Somatic hypermutation in T-independent and T-dependent immune responses to Haemophilus influenzae type b polysaccharide.

Secondary immune responses to T-independent antigens are characterized by little or no affinity maturation, a phenomenon attributed to limited somatic hypermutation. In the human immune response to Haemophilus influenzae type b capsular polysaccharide, however, there are numerous differences between rearranged heavy chain variable region gene segments and candidate germline genes, irrespective of antigen presentation in a T-independent or T-dependent form. To determine the characteristics of somatic hypermutation in this response, we analyzed rearranged heavy chain variable region segments and associated 3' untranslated JH4-JH5 introns from monoclonal anti-Hib PS antibodies. Mutation of untranslated introns and heavy chain variable segments in both T-independent and T-dependent responses resembles that described in murine and unselected human immune responses. Although mutation is frequent in both T-independent and T-dependent anti-Hib PS responses, there is little evidence of antigen-driven selection, suggesting that ongoing pressure to conserve the variable segment germline configuration limits affinity maturation in this immune response.

Haemophilus influenzae type b polysaccharides-protein conjugate vaccine elicits a more diverse antibody repertoire in infants than in adults.

Conjugation of bacterial polysaccharides (PS) to protein carriers confers the ability to elicit protective serum Ab in infants, who respond poorly to plain PS. The serum Ab of young children immunized with Haemophilus influenzae type b (Hib) PS conjugate vaccine varies with age and Ag formulation. To understand these age-related changes in human anti-Hib PS immune responses we determined the variable region gene sequences encoding anti-Hib PS mAbs of infants immunized with Hib oligosaccharide-diphtheria toxin vaccine. The anti-Hib PS repertoire of children differs from that of adults. A smaller proportion of mAbs from children have high affinity for Hib PS, and the overall variable region gene repertoire of infants is more diverse than that in adults. Variable region genes encoding high affinity mAbs of infants are similar to the restricted repertoire described in adults. Low affinity anti-Hib PS mAbs of infants are encoded by a heterogeneous group of genes that are uncommonly observed in the adult repertoire. Abs with high affinity for Hib PS from infants, like most mAbs from adults, react only with Hib PS and the structurally similar PS of Escherichia coli K100, whereas low affinity mAbs of infants are polyreactive. The low affinity anti-Hib PS mAbs of infants immunized with Hib oligosaccharide-diphtheria toxin vaccine vaccine are not reflected in serum Ab. However, the differences between the variable region gene repertoires of adults and infants may account for the distinct immunologic characteristics of the anti-Hib PS responses in young children immunized with other vaccine formulations.

Molecular heterogeneity in deficiency of complement protein C2 type I.

Deficiency of the complement protein C2 (C2D), one of the most common genetic deficiencies of the complement system, is associated with rheumatological disorders and increased susceptibility to infection. Two types of C2D have been recognized, each in the context of specific major histocompatibility complex (MHC) haplotypes; type I, a deletion, frameshift and premature stop codon resulting in absence of detectable C2 protein synthesis, and type II, missense mutations resulting in a block in secretion of C2 proteins. Analysis of C2 expression in a child with C2 deficiency, a MHC haplotype different from those associated with type I or II C2D, and recurrent infections revealed additional molecular heterogeneity among C2 deficient patients. No detectable C2 protein was synthesized in the child's fibroblasts under conditions supporting C2 synthesis and secretion in normals and the child's C2 mRNA was reduced to 42% of normal. Nucleotide sequencing of RT-PCR fibroblast mRNA and genomic DNA revealed a type I C2 deficiency (28 base-pair deletion) on one allele and a previously unrecognized two base-pair deletion in exon 2 on the other. Expression of the closely linked factor B gene was markedly decreased (Bf mRNA 25% of normal), though Bf was up-regulated appropriately by interferon-gamma and the flanking sequence containing the Bf promoter was normal in this C2-deficient patient. Moreover, the concentration of Bf protein was normal in the patient's plasma.

Autoantibody germ-line gene segment encodes VH and VL regions of a human anti-streptococcal monoclonal antibody recognizing streptococcal M protein and human cardiac myosin epitopes.

Cross-reactivity of anti-streptococcal Abs with human cardiac myosin may result in sequelae following group A streptococcal infections. Molecular mimicry between group A streptococcal M protein and cardiac myosin may be the basis for the immunologic cross-reactivity. In this study, a cross-reactive human anti-streptococcal/anti-myosin mAb (10.2.3) was characterized, and the myosin epitopes were recognized by the Ab identified. mAb 10.2.3 reacted with four peptides from the light meromyosin (LMM) tail fragment of human cardiac myosin, including LMM-10 (1411-1428), LMM-23 (1580-1597), LMM-27 (1632-1649), and LMM-30 (1671-1687). Only LMM-30 inhibited binding of mAb 10.2.3 to streptococcal M protein and human cardiac myosin. Human mAb 10.2.3 labeled cytoskeletal structures within rat heart cells in indirect immunofluorescence, and reacted with group A streptococci expressing various M protein serotypes, PepM5, and recombinant M protein. The nucleotide sequence of gene segments encoding the Ig heavy and light chain V region of mAb 10.2.3 was determined. The light chain V segment was encoded by a V kappa 1 gene segment that was 98.5% identical with germ-line gene humig kappa Vi5. The V segment of the heavy chain was encoded by a VH3a gene segment that differed from the VH26 germ-line gene by a single base change. VH26 is expressed preferentially in early development and encodes autoantibodies with anti-DNA and rheumatoid factor specificities. Anti-streptococcal mAb 10.2.3 is an autoantibody encoded by VH and VL genes, with little or no somatic mutation.

IgG subclasses: importance in pediatric practice.

Although many clinical studies provide evidence of a relation between IgG subclass deficiency and increased susceptibility to infection, a direct cause-and-effect relationship clearly does not exist. Each patient must be managed in the full context of clinical symptomatology, reliable measurement of IgG subclasses, and evaluation of specific antibody production. Future studies may clarify the factors that stimulate and regulate the production of IgG subclasses and improve our understanding of their role in the health of children.

The human VH3b gene subfamily is highly polymorphic.

We have previously shown that human antibody (Ab) directed against the capsular polysaccharide of the important bacterial pathogen, Haemophilus influenzae type b (Hib) is encoded by a small group of VH3 gene family members. The majority of anti-Hib PS Ab use members of the smaller VH3b subfamily. To examine directly the available human VH3 repertoire, we have used PCR to amplify and clone candidate germ-line VH3b H chain V region genes from two unrelated subjects from whom anti-Hib polysaccharide mAb had been previously obtained. A single functional VH3b germ-line gene was obtained from one subject. This gene is identical throughout the coding region to the previously identified gene 9.1. Twelve distinct VH3b germ-line sequences, 87.6-99.8% homologous to one another, were obtained from the second subject. One of these genes, LSG1.1, is also identical to the 9.1 germ-line gene, and a second, LSG6.1, is identical to a previously reported cDNA, M85. These germ-line VH3b genes are 82.7-94.1% homologous to rearranged anti-Hib PS VH3b segments obtained from these subjects. Our findings further demonstrate that considerable polymorphism of VH segments exists in the human population. Despite the presence of very highly homologous VH elements in the germ line, particular genes are highly conserved within the outbred human population.

Restricted immunoglobulin VH usage and VDJ combinations in the human response to Haemophilus influenzae type b capsular polysaccharide. Nucleotide sequences of monospecific anti-Haemophilus antibodies and polyspecific antibodies cross-reacting with self antigens.

To examine the human antibody repertoire generated against a biologically significant antigen we have obtained sequences of heavy chain variable region genes (IgVH) from 15 monoclonal antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b (Hib PS). All VH segments are members of the VH3 family and 9 of 15 are members of the smaller VH3b subfamily. Restriction is evident by the shared use of certain VDJ joints in independent hybridomas from different subjects. Two hybridomas generated from the same subject demonstrate identical heavy chain variable region gene sequences but differ in isotype and rearrange alternative light chain variable region genes (IgVL), suggesting that in a normal immune response, a single pre-B cell clone may use different light chain rearrangements and give rise to progeny capable of reacting with antigen. Using a polymerase chain reaction assay optimized to detect base pair differences among VH genes we demonstrate that at least a portion of expressed anti-Hib PS VH genes have undergone somatic mutation. Anti-Hib PS heavy chain genes are homologous to VH segments encoding autoantibodies and two hybridomas secrete anti-Hib PS antibody that cross-reacts with self antigens (double-stranded DNA and single-stranded DNA). Comparison of VH regions of self-reactive and monospecific anti-Hib PS Ab demonstrates no consistent structural feature correlating with fine antigen specificity. These data demonstrate significant restriction in VH usage and VDJ recombination in the anti-Hib PS response and confirm that autoantibodies may be elicited during normal immune responses.

Diversity of immunoglobulin light chain usage in the human immune response to Haemophilus influenzae type b capsular polysaccharide.

The response to the capsular polysaccharide of Haemophilus influenzae type b (Hib PS) has been used to determine the molecular basis of antibody gene diversity in humans. In contrast to the relatively restricted nature of anti-Hib PS heavy-chain variable region gene expression, a variety of light-chain variable region genes may encode this antibody (Ab) response. Light-chain variable region gene usage appears to determine the expression of certain Ab idiotypes and fine antigen specificity. To further define the role of light-chain variable region gene usage in important anti-Hib PS Ab subgroups, we have cloned and sequenced a number of immunoglobulin light-chain variable region genes (IgVL) from human monoclonal IgA anti-Hib PS Ab generated in response to Hib PS-protein conjugate vaccines. Three of these Ab are encoded by unusual variable segments. One kappa-Ab is encoded by the "predominant" V kappa II A2 germline gene but, in contrast to a previously reported A2-encoded IgVL sequence, differs from the A2 germline sequence. The IgVL sequence of a second Ab is the only sequence of a kappa-Ab that cross-reacts with the structurally related antigen Escherichia coli K100 polysaccharide reported to date. This IgVL is encoded by a V kappa III-segment most closely homologous to the Humhv328/L16 germline gene, whereas previous reports suggested V kappa III-encoded anti-Hib PS Ab might be exclusively encoded by the germline gene Humhv325/A27.(ABSTRACT TRUNCATED AT 250 WORDS)

Variable region expression in the antibody responses of infants vaccinated with Haemophilus influenzae type b polysaccharide-protein conjugates. Description of a new lambda light chain-associated idiotype and the relation between idiotype expression, avidity, and vaccine formulation. The Collaborative Vaccine Study Group.

Haemophilus influenzae b polysaccharide (Hib PS)-protein conjugate vaccines differ chemically and immunologically. To determine whether anti-Hib PS variable region expression might differ according to vaccine formulation, infants were vaccinated at 2, 4, and 6 mo of age with Hib PS coupled to either meningococcal outer membrane protein complex (Hib PS-OMPC) or tetanus toxoid (Hib PS-T), or Hib PS oligomers coupled to a mutant diphtheria toxin (Oligo-CRM). Two anti-Hib PS idiotypes were measured in sera obtained after the third injection: HibId-1, expressed by anti-Hib PS antibodies having the kappa II-A2 variable region, and HibId-2, a newly defined cross-reactive idiotype associated with a subset of anti-Hib PS antibodies having lambda VII variable regions. HibId-1 was present in 33, 68, and 64% of infants given either Hib PS-OMPC, Oligo-CRM, or Hib PS-T, respectively (P < 0.001). The respective values for HibId-2 were 47, 18, and 10% (P = 0.001). Subjects who were vaccinated with Hib PS-OMPC or Hib PS-T and who produced detectable HibId-1-positive antibody, had significantly higher mean antibody avidity than subjects who did not produce HibId-1 positive antibodies. In contrast, Oligo-CRM evoked high avidity anti-Hib PS antibodies, irrespective of the idiotypic profile. These findings indicate fundamental differences in both variable region content and antibody quality elicited by different Hib PS conjugate vaccines.

Immunoglobulin variable region gene expression in response to Haemophilus influenzae type b polysaccharide.

The mechanism(s) responsible for the ontogenic patterns of acquisition of the antibody repertoire is unknown. The immune response to Haemophilus influenzae type b (Hib) capsular polysaccharide provides an excellent model system in which to examine the ontogeny of immunoglobulin variable region expression. A panel of hybridomas secreting human antibodies specific for Hib capsular polysaccharide was developed using peripheral blood lymphocytes from donors immunized with Hib vaccines. Nucleotide sequence analysis of the heavy chain V regions expressed by four of these hybridomas suggests selective use of members of the VHIII gene family in combination with different D and J segments. The nucleotide sequences were highly homologous to two candidate germline gene sequences. Others have reported that these particular germline sequences are expressed in fetal liver, suggesting that the inability of young children to produce antibody to the Hib capsular polysaccharide is not due to failure to express these VH regions early in ontogeny.

Immunoglobulin light chain variable region gene sequences for human antibodies to Haemophilus influenzae type b capsular polysaccharide are dominated by a limited number of V kappa and V lambda segments and VJ combinations.

The immune repertoire to Haemophilus influenzae type b capsular polysaccharide (Hib PS) appears to be dominated by certain light chain variable region genes (IgVL). In order to examine the molecular basis underlying light chain bias, IgVL genes have been cloned from a panel of heterohybridomas secreting human anti-Hib PS (antibody) (anti-Hib PS Ab). One hybridoma, representative of the predominant serum clonotype of anti-Hib PS Ab in older children and adults following immunization or Hib infection, uses a V kappa II segment identical to the germline gene A2, and a JK3 segment. A second kappa hybridoma uses a member of the V kappa I family and a JK4 segment. Four lambda antibodies, all cross-reactive with the structurally related antigen Escherichia coli K100 PS, use V lambda VII segments which are 96-98% homologous to one another, and may originate from a single germline gene. Two additional lambda antibodies, not K100-cross-reactive, are encoded by members of the V lambda II family. All lambda antibodies use highly homologous J lambda 2 or J lambda 3 segments. The VJ joints of all lambda antibodies and the V kappa II-encoded antibody are notable for the presence of an arginine codon, suggesting an important role in antigen binding. Although more complex than heavy chain variable region gene usage, a significant portion of serum anti-Hib PS Ab is likely to be encoded by a limited number of V kappa and V lambda segments and VJ combinations, which may be selectively expressed during development, or following antigen exposure.

Restricted Ig H chain V gene usage in the human antibody response to Haemophilus influenzae type b capsular polysaccharide.

The mechanisms that govern the content of the human antibody repertoire are poorly understood. To investigate the antibody response to a clinically relevant Ag, we have produced heterohybridomas secreting human antibodies directed against the capsular polysaccharide of Haemophilus influenzae type b (Hib PS). Immune lymphocytes were harvested 7 days after immunization with either of two vaccine formulations, a plain polysaccharide vaccine (Hib PS) or a polysaccharide-protein conjugate of Hib PS and diphtheria toxoid (Hib PS-D). H chain V region gene nucleic acid sequences were determined for five stable hybridomas. All use members of the VHIII gene family and are 83% to 98% homologous to two candidate germ-line sequences. A variety of D and JH segments are used. Thus the Ig H chain repertoire appears to be restricted to a limited group of VHIII family members. The previously reported expression of homologous sequences in the human fetal repertoire suggests that the inability of young children to respond to this Ag is not caused by deficiencies of these important elements early in development. The restricted use of VHIII gene segments suggests that this gene family plays a pivotal role in the immune response to this important childhood pathogen.

Subnormal serum concentrations of IgG2 in children with frequent infections associated with varied patterns of immunologic dysfunction.

To characterize more fully the immunologic basis for increased susceptibility to infection in patients with low serum concentrations of IgG2, we identified eight infection-prone children, 1 to 2 years of age, with serum IgG2 concentrations greater than 2 SD below the mean for age and followed their serologic and clinical courses for 1 to 3 years. Two of the eight children became clinically and immunologically normal and may have had transient IgG2 deficiency with an exaggerated developmental delay of this late-maturing subclass. The remaining six subjects had persistently subnormal or low-normal serum IgG2 levels and continued to experience frequent infections. All six of these children responded poorly to Haemophilus influenzae type b (Hib) polysaccharide, and four of six responded poorly to Streptococcus pneumoniae type 3 polysaccharide. Both IgG1 and IgG2-specific antibody responses to these vaccines were abnormal. Three of these six children also responded poorly to tetanus toxoid, an antigen that normally induces a predominant IgG1 response. Although five of these six children produced antibodies in response to Hib polysaccharide protein conjugate vaccine, three of four given Hib oligosaccharide CRM conjugate vaccine required booster doses to respond, a pattern of response characteristic of infants less than 6 months of age. Further, although serum concentrations of IgG1 were normal, peripheral blood mononuclear cells from four of six children tested produced extremely small amounts of IgG1 and IgG3 as well as IgG2. Finally, varied patterns of abnormalities of IgG, IgA, IgM, and IgG4 became apparent in five of the six children with persistently low serum IgG2 values. This study demonstrates that subnormal serum concentrations of IgG2 may be associated with varied patterns of immunologic dysfunction, some of which are evolving and may be responsible for increased susceptibility of these children to infection.

Clinical and immunologic characteristics of healthy children with subnormal serum concentrations of IgG2.

To understand the relevance of subnormal serum concentrations of IgG2, we measured IgG2 in serum of 575 healthy children and identified 11 with concentrations greater than 2 SD less than the mean for age. The levels of IgG2 present were similar to those found in symptomatic children with IgG2 subclass deficiency associated with antibody deficiency. The 11 children ranged in age from 1 to 14 y (mean = 5.7). Detailed clinical information was available on 10 of the 11 children and each was matched for age with two controls. The median number of visits/y to the doctor for infectious illnesses was identical for the two groups (1.0). Nine of the children with subnormal IgG2 were followed for 1 to 5 y (mean = 2.3). All nine children had normal serum concentrations of IgA, IgG1, IgG3, and IgG4 but seven had persistently subnormal or low-normal serum IgG2 concentrations. One of these seven children also had a subnormal serum concentration of IgG, and one had subnormal IgM. Antibody responses to Haemophilus b polysaccharide vaccine were normal in five of six who were immunized. In vitro secretion of Ig by mitogen-stimulated peripheral blood mononuclear cells was measured in six of seven children with persistently subnormal or low-normal IgG2; five showed decreased secretion of IgG2, and two of the five also had subnormal secretion of IgG1 and IgG3. An important implication of this study is that the subnormal concentrations of serum IgG2 found in infection-prone children are not a sufficient explanation for their increased susceptibility to infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the molecular mechanisms of human Fc receptor-mediated phagocytosis. Amplification of ingestion is dependent on the generation of reactive oxygen metabolites and is deficient in polymorphonuclear leukocytes from patients with chronic granulomatous disease.

Human PMN and monocytes both possess a mechanism for amplifying Fc receptor-mediated phagocytic function, which is dependent on activation of the respiratory burst. The pathway for augmentation of phagocytosis requires superoxide anion, hydrogen peroxide, and lactoferrin and is independent of the hydrogen peroxide-MPO-halide system. In neither cell type is this mechanism induced upon exposure to the opsonized target. PMN require an additional signal for stimulation of the respiratory burst; this is not true of monocytes. On the other hand, monocytes require an exogenous source of lactoferrin in order to activate this pathway for enhanced ingestion. The dependence of this pathway for both PMN and monocytes on superoxide anion, hydrogen peroxide, and cell-bound lactoferrin is consistent with a role for locally generated reactive oxygen metabolites, possibly hydroxyl radicals, in phagocytosis amplification. Patients with chronic granulomatous disease, who are genetically deficient in the ability to activate the respiratory burst, are unable to amplify Fc receptor-mediated phagocytosis. Thus, these patients may have a previously unrecognized defect in the recruitment of phagocytic function at inflammatory sites.

IgG subclass response to immunization with Haemophilus influenzae type b polysaccharide-outer membrane protein conjugate vaccine.

We immunized 117 children with either Haemophilus influenzae type b polysaccharide vaccine or type b polysaccharide coupled to an outer membrane protein of group B Neisseria meningitidis (conjugate vaccine), and measured the IgG, IgG1, and IgG2 subclass composition of antibody to type b polysaccharide in postimmunization sera by ELISA. The IgG responses of 51 children, 24-83 months of age, immunized for the first time with the conventional type b vaccine consisted of both IgG1 and IgG2 antibody (respective geometric means of 2.24 and 0.77 micrograms/ml). In contrast, the IgG responses of 28 infants, 2-17 months of age, immunized with conjugate vaccine were predominantly or exclusively IgG1 (genometric mean IgG1 and IgG2 antibody concentrations of 1.92 and 0.19 micrograms/ml). A total of 38 children was primed with conjugate vaccine between 2 and 17 months of age and boosted approximately 1 yr later. The 28 children boosted with type b polysaccharide vaccine showed memory antibody responses consisting of both IgG1 and IgG2 (respective geometric means of 12.7 and 4.8 micrograms/ml); the 10 children boosted with conjugate vaccine showed a similar pattern of IgG subclass responses (respective geometric means of 20.8 and 5.1 micrograms/ml, p greater than 0.4 compared to the respective geometric mean IgG1 and IgG2 values of the group boosted with polysaccharide). Thus, in children 24-83 months of age, immunization with conventional type b polysaccharide vaccine generally elicits both IgG1 and IgG2 responses, with a slight predominance of IgG1.(ABSTRACT TRUNCATED AT 250 WORDS)