Abstract

Immune maturation is viewed as a Darwinian process that involves continuous selection of the structure (s) of best fit. The long term objective of this study is to understand the molecular basis of antigen recognition and its contribution as a selective force in shaping the antibody response to phosphocholine-protein (PC- KLH) conjugates. The evolution of antibodies (in particular Group II) recognizing different molecular aspects of the PCcarrier complex will be studied using a combination of molecular genetic and physical measurements of serum antibodies, hybridomas and monoclonal antibodies obtained at various times during the immune response to PC-KLH.
The specific aims are 1) Determine the molecular progression of the antibody response to PC-KLH via hybridoma analysis. The emergence of Group II antibodies will be determined and analyzed for isotype and/or clonotype restriction. The role of somatic mutation will be determined and specific gene usage will be assessed. 2) Determine the vulnerability of the T15+ clonotype to antigen-binding loss mutations. 3) Analyze the evolution of epitope recognition with regard to fine specificity, affinity, and physical interaction between structural features of phenyl-PC and the active site. 4) Analyze the relative contributions of PC-phenyl-tyrosine and PC-phenyl-histidine to evolution of the Group II response. The methods to be used include mRNA sequencing, in situ hybridoma lysis and mRNA hybridization to obtain gene usage information. Affinity of monoclonal antibodies will be measured by fluorescence spectroscopy. Antibody fine specificity will be assessed using a panel of hapten analogs. Physical interactions between antibody combining sites and structural components of the hapten will be assessed by nuclear magnetic resonance spectroscopy.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI026827-04
Application #
3140817
Study Section
Immunobiology Study Section (IMB)
Project Start
1988-07-01
Project End
1993-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
4
Fiscal Year
1991
Total Cost
Indirect Cost

  Institution

Name
Oregon Health and Science University
Department
Type
Schools of Medicine
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239

  Publications

Whitcomb, Elizabeth A; Martin, Tammy M; Rittenberg, Marvin B (2003) Restoration of Ig secretion: mutation of germline-encoded residues in T15L chains leads to secretion of free light chains and assembled antibody complexes bearing secretion-impaired heavy chains. J Immunol 170:1903-9
Wiens, Gregory D; Brown, McKay; Rittenberg, Marvin B (2003) Repertoire shift in the humoral response to phosphocholine-keyhole limpet hemocyanin: VH somatic mutation in germinal center B cells impairs T15 Ig function. J Immunol 170:5095-102
Wiens, G D; O'Hare, T; Rittenberg, M B (2001) Recovering antibody secretion using a hapten ligand as a chemical chaperone. J Biol Chem 276:40933-9
Wiens, G D; Lekkerkerker, A; Veltman, I et al. (2001) Mutation of a single conserved residue in VH complementarity-determining region 2 results in a severe Ig secretion defect. J Immunol 167:2179-86
Brown, M; Schumacher, M A; Wiens, G D et al. (2000) The structural basis of repertoire shift in an immune response to phosphocholine. J Exp Med 191:2101-12
Brown, M; Wiens, G D; O'Hare, T et al. (1999) Replacements in the exposed loop of the T15 antibody VH CDR2 affect carrier recognition of PC-containing pathogens. Mol Immunol 36:205-11
O'Hare, T; Wiens, G D; Whitcomb, E A et al. (1999) Cutting edge: proteasome involvement in the degradation of unassembled Ig light chains. J Immunol 163:11-4
Wiens, G D; Roberts, V A; Whitcomb, E A et al. (1998) Harmful somatic mutations: lessons from the dark side. Immunol Rev 162:197-209
Martin, T M; Wiens, G D; Rittenberg, M B (1998) Inefficient assembly and intracellular accumulation of antibodies with mutations in V(H) CDR2. J Immunol 160:5963-70
Heldwein, K A; Duncan, J E; Stenzel, P et al. (1997) Endotoxin regulates corticotropin-releasing hormone receptor 2 in heart and skeletal muscle. Mol Cell Endocrinol 131:167-72

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