Binding analysis of antibodies produced by precursor and branchpoint intermediates of an anti-influenza hemagglutinin B cell clone. Parallel replacement mutations do not confer increased avidity for hemagglutinin.
We have previously described a set of secondary (2 degrees) hybridomas that are specific for the Sb site of influenza hemagglutinin (HA(Sb)) and share the H37-68 ld (68ld). Cells producing 68ld Abs dominated the 2 degree response of one BALB/c mouse, but were virtually absent from the primary (1 degree) and 2 degree responses of other BALB/c mice. To understand the basis for the idiosyncratic nature of this response, we have assessed the functional importance of a conserved DJ junctional sequence. We find that substitutions of conserved residues within this sequence drastically reduced HA(Sb) binding, providing an explanation for the absence of this ld from 1 degree responses. We have also examined intraclonal affinity maturation by generating Abs inferred to have been produced by the unmutated precursors and branchpoint intermediates of the largest 68ld clone. Interestingly, unmutated Abs of this clone bound HA(Sb) well, comparable to that of some 2 degree 68ld hybridoma Abs. Abs produced by branchpoint intermediates of this clone have a lower affinity for HA(Sb) than their unmutated precursor, despite containing parallel amino acid replacement mutations. Thus, expansion of this clone did not follow the paradigm of stepwise increases in affinity. Together, these data underscore the interconnective nature of precursor frequency and somatic mutation in the formation of the 2 degree response to a given Ag.
Rickert
R C
RC
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill 27599.
Wloch
M K
MK
Hahn
R L
RL
Clarke
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000235:genetics / Q000378:metabolism*; D000915:Antibody Affinity / Q000235:genetics; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D005803:Genes, Immunoglobulin; D006389:Hemagglutinins, Viral / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009975:Orthomyxoviridae / Q000276:immunology*; D014162:Transfection
D000914:Antibodies, Viral; D017931:DNA Primers; D006389:Hemagglutinins, Viral