Variant proteins stimulate more IgM+ GC B-cells revealing a mechanism of cross-reactive recognition by antibody memory

Abstract

Vaccines induce memory B-cells that provide high affinity secondary antibody responses to identical antigens. Memory B-cells can also re-instigate affinity maturation, but how this happens against antigenic variants is poorly understood despite its potential impact on driving broadly protective immunity against pathogens such as Influenza and Dengue. We immunised mice sequentially with identical or variant Dengue-virus envelope proteins and analysed antibody and germinal-centre (GC) responses. Variant protein boosts induced GCs with a higher proportion of IgM+ B cells. The most variant protein re-stimulated GCs with the highest proportion of IgM+ cells with the most diverse, least mutated V-genes and with a slower but efficient serum antibody response. Recombinant antibodies from GC B-cells showed a higher affinity for the variant antigen than antibodies from a primary response, confirming a memory origin. This reveals a new process of antibody memory, that IgM memory cells with fewer mutations participate in secondary responses to variant antigens, demonstrating how the hierarchical structure of B-cell memory is used and indicating the potential and limits of cross-reactive antibody based immunity.

Keywords: B-cell; Dengue virus; antibody cross-reaction; antibody memory; immunology; inflammation; mouse.

Figure 1.. Serum antibody responses after boosting with Dengue envelope protein variants.

(A) Cross-reactivity of E3 primed serum IgG with E-protein variants. Red bar shows mean value. Serum used was from mice mock-boosted with PBS 37 days after E3 priming and obtained 7 days later; E3, Dengue-3 envelope protein; E2, Dengue-2 envelope protein; E4, Dengue-4 envelope protein; % identity, sequence identity between E3 envelope protein and respective protein; end-point titre (EPT) values plotted are log2 of 1/(end point dilution x 100), each unit increase represents a doubling of titre. (B) E3 primed mouse serum cross-reactivity with E2 versus E4. (C) Control. Anti-PR8 HA serum IgG titre of E3 day 7boost serum. (D) Anti-E3 serum IgG titre after boosting with respective proteins. Red bar shows mean value. n = 6 from two independent experiments for each group except boost only, n = 3; first set of data points reproduced from panel A for comparison; numbers 3, 2 and 4 refer to serotype of Dengue-envelope protein used for boost; BO, adjuvant primed, E3 boosted, analysed 7 days later; Day, days after boosting. p-values calculated using two-tailed Students t-test after testing for equality of variance. (E) Anti-E2 serum IgG titre after E2 boost. Red bar shows mean value. n = 6 from two independent experiments for each group; labeling and statistics as for panel D. (F) Anti-E4 serum IgG titre after E4 boost. Red bar shows mean value. n = 6 from two independent experiments for each group; labeling and statistics as for panel D.

Figure 2.. GC B-cell levels, isotypes, VH mutation and clonality after boosting with E-protein variants.

(A) FACS gating strategy used to identify and sort GC B-cells and determine isotype. (B) GC B-cell levels after E-variant boosting, expressed as % total lymphocytes; Red bar shows mean value; numbers 3,2 and 4 refer to serotype of Dengue-envelope protein used for boost; BO, boost only, adjuvant primed, E3 boosted day 37, analysed 7 days later; Day, days after boosting. (C) % IgM+ GC B-cells, of total GC B-cells, after boosting. Red bar shows mean value. n = 6 from two independent experiments for each group; labels as for panel B except % identity which refers to sequence identity between E3 and other variants; p-values calculated using two-tailed Students t-test after testing for equality of variance. (D) Levels of IgM+ and IgM- GC B-cells in individual boosted mice. (E) Number of mutations detected in VH of all isotypes of GC B-cells, from n = 3 mice except E4 boost day 17, n = 2. Red bar is median value. VH region sequenced is CDR1 to FR3; labeling as panel B. (F) Number of mutations detected in VH of IgM+ GC B-cells, from n = 3 mice except E4 boost day 17, n = 2. Red bar is median value. (G) Clonality of sequences from single GC B-cells 7 days after boosting; colours indicate different mice in each group; thin sectors, unique sequences; thicker sectors two or three clonal sequences according to sector size; black dots, VH 14–3 or VH14-4 sequences; numbers in circles, number of sequences from that mouse; Identical VH clones had the same: V-gene, CDR3 length, J-gene, D-gene if assigned, D-reading frame, three or fewer differences in CDR3 amino acid sequence.

Figure 3.. Relative serum affinity and avidity after boosting with E-protein variants, and T-cell re-stimulation.

(A) Relative avidity of E2 boost serum for E2, measured by resistance to 7M Urea. Red bar shows mean value; Day, days after E2 boosting; Day 0 sample was from mice mock-boosted with PBS 37 days after priming with E3 and obtained 7 days later. (B) Relative avidity of E4 boost serum for E4, measured by resistance to 7M Urea. Labeling as for panel A; Day 0 sample was from mice mock-boosted with PBS 37 days after priming with E3 and obtained 7 days later (C) Relative affinity of E2 boosted serum for E2. Inhibition by lower concentration of competitor implies higher affinity of serum for competitor. Maximum competitor amount 2 μg in 50 μl followed by six-fold dilutions of competitor; timepoint of samples and numbers of individuals in group indicated. Open circles, E2 boost day 17 serum competed with irrelevant His-tagged protein measured on E2 target (D) Relative affinity of E4 boosted serum for E4. Labeling as for panel A. (E) T-cell proliferation measured by ³H incorporation 96 hr after re-stimulation in vitro with indicated amounts of E-protein variants; error bars indicate standard error of the mean; n = 4 or five from two independent experiments (see source data). Closed symbols, E3 primed mouse splenocytes re-stimulated with indicated E-protein variant. Open symbols, adjuvant primed mouse splenocytes re-stimulated with indicated E-protein variant.

Figure 4.. Primary response to E4 and rAb binding.

(A) anti-E4 IgG titre after E4 priming; Red bars show mean titres; A, serum from adjuvant-only primed mice at day 45; d7, 7 days after E4 priming; d17, 17 days after E4 priming; EPT, end-point titre calculated as for Figure 1. (B) GC B-cell levels after E4 priming; Red bars indicate mean levels; A, cells from adjuvant-only primed mice 7 days after priming; other x-axis labels as for panel A. (C) %IgM + GC B-cells after E4 priming; Red bars show mean values; x-axis labels as for panel A. (D) Numbers of VH mutations in all isotypes of GC B-cells after E4 priming; Red bars show median values, from n = 3 mice (d7) and n = 2 mice (d17); x-axis labels as for panel A. (E) Numbers of VH mutations in IgM+ GC B-cells after E4 priming and boosting; Red bars show median values, from n = 3 mice (d7), n = 2 mice (d17) and n = 3 mice E4Bd7; x-axis labels as for panel A except E4Bd7, 7 days after E4 boosting which was 38 days after E3 priming. (F) ELISA screen of binding of all 48 rAbs. rAbs incubated at 100μgml⁻¹. Number of rAbs in each group indicated. P7, 7 days after E4 prime; P17, 17 days after E4 prime; B7, 7 days after E4 boost. As the antibodies were cloned as chimeric human IgG1 antibodies the background from non-specific human polyclonal IgG binding has been subtracted from O.D. readings. Values in supplementary file 2. (G) ELISA titration of rAbs that showed binding O.D. > 0.1 in panel F. All but one were IgM. IgG1 rAb indicated. Background subtraction as for panel F, using appropriate dilution of polyclonal IgG. (H) Anti-E4 end point titre of positive-binding rAbs, used as a proxy of rAB affinity. X-axis labels as for panel F. End-point titre values plotted are log2 of 1/end point dilution (undiluted = 100μgml⁻¹). Red bars show median values (excluding any IgG1 data). Stronger binding IgM rAb ‘B5’, and IgG1 rAb ‘G6’ EPT readings indicated. (I) anti-E3 versus anti-E4 endpoint titres. Star, E4 prime day 7 rAbs; Square, E4 prime day 17 rAbs; circle, E4 boost day 7 rAbs. IgG1 EPT reading indicated. End-point titre values plotted are log2 of 1/end point dilution (undiluted = 100μgml⁻¹).