Richard A. Insel - US grants

Haemophilus influenzae b (Hib) is the most common cause of meningitis and childhood sepsis in the US. The capsular polysaccharide (CP) is a major virulence factor of Hib. Vaccines composed of the purified CP prevent Hib disease in children older than l8 mo., but have proven poorly immunogenic in infants, the age-group with the highest attack rate. The delayed ontogeny of the antibody (Ab) response to the Hib CP in man may mimic the delayed ontogeny of the response to certain polysaccharides in mice. In addition to being delayed in ontogeny, the murine Ab response to certain polysaccharides is restricted in diversity and expresses cross-reactive idiotypes (IdX) that are found on a high proportion of Ab molecules in most individuals of a strain. We have found that the human Ab response to the Hib CP is also highly limited in diversity, with restriction in light chain type and Ab charge heterogeneity as detected on isoelectric focusing (IEF) gels. The limited diversity, the finding of identical Ab IEF patterns in unrelated individuals, and the ability to immunize older humans to induce high levels of an Ab that is directed to a clinically relevant antigen, makes it possible to define the structural basis, regulation, and ontogeny of the diversity of the human anti-Hib CP Ab response. The aims of the proposed research are to: l) determine the relative contribution of the Ab light (L) and heavy (H) chains to human Ab diversity and to binding of the Hib CP. Both human serum and hybridoma Ab will be prepared and purified. The L and H chains will be isolated and we will determine L chain charge heterogeneity by IEF, L and H chain subgroups by amino acid sequencing, and L and H chain contribution to antigen binding; 2) define the existence of IdX and its molecular basis by amino acid sequencing; 3) define the molecular basis of the Ab repertoire by amino acid sequencing; 4) determine the relative protective capacity of Ab with different V-regions using an animal model of the Hib disease; 5) define the normal ontogeny of Ab V-region expression and determine whether it is altered by passive immunization of the infant with Ab through maternal immunization or by active immunization with the Hib CP linked to a protein carrier; 6) determine whether autologous anit-idiotypic Ab is produced after immunization; and 7) determine whether Ab V-region expression is inherited and linked to immunoglobulin allotype markers. The long-range goal will be to apply the knowledge acquired of Ab V-region structure and regulation to develop an effective vaccine strategy to prevent Hib disease in man.

DESCRIPTION (Adapted from applicant's abstract): Antibody to bacterial capsular polysaccharides are the major form of protection against infections from pyogenic bacteria. The delay in ontogeny of human antibody responses to polysaccharides predisposes the healthy infant to infection and defective antibody responses renders patients with immunodeficiency, including the aged and patients with AIDS or malnutrition, at high risk. The overall goal of this proposal is to define the basis for the delayed ontogeny of human antibody responses to polysaccharides. We have found that the dominant Vk gene encoding high affinity human antibody to the Haemophilus influenzae b capsular polysaccharides is the A2 gene, which maps to the Jk-distal region of the locus, is used preferentially in germline form, and encodes antibody when expressed with an arginine at the Vk-Jk junction that arises by N region addition. We hypothesize that delayed rearrangement of critical Vk genes that encode these highly restricted antibody repertoires and delayed and infrequent N region addition contribute to this delay in ontogeny. This proposal will address whether: 1) there is a delay in a) ontogeny of immunoglobulin gene rearrangement of Vk genes located in the Jk-distal region of the human Vk locus, and b) N region addition at the Vk-Jk junction, which appears critical for the generation of an extended CDR3 loop for binding of serum antibody to polysaccharides; 2) rearrangements of Jk-distal Vk genes occur preferentially as secondary Vk-Jk rearrangements; 3) N region addition at Vk-Jk junctions occurs preferentially when L chain rearrangement occurs at an early state of B cell development; 4) Vk gene expression is affected by Ck light chain polymorphism; 5) pneumococcal polysaccharides that induce antibody responses late in infancy are encoded b map to the Jk-distal-region and require N region addition at their Vk-Jk junction; 6) deletion of the N region-derived junctional CDR3 residue of antibody to polysaccharides abolishes antibody binding. The results should provide novel insights into the ontogeny of human antibody responses and provide a background for developing vaccines that accelerate maturation of antibody responses.

Humoral immunity decreases with aging and contributes to the increased incidence and accompanying morbidity and mortality of infections and to the poor immunogenicity of vaccines in this age group. Although there are multiple etiologies of the decreased humoral immunity with aging, intrinsic defects of aged B lymphocytes and their precursors may make an important contribution to defective primary antibody responses, the low affinity antibody response, and poor persistence of memory. We hypothesize that contributing to these defects in aged humans are the poor generation of memory B cells that is associated with limited diversification of antibody responses and low levels of somatic hyper mutation of immunoglobulin(lg)variable (V) region genes and the poor persistence of memory B cells secondary to shortened telomere length that limits their longevity. We have recently developed an approach to activate and recover a diversified human antibody repertoire after immunization that can be analyzed at the molecular level and methods to up regulate expression of telomerase in memory B cells. Using these approaches, we propose to determine whether immunized healthy elderly adults generate diversified antibody repertoire with somatic hyper mutation and memory B cells to the same extent as young adults and to investigate whether telomerase is up-regulatable in- vitro and telomere shortening occurs in-vivo in B lymphocytes of elderly adults. The results should provide insight into the poor humoral immunity that occurs with aging and a background for developing interventions to reverse immunosenescence or improve, vaccines for the elderly.

DESCRIPTION (Adapted from Applicant's Description): Both premature and full term human neonates generate suboptimal antibody (Ab) responses to vaccines. The overall goal of this proposal is to investigate whether defects in the preimmune Ab repertoire of neonatal B cells and in the diversification of this repertoire after immunization contribute to defective Ab responses and whether immunization during pregnancy is able to alter the immunoglobulin repertoire and B cell memory of the neonate. In Aim 1, the investigators hypothesize that the preimmune repertoire of the neonate is restricted in diversity of the immunoglobulin heavy chain third complementarity determining region (CDR3), which arises from the joining of Ig gene segments and N region addition. To investigate this hypothesis, they will characterize and compare the diversity and structure of the CDR3 regions of preterm and full term neonatal and adult B lymphocytes. They will analyze the potential antigen binding activity of a CDR3 library expressed from each group with a single immunoglobulin heavy chain variable region gene (VH) and light chain gene in a Fab surface phage expression library for binding to self and non-self antigens. In Aim 2, we will investigate the hypothesis that the neonate fails to diversify postimmunization and Ab repertoires with somatic hypermutation of VH by analyzing somatic hypermutation in VH at birth and after immunization of the neonate. To explore the hypothesis that the neonatal B cell repertoire can be altered by maternal immunization during pregnancy with vaccines that induces a high titered clonally restricted IgG Ab response, they will immunize pregnant women in the third trimester of pregnancy with Haemophilus influenzae b vaccines, which generate a high-titered oligoclonal IgG Ab response, and analyze the offspring for evidence of immunologic priming with Ab production and generation of B cell memory. The results of these investigations should provide novel insights into the ontogeny of the neonatal B cell repertoire and in the potential to accelerate of immunity.