Secondary rearrangements and hypermutation generate sufficient B cell diversity to mount protective antiviral immunoglobulin responses

Abstract

Variable (V) region gene replacement was recently implicated in B cell repertoire diversification, but the contribution of this mechanism to antibody responses is still unknown. To investigate the role of V gene replacements in the generation of antigen-specific antibodies, we analyzed antiviral immunoglobulin responses of "quasimonoclonal" (QM) mice. The B cells of QM mice are genetically committed to exclusively express the anti-(4-hydroxy-3-nitrophenyl) acetyl specificity. However, approximately 20% of the peripheral B cells of QM mice undergo secondary rearrangements and thereby potentially acquire new specificities. QM mice infected with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus, or poliovirus mounted virus-specific neutralizing antibody responses. In general, kinetics of the antiviral immunoglobulin responses were delayed in QM mice; however, titers similar to control animals were eventually produced that were sufficient to protect against VSV-induced lethal disease. VSV neutralizing single-chain Fv fragments isolated from phage display libraries constructed from QM mice showed VH gene replacements and extensive hypermutation. Thus, our data demonstrate that secondary rearrangements and hypermutation can generate sufficient B cell diversity in QM mice to mount protective antiviral antibody responses, suggesting that these mechanisms might also contribute to the diversification of the B cell repertoire of normal mice.

Figure 1

QM mice mount VSV-specific Ig responses. Groups of three QM and C57BL/6 mice were immunized with 2 × 10⁶ PFU of VSV-IND intravenously. Blood was taken at the indicated time points, sera were separated and prediluted 40-fold, and the Ig responses were analyzed. (a) Kinetics of VSV-neutralizing Ab responses in QM and C57BL/6 mice. (b) Titration of the different VSV-specific Ig isotypes in infected QM and C57BL/6 mice. Titers were defined as 3 SD above mean values of negative controls. (c) Kinetics of VSV-neutralizing responses of 9- and 56-wk-old QM mice. C57BL/6 mice were used as controls.

Figure 2

Binding, neutralizing capacity, and sequence analysis of VSV-specific scFv fragments isolated from phage display libraries. (a) Phage clones displaying VSV-specific scFv fragments isolated from a naive QM mouse library (0B1 and 0G6) and libraries generated from QM mice after primary (1A10 and 1E12) or secondary (2F7 and 2H5) infections were tested for their capacity to bind VSV-coated plates in ELISA. (b) VSV-neutralizing titers of PEG-precipitated phage particles. (c) The nucleotide sequence of six VSV-specific clones is compared with the 17.2.25 transgene sequence (reference 16) shown in bold. Mutations in the DSP2.10 and J_H4 segments are underlined. The remaining 3′ sequence of the J_H4 segments did not contain somatic hypermutations. The sequences of the complete scFv fragments are available from EMBL/GenBank/DDBJ under accession nos. AF127092–AF127097.

Figure 3

QM mice mount neutralizing Ig responses against LCMV and PV. Groups of three mice were immunized with 200 PFU of LCMV-WE or 0.5 ml of PV (Salk vaccine) intravenously. (a) LCMV nucleoprotein–specific Ab titers were measured by ELISA. (b) LCMV-neutralizing Abs and (c) PV-neutralizing Abs were measured as described in Materials and Methods.