Subclass distribution of human antibodies to Haemophilus influenzae type b capsular polysaccharide

The polysaccharide (PS) capsule of Haemophilus influenzae type b (Hib) is a "simple" antigen, polyribosylribitolphosphate. Although similar carbohydrate antigens have been reported to elicit IgG antibodies relatively restricted to the IgG2 subclass in man, we report here that Hib PS elicits substantial quantities of both IgG1 and IgG2 serum antibodies in most individuals. Because the determination of IgG subclass distribution can be technically difficult, we used four different approaches to establish our finding. First, we used an IgG subclass-specific, antigen-specific "sandwich assay." Second, we measured IgG subclasses of purified antibodies to Hib PS. Third, we showed that significant amounts of IgG anti-PS can be absorbed with a monoclonal anti-IgG1 affinity column. Fourth, we showed that IgG1 and IgG2 fractions of immune sera have clonally restricted anti-Hib PS antibodies that are easily distinguishable by their isoelectric points. The data indicate that both IgG1 and IgG2 contribute substantially to the IgG antibody response of most adults to immunization with Hib PS.     

In vitro human antibody production to the Haemophilus influenzae type b capsular polysaccharide

In vitro production of human antibody to the Haemophilus influenzae type b capsular polysaccharide (PRP) and to tetanus toxoid (TT) and diphtheria toxoid was measured in culture supernatants of peripheral blood mononuclear cells and by enumeration of antibody secreting cells (AbSC) in an enzyme-linked immunosorbent-plaquing assay. Normal adult peripheral blood mononuclear cells stimulated with Epstein-Barr virus secreted anti-PRP antibody with a frequency of 1/552 to 1/1190 relative to total Ig secreting cells; the frequency of AbSC to tetanus toxoid (TT) was 7.5 times higher (p less than 0.05). These frequencies did not change significantly after in vivo immunization, although the isotype distribution shifted toward increased IgG for TT and increased IgG and IgA for PRP. At 8 days postimmunization, spontaneous AbSC to PRP and TT were detected; frequencies for total anti-TT AbSC again being higher than anti-PRP, but there were significantly more IgA plaques among anti-PRP AbSC. Spontaneous AbSC were suppressed in culture by pokeweed mitogen and enhanced by cyclosporine. Three wk after in vivo immunization with PRP and TT, in vitro stimulation with pokeweed mitogen, Staphylococcus aureus Cowan 1 bacteria, or antigen induced anti-TT but not anti-PRP in vitro antibody secretion, although Epstein-Barr virus induced both. These data suggest that PRP, a polysaccharide, and TT, a protein, differ in their requirements for in vitro activation with antigen and mitogens.     

Comparative evaluation of conjugate vaccines in the Haemophilus influenzae infection model

We used a murine model of Haemophilus influenzae type b (Hib) infection to analyze the immunologic response to two commercially available PRP conjugate vaccines (HbOC, PRP-T). The mortality rate in mice infected with a large dose of the bacteria after vaccination with HbOC or PRP-T at two and three doses was significantly lower than in non-vaccinated mice and mice vaccinated by one dose. Furthermore, for infections caused by a small bacterial dose, the mortality rate in mice vaccinated with one, two, or three doses was significantly lower than in non-vaccinated mice. The induction level of anti-PRP antibodies, especially IgG, in serum of mice vaccinated by two or three doses was higher than in those vaccinated with a single dose. Our results indicate that the dose of vaccine influences its efficacy in protecting against Hib infection. Our results also showed a lack of difference between two different PRP conjugate vaccines.     

In vitro induction of a Haemophilus influenzae type b polysaccharide-specific antibody response in human peripheral blood lymphocytes of individuals recently vaccinated with an oligosaccharide-protein conjugate.

In this paper an in vitro culture system for the induction of an antibody response to the Haemophilus influenzae type b polysaccharide (PRP) is described. Anti-PRP IgM and IgG antibody-secreting cells (ASC) and anti-diphtheria toxoid (DT) IgG ASC were detected in cultures of blood B and T cells derived from donors 4 to 6 wk after immunization with Haemophilus influenzae type b oligosaccharide-mutant diphtheria toxin (CRM197) conjugate (HbOC) and required in vitro restimulation with HbOC. When lymphocytes from HbOC-vaccinated donors were stimulated with PRP, anti-PRP IgM and IgG ASC could be detected in 50% offGe cases. Lymphocytes from PRP-vaccinated donors or non-vaccinated donors consistently failed to generate anti-PRP antibodies after in vitro stimulation with HbOC. Optimal in vitro responses were observed at concentrations of 0.06 to 0.6 micrograms/ml of Ag. At higher doses of Ag (6 micrograms/ml) anti-PRP and anti-DT antibody responses were suppressed. The in vitro generation of anti-PRP and anti-DT ASC, as detected by a spot-forming cell assay was shown to be T cell dependent, Ag dependent, and Ag specific. This culture system provides a model for the study of human B cell activation and immunoregulation by polysaccharide-protein conjugates and polysaccharides.     

ELISA方法测定血清中Hib多糖抗体含量的条件比较研究

分别以牛血清白蛋白和人血清白蛋白作为封闭液的主要成分测定7份血清中抗b型流感嗜血杆菌荚膜多糖的抗体含量,结果显示,两组数据之间没有显著性差异(P>0.05)。分别用HRP标记的羊抗人IgG和HRP标记的鼠抗人IgG测定该7份血清,结果显示两组结果无显著性差异(P>0.05)。同时,将血清反应的温度和时间由37℃1.5h改为4℃16h(过夜),结果显示两者无显著性差异(P>0.05)。从而优化了该方法。     

Isotype concentrations of human antibodies to Haemophilus influenzae type b polysaccharide (Hib) in young adults immunized with the polysaccharide as such or conjugated to a protein (diphtheria toxoid)

Antibody responses of young adults to Haemophilus influenzae type b polysaccharide (Hib) or its protein conjugate were studied with special attention to the isotype composition of the antibodies. Three conclusions of interest can be made: 1) Immunoglobulin G (IgG) antibodies in polysaccharide-immunized volunteers displayed the subclass pattern previously found in antibodies to meningococcal type A polysaccharide. IgG1 was the predominant subclass in IgG antibodies of some individuals, IgG2 in others. Still others had the two subclasses in varying but more even proportions. 2) The conjugate vaccine induced a geometric mean response 2 to 3 times higher and an IgG response 4 times higher to Hib than the polysaccharide vaccine. 3) Anti-Hib antibodies induced by the conjugate vaccine still had essentially the same IgG subclass composition as anti-Hib antibodies induced by the polysaccharide. This composition was strikingly different from the composition of the anti-diphtheria toxoid response induced by the same conjugate vaccine.     

The yeast PRP19 protein is not tightly associated with small nuclear RNAs, but appears to associate with the spliceosome after binding of U2 to the pre-mRNA and prior to formation of the functional spliceosome.

We have previously shown that the yeast PRP19 protein is associated with the spliceosome during the splicing reaction by immunoprecipitation studies with anti-PRP19 antibody. We have extended such studies by using extracts depleted of specific splicing factors to investigate the step of the spliceosome assembly process that PRP19 is involved in. PRP19 was not associated with the splicing complexes formed in U2- or U6-depleted extracts but was associated with the splicing complex formed in heat-inactivated prp2 extracts. This finding indicates that PRP19 becomes associated with the splicing complexes after or concomitant with binding of the U6 small nuclear ribonucleoprotein particle (snRNP) to the precursor RNA and before formation of the functional spliceosome. We further analyzed whether PRP19 is an integral component of snRNPs. We have constructed a strain in which an epitope of nine amino acid residues recognized by a well-characterized monoclonal antibody, 12CA5, is linked to the carboxyl terminus of the wild-type PRP19 protein. Immunoprecipitation of the splicing extracts with anti-PRP19 antibody or precipitation of the extracts prepared from the epitope-tagged strain with the 12CA5 antibody did not precipitate significant amounts of snRNAs. Addition of micrococcal nuclease-treated extracts to the PRP19-depleted extract restored its splicing activity. These results indicate that PRP19 is not tightly associated with any of the snRNAs required for the splicing reaction. No non-snRNP protein factor has been demonstrated to participate in either step of the spliceosome assembly pathway that PRP19 might be involved in. Thus, PRP19 represents a novel splicing factor.     

Human immunoglobulin-G subclass distribution in response to vaccination withVibria cholerae vaccine and procholeragenoid

Immunoglobulin-G (IgG) subclass-specific antibody to cholera toxin andVibrio cholerae lipopolysaccharide in 26 individuals vaccinated withV. cholerae whole cells and procholeragenoid was investigated. An ELISA assay with monoclonal IgG subclass-specific antibodies was employed. The response to vaccination was found to be predominantly of the IgGl (100%) and IgG2 (73%) subclass. The magnitude of the IgG response was greater for the antitoxic compared with the antilipopolysaccharide vaccine component. IgG subclass antibodies evoked in response to immunization had no influence on the overall IgG subclass serum concentrations.     

A newly described activity of guinea pig IgG1 antibodies: transfer of cutaneous basophil reactions.

Hapten-specific delayed time course skin reactions containing predominant accumulations of basophils and eosinophils were elicited in newborn guinea pigs after i.v. transfer of small amounts of oxazolone immune serum. The immune serum was fractionated by column chromatography procedures, and the fractions were examined for their ability in transferring this form of cutaneous basophil hypersensitivity (CBH). Only the 7S IgG-containing peak from Sephadex G-200 columns, and only the IgG1-containing fractions from DEAE columns, transferred CBH. An affinity column of bound oxazolone removed the activity from immune serum, and it could be recovered from the column by eluting with soluble oxazolone. About 35 microgram of purified IgG1 anti-oxazolone antibody could systemically transfer CBH reactivity. An immunoadsorbant column of anti-IgG1 removed this activity, but a column of anti-IgG2 did not. None of the procedures were able to separate activity in transferring CBH from passive cutaneous anaphylactic (PCA) activity classically associated with guinea pig IgG1 antibody. IgG1 from 8-day immune and 31-day hyperimmune donors were both effective. The average association constant of 8-day antibody was 8 X 10(-4) M-1. Transfer of cutaneous basophil reactions can be mediated by low affinity serum 7S IgG1 antibody.     

The subclass distribution of human IgG rheumatoid factor

The subclass distribution of IgG rheumatoid factor (RF) was determined by a sensitive ELISA assay in sera from patients with rheumatoid arthritis and from normal controls. In both instances, the most important subclasses were IgG1 and IgG4. The IgG4 RF was directed against the Fc region of IgG, and recognized human as well as rabbit IgG. Although human IgG4 myeloma proteins bound to rabbit IgG better than did myelomas of other IgG subclasses, the IgG4 RF activity in rheumatoid sera showed an additional specificity, because the fraction of IgG4 RF/total IgG4 for rheumatoid arthritis sera was far greater than for myelomas. This inference was supported by the observation that there was persistent, albeit diminished, IgG RF activity in pepsin-digested, RF-containing sera (but not myeloma proteins), indicating that a critical component of IgG4 RF activity was contained within the Fab region of the IgG4 molecule. The finding of large quantities of IgG4 RF was not due to a bias of the assay, because the preponderance of IgG4 did not extend to the subclass distribution of antibodies directed against other antigens. The demonstration of an important role for IgG4 as a RF is of special interest because of the relative inability of this subclass to fix complement or to bind to Fc receptors, and because of its potential role as a mediator of increased vascular permeability.     

Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen-binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (C_H1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between V_H-C_H1 and V_L-C_L was improved by the C_H1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91–62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked-and-recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.     

Immunoglobulin G3 from polyclonal human immunodeficiency virus (HIV) immune globulin is more potent than other subclasses in neutralizing HIV type 1.

Passive antibody prophylaxis against human immunodeficiency virus type 1 (HIV-1) has been accomplished in primates, suggesting that this strategy may prove useful in humans. While antibody specificity is crucial for neutralization, other antibody characteristics, such as subclass, have not been explored. Our objective was to compare the efficiencies of immunoglobulin G (IgG) subclasses from polyclonal human HIV immune globulin (HIVIG) in the neutralization of HIV-1 strains differing in coreceptor tropism. IgG1, IgG2, and IgG3 were enriched from HIVIG by using protein A-Sepharose. All three subclasses bound major HIV-1 proteins, as shown by Western blot assay and enzyme-linked immunosorbent assay. In HIV-1 fusion assays using X4, R5, or X4R5 envelope-expressing effector cells, IgG3 more efficiently blocked fusion. In neutralization assays with cell-free viruses using X4 (LAI, IIIB), R5 (BaL), and X4R5 (DH123), a similar hierarchy of neutralization was found: IgG3 > IgG1 > IgG2. IgG3 has a longer, more flexible hinge region than the other subclasses. To test whether this is important, IgG1 and IgG3 were digested with pepsin to generate F(ab')(2) fragments or with papain to generate Fab fragments. IgG3 F(ab')(2) fragments were still more efficient in neutralization than F(ab')(2) of IgG1. However, Fab fragments of IgG3 and IgG1 demonstrated equivalent neutralization capacities and the IgG3 advantage was lost. These results suggest that the IgG3 hinge region confers enhanced HIV-neutralizing ability. Enrichment and stabilization of IgG3 may therefore lead to improved HIVIG preparations. The results of this study have implications for the improvement of passive immunization with polyclonal or monoclonal antibodies and suggest that HIV-1 vaccines which induce high-titer IgG3 responses could be advantageous.     

Monoclonal antibodies against the surface antigens of Shigella flexneri serotype 1b and Shigella dysenteriae serotype 1

Monoclonal antibodies against the surface antigens of Shigella flexneri 1b and S. dysenteriae 1 were prepared. The specificities of the antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), and quantitative agglutination using microtiter plate. Monoclonal antibodies against S. flexneri 1b, designated Sf2B2 and Sf2G4, belonged to IgG2a and IgG1 subclass, respectively. The former was specific for S. flexneri 1b, whereas the latter was reactive not only to S. flexneri 1b, but also weakly to 3a and 4b. Monoclonal antibody against S. dysenteriae 1, Sd5E1 (IgM), reacted with S. dysenteriae 1, 3, 6, 7, and S. boydii 2.     

A dominant negative mutation in the conserved RNA helicase motif ''SAT'' causes splicing factor PRP2 to stall in spliceosomes.

To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibited cell growth by causing a defect in pre-mRNA splicing. This defect was partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2-dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild-type level of RNA-stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein.     

Serum immunoglobulin IgG subclass distribution of antibody responses to Yop proteins and lipopolysaccharide of Yersinia enterocolitica in patients with yersiniosis.

To assess the humoral immunological responses at the IgG subclass level in yersiniosis specific antibody responses against lipopolysaccharide of Yersinia enterocolitica 03 (LPS) and Yersinia Yop proteins were analyzed by ELISA. Thirty five patients with arthritis and forty nine patients with uncomplicated yersiniosis were included in the study. Analysis of the IgG subclass responses to the LPS revealed that the subclass distribution for both groups of patients was IgG2>IgG1>IgG3. The concentration of IgG4 was below detection level. The predominant antibody responses to Yop proteins were IgG1>IgG3>IgG2>IgG4 but the frequency of detection of particular IgG subclass antibodies were dependent on the age of patients. Generally, the frequency of occurrence of IgG2 antibodies for Yop proteins of Yersinia together increased with age reaching its peak among individuals aged above 40 years. On the other hand, IgG1 for Yop proteins and IgG3 for Y. enterocolitica LPS were diagnosed more often in serum samples obtained from children than from adults. We also found significantly higher frequency of IgG4 to Yop proteins of Y. enterocolitica in men than in women.     

Biological characterization of Campylobacter fetus lipopolysaccharides

Abstract Ten patients with chronic liver disease, seven healthy seropositive individuals with a remote history of rubella, and three patients with acute rubella were examined for serum levels of IgG subclasses and subclass antibodies against rubella virus structural proteins. One patient with AICAH had no detectable total or rubella specific IgG3 or IgG4. The liver disease patients were hypergammaglobulinemic and had greatly raised IgG1 levels. Patients with acute rubella lacked antibodies to the rubella virus E2 protein and showed no IgG4 antibody response. The liver disease patients showed a somewhat weaker IgG4 antibody response against the core (C) protein than healthy controls. However, differences are suggested within the subclasses in antibody reactivity against the individual rubella virus antigens. It is concluded that test systems that discriminate reactivities against individual antigens have to be used for characterization of viral antibody subclass profiles.     

Humoral immunity aspects of meningococcal infection. I. A method of performing and the characteristics of the immune bacteriolysis reaction]

Immune bacteriolysis test with meningococcus, group A, was used for the purpose of serum antibody study. Meningococcus cultures with a bright orange fluorescence of the colonies in oblique illumination (the I type) proved to possess the greatest lysability. Guinea pig serum sorbed with meningococcus suspension was found to be the best source of the complement. Sera obtained after 1 to 3 days of rabbit immunization, containing mostly IgM antibodies, had the greatest bactericidal capacity. Only those fractions which contained IgM possessed bactericidal activity in the hyperimmune rabbit sera with a high IgG antibody concentration. No lytic activity was displayed against meningococcus by unfractionated hyperimmune sera.     

The effects of a stable analogue of PGE1 on the IgG subclass response to particulate bovine tubular basement membrane in the brown-Norway rat

The IgG subclass and the IgM isotype response to immunization with particulate bovine tubular basement membrane (TBM) and adjuvants was studied in Brown-Norway rats receiving daily injections of a stable analogue of PGE1 (M-PGE1). M-PGE1 slightly reduced the average quantity of circulating TBM antibody as well as the average quantity of eluted IgG per gram of renal tissue as compared to controls. However, M-PGE1 did not qualitatively affect the distribution of the IgG subclass or IgM isotype response to TBM. The IgG response, which occurred predominantly in the IgG1 and IgG2a subclasses, increased from Days 8 to 14 after immunization, while the IgM response decreased over the same time period. The percentage of TBM antibody in the IgG2b subclass was markedly decreased as compared to the percentage of IgG2b antibody in total IgG. A substantial heterogeneity in the IgG subclass response was noted among individual rats with IgG1 constituting from 46 to 82% of circulating TBM antibody. Although no correlation between the IgG subclass response and the severity of tubulointerstitial nephritis was noted, heterogeneity in the IgG subclass response to autoantigens may, nevertheless, theoretically play an important role in the pathogenesis of autoimmune inflammatory phenomena.     

Immunoglobulin classes of anti-Sendai virus antibody detected by ELISA in infected nude mouse serum

Sendai virus-infected nude mouse sera obtained on the seventh day after infection or later, in which anti-Sendai virus antibodies were undetectable by hemagglutination-inhibition and neutralization tests, were found to be reactive with the virus antigen by ELISA using horseradish peroxidase-conjugated anti-mouse IgG rabbit IgG. The reactivity was blocked by rabbit anti-Sendai virus antiserum and was not observed against influenza virus which served as a control antigen. Anti-Sendai virus antibody activity of fractions from Sephadex G-200 gel filtration was detected in the IgM fraction when anti-mouse mu chain-specific antiserum was used and in both IgG and IgM fractions when heavy and light chain-specific anti-mouse IgG serum was employed in ELISA. ELISA of the fractions from protein A-Sepharose affinity chromatography of Sendai virus-infected nude mouse sera showed that the eluates at pH 6.0 and pH 3.5 contained IgG1 and IgG2b anti-Sendai virus anti-bodies, respectively, and that the eluate at pH 4.5 contained both IgG2a and IgG3 antibodies.     

Detection of monoclonal antibody to high-mobility-group protein 17 from chick oviduct.

Total chromosomal HMG (high-mobility-group) proteins have been isolated from oestrogen-stimulated chick oviduct. The antibodies against these proteins were induced in mice and subsequently their spleen cells were fused with myeloma cells to form hybridomas. A highly purified HMG protein, 17, was used to select for the hybridomas that produce antibody against HMG protein 17. The hybridomas were cultured and injected into mice to produce ascites. The antibody against HMG protein 17 in the IgG (immunoglobulin G) fraction of the ascites fluid was obtained by Protein A-Sepharose column chromatography. We have devised a solid-phase radioimmunoassay and enzyme-linked serological assay for the detection and characterization of this antibody directed against HMG protein 17. This anti-(HMG protein 17) IgG interacted only with HMG protein 17, but not with other chromosomal proteins, e.g. histone H1, "95K protein' (a chick oviduct-specific chromosomal protein) and HMG proteins 1, 2 and 14. The monospecific nature of this anti-(HMG protein 17) IgG fraction is confirmed.