Antibody sequence determinants of viral antigen specificity

Abstract

Throughout life, humans experience repeated exposure to viral antigens through infection and vaccination, resulting in the generation of diverse, antigen-specific antibody repertoires. A paramount feature of antibodies that enables their critical contributions in counteracting recurrent and novel pathogens, and consequently fostering their utility as valuable targets for therapeutic and vaccine development, is the exquisite specificity displayed against their target antigens. Yet, there is still limited understanding of the determinants of antibody-antigen specificity, particularly as a function of antibody sequence. In recent years, experimental characterization of antibody repertoires has led to novel insights into fundamental properties of antibody sequences but has been largely decoupled from at-scale antigen specificity analysis. Here, using the LIBRA-seq technology, we generated a large data set mapping antibody sequence to antigen specificity for thousands of B cells, by screening the repertoires of a set of healthy individuals against 20 viral antigens representing diverse pathogens of biomedical significance. Analysis uncovered virus-specific patterns in variable gene usage, gene pairing, somatic hypermutation, as well as the presence of convergent antiviral signatures across multiple individuals, including the presence of public antibody clonotypes. Notably, our results showed that, for B-cell receptors originating from different individuals but leveraging an identical combination of heavy and light chain variable genes, there is a specific CDRH3 identity threshold above which B cells appear to exclusively share the same antigen specificity. This finding provides a quantifiable measure of the relationship between antibody sequence and antigen specificity and further defines experimentally grounded criteria for defining public antibody clonality.IMPORTANCEThe B-cell compartment of the humoral immune system plays a critical role in the generation of antibodies upon new and repeated pathogen exposure. This study provides an unprecedented level of detail on the molecular characteristics of antibody repertoires that are specific to each of the different target pathogens studied here and provides empirical evidence in support of a 70% CDRH3 amino acid identity threshold in pairs of B cells encoded by identical IGHV:IGL(K)V genes, as a means of defining public clonality and therefore predicting B-cell antigen specificity in different individuals. This is of exceptional importance when leveraging public clonality as a method to annotate B-cell receptor data otherwise lacking antigen specificity information. Understanding the fundamental rules of antibody-antigen interactions can lead to transformative new approaches for the development of antibody therapeutics and vaccines against current and emerging viruses.

Keywords: B-cell repertoire; B-cell responses; antibody repertoire; antigen specificity; public clonotype; virus.

Fig 1

Identification of antigen-specific B cells from healthy donors. (A) Schematic of sample processing and flow cytometric staining to identify antigen specific B cells. Fluorescence-activated cell sorting of lymphocytes, B cells, and antigen-specific IgG cells. Cells were revealed by anti-CD14 allophycocyaninin cyanine dye (APC-Cy7), anti-CD19 brilliant violet 711 (BV711), anti-CD3 fluorescein isothiocyanate (FITC), and anti-IgG phycoerythrin cyanine dye (PE-Cy5). LIBRA-seq antigens were biotinylated and conjugated to streptavidin-phycoerythrin (Strep-PE) and streptavidin brilliant violet 421 (BV421). (B) Number of B cells identified for each antigen specificity category after computational filtering. Proportion of immunoglobulin isotypes identified within each antigen specificity category.

Fig 2

Diversity of viral antigen-specific B cells. (A) IGHV and IGL(K)V gene usage in B cells specific to Coronaviridae (blue), Pneumoviridae (purple), Paramyxoviridae (pink), and Orthomyxoviridae (orange) antigens. IGHV gene usage from previously published, unselected repertoires shown in black. Gene usage is represented as frequency of gene use within each antigen specificity category. Circles are superimposed. Zeros are not plotted. (B) The frequency of different IGHV:IGL(K)V gene pairs for B cells specific to Coronaviridae (blue circle), Pneumoviridae (purple cross), Paramyxoviridae (pink square), and Orthomyxoviridae (orange plus) antigens. The size of each data point represents the frequency of the corresponding IGHV:IGK(L)V pair within its antigen specificity category. Frequencies above 0.5% are represented. (C) Frequency of heavy chain variable (VH) somatic hypermutation represented as 1-VH identity calculated at the nucleotide level. Violin plot width is proportional to the fraction of B cells with the indicated proportion of VH somatic hypermutations. Two-sided Mann-Whitney-Wilcoxon test with Bonferroni correction used to calculate significance. Coronaviridae vs Pneumoviridae: P = 5.626e⁻³⁴, Pneumoviridae vs Paramyxoviridae: P = 3.990e⁻⁰², Paramyxoviridae vs Orthomyxoviridae: P = 1.000 Coronaviridae vs Paramyxoviridae: P = 1.903e⁻⁰³, Pneumoviridae vs Orthomyxoviridae: P = 2.173e⁻⁰⁶, Coronaviridae vs Orthomyxoviridae: P = 2.751e⁻⁰⁷. Orthomyxoviridae vs Unselected: P = 1.969e⁻²⁴, Paramyxoviridae vs Unselected: P = 5.978e⁻⁰⁶, Pneumoviridae vs Unselected P = 2.3853e⁻⁰⁵, Coronaviridae vs Unselected: P = 1.522e⁻¹⁵⁷.

Fig 3

Germline gene usage and CDR3 identity thresholds among viral public clonotypes. (A) CDRH3 and CDRL3 identity in pairs of B cells encoded by identical IGHV and IGL(K)V genes with same length CDRH3 region (left) or identical IGHV and IGL(K)V genes without consideration of CDRH3 length (right). Pairs of B cells with the same antigen specificity are shown in orange; pairs of B cells with different antigen specificities are shown in blue. Identity calculated at the amino acid level. Matrix represents the percentage of events occurring with greater than or equal to 70% CDRH3 identity for both same and different antigen specificities with the listed genetic conditions. (B) The frequency of different IGHV:IGL(K)V gene pairs for predicted public clonotypes specific to Coronaviridae (blue circle), Pneumoviridae (purple cross), Paramyxoviridae (pink square), Orthomyxoviridae (orange plus), and Flaviviridae (red diamond) antigens. The size of each data point represents the frequency of the corresponding IGHV:IGK(L)V pair within its antigen specificity category. Frequencies above 0.5% are represented.

Fig 4

Molecular characteristics of mono-reactive and cross-reactive B cells. (A) IGHV and IGL(K)V gene usage in mono-reactive (orange) and cross-reactive (purple) B cells. Gene usage is represented as frequency of gene use within each reactivity category. Circles are superimposed. Zeros are not plotted. (B) The frequency of different IGHV:IGL(K)V gene pairs for B cells of mono- and cross-reactivity. The size of each data point represents the frequency of the corresponding IGHV:IGK(L)V pair within its reactivity category. Frequencies above 0.5% are represented. (C) Frequency of heavy chain variable (VH) somatic hypermutation represented as 1-VH identity calculated at the nucleotide level. Violin plot width is proportional to the fraction of antibodies with the indicated proportion of VH somatic hypermutation. Kruskal-Wallis test with Bonferroni correction used to calculate significance. P = 1.808^-36.