This proposal represents our continuing efforts to understand the genetics, cellular basis and somatic evolution of protective polysaccharide (PS) antibody (Ab) repertoires. We will focus upon Ab repertoires specific for Streptococcus pneumoniae polysaccharide serotypes 14 (PPS 14) and 23F (PPS 23F) and the Haemophilus influenzae type b capsular polysaccharide (Hib PS). Preliminary studies lead us to hypothesize that the primary PPS B cell repertoire is of low affinity and relies upon hypermutation, driven by conjugate vaccination, to generate a high affinity, protective response. The molecular ontogeny of the PPS Ab repertoire will be examined by isolating PPS-specific B cells from infants following immunization with PPS-protein conjugate vaccines. The variable (V) region genes used by individual PPS-specific B cells or their clonal products will be sequenced and affinity analyses will be performed on monoclonal Fab fragments. Selected Fab fragments will be converted to full-length recombinant Abs in order to evaluate opsonophagocytic activity. These studies will identify the V genes used by infants in response to PPS 14 and PPS 23F, document the extent to which somatic hypermutation and/or V region shifts occur in the maturation of the response and determine the functional consequences of this mutation. Our in vitro studies indicate that IgH allelic polymorphisms can dramatically influence PS Ab function. Accordingly, we will perform vaccination and B cell cloning studies in infants and adults to test the hypothesis that individuals utilizing the V3-23*03 allele will produce higher quality canonical Hib PS Abs and PPS 23F Abs than individuals using the V3-23*01 allele. Little is known about the specificity repertoire of peripheral blood IgM memory B cells or their role in PS immunity. We will identify the V region repertoire of PS-specific IgM 'memory'B cells isolated from vaccinated adults. V gene usage and mutation will be evaluated in individual IgM memory B cells or expanded clones, and PS binding studies will performed on Fab fragments derived from these clones. We will use a stable isotope in vivo labeling method to determine the DMA synthetic rates (half lives) of IgM memory B cells and other blood B cell subsets both before and after vaccination to determine whether vaccination induces DNA synthesis (clonal proliferation) in these B cells. These studies address clinically relevant topics. They will identify genetic determinants of Ab efficacy and disease susceptibility, elucidate the cells and processes involved in the immune response to pediatric vaccines, and delineate the mechanisms underlying immunity to encapsulated pathogens. This research will deepen our understanding of the human immune system and may contribute toward the design of better vaccines.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI025008-23
Application #
7793442
Study Section
Immunity and Host Defense Study Section (IHD)
Program Officer
Khambaty, Farukh M
Project Start
1988-09-01
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2012-04-30
Support Year
23
Fiscal Year
2010
Total Cost
$514,706
Indirect Cost
Name
Children's Hospital & Res Ctr at Oakland
Department
Type
DUNS #
076536184
City
Oakland
State
CA
Country
United States
Zip Code
94609
Colino, Jesus; Duke, Leah; Snapper, Clifford M (2013) Noncovalent association of protein and capsular polysaccharide on bacteria-sized latex beads as a model for polysaccharide-specific humoral immunity to intact gram-positive extracellular bacteria. J Immunol 191:3254-63
Colino, Jesus; Duke, Leah; Arjunaraja, Swadhinya et al. (2012) Differential idiotype utilization for the in vivo type 14 capsular polysaccharide-specific Ig responses to intact Streptococcus pneumoniae versus a pneumococcal conjugate vaccine. J Immunol 189:575-86
Wang, Taia T; Fellows, Patricia F; Leighton, Terrance J et al. (2004) Induction of opsonic antibodies to the gamma-D-glutamic acid capsule of Bacillus anthracis by immunization with a synthetic peptide-carrier protein conjugate. FEMS Immunol Med Microbiol 40:231-7
Wang, Taia T; Lucas, Alexander H (2004) The capsule of Bacillus anthracis behaves as a thymus-independent type 2 antigen. Infect Immun 72:5460-3
Liu, Leyu; Lucas, Alexander H (2003) IGH V3-23*01 and its allele V3-23*03 differ in their capacity to form the canonical human antibody combining site specific for the capsular polysaccharide of Haemophilus influenzae type b. Immunogenetics 55:336-8
Lucas, Alexander H; McLean, Gary R; Reason, Donald C et al. (2003) Molecular ontogeny of the human antibody repertoire to the Haemophilus influenzae type B polysaccharide: expression of canonical variable regions and their variants in vaccinated infants. Clin Immunol 108:119-27
Zhou, Jianhui; Lottenbach, Kathleen R; Barenkamp, Stephen J et al. (2002) Recurrent variable region gene usage and somatic mutation in the human antibody response to the capsular polysaccharide of Streptococcus pneumoniae type 23F. Infect Immun 70:4083-91
Lucas, A H; Granoff, D M (2001) Imperfect memory and the development of Haemophilus influenzae type B disease. Pediatr Infect Dis J 20:235-9
Reason, D C; O'Connor, A P; Lucas, A H (2001) Structural determinants of the human idiotype HibId-1. J Mol Recognit 14:393-400
Lucas, A H; Moulton, K D; Tang, V R et al. (2001) Combinatorial library cloning of human antibodies to Streptococcus pneumoniae capsular polysaccharides: variable region primary structures and evidence for somatic mutation of Fab fragments specific for capsular serotypes 6B, 14, and 23F. Infect Immun 69:853-64

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