Relationship of in vitro phagocytosis of serotype 14 Streptococcus pneumoniae to specific class and IgG subclass antibody levels in healthy adults

The role of specific IgG2 antibody in the protection against serious infection with Streptococcus pneumoniae is unclear. We therefore decided to investigate the relationship between serum antibody levels and opsonization and phagocytosis of this microorganism. We have measured serum IgM, IgA and IgG subclass antibody specific for pneumococcal capsular polysaccharide and in vitro phagocytosis of serotype 14 pneumococcus by polymorphs, in healthy adults before and after immunization with Pneumovax II. IgM and IgG2 were the predominant anti-pneumococcal antibodies seen, IgA and IgGl being present at low titre. No significant relationship of phagocytosis with specific IgM and IgA antibodies was found. However, both specific IgG 1 and IgG2 antibodies in post-immunization sera correlated significantly with phagocytosis of the pneumococcus in the presence of complement (r= 0.57, P= 0.029 and r= 0.59, P= 0.022 respectively). After heat-inactivation, the remaining opsonic activity of sera correlated only with levels of specific IgG2 antibody (r= 0.61, P = 0.0006). Whereas phagocytosis supported by specific IgG 1 and IgG2 antibody to serotype 14 pneumococcus after immunization is mediated by complement activation, IgG2-specific antibody in high titre may also be able to function by complement-independent interaction with Fcγ receptors on polymorphs.     

Recurrent sinopulmonary infection and impaired antibody response to bacterial capsular polysaccharide antigen in children with selective IgG-subclass deficiency

We studied 20 children with recurrent sinopulmonary infections and serum IgG levels within the normal range, who had selective IgG-subclass deficiency. Twelve of the children were IgG2 deficient, five were IgG3 deficient, and three were deficient in both IgG2 and IgG3. IgA deficiency was present in 3 of the 20 patients. In the children with IgG2 deficiency, serum antibody concentrations to the capsular polysaccharide of Hemophilus influenzae type B (Hib) were significantly lower than those in age-matched controls, both before and after immunization with the Hib capsular polysaccharide antigen, which elicits antibody predominantly of the IgG2 subclass. In contrast, their serum antibody titers to the tetanus and diphtheria toxoid protein antigens, which elicit antibody predominantly of the IgG1 subclass, were normal in comparison with those of age-matched controls. These results suggest that impairment of the antibody response to specific microbial antigens predisposes patients with selective IgG-subclass deficiencies to recurrent infections. Thus, as an aid in determining therapy, children with recurrent infections and normal total serum IgG should be evaluated for this condition.     

Evolution of the subclass of IgG antibody to type 3 pneumococcal polysaccharide during childhood

Human antibodies to bacterial polysaccharides consist primarily of IgG and are largely restricted to the IgG2 subclass in adults. We examined the ontogeny of the IgG subclass response to pneumococcal polysaccharide type 3 to determine if the poor response of infants to immunization with polysaccharide antigens is due to a diminished capacity to form this subclass of antibodies. Sera from 33 patients aged 2 months to 25 years who had previously been shown to respond to polyvalent pneumococcal polysaccharide vaccine by producing IgG antibodies, were assayed for pneumococcal type 3 specific antibodies of the IgG1, IgG2, IgG3, or IgG4 subclass. IgG1 antibodies to pneumococcal polysaccharide type 3 were uniformly low in all age groups. In contrast, IgG2 antibody activity was lowest in children less than the age of 2 years (170 +/- 20 ng/ml), but rose progressively in the age group 2-5 years (210 +/- 40 ng/ml), 5-10 years (330 +/- 30 ng/ml), and over the age of 10 (390 +/- 30 ng/ml) (differences significant at P less than 0.0005 by ANOVA). Thus, even in infants, pneumococcal polysaccharide responses are confined largely to the IgG2 subclass. Our findings are consistent with the hypothesis that purified bacterial capsular polysaccharide antigens preferentially activate IgG2-committed B cell clones at all ages.     

Class- and subclass-specific pneumococcal antibody levels and response to immunization after bone marrow transplantation.

Immunoglobulin class- and subclass-specific antibodies to a polyvalent pneumococcal capsular polysaccharide vaccine (Pneumovax II) were measured before and after immunization in children, 1 year or more after bone marrow transplantation for a variety of genetic disorders. The median titres of specific IgG, IgG1 and IgG2 pneumococcal antibodies fell significantly (P less than 0.05) from pre-transplantation levels. The levels of pneumococcal antibodies in the patients before immunization were markedly lower than those in control children of comparable age, for antibodies of IgM, IgG, IgG1 and IgG2 classes (P = less than 0.001 in each case). Apart from IgG2 antibodies, the median response to immunization with Pneumovax II was not significantly different from the controls (P greater than 0.05). However, because of the lower pre-immunization levels, the patients did not achieve a high post-immunization-specific antibody titre in any immunoglobulin class or subclass, when compared with normal children. Neither the pre-immunization specific antibody levels nor the response to immunization were affected by splenectomy or the presence of chronic graft-versus-host disease. Immunization of the donor before bone marrow harvest did not influence the level of specific antibody 1 year or more after transplantation. No significant correlation was found between the total serum IgG2, the patients' age at the time of assessment, or time after transplantation, and the IgG2-specific antibody response. The lack of specific antibodies and the poor IgG2 response to pneumococcal antigens may contribute towards the occurrence of infection with Streptococcus pneumoniae in the late post-transplantation period.     

Opsonization of Cryptococcus neoformans by a family of isotype-switch variant antibodies specific for the capsular polysaccharide

A family of immunoglobulin isotype-switch variants was isolated by sib selection from a murine hybridoma which produced an immunoglobulin G subclass 1 (IgG1) antibody specific for the capsular polysaccharide of Cryptococcus neoformans. Antibodies of the IgG1, IgG2a, and IgG2b isotypes had similar serotype specificity patterns in double immunodiffusion assays which used polysaccharides of the four cryptococcal serotypes as antigens. A quantitative difference in the ability of the isotypes to form a precipitate with the polysaccharide was observed in a double immunodiffusion assay and confirmed in a quantitative precipitin assay. The relative precipitating activity of the antibodies was IgG2a greater than IgG1 much greater than IgG2b. Analysis by enzyme-linked immunosorbent assay of the reactivity of the three isotypes with cryptococcal polysaccharide showed identical titers and slopes, suggesting that the variable region of the class-switch antibodies was unaltered. This system allowed us to examine the effect of the Fc portion of the antibody on opsonization of encapsulated cryptococci. Yeast cells were precoated with antibodies of each isotype and incubated with murine macrophages or cultured human monocytes. Antibodies of all three isotypes exhibited a dose-dependent opsonization for phagocytosis by both human and murine phagocytes. The relative opsonic activity of the antibodies was IgG2a greater than IgG1 greater than IgG2b.     

Clinical and immunological evaluation of patients with mild IgG1 deficiency.

Serum IgG subclass concentrations were determined in patients visiting, the pulmonology out-patient clinic with chronic respiratory tract problems. A total of 24 patients with a serum IgG1 concentration < 4.9 g/l (i.e. below the reference range) and normal values for IgG2, IgM and IgA were included. Patients with a selective IgG1 deficiency were vaccinated with a 23-valent pneumococcal polysaccharide vaccine. There were nine patients with a poor antibody response to pneumococcal capsular polysaccharide antigens. Responsiveness to protein antigens was intact in all patients. Patients with pneumonia showed a significantly lower anti-polysaccharide response in the IgG2 subclass than patients without pneumonia. Patients with recurrent sinusitis showed a significantly lower response in the IgA isotype after vaccination with pneumococcal polysaccharide vaccine compared with non-sinusitis patients. It can be concluded that patients with recurrent sinopulmonary infections and a mild IgG1 subclass deficiency have an impaired IgG1 anti-polysaccharide response, which can extend to decreased IgG2 and IgA anti-polysaccharide responses.     

The use of human antigen-specific monoclonal antibodies in an enzyme-linked immunosorbent assay for the determination of anti-HBsAg antibody subclasses

A human monoclonal anti-HBsAg antibody (IgGl, λ) was used as a reference for an ELISA determination of the serum levels of anti-HBsAg antibodies in human volunteers after vaccination with H-B vax. The IgG subclass distribution of specific antibodies showed a marked dominance of IgGl antibodies. In addition, small amounts of specific IgG4 antibodies were occasionally found, suggesting a different pattern from that found after natural disease. The novel use of a human monoclonal antibody to measure specific antibodies may give a more accurate determination of specific IgG subclass levels than previously available methods.     

IgG2 subclass restriction of antibody to pneumococcal polysaccharides

Studies in experimental animals suggest that antibody responses to certain polysaccharide antigens may be restricted in IgG subclass distribution. To determine if human antibodies to pneumococcal polysaccharides are similarly restricted we measured the IgG subclass specific response to immunization with purified polyvalent pneumococcal polysaccharide vaccine. For the type 3 pneumococcal antigen, the geometric mean titre of IgG2 antibody was significantly greater than that of IgG1, IgG3 or IgG4, in both pre-immunization and post-immunization sera. A significant rise in mean titre, comparing pre- to post-immunization sera was observed only for IgG2 antibody. Similar predominance of IgG2 antibody was found for pneumococcal polysaccharides types 6, 18, 19 and 23. In contrast, antibody to the protein antigen tetanus toxoid was exclusively of the IgG1 subclass. Patients with IgA/IgG2 deficiency demonstrated a normal IgG response to tetanus, a normal IgM response to pneumococcal polysaccharides, but no IgG antibody to pneumococcal antigens. IgG2 subclass restriction of antibody to pneumococcal polysaccharides suggest that these antigens may elicit an immune response analogous to the murine type 2 T-cell independent immunogens which show IgG subclass restriction and the requirement of a mature B cell subset defined by the Lyb5+ alloantiserum. Our findings support the possibility of subclass-specific inducing or regulating mechanisms for human responses to carbohydrate or polysaccharide antigens.     

Impaired antibody responses in the hyperimmunoglobulin E syndrome

Patients with the hyper-IgE (HIE) syndrome have recurrent bacterial infections with Staphylococcus aureus and other polysaccharide encapsulated organisms. To determine whether an impairment of the antibody response to polysaccharide antigens contributes to infections in this syndrome, we measured serum antibody to the teichoic acid of S. aureus and to the capsular polysaccharide of Haemophilus influenzae type b. Compared to control subjects who had no history of S. aureus infections (N = 14), sera from patients with HIE (N = 9) lacked the expected elevation of serum antibody to teichoic acid (p greater than 0.05) and had significantly lower levels of this antibody than sera from 14 patients with atopic dermatitis, complicated by recurrent cutaneous S. aureus infections (p less than 0.01). After immunization with the capsular polysaccharide of Haemophilus influenzae type of vaccine, the antibody response of patients with HIE was significantly impaired compared to that of age-matched control subjects (p = 0.01). Although patients with HIE syndrome had normal total IgG levels, most patients with HIE but not patients with atopic dermatitis had IgG2 subclass deficiency. Defective antibody responses in patients with HIE were not restricted to polysaccharide antigens because the serum levels of antitetanus toxoid antibody in these patients were significantly lower than that of control subjects (p less than 0.001). Impaired antigen-specific antibody responses in patients with HIE syndrome may contribute to their increased susceptibility to infection.     

Class and subclass antibodies to Haemophilus influenzae type b capsule: comparison of invasive disease and natural exposure

Recently, the role of immunoglobulin G (IgG) subclasses in the immune responses to organisms with polysaccharide capsules, particularly Haemophilus influenzae type b, has been of interest. We developed assays to measure IgG2- and IgG4-specific antibodies to the polyribosylribitol phosphate (PRP) polysaccharide antigen of H. influenzae type b and demonstrated that these assays were subclass specific. Relative levels of subclass-specific antibody were assayed in serum from 30 Alaskan Eskimo children who had invasive H. influenzae type b disease and 30 healthy controls that were matched for age and village of residence. We also measured total PRP antibody and total serum IgG4. The group with invasive H. influenzae type b disease had a significantly higher mean level of IgG4-specific PRP antibody than did the controls (P = 0.0006). However, we found no significant difference between cases and controls for IgG2-specific PRP antibody, total IgG4, or total PRP antibody. The data suggest that IgG4-specific PRP antibody is elicited by invasive H. influenzae type b disease, independent of age. The IgG4 subclass thus may be a critical determinant of the immune response to invasive infection caused by H. influenzae type b, especially for young infants who generally have a weak immune response to this organism.     

Antibody-Induced Hemolytic Activity of Human Blood Monocytes

The hemolytic effect of purified human blood monocytes was quantitated as the release of isotope from human erythrocytes labeled with ⁵¹Cr-chromate. We here demonstrate that IgM antibody (anti-A or cold agglutinin) was unable to confer monocyte-mediated hemolysis, and that lysis was induced by IgG anti-A antibody. In addition, IgG anti-D sera containing antibodies belonging to subclass IgG1 or IgG3 had approximately equal hemolytic efficiency, as calculated from the antigen-binding capacity of the sera at the concentrations inducing 50% monocyte-mediated hemolysis. The lytic reaction was suppressed by pooled IgG or by individual myeloma proteins of subclass IgGl or IgG3. The inhibitory effect of heat-aggregated IgG was not different from that of native IgG. IgG2 or IgG I had no or weak effects. Cross-inhibition of lysis induced by IgG1 and IgG3 anti-D antibody showed strong and equal suppression by IgG1 and IgG3 myeloma proteins. Hundredfold higher concentrations of IgG2 or IgG4 was required for inhibition of lysis, which may be an effect of contaminating IgGl or IgG3. Our data strongly suggest that the receptors on human blood monocytes for IgGl and IgG3 that participate in monocyte-mediated hemolysis are identical.     

Avidity as a determinant of the protective efficacy of human antibodies to pneumococcal capsular polysaccharides

Antibodies reactive with capsular polysaccharides are considered the principal mediators of immunity against invasive diseases caused by Streptococcus pneumoniae. In this study, we tested the hypothesis that anti-pneumococcal capsular polysaccharide (PPS) antibody avidity can influence protective efficacy. We measured the avidities of individual adult postvaccination immunoglobulin G2 (IgG2) antibodies to PPS serotypes 6B and 23F and examined the relationship between avidity and opsonophagocytic and mouse-protective activities. The avidities of PPS 6B- and PPS 23F-specific IgG2 antibodies ranged from 6 to 31 nM-1 and from 3 to 20 nM-1, respectively. We observed an inverse correlation between the magnitude of avidity and the amount of antibody required to protect mice against lethal bacteremia caused by serotype 6B pneumococci. Similarly, higher-avidity antibodies were more effective than lower-avidity antibodies in vitro in mediating complement-dependent opsonophagocytosis of both 6B and 23F pneumococci. These data suggest that in adults, PPS antibodies are sufficiently polymorphic to possess biologically significant variations in avidity. We conclude that avidity functions as an important determinant of anticapsular antibody protective efficacy against pneumococci.     

Human IgG and IgA subclass response following immunization with a polyvalent Klebsiella capsular polysaccharide vaccine

The human IgG and IgA response was determined after vaccination with an experimental 24-valent Klebsiella capsular polysaccharide vaccine. The majority of natural antibody acquired prior to immunization was found in the IgG1, IgG2 and IgA1 subclasses. The immune response to vaccination was concentrated within the IgG1, IgG2, IgA1 and IgA2 subclasses with greater than or equal to 70% of subjects responding with a significant (greater than or equal to fourfold) rise in titer. The IgG3 and IgG4 response was meager with few volunteers (0%-40%) showing a significant titer rise. Therefore, vaccination with Klebsiella capsular polysaccharide induces a vigorous IgG and IgA response in humans which is not restricted to single antibody class or subclass.     

Bindine affinities of allergens from pollen, mites, and house dust for specific IgG subclass antibodies

The distribution and affinity of IgG subclasses against various aeroallergens were assessed by inhibition of specific antibody binding. Two parameters from the dose-response curves were taken as indicative of antibody affinity: the point of 50% inhibition and the value of the slopes on double-log plots. It was found that IgG4 antibody specific for aeroallergens (i.e., from pollens of several species of Gramineae, Olea europaea , and Parietaria judaica and from house dust) usually exhibits high affinity, except for Dermatophagoides pteronyssinus . High binding affinity was also displayed by IgGl subclass antibodies against the allergens of O. europaea and P. judaica . Distinct IgG subclass affinity profiles were observed for the allergens of grass pollen (i.e., Holcus lanatus ) and dust mites (i.e., D. pteronyssinus ). These results demonstrate that IgG subclass distribution, as well as antibody affinity, depends on the nature of the sensitizing allergen.     

Humoral immunity and bronchiectasis

Bronchiectasis is a common complication of primary antibody deficiency but the incidence of antibody deficiency as an underlying cause of bronchiectasis is largely undefined. In this study the humoral immune status of a cohort of bronchiectatic patients was investigated to detect the frequency of significant antibody deficiency and to determine the extent of immunological investigation which is appropriate for routine assessment of bronchiectasis patients. Fifty-six out-patients (with a mean age of 59.6 years) had serum immunoglobulins, IgG subclasses and specific antibodies to capsular polysaccharides of Haemophilus influenzae and Streptococcus pneumoniae measured. Where specific antibody -levels were low, where possible, appropriate immunization with pneumococcal or conjugated Haemophilus polysaccharide vaccines was offered and the responses quantified. Three of 56 patients had low total serum IgG levels. Thirteen of 56 had deficiencies of either a single IgG subclass or combinations of two or more subclasses, with IgG4 being most frequently implicated (9/56). Twenty-nine of 56 had low basal specific polysaccharide antibody levels. Test immunization, where performed, produced satisfactory responses in all cases except one, where a specific defect of responsiveness to pneumococcal polysaccharide was identified. This study indicates that antibody deficiency is an uncommon aetiological/underlying factor in the causation of bronchiectasis beyond the fourth decade and that detailed investigation of humoral immune status as a routine in bronchiectasis patients, at least at this age, is not generally justified.     

Measurement of the IgG2 response to Pneumococcal capsular polysaccharides may identify an antibody deficiency in individuals referred for immunological investigation

IgG2 is the most efficient subclass for providing protection against pneumococcal pathogens. We hypothesised that some individuals may be unable to mount an effective pneumococcal capsular polysaccharide (PCP) IgG2 response despite having a normal PCP IgG concentration (PCP IgG2 deficient). The median pre-vaccination PCP IgG2 concentration was significantly lower in individuals referred for immunological investigation compared to healthy controls (2.8 mg/L range, 95% CI 1.1–88 vs. 29.5mg/L, 95% CI 13.5–90, p = 0.0002). PCP IgG:IgG2 ratios were significantly higher for the referral population than for healthy controls suggesting the increased production of PCP specific subclasses other than IgG2. The percentage of individuals with PCP IgG2 deficiency was significantly higher in referral groups compared to controls (31% vs. 5%; p = 0.0009) and in an individual with PCP IgG2 deficiency, the balance of PCP specific IgG subclass antibodies post vaccination changed from IgG2>IgG1>IgG3>IgG4 to IgG1>IgG3>IgG2>IgG4. The median PCP IgG2 concentration in those with PCP IgG2 deficiency was significantly lower in the referral groups compared to controls (7.8 mg/L, 95% CI 1.1–12 vs. 12.7 mg/L, 95% CI 11.8–13.1; p = 0.006). The data suggests a defect in the production PCP IgG2 may be present in individuals with normal PCP IgG referred for immunological investigation.     

Gliadin IgG subclass antibodies in patients with coeliac disease

We have measured IgG1, 2, 3 and 4 subclass antibodies to gliadin and casein in normal controls, treated and untreated coeliac patients. The amount of antibody in each subclass was determined separately as a titre and as a concentration, by reference to a standard curve. Both methods of quantitation, carried out in separate assays, yielded comparable results (IgG antibody r = 0.86, IgG1 antibody r = 0.89). In all the coeliac patients, the majority of antibody was IgG1, and little IgG2, 3, 4 antibody was detected. Untreated coeliac patients had higher titres of IgG and IgG1 antibodies to gliadin and casein compared with normal controls. In the treated coeliac patients compared with the normal controls, there was no significant difference between the titres of IgG and IgG1 antibodies to gliadin but there were higher titres to casein.     

Distribution of virus antibody activity among human IgG subclasses.

Human IgG subclass antibody activity against viruses was studied by separating the IgG3 fractions from sera exhibiting high titres for rubella, polio types I, II and III, and herpes I viruses. The sera were fractionated on DEAE Affi-Gel Blue and protein A-Sepharose CL-4B columns using specific subclass antisera for identification. All IgG3 fractions exhibited a molecular weight of 164,000 daltons, a pI mean of 8.21 and S20,W1% = 6.2 as determined by polyacrylamide gel electrophoresis, isoelectric focusing and analytical centrifugation. Quantitative determination of the individual subclass concentrations by nephelometry showed them to be within the biological norm. The concentration and distribution of IgG in the sera and that of the IgGl, -2 and -4 and IgG3 fractions were used as a basis for studying antiviral activity. The IgG3 fractions showed a greater ratio of IgG concentration to antibody titre than the IgGl, -2 and -4 fractions as determined in neutralization and haemagglutination inhibition tests. The IgG3 fraction from anti-rubella serum bound 96.6% 125I-labelled rubella virus (HP 77/DE5). The IgG 3-125I-rubella immune complex was separated over a protein A-Sepharose CL-4B column and confirmed with subclass-specific antisera in radial immunodiffusion plates. Individual Gm allotype analyses showed markers distributed as follows: G3m(5,-6,10,11,13,-16,21,-24) for all the serum donors indicating similar genotypic expression of IgG3s.     

Evaluation of antibody responses to pneumococcal vaccines with ELISA and opsonophagocytic assay

Antibodies to a capsular polysaccharide (PS) provide protection against Streptococcus pneumoniae which express the homologous capsular serotype, and pneumococcal vaccines are designed to induce antibodies in the capsular PS. Levels and opsonophagocytic capacity of antibodies to the capsular PS of S. pneumoniae serotype 19F were determined by sera from adults immunized with 23-valent S. pneumoniae capsular PS vaccines. Geometric means of IgG anti-19F antibody level and specific opsonic titer rise significantly after immunization. The level of anticapsular PS antibodies for S. pneumoniae 19F serotype is fairly well correlated (r2=O.63) with the opsonophagocytic activities of sera. However, 3.7% (1/27) of serum samples display strikingly less opsonophagocytic activity than expected on the basis of their antibody level. Thus, antibody level may be of general use in predicting vaccine-induced protection among adults for 19F serotype. However, the opsonic activity data suggest that antibody levels are not always indicative of functional antibody.     

Illustration of pneumococcal polysaccharide capsule during adherence and invasion of epithelial cells

The capsular polysaccharide of Streptococcus pneumoniae represents an important virulence factor and protects against phagocytosis. In this study the amount of capsular polysaccharide present on the bacterial surface during the infection process was illustrated by electron microscopic studies. After infection of A549 cells (type II pneumocytes) and HEp-2 epithelial cells a modified fixation method was used that allowed visualization of the state of capsule expression. This modified fixation procedure did not require the use of capsule-specific antibodies. Visualization of pneumococci in intimate contact and invading cells demonstrated that pneumococci were devoid of capsular polysaccharide. Pneumococci not in contact with the cells did not show alterations in capsular polysaccharide. After infection of the cells, invasive pneumococci of different strains and serotypes were recovered. Single colonies of these recovered pneumococci exhibited an up-to-10(5)-fold-enhanced capacity to adhere and an up-to-10(4)-fold-enhanced capacity to invade epithelial cells. Electron microscopic studies using a lysine-ruthenium red (LRR) fixation procedure or cryo-field emission scanning electron microscopy revealed a reduction in capsular material, as determined in detail for a serotype 3 pneumococcal strain. The amount of polysaccharide in the serotype 3 capsule was also determined after intranasal infection of mice. This study illustrates for the first time the phenotypic variation of the polysaccharide capsule in the initial phase of pneumococcal infections. The modified LRR fixation allowed monitoring of the state of capsule expression of pathogens during the infectious process.