Virus neutralization by germ-line vs. hypermutated antibodies - PubMed (original) (raw)

Virus neutralization by germ-line vs. hypermutated antibodies

U Kalinke et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Mice infected with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, mount a virus-neutralizing antibody response protecting against lethal disease. VSVneutralizing monoclonal IgGs isolated from primary immune responses were devoid of somatic mutations, whereas most secondary and all hyperimmune response IgGs tested were hypermutated. A comparative analysis of recombinant single-chain antibody fragments (scFv-Ckappa) revealed that even the germ-line precursor of one hypermutated antibody bound and neutralized VSV. Four somatic amino acid substitutions in V(H) increased by 300-fold the binding strength of monovalent scFv-Ckappa. The multivalent binding avidity of germ-line scFv-Ckappa was increased by more than 10-fold compared with the monovalent binding strength. In contrast, hypermutated scFv-Ckappa did not show such avidity effects. Thus the overall binding difference between the germ-line and the hypermutated VSV-neutralizing antibody was only 10- to 15-fold. This may explain why primary germ-line antibodies and secondary hypermutated antibodies directed against pathogens such as viruses and bacteria expressing repetitive antibody determinants show rather similar binding qualities, whereas monovalently binding hapten-specific antibodies can show "affinity maturation" effects of up to 1000-fold.

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Figures

Figure 1

Comparison of VH and VL sequences of VH7183/JH2-positive VSV-neutralizing antibodies. VH and some VL sequences of hybridomas are shown isolated from BALB/c mice 4 days after infection (41.6), isolated 12 days after primary infection and a booster infection on day 9 (VI26, VI30, VI22, VI20, and VI24), and isolated on day 150 after repeated booster infections every 3 weeks (VI53, VI51, VI55, VI52, and VI43). CDR, complementarity-determining region. (A) VH sequences are aligned with germ-line sequences of the VH61–1P gene (20) and the JH2 segment (39). Only codons differing from the germ-line sequence are shown. VH nucleotide sequence identity with reference sequences is indicated by - - -. Differences are marked by the respective nucleotide, and a change in the deduced amino acid is shown by the single-letter code. Codons are numbered according to ref. . (B) VL sequences are aligned with the germ-line sequences of the Vκ21B16 gene (23), the Vκ8–220GL gene (24), and the Jκ2 segment (21). The Vκ consensus sequence of VI20L and VI22L (VκVI20/22) is identical to an unrelated Vκ sequence (accession no. AF045518) and presumably represents a previously undescribed germ-line gene of the Vκ21 family. The sequence data are available from the European Molecular Biology Laboratory nucleotide sequence database (accession nos. AJ400966–81).

Figure 2

Structural characteristics of the IgG VI24 and of recombinant scFv-Cκ. (A) Schematic diagram of a prototypical IgG (heavy chain, black; light chain, white) and of recombinant scFv-Cκ. In scFv-Cκ the carboxyl terminus of the VH domain (black) is linked via the 18-aa linker [VE(GGS)4GGVD] to the amino terminus of the κ light chain, consisting of the Vκ and the Cκ domains (white). (B) Sequence alignment of scFv24-Cκ derived from the parental antibody VI24 and of the scFv-Cκ derivatives M0, M31, M55, and M31.55. With the exception of the shown VH codons 31, 35, 55, 58, and 99, sequences were identical (for notation and numbering see the legend of Fig. 1). The sequence data are available from the European Molecular Biology Laboratory nucleotide sequence database (accession nos. AJ400982–6). (C) SDS/PAGE analysis of purified VI24 and of purified scFv-Cκ proteins. Four micrograms of purified protein was separated by SDS/12% PAGE under reducing conditions and was visualized by silver staining (Bio-Rad). The IgG2a antibody VI24 gave rise to two bands, at 50 kDa for the heavy chain and at 25 kDa for the light chain. In contrast, the different scFv-Cκ proteins showed one major band at approximately 38 kDa, verifying the single-chain character of the proteins. In the last lane a prestained low-molecular-mass protein standard (Bio-Rad) was run.

Figure 3

VSV-specific binding and neutralization of scFv-Cκ antibody fragments. Supernatant containing scFv-Cκ protein was serially 2-fold diluted and transferred to VSV-coated plates. Bound scFv-Cκ was detected by horseradish peroxidase-labeled anti-Cκ antibody (closed symbols). To determine avidity effects, supernatant containing scFv-Cκ was crosslinked by horseradish peroxidase-labeled anti-Cκ antibody before serial 2-fold dilution and then applied to VSV-coated plates (open symbols). (A) VSV-specific binding of hypermutated scFv24-Cκ (circles) and of germ-line scFv-Cκ M0 (squares). (A–D) Half-maximal binding of scFv24-Cκ and M0 is indicated by dotted lines. (B) The VH Ser31-to-Asn substitution did not improve scFv-Cκ binding. (C) Despite the fact that the VH Ser55-to-Arg substitution was not expressed by the parental antibody VI24, it improved the binding of M55. (D) Coexpression of the VH Ser31-to-Asn and the VH Ser55-to-Arg substitutions improved binding of M31.55. (E) VSV-IND neutralization by various scFv-Cκ antibody fragments was tested with untreated reagents (black bars) and after crosslinking (open bars). For crosslinking, 2.5 μg of purified scFv-Cκ protein was incubated with 1.25 μg of anti-Cκ antibody in a total volume of 100 μl for 30 min at room temperature. The crosslinked and untreated scFv-Cκ protein and the purified antibody VI24 were serially 2-fold diluted and analyzed in a standard neutralization assay. The protein dilution reducing the number of plaques by 50% is indicated as the titer.

Figure 4

Protective capacity of hypermutated and germ-line IgGs in mice. SCID mice were reconstituted with graded doses of monoclonal antibodies of the IgG2a subclass, which were either devoid of somatic mutations (open symbols) or hypermutated (filled symbols). Five hours later mice were i.v. challenged with 108 pfu of VSV-IND. After 4 days the brains of surviving mice were assessed for the presence of virus. Mice without detectable virus were scored as being protected. Groups of three mice were tested; deviating numbers of tested animals are given in parentheses. (A) Analysis of IgGs using V gene fragments belonging to the VHQ52 and the Vκ19–28 families. The germ-line antibodies 41.11 (□), 41.2 (▵), and 41.9 (○) were isolated from primary responses 4 days after infection. The hypermutated antibodies VI10 (■) and VI49 (●) were isolated, respectively, from a secondary response 12 days after infection and from a hyperimmune response 150 days after infection. (B) Analysis of IgGs using the VH61–1P germ-line gene fragment belonging to the VH7183 family and the JH2 segment. The germ-line antibodies 41.6 (□) and 51.3 (▵) were isolated from primary responses 4 and 5 days after infection, respectively. The germ-line antibody VI26 (□) was isolated from a secondary response 12 days after infection. The hypermutated antibody VI43 (●) was isolated from a hyperimmune response 150 days after infection.

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Virus neutralization by germ-line vs. hypermutated antibodies - PubMed (original) (raw)