, but they ignore epitope sequences recognized by T-cell receptor (TCR). Immunogenic determinant idiotopes are present on the variable regions of immunoglobulin molecules expressed on and secreted by B-cells. In idiotope-driven T-cell/B-cell collaboration, B-cells present the idiotopes on MHC molecules for recognition by idiotope-specific T-cells. According to the idiotype network theory formulated by Niels Jerne, such idiotopes found on anti-idiotypic antibodies exhibit molecular mimicry of antigens. Here, by combining these concepts and defining the patterns of TCR-recognized epitope motifs (TREMs), we developed a T-cell epitope prediction method that identifies T-cell epitopes derived from antigen proteins by analyzing B-cell receptor (BCR) sequences. This method allowed us to identify T-cell epitopes that contain the same TREM patterns between BCR and viral antigen sequences in two different infectious diseases caused by dengue virus and SARS-CoV-2 infection. The identified epitopes were among the T-cell epitopes detected in previous studies, and T-cell stimulatory immunogenicity was confirmed. Thus, our data support this method as a powerful tool for the discovery of T-cell epitopes from BCR sequences.

A spike-targeting bispecific T cell engager strategy provides dual layer protection against SARS-CoV-2 infection in vivo.

Neutralizing antibodies exert a potent inhibitory effect on viral entry; however, they are less effective in therapeutic models than in prophylactic models, presumably because of their limited efficacy in eliminating virus-producing cells via Fc-mediated cytotoxicity. Herein, we present a SARS-CoV-2 spike-targeting bispecific T-cell engager (S-BiTE) strategy for controlling SARS-CoV-2 infection. This approach blocks the entry of free virus into permissive cells by competing with membrane receptors and eliminates virus-infected cells via powerful T cell-mediated cytotoxicity. S-BiTE is effective against both the original and Delta variant of SARS-CoV2 with similar efficacy, suggesting its potential application against immune-escaping variants. In addition, in humanized mouse model with live SARS-COV-2 infection, S-BiTE treated mice showed significantly less viral load than neutralization only treated group. The S-BiTE strategy may have broad applications in combating other coronavirus infections.

CD71+ erythroid cells suppress T-cell effector functions and predict immunotherapy outcomes in patients with virus-associated solid tumors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer. However, only a portion of patients respond to such treatments. Therefore, it remains a prevailing clinical need to identify factors associated with acquired resistance or lack of response to ICIs. We hypothesized that the immunosuppressive CD71+ erythroid cells (CECs) within the tumor and/or distant 'out-of-field' may impair antitumor response. METHODS: We studied 38 patients with cancer through a phase II clinical trial investigating the effects of oral valproate combined with avelumab (anti-programmed death-ligand 1 (PD-L1)) in virus-associated solid tumors (VASTs). We quantified the frequency/functionality of CECs in blood and biopsies of patients. Also, we established an animal model of melanoma (B16-F10) to investigate the possible effects of erythropoietin (EPO) treatment on anti-PD-L1 therapy. RESULTS: We found a substantial expansion of CECs in the blood of patients with VAST compared with healthy controls. We noted that the frequency of CECs in circulation was significantly higher at the baseline and throughout the study in non-responders versus responders to PD-L1 therapy. Moreover, we observed that CECs in a dose-dependent manner suppress effector functions of autologous T cells in vitro. The subpopulation of CD45+CECs appears to have a more robust immunosuppressive property compared with their CD45- counterparts. This was illustrated by a stronger expression of reactive oxygen species, PD-L1/PD-L2, and V-domain Ig suppressor of T-cell activation in this subpopulation. Lastly, we found a higher frequency of CECs in the blood circulation at the later cancer stage and their abundance was associated with anemia, and a poor response to immunotherapy. Finally, we report the expansion of CECs in the spleen and tumor microenvironment of mice with melanoma. We found that although CECs in tumor-bearing mice secret artemin, this was not the case for VAST-derived CECs in humans. Notably, our results imply that EPO, a frequently used drug for anemia treatment in patients with cancer, may promote the generation of CECs and subsequently abrogates the therapeutic effects of ICIs (eg, anti-PD-L1). CONCLUSIONS: Our results demonstrate that anemia by the expansion of CECs may enhance cancer progression. Notably, measuring the frequency of CECs may serve as a valuable biomarker to predict immunotherapy outcomes.

[Assesment of specific T-cell immunity to SARS-CoV-2 virus antigens in COVID-19 reconvalescents].

INTRODUCTION: The development of the COVID-19 pandemic has stimulated the scientific research aimed at studying of the mechanisms of formation the immunity against SARS-CoV-2. Currently, there is a need to develop a domestic simple and cost-effective specific method suitable for monitoring of T-cell response against SARS-CoV-2 in reconvalescents and vaccinated individuals. AIM: Development of a screening method for evaluation specific T-cell immunity against SARS-CoV-2. MATERIALS AND METHODS: Total 40 individuals who had mild to moderate COVID-19 and 20 healthy volunteers who did not have a history of this disease were examined. The presence and levels of IgG and IgM antibodies to SARS-CoV-2 were identified in participants sera by ELISA using the diagnostic kits from JSC Vector-Best (Novosibirsk, Russian Federation). Antigenic stimulation of mononuclear cells was carried out on commercial plates with adsorbed whole-virion inactivated SARS-CoV-2 antigen (State Research Center of Virology and Biotechnology VECTOR Novosibirsk, Russian Federation). The concentration of IFN- was measured in ELISA using the test systems from JSC Vector-Best (Novosibirsk, Russian Federation). The immunophenotyping of lymphocytes was performed on a flow cytometer Cytomics FC500 (Beckman Coulter, USA). Statistical data processing was carried out using the Microsoft Excel and STATISTICA 10 software package. RESULTS: Stimulation of mononuclear cells isolated from the peripheral blood with whole-virion inactivated SARS-CoV-2 antigen fixed at the bottom of the wells of a polystyrene plate showed a significantly higher median response in terms of IFN- production in 40 people who had history of COVID-19 compared to 20 healthy blood donors (172.1 [34.3575.1] pg/ml versus 15.4 [6.925.8] pg/ml, p 0.0001). There was no difference in median IFN- levels in supernatants collected from unstimulated mononuclear cells from COVID-19 reconvalescents and healthy donors (2.7 [0.411.4] pg/ml versus 0.8 [0.023.3] pg/ml, p 0.05). The overall sensitivity and specificity of this method were 73% (95% CI 5888%) and 100% (95% CI 100100%), respectively, at a cut-off of 50 pg/ml. CONCLUSION: The developed method for assessment of the cellular immune response to SARS-CoV-2 can be used as a screening method for monitoring the T-cell response in a population against a new coronavirus infection in recovered people.

Targets and cross-reactivity of human T cell recognition of common cold coronaviruses.

The coronavirus (CoV) family includes several viruses infecting humans, highlighting the importance of exploring pan-CoV vaccine strategies to provide broad adaptive immune protection. We analyze T cell reactivity against representative Alpha (NL63) and Beta (OC43) common cold CoVs (CCCs) in pre-pandemic samples. S, N, M, and nsp3 antigens are immunodominant, as shown for severe acute respiratory syndrome 2 (SARS2), while nsp2 and nsp12 are Alpha or Beta specific. We further identify 78 OC43- and 87 NL63-specific epitopes, and, for a subset of those, we assess the T cell capability to cross-recognize sequences from representative viruses belonging to AlphaCoV, sarbecoCoV, and Beta-non-sarbecoCoV groups. We find T cell cross-reactivity within the Alpha and Beta groups, in 89% of the instances associated with sequence conservation >67%. However, despite conservation, limited cross-reactivity is observed for sarbecoCoV, indicating that previous CoV exposure is a contributing factor in determining cross-reactivity. Overall, these results provide critical insights in developing future pan-CoV vaccines.

T cell immunity following COVID-19 vaccination in adult patients with primary antibody deficiency - a 22-month follow-up.

Primary antibody deficiencies, such as common variable immunodeficiency (CVID), are heterogenous disease entities consisting of primary hypogammaglobulinemia and impaired antibody responses to vaccination and natural infection. CVID is the most common primary immunodeficiency in adults, presenting with recurrent bacterial infections, enteropathy, autoimmune disorders, interstitial lung diseases and increased risk of malignancies. Patients with CVID are recommended to be vaccinated against SARS-CoV-2, but there are relatively few studies investigating humoral and cellular responses to immunization. We studied the dynamics of humoral and cell-mediated immunity responses up to 22 months in 28 patients with primary immunodeficiency and three patients with secondary immunodeficiency receiving ChAdOx1, BNT162b2 and mRNA-1273 COVID-19 vaccines. Despite inadequate humoral response to immunization, we demonstrate a robust T cell activation likely protecting from severe COVID-19.

Prevalence and functional profile of SARS-CoV-2 T cells in asymptomatic Kenyan adults.

BackgroundSARS-CoV-2 infection in Africa has been characterized by a less severe disease profile than what has been observed elsewhere, but the profile of SARS-CoV-2-specific adaptive immunity in these mainly asymptomatic patients has not, to our knowledge, been analyzed.MethodsWe collected blood samples from residents of rural Kenya (n = 80), who had not experienced any respiratory symptoms or had contact with individuals with COVID-19 and had not received COVID-19 vaccines. We analyzed spike-specific antibodies and T cells specific for SARS-CoV-2 structural (membrane, nucleocapsid, and spike) and accessory (ORF3a, ORF7, ORF8) proteins. Pre-pandemic blood samples collected in Nairobi (n = 13) and blood samples from mild-to-moderately symptomatic COVID-19 convalescent patients (n = 36) living in the urban environment of Singapore were also studied.ResultsAmong asymptomatic Africans, we detected anti-spike antibodies in 41.0% of the samples and T cell responses against 2 or more SARS-CoV-2 proteins in 82.5% of samples examined. Such a pattern was absent in the pre-pandemic samples. Furthermore, distinct from cellular immunity in European and Asian COVID-19 convalescents, we observed strong T cell immunogenicity against viral accessory proteins (ORF3a, ORF8) but not structural proteins, as well as a higher IL-10/IFN-γ cytokine ratio profile.ConclusionsThe high incidence of T cell responses against different SARS-CoV-2 proteins in seronegative participants suggests that serosurveys underestimate SARS-CoV-2 prevalence in settings where asymptomatic infections prevail. The functional and antigen-specific profile of SARS-CoV-2-specific T cells in African individuals suggests that environmental factors can play a role in the development of protective antiviral immunity.FundingUS Centers for Disease Control and Prevention, Division of Global Health Protection; the Singapore Ministry of Health's National Medical Research Council (COVID19RF3-0060, COVID19RF-001, COVID19RF-008, MOH-StaR17Nov-0001).

MERS-CoV‒Specific T-Cell Responses in Camels after Single MVA-MERS-S Vaccination.

We developed an ELISPOT assay for evaluating Middle East respiratory syndrome coronavirus (MERS-CoV)‒specific T-cell responses in dromedary camels. After single modified vaccinia virus Ankara-MERS-S vaccination, seropositive camels showed increased levels of MERS-CoV‒specific T cells and antibodies, indicating suitability of camel vaccinations in disease-endemic areas as a promising approach to control infection.

Evidence for broad cross-reactivity of the SARS-CoV-2 NSP12-directed CD4+ T-cell response with pre-primed responses directed against common cold coronaviruses.

Introduction: The nonstructural protein 12 (NSP12) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has a high sequence identity with common cold coronaviruses (CCC). Methods: Here, we comprehensively assessed the breadth and specificity of the NSP12-specific T-cell response after in vitro T-cell expansion with 185 overlapping 15-mer peptides covering the entire SARS-CoV-2 NSP12 at single-peptide resolution in a cohort of 27 coronavirus disease 2019 (COVID-19) patients. Samples of nine uninfected seronegative individuals, as well as five pre-pandemic controls, were also examined to assess potential cross-reactivity with CCCs. Results: Surprisingly, there was a comparable breadth of individual NSP12 peptide-specific CD4+ T-cell responses between COVID-19 patients (mean: 12.82 responses; range: 0-25) and seronegative controls including pre-pandemic samples (mean: 12.71 responses; range: 0-21). However, the NSP12-specific T-cell responses detected in acute COVID-19 patients were on average of a higher magnitude. The most frequently detected CD4+ T-cell peptide specificities in COVID-19 patients were aa236-250 (37%) and aa246-260 (44%), whereas the peptide specificities aa686-700 (50%) and aa741-755 (36%), were the most frequently detected in seronegative controls. In CCC-specific peptide-expanded T-cell cultures of seronegative individuals, the corresponding SARS-CoV-2 NSP12 peptide specificities also elicited responses in vitro. However, the NSP12 peptide-specific CD4+ T-cell response repertoire only partially overlapped in patients analyzed longitudinally before and after a SARS-CoV-2 infection. Discussion: The results of the current study indicate the presence of pre-primed, cross-reactive CCC-specific T-cell responses targeting conserved regions of SARS-CoV-2, but they also underline the complexity of the analysis and the limited understanding of the role of the SARS-CoV-2 specific T-cell response and cross-reactivity with the CCCs.

Cellular and humoral responses after second and third SARS-CoV-2 vaccinations in patients with autoimmune diseases treated with rituximab: specific T cell immunity remains longer and plays a protective role against SARS-CoV-2 reinfections.

Background: Humoral and cellular immune responses are known to be crucial for patients to recover from COVID-19 and to protect them against SARS-CoV-2 reinfection once infected or vaccinated. Objectives: This study aimed to investigate humoral and T cell responses to SARS-CoV-2 vaccination in patients with autoimmune diseases after the second and third vaccine doses while on rituximab and their potential protective role against reinfection. Methods: Ten COVID-19-naïve patients were included. Three time points were used for monitoring cellular and humoral responses: pre-vaccine to exclude virus exposure (time point 1) and post-second and post-third vaccine (time points 2 and 3). Specific IgG antibodies were monitored by Luminex and T cells against SARS-CoV-2 spike-protein by ELISpot and CoVITEST. All episodes of symptomatic COVID-19 were recorded. Results: Nine patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis and one with an undifferentiated autoimmune disease were included. Nine patients received mRNA vaccines. The last rituximab infusion was administered for a mean (SD) of 15 (10) weeks before the first vaccine and six patients were CD19-B cell-depleted. After a mean (SD) of 19 (10) and 16 (2) days from the second and third vaccine dose, IgG anti-SARS-CoV-2 antibodies were detected in six (60%) and eight (80%) patients, respectively. All patients developed specific T cell responses by ELISpot and CoVITEST in time points 2 and 3. Previous B cell depletion correlated with anti-SARS-CoV-2 IgG levels. Nine (90%) patients developed mild COVID-19 after a median of 7 months of the third dose. Conclusion: Rituximab in patients with autoimmune diseases reduces humoral responses but does not avoid the development of T cell responses to SARS-CoV-2 vaccination, which remain present after a booster dose. A steady cellular immunity appears to be protective against subsequent reinfections.

An ACAT inhibitor suppresses SARS-CoV-2 replication and boosts antiviral T cell activity.

The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 pseudoparticle infection and disrupts the association of ACE2 and GM1 lipid rafts on the cell membrane, perturbing viral attachment. Imaging SARS-CoV-2 RNAs at the single cell level using a viral replicon model identifies the capacity of Avasimibe to limit the establishment of replication complexes required for RNA replication. Genetic studies to transiently silence or overexpress ACAT isoforms confirmed a role for ACAT in SARS-CoV-2 infection. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled during the acute phase of infection. Thus, re-purposing of ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects. Trial registration: NCT04318314.

Impaired SARS-CoV-2 specific T-cell response in patients with severe COVID-19.

Cellular immune responses are of pivotal importance to understand SARS-CoV-2 pathogenicity. Using an enzyme-linked immunosorbent spot (ELISpot) interferon-γ release assay with wild-type spike, membrane and nucleocapsid peptide pools, we longitudinally characterized functional SARS-CoV-2 specific T-cell responses in a cohort of patients with mild, moderate and severe COVID-19. All patients were included before emergence of the Omicron (B.1.1.529) variant. Our most important finding was an impaired development of early IFN-γ-secreting virus-specific T-cells in severe patients compared to patients with moderate disease, indicating that absence of virus-specific cellular responses in the acute phase may act as a prognostic factor for severe disease. Remarkably, in addition to reactivity against the spike protein, a substantial proportion of the SARS-CoV-2 specific T-cell response was directed against the conserved membrane protein. This may be relevant for diagnostics and vaccine design, especially considering new variants with heavily mutated spike proteins. Our data further strengthen the hypothesis that dysregulated adaptive immunity plays a central role in COVID-19 immunopathogenesis.

Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is lethal in 88% of patients1, yet harbours mutation-derived T cell neoantigens that are suitable for vaccines 2,3. Here in a phase I trial of adjuvant autogene cevumeran, an individualized neoantigen vaccine based on uridine mRNA-lipoplex nanoparticles, we synthesized mRNA neoantigen vaccines in real time from surgically resected PDAC tumours. After surgery, we sequentially administered atezolizumab (an anti-PD-L1 immunotherapy), autogene cevumeran (a maximum of 20 neoantigens per patient) and a modified version of a four-drug chemotherapy regimen (mFOLFIRINOX, comprising folinic acid, fluorouracil, irinotecan and oxaliplatin). The end points included vaccine-induced neoantigen-specific T cells by high-threshold assays, 18-month recurrence-free survival and oncologic feasibility. We treated 16 patients with atezolizumab and autogene cevumeran, then 15 patients with mFOLFIRINOX. Autogene cevumeran was administered within 3 days of benchmarked times, was tolerable and induced de novo high-magnitude neoantigen-specific T cells in 8 out of 16 patients, with half targeting more than one vaccine neoantigen. Using a new mathematical strategy to track T cell clones (CloneTrack) and functional assays, we found that vaccine-expanded T cells comprised up to 10% of all blood T cells, re-expanded with a vaccine booster and included long-lived polyfunctional neoantigen-specific effector CD8+ T cells. At 18-month median follow-up, patients with vaccine-expanded T cells (responders) had a longer median recurrence-free survival (not reached) compared with patients without vaccine-expanded T cells (non-responders; 13.4 months, P = 0.003). Differences in the immune fitness of the patients did not confound this correlation, as responders and non-responders mounted equivalent immunity to a concurrent unrelated mRNA vaccine against SARS-CoV-2. Thus, adjuvant atezolizumab, autogene cevumeran and mFOLFIRINOX induces substantial T cell activity that may correlate with delayed PDAC recurrence.