Antibody V(h) repertoire differences between resolving and chronically evolving hepatitis C virus infections

Abstract

Despite the production of neutralizing antibodies to hepatitis C virus (HCV), many patients fail to clear the virus and instead develop chronic infection and long-term complications. To understand how HCV infection perturbs the antibody repertoire and to identify molecular features of antibody genes associated with either viral clearance or chronic infection, we sequenced the V(D)J region of naïve and memory B cells of 6 persons who spontaneously resolved an HCV infection (SR), 9 patients with a newly diagnosed chronically evolving infection (CE), and 7 healthy donors. In both naïve and memory B cells, the frequency of use of particular antibody gene subfamilies and segments varied among the three clinical groups, especially between SR and CE. Compared to CE, SR antibody genes used fewer VH, D and JH gene segments in naïve B cells and fewer VH segments in memory B cells. SR and CE groups significantly differed in the frequency of use of 7 gene segments in naïve B cell clones and 3 gene segments in memory clones. The nucleotide mutation rates were similar among groups, but the pattern of replacement and silent mutations in memory B cell clones indicated greater antigen selection in SR than CE. Greater clonal evolution of SR than CE memory B cells was revealed by analysis of phylogenetic trees and CDR3 lengths. Pauciclonality of the peripheral memory B cell population is a distinguishing feature of persons who spontaneously resolved an HCV infection. This finding, previously considered characteristic only of patients with HCV-associated lymphoproliferative disorders, suggests that the B cell clones potentially involved in clearance of the virus may also be those susceptible to abnormal expansion.

Figure 1. Frequency of usage of V(D)J subfamilies in molecular clones of naïve and memory B cells from 7 healthy donors (HD), 6 persons who spontaneously resolved an HCV infection (SR) and 9 patients with chronically evolving HCV infection (CE).

For each subject, 32 clones from naïve B cells and another 32 from memory B cells were analyzed. Values represent the percentage of subfamily use for all clones in a particular clinical group and B cell subset. Panels A , C and E, naïve B cell clones; panels B , D and F, memory B cell clones. Associations between clinical group and individual subfamily were tested for significance with chi-square or Fisher's exact test only when a previous chi-square test indicated a significant association of gene family usage in a pairwise comparison of groups; p values are shown only when ≤0.05.

Figure 2. Naïve B cells: fractional usage of individual V(D)J gene segments in molecular clones from healthy donors (HD), persons who spontaneously resolved an HCV infection (SR) and patients with chronically evolving HCV infection (CE).

Figure 3. Memory B cells: fractional usage of individual V(D)J gene segments in molecular clones from healthy donors (HD), persons who spontaneously resolved an HCV infection (SR) and patients with chronically evolving HCV infection (CE).

Figure 4. Mutation frequencies of V_H gene segments in molecular clones of naïve B cells (A) and memory B cells (B), by clinical group.

Values are mean (SD) of the per-subject average (32 clones per subject). p<0.001, all naïve vs. all memory clones, chi-squared test.

Figure 5. Evidence for antigen selection of V_H gene segments in molecular clones from memory B cells.

A Distribution of replacement (R) and silent (S) mutations in each framework region (FR) and complementarity-determining region (CDR), by clinical group. Statistical significance by ANOVA is indicated. B Percentage of V_H gene segments with evidence of antigen selection, according to Lossos et al. . Values are mean (SD) of the per-subject average (32 clones per subject). C Representative phylogenetic trees from one healthy donor (HD), one patient who spontaneously resolved an HCV infection (SR), and one patient with chronically evolving HCV infection (CE). The pattern of branching and clustering of sequences in the SR tree is evidence of clonal evolution. D Length of CDR3 peptide loops deduced from V_H gene nucleotide sequences of memory B cells, by clinical group. Values are mean (SD) of the average value per subject. ANOVA p = 0.034; Newman-Keuls post test: HD vs. SR, p = 0.0.017; HD vs. CE, p = 1.0; SR vs. CE, p = 0.042. E Representative CDR3 length distribution profiles determined by spectratyping on CDR3 PCR products reveal evidence of clonal selection in SR.

Figure 6. In vitro proliferation of CFSE-labelled B cells in response to 7 days' stimulation with different recombinant HCV-SOD fusion proteins, by clinical group.

A Stimulation index, calculated as the percentage of cells with diluted CFSE staining (indicating cell division) after HCV-SOD treatment relative to that of SOD-treated control cells. SR vs. CE, p = 0.011 Mann-Whitney U test after ANOVA. B Representative FACS scatter plots of B cells from SR (left) and CE (right) stimulated with NS5 HCV-SOD fusion protein show greater CFSE dilution (hence proliferation) in SR.