key: cord-0957289-gk7q4hu2
authors: Papayanni, Penelope-Georgia; Chasiotis, Dimitrios; Koukoulias, Kiriakos; Georgakopoulou, Aphrodite; Iatrou, Anastasia; Gavriilaki, Eleni; Giannaki, Chrysavgi; Bitzani, Militsa; Geka, Eleni; Tasioudis, Polychronis; Chloros, Diamantis; Fylaktou, Asimina; Kioumis, Ioannis; Triantafyllidou, Maria; Dimou-Besikli, Sotiria; Karavalakis, Georgios; Boutou, Afroditi K; Siotou, Eleni; Anagnostopoulos, Achilles; Papadopoulou, Anastasia; Yannaki, Evangelia
title: Vaccinated and convalescent donor-derived SARS-CoV-2-specific T cells as adoptive immunotherapy for high-risk COVID-19 patients
date: 2021-04-27
journal: Clin Infect Dis
DOI: 10.1093/cid/ciab371
sha: 5a46e32e5095c900f41f9551d0f28ab252cc5983
doc_id: 957289
cord_uid: gk7q4hu2

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic poses an urgent need for the development of effective therapies for Coronavirus Disease 2019 (COVID-19). METHODS: We first tested SARS-CoV-2-specific T-cell (CοV-2-ST) immunity and expansion in unexposed donors, COVID-19 infected individuals (convalescent), asymptomatic PCR-positive subjects, vaccinated individuals, non-ICU hospitalized patients and ICU patients who either recovered and were discharged (ICU recovered) or had a prolonged stay and/or died (ICU critical). CoV-2-STs were generated from all types of donors and underwent phenotypic and functional assessment. RESULTS: We demonstrate causal relationship between the expansion of endogenous CoV-2-STs and the disease outcome; insufficient expansion of circulating CoV-2-STs, identified hospitalized patients at high-risk for an adverse outcome. CoV-2-STs with a similarly functional and non-alloreactive, albeit highly cytotoxic, profile against SARS-CoV-2 could be expanded from both convalescent and vaccinated donors generating clinical-scale, SARS-CoV-2-specific T-cell products with functional activity against both the unmutated virus and its B.1.1.7 variant. In contrast, critical COVID-19 patient-originating CoV-2-STs failed to expand, recapitulating the in vivo failure of CoV-2-specific T-cell immunity to control the infection. CoV-2-STs generated from asymptomatic PCR+ individuals presented only weak responses whereas their counterparts originating from exposed to other seasonal coronaviruses subjects failed to kill the virus, thus disempowering the hypothesis of protective cross-immunity. CONCLUSIONS: Overall, we provide evidence on risk stratification of hospitalized COVID-19 patients and the feasibility of generating powerful CoV-2-ST products from both convalescent and vaccinated donors as an “off-the shelf” T-cell immunotherapy for high-risk patients.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the devastating outbreak of Coronavirus Disease 2019 (COVID- 19) pandemic. Despite that several repurposed or novel agents have been evaluated as COVID-19 treatment, no proven therapeutic strategy exists. [1] Similar to the essential role in viral clearance of the related virus SARS-CοV T-cells shown to persist for >10years after exposure, [2] T-cell responses play a significant role in recovering from SARS-CoV-2. [3, 4] A c c e p t e d M a n u s c r i p t

Following the temporal evolution of CoV-2-STs for up to 2 weeks post-admission, we observed that the majority of patients unable to expand their CoV-2-STs in vivo, failed to control the infection and either had a prolonged/complicated ICU stay or succumbed (ICU-critical), whereas patients with CoV-2-ST rebounds, cleared the infection and were discharged (non-ICU and ICU-recovered) (Figure 2A-C) . The latter presented significant expansion of CoV-2-T-cell immunity over baseline as opposed to ICU-critical patients ( Figure 2D-E) .

The magnitude of CoV-2-ST expansion two weeks post admission (ΔSFCs), rather than baseline CoV-2-STs, was predictive of the patient outcome by receiver operating characteristics (ROC) curve analysis; a threshold of ΔSFC>35 and ΔSFC>101 of IFN-γ-and TNF-α-secreting CoV-2-STs, respectively, could predict with high sensitivity and specificity a favorable outcome (Figure 2F-G;S3; Table S2 ), suggesting that the magnitude of CoV-2-ST expansion could serve as a risk stratification tool.

To generate CoV-2-STs for adoptive immunotherapy (AI), donor PBMCs were stimulated with pepmixes spanning NCAP and spike antigens and cultured as described [5, 6, 9, 10] . Convalescent or vaccinated donor-derived T-cells robustly expanded upon antigen exposure, providing multiple clinical-scale doses per T-cell A c c e p t e d M a n u s c r i p t product whereas virus-naïve-or asymptomatic donor-derived CoV-2-STs had considerably lower expansion (Figure 3A-B) .

Convalescent-or vaccinated-donor-derived CoV-2-STs were predominantly CD4+ but also CD8+ T-cells, expressing memory and only at a minimum regulatory T-cell markers ( Figure 3C ) and presenting an activated and non-exhausted profile ( Figure   3D A c c e p t e d M a n u s c r i p t

As the mortality due to COVID-19 continues to rise, developing therapeutic modalities against SARS-CoV-2 remains mandatory. Reasonably, vaccination has generated great optimism, [11] [12] [13] [14] however, specific and effective therapeutic approaches are lacking. Even after herd immunity is achieved, vaccine breakthrough cases will exist, emerging mutations may escape antibody binding, vaccine-deniers will be vulnerable and immunocompromised patients always at risk for severe COVID-19.

Based on the safety and the high response rates of post-transplant adoptive immunotherapy (AI) with donor-or third-party-derived VSTs against, most commonly, the human herpes viruses (HHVs) family, [5] [6] [7] [8] and by leveraging a previous protocol to generate multi-virus-, [5, 6] Aspergillus fumigatus-[9] and multipathogen-specific T-cells, [10, 15] we explored the possibility of producing CoV-2-STs from COVID-19 convalescent donors and, for first time, BNT162b2-vaccinated donors. Furthermore, by monitoring the endogenous CoV-2-ST kinetics, we could identify appropriate candidates for T-cell immunotherapy, i.e. patients at high risk for an adverse outcome.

We here confirmed the observed lymphopenia [16, 17] in severely affected patients and the skewing of surviving T cells towards an ineffective differentiation status of exhausted or/and terminally differentiated cells, at the expense of functional memory and naïve subpopulations.

T-cell immunity plays a major role in COVID-19 resolution, [18] but whether protective memory provides long-lasting immunity as with the related SARS-CoV, [2] is still A c c e p t e d M a n u s c r i p t unclear. By contrast, there is increasing evidence that antibody-based immunity wanes over time. [19, 20] We here, further supporting recent findings, [21] demonstrate that the majority of recovered donors maintained SARS-CoV-2 T-cell responses for ≥8 months post-infection, suggesting that this branch of immunity is not compromised whereas decreasing antibodies in the same donors, implicated a rather short-lived humoral immunity. [19, 22] It remains to be proven however, controversial and inconclusive. [24, 25] Given that no curative therapy exists for COVID-19, adoptive transfer of immunity has emerged as a promising alternative. In this context, convalescent plasma did not reduce mortality over placebo [26] probably reflecting the inherent heterogeneity of plasma therapy providing different immune signatures, and the waning antibodymediated immunity in convalescent plasma donors. [20] We here pursued the generation of convalescent and vaccinated donor-derived CoV- 

The protocol and informed consent forms were approved by the Institutional Review

Board.

The study subjects were unexposed donors with no COVID-19 history or contact with affected individuals, vaccinated subjects, asymptomatic PCR-COV-2-positive subjects and SARS-CoV-2 infected individuals; with ≥1 month recovery before sampling (convalescent), non-ICU hospitalized patients, ICU-recovered or ICU critical patients having a prolonged/complicated stay (>25 days) and/or who died (Table SI) .

A c c e p t e d M a n u s c r i p t

Circulating CoV-2-STs were measured post hospitalization or ICU admission weekly.

Peripheral blood monocytes (PBMCs) or T-cell products were pulsed with spike, 

PBMCs pulsed with 0.5μg/ml of spike and NCAP pepmixes were cultured as described, [5, 6, 9] in G-Rex10, in media supplemented with 10ng/ml interleukin-7 and 400U/ml interleukin-4 until day 9-11.

Results are expressed as mean±standard error of the mean (SEM). Differences between data sets were analyzed using nonparametric Kruskal-Wallis test for M a n u s c r i p t Each dot represents an individual donor. Differences between data sets were analyzed using Kruskal-Wallis test. *p≤0·04; **p≤0·0085; ***p≤0·0006; ****p<0·0001. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

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