Antigen Receptor Gene Repertoire Profiling in Kidney Transplant Recipients Post-Sars-Cov-2 Vaccination

Kidney transplant recipients (KTRs) are highly susceptible to viral infections, while also exhibiting a higher risk of developing severe COVID-19. Recent studies have demonstrated a suboptimal humoral response to current COVID-19 vaccines in this cohort, necessitating multiple vaccine doses. Nevertheless, low rates of seroconversion have been observed even after booster vaccinations, highlighting the need for the in-depth characterization of both humoral and cellular immune responses towards the development of innovative and effective immunoprophylactic strategies tailored for KTRs.

Here, we investigated the immunogenetic features of the T cell receptor (TR) and B cell receptor immunoglobulin (BcR IG) gene repertoires in KTRs using targeted Next Generation Sequencing (NGS). In more detail, we studied 19 KTRs at two consecutive timepoints, after the 2 ^nd (T1) and multiple vaccination doses (T2), respectively. We included as a control group 19 healthy individuals (HIs) sampled either after two mRNA vaccination doses or after a documented SARS-CoV-2 infection. Seroconversion (>1300 AU/ml of anti-RBD IgG) was measured by the Abbott SARS-CoV-2 IgG assay: all HIs tested positive, in sharp contrast to KTRs who failed to exhibit humoral responses after the 2 ^nd dose (T1). Immunogenetic characterization of TR/BcR IG gene repertoires was performed by paired-end NGS; resultant data were processed through the TRIP software. Only productive V(D)J gene rearrangements were considered for the computation of clonotypes (i.e. TR/BcR IG gene rearrangements with identical V gene usage and complementarity-determining region 3 amino acid sequence-CDR3 aa). Starting with the TR gene repertoire, diversity significantly increased at T2 (average Shannon diversity: KTR-T1=638 vs KTR-T2=3212, p<0.0005| KTR-T2=3212 vs HI=2146, p<0.05). TR clonality (mean cumulative frequency of the 10 dominant TR clonotypes/sample) was significantly increased at T1 in KTRs vs HIs (KTR-T1=37% vs HI=25%, p<0.05), eventually decreasing at T2, reaching comparable levels to those measured in HIs (KTR-T2=27%). At T1, a median of 7.6% of TR clonotypes/sample in KTRs were SARS-specific (high-affinity TR clonotypes to SARS-specific peptide-MHC complexes, >0.9 binding score determined by the ERGO-II algorithm), almost identical to what we found for HIs (7.7%). Although the TR gene repertoire was renewed in T2, the percentage of SARS-specific clonotypes remained essentially identical (median of 7.8%). Turning to the BcR IG gene repertoire, despite the absence of a serological response at T1, the percentage of the predicted SARS-specific BcR IG clonotypes (high-homology with SARS-specific CDR3 aa sequences published at CoV-AbDab, >0.9 identity score) did not differ between KTRs (0.72% at T1 and 0.65% at T2) vs HIs (0.63%), raising the intriguing possibility of B cell anergy in the former. Overtime assessment revealed that BcR IG clonotype diversity significantly increased at T2 vs T1 in KTRs (average Shannon diversity: KTR-T1=12006 vs KTR-T2=38576 p<0.05) and strongly correlated with the presence of SARS-specific BcR IG clonotypes (r=0.65, p<0.001). Nevertheless, the cumulative fraction of SARS-specific BcR IG clonotypes at KTR-T2 decreased (median cumulative frequency of SARS-specific clonotypes: KTR-T2=0.45% vs HI=0.67%, p<0.05). Of note, multiple immunizations resulted to SARS-specific BcR IG clonotypes with a significantly increased number of somatic hypermutations (SHM) compared to either HIs (p<0.0005) or T1 (p<0.05).

In conclusion, our findings suggest that the lack of seroconversion at T1 in KTRs is potentially counteracted by cellular sensitization, likely mediated by SARS-specific T cells, that is sustained by booster vaccination. The latter also drives the accumulation of SHM in BcR IG clonotypes, suggesting distinct differentiation trajectories (germinal center-independent at T1 vs germinal center-dependent at T2) that could give rise to new B cell clones with increased neutralizing potency.