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Immune Response After a Third Dose of the mRNA-1273 SARS-CoV-2 Vaccine in Liver Transplant Recipients

1) MATERIALS AND METHODS Patients The cohort comprised 129 adult LTRs follow-up at the Liver Transplant Unit of University Hospital Marqués de Valdecilla, Santander (Spain) with a negative history for COVID-19 who had previously received two doses of mRNA-1273 SARS-CoV-2 and were subsequently vaccinated 4 moths after the second dose (IQR= 133-139 days) with a homologous third dose of the mRNA1273 SARS-CoV-2 vaccine between September 27, 2021, and October 10, 2021. Neither severe side-effect nor rejection event was documented after third dose. Mild local pain and self-limited fever were reported as the main side-effects. No adjustment of immunosuppression was performed within vaccination procedure. Clinical data including comorbidities and immunosuppressive regimen were obtained from patient ́s medical records and routine blood test up to one month after the third vaccine. This study was approved by the Institutional Ethics Committee of Cantabria (internal code: 2021.391) and complied with the provisions of the Good Clinical Practice guidelines and the Declaration of Helsinki.

side-effect nor rejection event was documented after third dose. Mild local pain and self-limited fever were reported as the main side-effects. No adjustment of immunosuppression was performed within vaccination procedure. Clinical data including comorbidities and immunosuppressive regimen were obtained from patient´s medical records and routine blood test up to one month after the third vaccine. This study was approved by the Institutional Ethics Committee of Cantabria (internal code: 2021.391) and complied with the provisions of the Good Clinical Practice guidelines and the Declaration of Helsinki.

Measurement of SARS-CoV-2 spike antibodies
The IgG antibodies directed against the receptor binding domain (RBD) of the spike glycoprotein [IgG(S-RBD)] of the SARS-CoV-2 was assessed using the ARCHITECT IgG II Quant test (Abbot), measured a median of 43 days (IQR, 37-49 days) after the second vaccine dose and a median of 30 days (IQR, 30-33.7 days) after the third vaccine injection (the sensitivity with the test is near 100% in this range of analysis (1). Values higher than 50 AU/mL were considered positive as specified by the manufacturer. The results of this assay have been shown to correlate with in vitro neutralization of SARS-CoV-2 (2). This interpretation is limited by its lack of association with vaccine effectiveness and its lack of comparability with other assays, using different cut-offs (3).
Thus, for surrogate measures of antibody neutralization, we examined IgG(S-RBD) levels at or above 4,160 AU/mL given that this threshold is recommended by the manufacturer and is used a surrogate measure of vaccine protection because it corresponds to a 0.95 probability of obtaining an in-vitro plaque reduction neutralization test. In addition, such cut-off was chosen according to a recent study that reported that lower anti-S1 titers were associated with significantly more breakthrough infection in vaccinated recipients (4).

Definitions
Groups were defined based on the level of anti-S1 IgG antibodies measured after the second vaccine dose: seronegative; low responders and high responders. Seronegatives were defined as levels <50 AU/mL, low responders with levels between 50 and 4,159 AU/mL and high responders with levels ≥4160 AU/mL as estimate neutralizing antibody.
The primary composite endpoint was defined as the cumulative percentage of patients switching from no-to low-responder status, from low-to high responders status or doubling their anti-S1 IgG level.

Assessment of SARS-CoV-2 T-Specific Response by Flow Cytometry
The assay was validated by the Spanish Society of Immunology and based on the surface expression of activation-induced markers (AIM) after exposure with specific SARS-CoV-2 peptide pools (5). This procedure has been previously published by our group (6). Briefly, peripheral blood mononuclear cells (PBMCs) from heparinized blood were isolated by Ficoll gradient and Student´s t-test, the Mann-Whitney test or Fisher's exact test as appropriate. A two-sided pvalue < 0.05 was considered statistically significant.

2) Patients and Results
Baseline characteristics of the 129 LTRs included in this study are summarized in Table S1.

Antibody response and levels after SAS-Cov2
The overall an antibody response developed in 113 (87.6%) of patients after two doses of mRNA-

Specific T-Cell Immune Response
To examine the specific T-cell response, PBMCs were exposed to anti-SARS-CoV-2 peptides using an unstimulated negative control and CD3/CD28 as positive control, as showed in the Material and Methods section. The assessment of vaccine-specific T-cell response has been proposed to fully evaluate the immune response against the vaccine. The assay was addressed in those 16 seronegative to 2 dose vaccination.
3. Table S1. Baseline characteristics of Liver Transplant Recipients (LTRs) and comparison of LTRs with positive and negative antibody response after third dose of mRNA-1273 SARS-CoV-2 Vaccine

Variables
All