key: cord-0962073-krftllor authors: D’Offizi, Gianpiero; Agrati, Chiara; Visco‐Comandini, Ubaldo; Castilletti, Concetta; Puro, Vincenzo; Piccolo, Paola; Montalbano, Marzia; Meschi, Silvia; Tartaglia, Eleonora; Sorace, Chiara; Leone, Sara; Lapa, Daniele; Grassi, Germana; Goletti, Delia; Ippolito, Giuseppe; Vaia, Francesco; Ettorre, Giuseppe Maria; Lionetti, Raffaella title: Coordinated cellular and humoral immune responses after two‐dose SARS‐CoV2 mRNA vaccination in liver transplant recipients date: 2021-11-17 journal: Liver Int DOI: 10.1111/liv.15089 sha: 205ef28ed9b95b3942df68c4e5375aa8639f8bd2 doc_id: 962073 cord_uid: krftllor Limited data are available on risks and benefits of anti‐SARS‐CoV2 vaccination in solid organ transplant recipients, and weaker responses have been described. At the Italian National Institute for Infectious Diseases, 61 liver transplant recipients underwent testing to describe the dynamics of humoral and cell‐mediated immune response after two doses of anti‐SARS‐CoV2 mRNA vaccines and compared with 51 healthy controls. Humoral response was measured by quantifying both anti‐spike and neutralizing antibodies; cell‐mediated response was measured by PBMC proliferation assay with IFN‐γ and IL‐2 production. Liver transplant recipients showed lower response rates compared with controls in both humoral and cellular arms; shorter time since transplantation and multi‐drug immunosuppressive regimen containing mycophenolate mofetil were predictive of reduced response to vaccination. Specific antibody and cytokine production, though reduced, were highly correlated in transplant recipients. the Italian National Institute for Infectious Diseases, 61 liver transplant recipients underwent testing to describe the dynamics of humoral and cell-mediated immune response after two doses of anti-SARS-CoV2 mRNA vaccines and compared with 51 healthy controls. Humoral response was measured by quantifying both anti-spike and neutralizing antibodies; cell-mediated response was measured by PBMC proliferation assay with IFNγ and IL-2 production. Liver transplant recipients showed lower response rates compared with controls in both humoral and cellular arms; shorter time since transplantation and multi-drug immunosuppressive regimen containing mycophenolate mofetil were predictive of reduced response to vaccination. Specific antibody and cytokine production, though reduced, were highly correlated in transplant recipients. anti-spike titre, liver transplant, SARS-CoV2 vaccination, T-cell immune response mortality rate up to 20%. 2 A position paper from the European Association for the Study of the Liver, consequently, recommended vaccination for liver transplant recipients (LTRs). 3 Two novel mRNAtechnology-based anti-SARS-CoV2 vaccines, developed by Pfizer-BioNTech and Moderna, have been safely administered in the general population, with efficacy reaching 94%-95%. 4, 5 . SOTRs with mRNA vaccines (BNT162b2 or mRNA-1273) with two doses administered 3 or 4 weeks apart, respectively. Preliminary studies suggested a poorer response to SARS-CoV2 vaccination in LTRs with a significantly lower antibody titre and faster decline in antibody levels than the general population. 6 Treatment factors significantly related to non-response were high-dose prednisone in the previous 12 months and mycophenolate mofetil (MMF) treatment. SOTRs, with only four LTRs, were assessed after the second dose of BNT162b2 vaccine. Cellular response rate in SOTRs was 56.2%, and humoral response was significantly lower than in immunocompetent controls. 10 The objective of our study was assessment of humoral and cellular responses after two doses of mRNA anti-SARS-CoV2 vaccine in a larger cohort of LTRs, compared with healthy controls, and investigating clinical features associated with non-response. Consecutive 61 LTRs who received anti-SARS-CoV2 vaccination between March and April 2021 underwent testing for humoral and cell-mediated immune response at three time points: before 1st dose (T0), 2nd dose (T1) and 2 weeks after 2nd dose (T2). Results were compared with a healthy control (HC) group of hospital employees with no major co-morbidities who underwent the same protocol. Subjects who tested positive for anti-nucleoprotein IgG at T0 (indicating previous SARS-CoV2 natural infection) were excluded. All subjects tested negative for SARS-CoV2 anti-receptorbinding domain (anti-Spike) IgG before vaccination. All subjects received either BNT162b2 or mRNA-1273 anti-SARS-CoV2 vaccine. Committee 3580 (March 17, 2021) and all participants signed a written informed consent. A chemiluminescence microparticle antibody assay (ARCHITECT ® i2000sr Abbott Diagnostics) was used to detect anti-spike IgG to quantify response to mRNA vaccination. Positive anti-spike response was defined as ≥7.2 Binding Arbitrary Units (BAU)/ml. The assay was performed according to, 6 using SARS-CoV2/Human/ ITA/ PAVIA10734/2020, provided by F. Baldanti, Pavia, as a challenging virus. First, heat-inactivated and 7 twofold serial diluted sera (starting dilution 1:10) were mixed and incubated at 37°C and 5% CO 2 for 30 min with equal volumes of 100 TCID50 SARS-CoV2. Next, 96-well tissue culture plates with sub-confluent Vero E6 cell monolayers were infected with 100 µl/well of virus-serum mixture and incubated at 37°C and 5% CO 2 . To standardize inter-assay pro- Peripheral blood was collected in heparin tubes and stimulated with a pool of peptides spanning the Spike antigen (S-peptides, Miltenyi Biotech) at 37°C (5% CO 2 ), according to. 7 A superantigen was used as positive control. Cultured plasma was harvested after 16-20 h of stimulation and stored at −80°C. Th1 cytokine production of interferonγ (IFNγ) and interleukin-2 (IL-2) were quantified in plasma using an automatic ELISA (ELLA, Protein Simple). Detection limits of these assays were 0.17 pg/ml and 0.54 pg/ml for IFNγ and IL-2, respectively. Positive response was defined as >10 pg/ml for IFNγ and >25 pg/ml for IL-2. 11 LTRs were evaluated for obesity, diabetes mellitus, chronic renal disease with estimated glomerular filtration rate (eGFR). Immunosuppressive regimens were defined as containing calcineurin inhibitors (CNIs), MMF or steroids. Continuous variables including anti-spike, IFNγ and IL-2 levels were reported as median and interquartile range (IQR). Comparisons of medians across groups were evaluated using Kruskal-Wallis analysis with the Mann-Whitney U-test with Bonferroni correction for pairwise comparisons. Categorical variables including dichotomous anti-spike, N-Ab, IFNγ and IL-2 response were summarized as counts and percentages and compared with Chi-square test or Fisher's Exact test. Correlations between assays were assessed by non-parametric Spearman's rank tests. To identify significant variables that could contribute to the anti-spike, N-Ab and IFNγ response, a multivariate regression analysis model was constructed including gender, age, years since transplant, immunosuppression and comorbidities. Analyses were performed using R software (version 4.0.3). A two-sided P value <.05 was considered to be statistically significant. In LTRs, median time from transplant was 6 years (IQR 3-10, range 1-26). CNIs were used as immunosuppressive regimen backbone in 59 (96%) and 29 (47.5%) received MMF in combination with CNIs. Among LTRs, diabetes was present in 15 patients (24.6%) and obesity defined as Body Mass Index>30 in 14 (23%). Only 9 patients (14.5%) showed eGFR<51 ml/min. Specific T-Cell cytokine production at T0 did not differ between the two groups. 160 (IQR 80-320), P < .0001 ( Figure 1B) . A strong correlation between anti-spike and N-Ab titres was observed at T2 in LTRs (r = 0.9047, P <.0001, Figure 1C ). Specific T-cell response to S-peptides was measured via Th1 cytokine production of IFNγ and IL-2 released after in vitro stimulation ( Figure 1D,E) . Before vaccination, 25.4% of LTRs and 17.6% of HCs had detectable levels of IFNγ. In LTRs, median IFNγ level was 3.5 pg/ml (IQR 0.1-10.8) at T0, 9.5 pg/ml (IQR 2.1-29.9) at T1 and 49.1 pg/ml (IQR 5.3-189.3) at T2 (T0 vs. T2: P < .0001). In HCs, median IFNγ level was 0.85 pg/ml (IQR 0.1-5.7) at T0, 112.2 pg/ml (IQR 53. .0) at T1 and 344.0 pg/ml (IQR 193.6-699.6) at T2 (T0 vs. T1: P < .0001; T1 vs. T2: P < .0001). Positive IFNγ response at T2 was significantly lower in LTRs compared with HCs (72.1% vs. 100%, P < .0001). Median IFNγ levels were significantly lower in LTRs compared with HCs at T1 and T2 (P < .0001, Figure 1D ). In LTRs, median IL-2 level was 2.6 pg/ml (IQR 0.9-1.6) at T0, Positive IL-2 response at T2 was significantly lower in LTRs compared with HCs (50.8% vs. 100%, P < .0001). Median IL-2 levels were significantly lower in LTRs compared with HCs at T1 and T2 (P < .0001, Figure 1E ). In LTRs the amount of IFNγ released by S-specific T cells correlated with anti-spike titre (r = 0.4850, P < .0001), N-Ab titre (r = 0.5272, P < .0001) and IL-2 levels (r = 0.7535, P < .0001, Figure 1F ). IFNγ production, respectively. At least one comorbidity was not associated with humoral or T-cell non-response, although a trend was observed for obesity and GFR<51 ml/min (P = .07). Significant associations are depicted in Table 1 . Among those who developed antibodies, anti-spike titre was lower than in HCs. 12 Regarding specific T-cell response, positive IFNγ testing after stimulation with S-peptides was observed at T2 in 77% of LTRs; a single previous report of a small series of 16 SOTRs found a detectable cellular response in 56%, although with a different cut-off. 10 This difference could be explained by stronger immunosuppression used in non-liver transplant recipients. In our study, 25.4% of LTRs and 17.6% of HCs had a positive IFNγ test at T0. This could be due to a cross-reactivity with seasonal coronaviruses. HCs. Panel A: anti-RBD/spike Ig G titres at T0, T1 and T2. Panel B: Neutralizing Ab titres at T2. Panel C: correlation between anti-RBD/spike titres anti neutralizing Ab titres at T2. Panel D: IFNγ production at T0, T1 and T2. Panel E: IL-2 production at T0, T1 and T2. Panel F: Correlation of humoral responses and IL-2 production with IFNγ production at T2. *P < .05; **P < .01; ***P < .001; ****P < .0001. T0, before first vaccination dose; T1, before second vaccination dose; T2, two weeks after second vaccination dose; LTRs, liver transplant recipients; HCs, healthy controls; RBD, receptor binding domain; MNA90, 90% micro-neutralization assay; IFN, interferon; IL-2, interleukin-2 Furthermore, we tested IL-2 production, representing a key cytokine in the Th1 response with homeostatic functions. Correlation between IFNγ and IL-2 produced by S-specific T cells highlights the ability of anti-SARS-CoV2 vaccine to shape a balanced immune response. The pivotal role of immunosuppression is demonstrated by the negative effect of MMF/CNI combination on both antibody and cytokine production. Treatment with MMF was shown to have a negative effect on influenza vaccine immunogenicity in other SOTRs: in particular, SOTRs receiving MMF ≥2 g/day showed significantly lower mean antibody titres than those receiving <2 g/day, and MMF reduced IL-4 + CD4 + T-cell frequencies and B-cell activation. 13 In this setting, a temporary suspension of MMF during the vaccination period could be proposed in selected LTRs. The correlation between humoral and T-cell response may support using only anti-spike titre, a simple and inexpensive test, as the ideal parameter to assess immunological response in upcoming large-scale booster dose vaccination studies in LTRs. In conclusion, despite limitations related to small sample size and brief clinical follow-up, our study in LTRs demonstrated a blunted but coordinated humoral and T-cell-mediated response after two standard doses of mRNA anti-SARS-CoV2 vaccine compared with HCs. Longer follow-up studies are needed to assess durability of immune response in this population. None declared. All authors contributed substantially to the conception of the work, to the acquisition, analysis, and interpretation of data, and to drafting and critically revising the manuscript. All authors have approved the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Permission to reproduce material from other sources was not required for the present work. 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