Oligosaccharide-protein conjugate vaccines induce and prime for oligoclonal IgG antibody responses to the Haemophilus influenzae b capsular polysaccharide in human infants.

The diversity of the IgG antibody induced by immunization of human infants and children with conjugate vaccines, composed of oligosaccharides prepared from the Haemophilus influenzae b capsular polysaccharide (CP) and covalently linked to diphtheria toxoids, was studied by analytical IEF. The antibody response was similar, in the degree of restriction, to that observed in the antibody response of older children to immunization with the CP alone. The booster responses induced by reimmunization with conjugate vaccines were accompanied by increases predominantly in the IgG antibody clonotypes expressed after the priming dose of vaccine. After a series of conjugate immunizations, immunization with isolated CP boosted the antibody titer and increased expression from all the clonotypes that were expressed after conjugate immunization. These findings suggest that the conjugate vaccines are acting on a limited number of human B cell clones that are preferentially restimulated after reimmunization. Little evidence of antigen-specific B cell recruitment was found. In addition, the ability of isolated CP immunization to restimulate the same B cell clone indicates that the responding B cell has matured and suggests a linear rather than a dual developmental pathway for the B cell participating in this human antibody response.

Protective human hybridoma antibody to tetanus toxin.

Postimmunization human B lymphocytes and mouse myeloma cells were fused to produce interspecies hybridomas secreting human antibody of predefined specificity with an initial frequency comparable to intraspecies fusion. After 13 mo in culture, one clone continued to secrete high titers of human IgG antitetanus toxin antibody. This antibody binds to the B fragment of tetanus toxin and protects mice against tetanus. The demonstration of in vivo protection with a human monoclonal antibody is an important first step towards the ultimate goal of human administration of monoclonal antibodies for the prevention and therapy of human infections.

Isolation of germinal centerlike events from human spleen RNA. Somatic hypermutation of a clonally related VH6DJH rearrangement expressed with IgM, IgG, and IgA.

12 rearranged human VH6 immunoglobulin heavy chain genes arising from the same rearrangement were isolated without preselection from the RNA of a fragment of human spleen. The 12 clones were isolated from a pool of 31 unique VH6 clones arising from 18 unique rearrangements. 2 of the 12 related clones were expressed with IgM, 2 with IgG, and 8 with IgA1. All the clones, including those expressing IgM, showed extensive somatic mutation of germline bases (5.6%), which was consistent with antigen-driven activation of these VH6-expressing clones with recruitment into the immune repertoire. On the basis of significant sharing of somatic mutations between the IgM clones and clones expressing the other isotypes (six mutations shared with IgG clones and eight mutations shared with IgA clones), it was apparent that the IgM-expressing precursor in this diversified family had undergone extensive antigen-driven somatic mutation prior to isotype switching. This family of related clones suggests that a germinal centerlike event had been sampled. The highly mutated IgM clones suggest that there may exist memory B cells capable of further somatic mutation and differential isotype-switching depending on the specific antigenic stimulus.

A mucosal antibody response following systemic Haemophilus influenzae type B infection in children.

The possibility that mucosal antibody is produced as a host response to Haemophilus influenzae type b (Hib) infection was examined in this study. 17 of 18 prospectively evaluated children ranging in age from 2 mo to 7 yr developed a detectable level of anticapsular antibody in their nasopharyngeal secretions after systemic Hib infection. The mean concentration of nasal anti-capsular antibody of the 18 children was 554 ng/mg IgA (SD = 35-8,863) during the acute phase of illness and declined to 224 ng/mg IgA (SD = 19-2,688) in convalescence. Some children had mucosal antibody detectable at least 10 mo after infection. The mucosal antibody levels were not affected by the length of illness before diagnosis, type of disease, age of the patient, sex, or presence of detectable capsular antigen or viable bacteria in the nasopharynx. The mucosal antibody was predominantly of the IgA class and occurred independent of the serum antibody. Six of the children aged less than 1 yr who did not produce and/or sustain a serum antibody level correlated with protection demonstrated a persistent mucosal antibody response. These findings suggest that the mucosal immune system may have the ability to respond at an earlier age than the serum immune system and lead us to postulate that protective secretory antibodies to prevent systemic Hib disease may be inducible in young infants in spite of the poor serum antibody response occurring at this age.

Immunogens consisting of oligosaccharides from the capsule of Haemophilus influenzae type b coupled to diphtheria toxoid or the toxin protein CRM197.

Haemophilus influenzae type b (Hib) capsular polysaccharide (PRP) was selectively hydrolyzed to reducing oligosaccharides, and the fraction containing 3-10 ribosylribitolphosphate repeating units (VS) was conjugated by reductive amination to diphtheria toxin (DTx), its nontoxic derivative CRM197 (Dcr), or diphtheria toxoid (DTd). Conjugate DTx-VS retained approximately 1% of native toxicity, which was eliminated by treatment with formalin. Immunization of rabbits with the conjugates elicited antibody (Ab) to PRP and to DTx but not to a model for the linkage determinant. Human adults given single subcutaneous injections had rises in serum Ab to PRP and in bactericidal activity in vitro; the Ab protected infant rats challenged with Hib. Adults had rises also in Ab to DTd, and these Ab protected rabbits against DTx. A series of two injections of the conjugates Dcr-VS and DTd-VS was tested in infants beginning at 19-23 mo of age. Rises in anti-PRP Ab after the primary resembled the rises after PRP vaccine. In contrast to PRP, the conjugates elicited large rises after the secondary vaccinations and a substantial IgG component. Development of bactericidal activity paralleled the rises in anti-PRP Ab. Secondary rises after Dcr-VS were higher than after DTd-VS. In infants 12-16 mo of age, Dcr-VS (but not DTd-VS) elicited strong primary and secondary Ab responses that included IgG and bactericidal activity. Both conjugates produced consistent rises in Ab to DTd.

Somatic mutation of human immunoglobulin V genes in the X-linked HyperIgM syndrome.

Somatic mutation of Ig variable regions occurs prominently in germinal centers, but it has been debated whether the mutation process initiates in germinal centers or is activated before germinal center entry of B cells. We have analyzed for the presence of somatic mutation in Ig gene rearrangements of the nonpolymorphic human VH6 gene in the X-linked HyperIgM syndrome, which is associated with defective CD40 ligand expression and absence of germinal centers and generation of memory B lymphocytes. IgM and rare IgG VH6 productive rearrangements were isolated from PBL of patients with X-linked HyperIgM syndrome. Although the majority of both the IgM and IgG VH6 rearrangements had a germline VH6 sequence, 7 of 102 VH6 IgM and 1 of 6 IgG rearrangements had a mutated VH6 gene. The mutation frequency (mutations/bp) was 1.4% with a range of 2-9 mutations per clone, a mutation frequency lower, however, than that observed in IgM (3.2%) and IgG (5.4%) VH6 rearrangements of normal individuals. These results suggest that somatic mutation may be initiated in a CD40 ligand-independent pathway before entry of B cells into germinal centers, but fails to achieve the high mutation frequency observed in the presence of germinal centers.

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 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.

Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance.

The study of human B cell tolerance has been hampered by difficulties in identifying a sizable population of autoreactive B lymphocytes whose fate could be readily determined. Hypothesizing that B cells expressing intrinsically autoreactive antibodies encoded by the VH4-34 heavy chain gene (VH4-34 cells) represent such a population, we tracked VH4-34 cells in healthy individuals. Here, we show that naive VH4-34 cells are positively selected and mostly restricted to the follicular mantle zone. Subsequently, these cells are largely excluded from the germinal centers and underrepresented in the memory compartment. In healthy donors but not in patients with systemic lupus erythematosus (SLE), these cells are prevented from differentiating into antibody-producing plasma cells. This blockade can be overcome ex vivo using cultures of naive and memory VH4-34 cells in the presence of CD70, IL-2, and IL-10. VH4-34 cells may therefore represent an experimentally useful surrogate for autoantibody transgenes and should prove valuable in studying human B cell tolerance in a physiological, polyclonal environment. Our initial results suggest that both positive and negative selection processes participate in the maintenance of tolerance in autoreactive human B cells at multiple checkpoints throughout B cell differentiation and that at least some censoring mechanisms are faulty in SLE.

Human splenic IgM immunoglobulin transcripts are mutated at high frequency.

Human spleen immunoglobulin gene rearrangements that used the VH6 gene and were expressed with IgM were characterized for their frequency of somatic hypermutation from PCR amplified spleen cDNA. A high frequency of rearrangements that were somatically mutated was demonstrated by restriction endonuclease analysis and sequencing of cloned rearrangements. The 24 rearrangements cloned from three different spleens had an overall mutation frequency of 3.1% mutations/bp sequenced and ranged from 0.4 to 6.0%. These mutations appeared to have been antigenically selected based on both the high frequency and high amino acid replacement to silent (R/S) ratios in the complementarity determining regions. Five clones that arose from two different rearrangements showed evidence of intraclonal diversification with both shared and unique mutations. The mutated clones of one spleen donor were lower in frequency and were not concentrated in the CDR, which suggested these mutations had not been antigenically selected. These findings support the dissociation of somatic mutation and isotype switching and the possibility that IgM-expressing B cells may serve as human memory B cells.

Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens.

A combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to identify phage clones capable of binding to the surface of Candida albicans blastoconidia. Single chain antibody variable fragments (scFv) derived from three clones detected C. albicans antigens by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blotting. The antigens detected were conserved among different strains of C. albicans and several other Candida species. Two scFv clones detected antigens specifically expressed by C. albicans blastoconidia; the third detected antigens in both blastoconidia and filamentous forms of C. albicans. The antigens containing the epitopes recognized by all three scFv could be extracted from blastoconidia by dithiothreitol, suggesting attachment to the cell wall via sulfhydryl bonds. Epitope detection by the scFv was sensitive to treatment of C. albicans blastoconidia with sodium periodate, but not proteinase K, indicating the cognate epitopes were composed of carbohydrate. Antigenic determinants for each of the three scFv were detected by immunohistochemical staining of skin sections from a model of cutaneous candidiasis, demonstrating expression in vivo. Through selection for the ability to bind intact organisms, the phage display system provides a means to rapidly identify monoclonal binding ligands to Candida surface antigens. Being entirely human, mature antibodies generated from the scFv have potential utility in the treatment of candidiasis.

Autologous mixed lymphocyte reaction in human neonatal lymphocytes.

Human neonatal mononuclear cells were examined to determine their ability to participate in an autologous mixed lymphocyte reaction (AMLR). Stimulator cells were isolated by plastic adherence and nylon wool adherence. The nylon wool-nonadherent cells were used as responder cells. In 10 of 10 neonatal samples and 6 of 7 adult samples, a significant AMLR was present when plastic-adherent cells were used as stimulators. Neonatal blood showed a mean increase in proliferation of 7.6 (3.6-14.9), while adult cultures showed a mean stimulation index of 11.8 (1.0-39.0). When nylon wool-adherent cells were used as stimulator cells, only 2 of 7 neonatal blood samples and 1 of 5 adult blood samples showed a significant AMLR. When recombinant interleukin 2 (IL-2) was added to AMLR cultures of plastic-adherent cells and nylon wool-nonadherent cells, a mean augmentation of 12.0 was seen in the neonatal AMLR, while the adult cultures were augmented by a mean response of 4.1. Addition of IL-2 to nylon wool-nonadherent cells alone produced a 5.9-fold increase in adult cells, while neonatal cells showed an 85.8-fold mean increase in proliferation. The results suggest that autoreactive T cells are present in neonatal blood and that these cells can be activated by plastic-adherent autologous cells. However, neonatal and adult nylon wool-adherent cells do not consistently activate autoreactive T cells.

The repertoire of human antibody to the Haemophilus influenzae type b capsular polysaccharide.

Human antibody to the Haemophilus influenzae capsular polysaccharide (Hib CP) is restricted in diversity in the individual and the population with a limited number of variable region genes encoding antibody. Antibody to the Hib CP shows restricted isoelectric focusing gel patterns and light chain usage with frequent restriction to use of only kappa light chains. Shared cross-reactive idiotypes are expressed on antibody. The heavy chain of antibody to the Hib CP is predominantly encoded by two members of the VH3 family--LSG 6.1/M85-like and VH26/30P1-like. In VH the CDR1, based on complete identity in LSG 6.1/M85-like antibodies, CDR2, based on the suggestion of mutation in this region, and CDR3, based on conserved CDR3 usage in unrelated individuals, may be important for antigen binding. Six or more different VL gene families encode antibody. The predominant antibody of the majority of individuals uses the A2-V kappa II gene in germline or near germline configuration, which encodes an idiotype designated HibId-1. Antibody can also be encoded by V kappa I, non-A2 V kappa II, V kappa III, V kappa IV, V lambda II, and V lambda VII genes. Although different VL genes can be used, unrelated individuals appear to use the same V kappa III (A27), V lambda II (V lambda 2.1 and V lambda VII (4A) genes. The VL diversity accounts for differences in fine binding specificity, with A2-V kappa II genes not encoding E. coli K100 CP cross-reactive antibodies and V lambda VII genes and some of the non-A2 V kappa genes encoding cross-reactive antibodies. The arginine in CDR3 of both antibody kappa and lambda light chains and the asparagine in CDR2 of VL sequences and in CDR1 of LSG6.1-M85 VH sequences of antibody appear to be important residues for antigen binding. A relatively limited degree of somatic mutation has occurred in the non-A2 VL genes, V lambda VII, and the VH genes. Further studies comparing the polymorphism of germline V genes to antibody-encoding V genes are needed to clarify this issue. Research comparing this repertoire to repertoires directed to other bacterial CP and to self antigens and defining structure-antigen binding relationships is in progress.

Neonatal passive immunization by maternal vaccination.

An old concept--passively immunizing a fetus by actively immunizing (vaccinating) its mother--is reevaluated in light of 50 years of data. The history and data reviewed here suggest that this concept is one whose time has come for active modern research and clinical use. In Third World countries, this concept already has provided significant reduction in morbidity and mortality from neonatal infections such as tetanus. Some other neonatal and infant infections--heretofore life-threatening--may now have a practical method for prevention. These include group B beta-streptococcal sepsis and Hemophilus influenzae meningitis.

Suppression of tumorigenicity in human ovarian carcinoma cell line SKOV-3 by microcell-mediated transfer of chromosome 11.

To determine the pathogenic role of chromosomes 11 and 17 in the carcinogenesis of human ovarian cancers, neo(R)-tagged chromosome 11 or 17 was transferred from cell lines A9H11 or A9H17, respectively, into the ovarian carcinoma cell line SKOV-3 using microcell-mediated chromosome transfer. The chromosome transfer was verified by polymerase chain reaction detection of the neo(R) gene, fluorescence in situ hybridization detection of an extra chromosome 11, and microsatellite polymorphism detection of an exogeneous chromosome 11. Five SKOV-3/A9H11 hybrids and five SKOV-3/A9H17 hybrid clones were generated. For the chromosome 11 transfer, complete suppression of tumorigenicity was observed in four clones, (11)9-8 and 11(H)7-2, 11(H)8-3, and 11(H)7-2, 100 days post implantation. For the chromosome 17 transfer, no complete suppression of tumorigenicity was observed. However, an increased latency period ranging from 25 to 49 days in contrast to 7 days for the SKOV-3 parental line, and a significant reduction in tumor size was observed. There was no correlation between the in vitro growth rate and the tumorigenicity or length of latency period. Our results demonstrate functionally that chromosome 11 may carry a tumor suppressor gene(s) while chromosome 17 may carry a tumor growth-inhibitor gene(s) for the ovarian carcinoma cell line, SKOV-3.

Oncogene elements within an endogenous retrovirus.

The human genome contains a large number of endogenous retroviral-related sequences. While the function of these sequences is unknown, they may contribute to disease processes through their regions of homology with infectious retroviruses. We have been further characterizing a recently reported HTLV-1 related endogenous retroviral sequence cloned from T lymphocytes isolated from a patient with essential cryoglobulinemia. We here report further detailed transcriptional analysis of the sequence for tissue and cell-cycle specificity and a novel finding of an association between the endogenous retrovirus and a ras-related gene.