key: cord-1030987-30v03o8l authors: Harrington, Patrick; Doores, Katie J.; Saha, Chandan; Saunders, Jamie; Child, Fiona; Dillon, Richard; Saglam, Sukran; Raj, Kavita; McLornan, Donal; Potter, Victoria; Kordasti, Shahram; O’Reilly, Amy; Espehana, Andreas; Lechmere, Thomas; Khan, Hataf; Malim, Michael H.; Harrison, Claire; Mehra, Varun; de Lavallade, Hugues title: Repeated vaccination against SARS-CoV-2 elicits robust polyfunctional T cell response in allogeneic stem cell transplantation recipients date: 2021-10-12 journal: Cancer Cell DOI: 10.1016/j.ccell.2021.10.002 sha: 3e9b08efd5ab76e7808a9021d28e000acf82ca1e doc_id: 1030987 cord_uid: 30v03o8l nan SARS-CoV-2 has led to unprecedented global healthcare challenges, with poor outcomes observed in groups with immune deficiency, including allogeneic stem cell transplantation (allo-SCT) recipients (Bakouny et al., 2020) . T cell and B cell responses following vaccination against SARS-CoV-2 are important in reducing the risk of severe COVID-19, but the T cell response has not been extensively investigated in this population. We designed a prospective study to evaluate response to vaccination in patients with hematologic malignancies. Herein we report analysis of T cell and humoral response to sequential dosing of vaccination against SARS-CoV-2 in allo-SCT recipients. Anti-SARS-CoV-2 Spike protein (S) IgG ELISA and neutralizing antibody testing were performed as described previously. The induction of virus-specific T cell responses by vaccination was assessed by flow-cytometric enumeration of antigen-specific CD8 + and CD4 + T lymphocytes using an intracellular cytokine assay for IFN and TNF. A total of 23 patients were analyzed at one or more time point around the two-dose vaccination schedule (Table S1 ). Median age was 55 years (range 25-74), and 69.6% (16) were male. Median time from allo-SCT was 55 months (19-172), and BNT162b2 vaccine was given to 81% (21) of patients, while others received ChAdOx1-S. Following a first dose of vaccine, an anti-S IgG response was assessed in 18 patients at a median of 4.2 weeks after vaccination. Anti-S IgG was detectable in only 38.9% (7), with 4 of these having weak positive results ( Figure S1A ). A mean anti-S IgG EC50 of 76 (range 0-526) was observed at this time point ( Figure S1B ). Neutralizing antibody analysis was performed in all 7 patients with detectable anti-S IgG at this time point, with a mean ID50 of 292 observed (32-968) ( Figure S1C ). Antibody testing was performed in 16 patients following two doses of vaccine, at a median of 12 weeks after the second dose. A detectable anti-S IgG was observed in 81% (13) of patients (p ≤ 0.017) (Figure S1A), with a mean anti-S IgG of 1043 (0-5594) (p = 0.025) ( Figure S1B ). Neutralizing antibody testing performed in 13 patients with detectable IgG showed a mean ID50 of 747 (107-4707) ( Figure S1C ). After two doses of vaccine, antibody testing was performed in 10 patients with chronic graft-versus-host disease (GvHD) receiving extracorporeal photopheresis (ECP) and 6 patients not receiving ECP, with a mean EC50 of 574 in ECP group, compared with 1826 non-ECP (p = 0.17). Similarly, mean neutralizing antibody ID50 was 312 in those requiring ECP compared with 719 in non-ECP. There was a significant correlation between anti-S IgG level and neutralizing ability from paired samples, with r value of 0.83 (p < 0.0001) ( Figure S1D ). T cell analysis was performed in 17 patients after a single dose of vaccine and in 17 patients after two doses. A T cell response was observed in 35.3% (6) of patients after one dose and in 82.3% (14) of patients after two doses (p = 0.013) ( Figure S1E ). A CD4 + T cell response was observed in 29.4% (5) of patients after one dose and 70.6% (12) of patients after two doses (p = 0.0.38), while a CD8 + T cell response was only seen in 17.6% (3) after one dose but 52.3% (9) after two doses (p = 0.07). Mean CD4 + /CD8 + TNF expression after a single dose was 0.12%/0.04%, which increased to 0.42%/0. CD4 + /CD8 + IFN expression after a single dose was 0.06%/0.03%, which again increased to 0.07%/0.17% (p = 0.8/0.1). A polyfunctional T cell response, with dual expression of more than one proinflammatory cytokine within the same cell, was observed in 29.4% (5) of patients after one dose and 70.6% (12) after two doses (p = 0.038) ( Figures S1F and S1G) . After a single dose, mean CD4 + polyfunctional T cell response was 0.009%, with an increase to 0.026% after 2 doses (p = 0.068) ( Figure S1H ). Consistently, more than 90% of reactive T cells expressing pro-inflammatory cytokines showed co-expression of CD45RO, a surface protein marker for memory T cells. After a second dose, patients with chronic GvHD requiring ECP had a mean CD4 + TNF expression of 0.18% compared with 0.86% in those not requiring ECP (p = 0.09) ( Figure S1I ). Patients with prior allo-SCT who contract COVID-19 infection have poor outcomes, with overall survival reported at 68% at 30 days post diagnosis (Sharma et al., 2021) . Therefore, the development of immunity is particularly important in this patient group. We have previously reported that a single dose of BNT162b2 is sufficient to generate both a humoral and a T cell response in most patients with chronic myeloid malignancies (Harrington et al., 2021) . This is in contrast to the response observed in many cancer-patient groups, particularly those with lymphoid malignancies who have received anti-CD20 targeted therapy (Addeo et al., 2021 , Greenberger et al., 2021 , Thakkar et al., 2021 . We demonstrate here how a second dose is required for a significant increase in seroconversion rates and detectable memory T cells in allo-SCT recipients. Through analysis of samples at consecutive time points, including sequential samples from the same patients, we were able to observe the longitudinal response to vaccination and show that a second dose is required for adequate immunogenicity in this population. Our findings are in keeping with that from two studies on isolated antibody responses in allo-SCT patients which reported an anti-S IgG response after a second injection in 83% and 78% of participants, respectively (Redjoul et al., 2021 , Le Bourgeois et al., 2021 . Our data report the T cell response to SARS-CoV-2 vaccination in patients with previous allo-SCT. Despite a poor T cell response after a first vaccine injection, a second dose elicited anti-S reactive T cells in most patients. Moreover, a polyfunctional T cell response was also elicited by a second dose, which may have particular functional relevance with regards to anti-viral immunity, with these cells recognized as providing a more effective anti-viral response in the context of COVID-19 infection (Peng et al., 2020) . A memory T cell response may play a particularly important role in providing immunity to COVID-19, as studies have shown significant decline in antibody levels in the general population at 3 months post natural infection (Seow et al., 2020) . We have also focused our analysis on patients considered to be particularly immune suppressed with regards to chronic GvHD and ongoing systemic immune suppression. While these patients did show a reduced T cell and antibody response when compared with patients off immune suppression, this was not significant, and most showed an adequate neutralizing antibody response after a second injection. Our study is, however, limited by small sample size, and further longitudinal data are required to evaluate whether the response generated is adequate to provide anti-viral protection. Immunogenicity of SARS-CoV-2 messenger RNA vaccines in patients with cancer Antibody response to SARS-CoV-2 vaccines in patients with hematologic malignancies Single dose of BNT162b2 mRNA vaccine against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) induces neutralising antibody and polyfunctional T-cell responses in patients with chronic myeloid leukaemia Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans Seroconversion rates following COVID-19 vaccination among patients with cancer The authors acknowledge a Blood Cancer UK award to P.H. and H.