Imprints of somatic hypermutation on B-cell receptor immunoglobulins post-infection versus post-vaccination against SARS-CoV-2

Abstract

Published evidence supports significant heterogeneity of immune responses among individuals infected with or vaccinated against SARS-CoV-2. This highlights the need for in-depth investigation of the implicated processes toward refined understanding and improved management of COVID-19. The main objective of the present study was to investigate the dynamics of B cell responses to SARS-CoV-2, focusing on how initial infection and subsequent vaccination influence the immunoglobulin gene repertoire, with special emphasis on the impact of somatic hypermutation (SHM) on antibody maturation. Samples were collected from 81 individuals infected by SARS-CoV-2 in the municipality of Vo' during the first pandemic wave in 2020. For 25 of them, sampling was repeated 7 d after completing the primary vaccination series. Deep immunogenetic analysis of the B-cell receptor immunoglobulin (BcR IG) gene repertoire was performed using targeted next-generation sequencing. Bioinformatics analysis focused on repertoire metrics, prediction of IG antigen specificity, and detailed profiling of the SHM patterns. Significant expansions of unmutated sequences early post-infection suggest extrafollicular B cell maturation. In contrast, vaccination promoted SHM acquisition, indicating a germinal center-dependent response, and pronounced repertoire renewal. Restricted SHMs in SARS-homologous clonotypes along with preferential targeting of specific codons within the VH domain post-vaccination support ongoing affinity maturation within germinal centers. Differences in the BcR IG profiles post-infection versus post-vaccination allude to distinct trajectories in B cell maturation. Distinct profiles of SHM targeting reflect ongoing affinity maturation post-vaccination, with implications for optimizing preventive and therapeutic interventions against COVID-19.

Keywords: B-cell receptor gene repertoire; COVID-19; adaptive immunity; somatic hypermutation.

Figure 1.

Comparison of BcR IG gene repertoire features post-infection versus post-vaccination. (A) Bar plots depict the frequency of the 10 immunodominant (major) clonotypes in the post-infection and post-vaccination BcR IG gene repertoires, emphasizing the oligoclonal profile of the post-infection repertoire. (B) Density box plots show the distribution of clonality across paired samples at the two time points: clonality was found to be increased post-infection versus post-vaccination (P = 0.0007). Clonality was estimated as the MCF-10 and is represented as points, indicating clonality per sample. (C) Bar plots illustrate the frequency of the utilized IGHV genes, revealing differences between the two time points (P < 0.05). (D) Circos diagrams depict IGHV-IGHJ gene rearrangements at the two time points, with colored connections indicating statistically significant signatures (***P < 0.05).

Figure 2.

SHM status and clonality. Lollipop graphs depict clonality, showing cumulative frequencies of mutated and unmutated clonotypes per sample at the two time points. Bar plots illustrate the percentage of mutated and unmutated clonotypes per sample. The binomial test was employed to assess potential differences in the fraction occupied by the mutated and unmutated clonotypes in post-infection versus post-vaccination repertoires, revealing statistically significant results (P < 0.0001) between the two time points.

Figure 3.

SARS-homologous clonotypes. (A) Density box plots illustrate the distributions of post-infection BcR IG gene clonotypes binding to SARS-derived epitopes. The colors represent the number of clonotypes, and the density indicates the percentage of values, with the darkest region encompassing 50% of the observations. (B) Points represent clonotypes that exhibit more than 85% similarity to known neutralizing antibodies in CDR3 amino acid sequences. The colors indicate different time points.

Figure 4.

Frequency of SARS-homologous clonotypes with respect to SHM status. Points depict the frequency of all mutated and unmutated SARS-homologous clonotypes for the paired samples at (A) post-infection versus (B) post-vaccination, revealing a statistically significant expansion of the unmutated clonotypes post-infection (P = 0.0002).

Figure 5.

Significant amino acid substitutions observed in SARS-homologous clonotypes as a string of co-existing SHMs in rearrangements of a particular IGHV gene.

Figure 6.

Example of localization of frequent mutations post-vaccination or infection by SARS-CoV-2. The structure of a BcR IG encoded by the IGHV3-30-3 gene (PDB code 6KYZ) is shown through its molecular surface, with the heavy chain in white and the light chain in gray. The HCDR1, HCDR2, and HCDR3 regions are colored pink, blue, and orange, respectively. (A) The position of residue G27 is shown in red. (B) The effect of the mutation to a glutamic acid, a frequent mutation in antibodies from individuals post-vaccination, has a minor effect on the combining site but introduces a negative charge at the edge of the region devoted to antigen binding. (C) The simultaneous introduction of the two Ser36Asn and Asn59Lys mutations observed in antibodies from individuals post-infection affects directly the character of the surface involved in antigen recognition.

Figure 7.

Mutational analysis of SARS-homologous clonotypes. Stacked bar plots illustrate amino acid substitutions resulting from SHM on the FR1-FR3 part of the VH domain of all SARS-homologous IG clonotypes sharing the same IGHV gene in the post-infection and post-vaccination BcR IG gene repertoires. The x-axis letters represent the amino acids encoded by the germline gene sequence and the letters in red depict mutational hotspots, while the named bars indicate amino acid substitutions with a frequency exceeding 5%. For the sake of clarity, SHMs called by a single sequencing read were excluded from the figure. The color code employed to describe the physicochemical properties of each amino acid per position is based on the IMGT database classification: dark blue for aliphatic (A, V, I, L, G, P), yellow for sulfur (C, M), light blue for hydroxyl (S, T), red for acidic (D, E), orange for amide (N, Q), purple for basic (H, K, R), and green for aromatic (F, W, Y) amino acids.