Cytoplasmic immunofluorescence of bone marrow plasma cells producing immunoglobulins of the four IgG subclasses.

Immunofluorescence studies were performed on bone marrow samples from 21 individuals with normal or slightly elevated serum IgG levels. On the average, 40.7 percent of the IgG-positive plasma cells contained molecules of the subclass IgG; 30.6 percent had intracellular IgG2; 21.7 percent, IgG3, and 7.0 percent, IgG4. These figures were compared to the relative serum IgG subclass concentrations. The principal finding was the discrepancy between the high relative number of IgG-3 producing bone marrow plasma cells and the low relative IgG3-producing bone marrow plasma cells and the low relative IgG3 serum concentration (5.5 percent of the total IgG). It was explained by the short biologic half-life of molecules of this subclass.     

Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4(+) T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4(+) Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.     

Isotype concentrations of human antibodies to group A meningococcal polysaccharide

Antibodies to meningococcal group A polysaccharide (MenA) in the sera of 34 vaccinated adults were quantitated by an isotype-resolving solid-phase RIA (IgA, IgM, IgG1, IgG2, IgG3, and IgG4). All individuals had antibodies before vaccination. The geometric mean concentration was 2.9 micrograms/ml. Two weeks after vaccination the mean antibody concentration had trebled. Average proportions of the three isotypes were then as follows: IgA 15%, IgM 48%, IgG 37%. No differences were found between individuals who had been immunized with the polysaccharide 7 to 8 yr earlier and "primary responders." The subclass composition of IgG antibodies was determined in the 24 postvaccination samples with a definite IgG response (greater than 2-fold increase). IgG1 was the predominant subclass in antibodies of some sera and IgG2 in others, but the average proportions of both subclasses were nearly the same. IgG3 and IgG4 were only found in occasional sera, but when present, each subclass accounted for up to 6%. Although the ratio of kappa and lambda chains could not be determined, there was evidence to suggest that it was higher in anti-MenA antibodies than in antibodies to protein antigens.     

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.     

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.     

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.     

IgG subclass responses to a transmembrane protein (gp41) peptide in HIV infection

The IgG subclass distribution to the E34/E32 peptides, derived from the HIV-1 glycoprotein 41 transmembrane protein, was analyzed in ELISA. Sera from individuals at different stages of the disease were assayed. A restricted subclass response of mainly IgG1 and IgG2 was found. The subclass response was of a different type than the one observed to HIV whole Ag and to a synthetic peptide from the C'-terminal part of the HIV-1 p24 core protein. An increased subclass restriction was observed in progressed stages of the disease.     

IgG Subclass and Heavy Chain Domains Contribute to Binding and Protection by mAbs to the Poly γ-D-glutamic Acid Capsular Antigen of Bacillus anthracis

Bacterial capsules are common targets for antibody-mediated immunity. The capsule of Bacillus anthracis is unusual among capsules because it is composed of a polymer of poly-γ-d-glutamic acid (γdPGA). We previously generated murine IgG3 monoclonal antibodies (mAbs) to γdPGA that were protective in a murine model of pulmonary anthrax. IgG3 antibodies are characteristic of the murine response to polysaccharide antigens. The goal of the present study was to produce subclass switch variants of the γdPGA mAbs (IgG3→IgG1→IgG2b→IgG2a) and assess the contribution of subclass to antibody affinity and protection. Subclass switch antibodies had identical variable regions but differed in their heavy chains. The results showed that a switch from the protective IgG3 to IgG1, IgG2b or IgG2a was accompanied by i) a loss of protective activity ii) a change in mAb binding to the capsular matrix, and iii) a loss of affinity. These results identify a role for the heavy chain constant region in mAb binding. Hybrid mAbs were constructed in which the CH1, CH2 or CH3 heavy chain constant domains from a non-protective, low binding IgG2b mAb were swapped into the protective IgG3 mAb. The IgG3 mAb that contained the CH1 domain from IgG2b showed no loss of affinity or protection. In contrast, swapping the CH2 or CH3 domains from IgG2b into IgG3 produced a reduction in affinity and a loss of protection. These studies identify a role for the constant region of IgG heavy chains in affinity and protection against an encapsulated bacterial pathogen.     

Human antibodies to phosphocholine. IgG anti-PC antibodies express restricted numbers of V and C regions

We examined the IgG subclass composition and isoelectric focusing (IEF) spectrotype pattern of naturally occurring human IgG antibodies that bind phosphocholine (PC) and found direct evidence for restricted expression of both V and C regions among these antibodies. In most individuals, the isotype of these IgG anti-PC antibodies was primarily IgG2. However, serum from some individuals contained significant amounts of IgG1 and IgG3 anti-PC antibodies. We also found that in individual sera, anti-PC antibodies are pauciclonal, as demonstrated by restricted spectrotypic patterns of the anti-PC antibodies. The IEF pattern of these antibodies were for the most part unique for each individual. In some sera, certain anti-PC antibodies with isoelectric points of basic pH bound PC conjugated to bovine serum albumin (PC-BSA) but did not bind pneumococcal C-carbohydrate bearing PC determinants. In two individuals, we found that the spectrotypes that bound only PC-BSA were of the IgG1 subclass. Taken together, these findings demonstrate that within individual sera, human antibodies to PC are quite restricted in both V and C region expression, and furthermore, these V and C regions of human Ig may not randomly associate.     

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.     

The early humoral immune response to Bacillus anthracis toxins in patients infected with cutaneous anthrax

Bacillus anthracis, the causative agent of anthrax, produces a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF), which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin and edema toxin, respectively. In this preliminary study, we characterized the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody responses observed following infection were directed against LF, with immunoglobulin G (IgG) detected as early as 4 days after the onset of symptoms in contrast to the later and lower EF- and PA-specific IgG responses. Unlike the case with infection, the predominant toxin-specific antibody response of those immunized with the US anthrax vaccine absorbed and UK anthrax vaccine precipitated licensed anthrax vaccines was directed against PA. We observed that the LF-specific human antibodies were, like anti-PA antibodies, able to neutralize toxin activity, suggesting the possibility that they may contribute to protection. We conclude that an antibody response to LF might be a more sensitive diagnostic marker of anthrax than to PA. The ability of human LF-specific antibodies to neutralize toxin activity supports the possible inclusion of LF in future anthrax vaccines.     

Inhibitors to factor IX contain all IgG subclasses except IgG3.

Five per cent of patients with haemophilia B develop inhibitors to factor IX. It is of interest to know the immunoglobulin subclass of these IgG antibodies. We have developed a sensitive method for the characterization of the subclass nature of inhibitors to factor IX. The technique is a crossed immunoelectrophoresis for the isolation of factor IX-inhibitor complexes followed by an enzyme-linked immunoassay using monoclonal antibodies to IgG subclasses for the subclass identification. We studied seven inhibitors with both low and high titres. One patient was studied at a very early stage of inhibitor development. All inhibitors gave a strong reaction with antibody to IgG4. Depending on the titre of the inhibitor, a reaction was also found with antibodies to IgG1 and IgG2. No inhibitor contained any detectable IgG3. IgG4 does not bind complement and it is therefore of importance that IgG4 is the main subclass both in high-titred and in low-titred inhibitors. The inhibitors are polyclonal antibodies, also at an early stage of inhibitor development.     

Malaria resistance genes are associated with the levels of IgG subclasses directed against Plasmodium falciparum blood-stage antigens in Burkina Faso

ABSTRACT: BACKGROUND: HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms have been associated with malaria resistance in humans, whereas cytophilic immunoglobulin G (IgG) antibodies are thought to play a critical role in immune protection against asexual blood stages of the parasite. Furthermore, HBB, IL4, TNF, and FCGR2A have been associated with both malaria resistance and IgG levels. This suggests that some malaria resistance genes influence the levels of IgG subclass antibodies. METHODS: In this study, the effect of HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms on the levels of IgG responses against Plasmodium falciparum blood-stage extract was investigated in 220 individuals living in Burkina Faso. The Pearson's correlation coefficient among IgG subclasses was determined. A family-based approach was used to assess the association of polymorphisms with anti-P. falciparum IgG, IgG1, IgG2, IgG3 and IgG4 levels. RESULTS: After applying a multiple test correction, several polymorphisms were associated with IgG subclass or IgG levels. There was an association of i) haemoglobin C with IgG levels; ii) the FcgammaRIIa H/R131 with IgG2 and IgG3 levels; iii) TNF-863 with IgG3 levels; iv) TNF-857 with IgG levels; and, v) TNF1304 with IgG3, IgG4, and IgG levels. CONCLUSION: Taken together, the results support the hypothesis that some polymorphisms affect malaria resistance through their effect on the acquired immune response, and pave the way towards further comprehension of genetic control of an individual's humoral response against malaria.     

IgG subclass expression by human B lymphocytes and plasma cells: B lymphocytes precommitted to IgG subclass can be preferentially induced by polyclonal mitogens with T cell help

The human IgG subclasses expressed by circulating B lymphocytes, tissue plasma cells, and plasma cells generated from B cell precursors in response to the polyclonal mitogens LPS and PWM were examined by immunofluorescence using subclass-specific monoclonal antibodies. The subclass distribution observed for circulating B lymphocytes was IgG2 (48%) greater than IgG1 (40%) greater than IgG3 (8%) greater than IgG4 (1%), while the distribution among IgG plasma cells in bone marrow, blood, spleen, and tonsils was IgG1 (64%) greater than IgG2 (26%) greater than IgG3 (8%) greater than IgG4 (1%). Multiple IgG isotypes were not observed on B cells or in plasma cells. Although IgG plasma cell responses to both LPS and PWM were T cell dependent, the distributions of IgG subclasses elicited were strikingly different. In control and LPS-stimulated cultures of blood mononuclear cells, the induced plasma cells expressed the IgG subclass distribution: IgG2 greater than 80%, IgG1 less than 20%, IgG3 less than 1%, IgG4 less than 1%. In PWM-stimulated cultures, the subclass distribution, IgG1 approximately 65%, IgG2 approximately 25%, IgG3 approximately 7%, IgG4 approximately 1%, was in perfect concordance with the in vivo subclass distribution of IgG plasma cells. Selective inhibition of suppressor T cell activity by x-irradiation and mitomycin C treatment did not alter the IgG subclass distribution pattern induced by LPS and PWM. Monoclonal antibodies were used to deplete selectively the B cell precursors bearing IgG1, IgG2, or IgG3 before PWM stimulation of blood mononuclear cells. In each instance, a reduction was observed only in the subpopulation of plasma cells producing the homologous IgG subclass. The results indicate that T cells can preferentially influence the terminal differentiation of B cells that are precommitted to different IgG subclasses.     

A field usable qualitative anti‐protective antigen enzyme‐linked immunosorbent assay for serodiagnosis of human anthrax

Although all mammals, including humans, are vulnerable when they come into direct contact with infected animals, anthrax is primarily a disease of herbivorous animals. In countries like India, cutaneous anthrax is a public health problem in several regions. Hence, a simple and efficacious serodiagnostic assay for large scale surveillance of endemic populations is required. In the present study, a field‐usable, qualitative ELISA was developed for serodiagnosis of human anthrax. Results are assessed on a visual basis and no sophisticated instruments are required. Anti‐protective antigen (PA) IgG was determined by visual examination of ELISA results of 225 human serum samples (160 from healthy humans, 5 from PA vaccinated individuals and 60 from confirmed anthrax cases). Comparison of the ELISA results with the results obtained from optical density values showed compatible sensitivity and specificity. Assay sensitivity, specificity, and positive and negative predictive values were found to be 100%. The developed assay could be a very useful tool for serological diagnosis of anthrax infection in humans.     

Subclass restriction of murine anti-carbohydrate antibodies.

Examination of the subclass distribution of murine antibodies directed against groups A and C streptococcal carbohydrate, alpha-(1 leads to 3) dextran and phosphocholine yields the surprising observation that these carbohydrate antigens stimulate IgG responses largely restricted to the rare IgG3 subclass. This subclass restriction is particularly impressive in light of the low circulating levels of IgG3 in nonimmune mouse serum and the failure of a variety of other antigens including proteins and aromatic haptens to stimulate IgG3 antibody production. Attempts to alter the subclass restriction of antibodies with carbohydrate specificity by immunization with carbohydrate-coupled protein have been unsuccessful and indicate that immunoregulation of subclass expression probably occurs at the level of the antibody forming (B) cell. It is therefore conceivable that VH regions of murine immunoglobulins may be restricted to particular IgG subclasses. A similar type of subclass restriction has been reported in human and rat anti-carbohydrate antibodies. This recruitment of a minor immunoglobulin isotype by carbohydrate antigens in several species further supports the concept of immunoregulation at the level of subclass, and suggests that these and other mammals may share a structurally similar isotype with perhaps a common evolutionary origin.     

Characterization of the human immune response to the UK anthrax vaccine

The anthrax bipartite lethal toxin (protective antigen (PA) and lethal factor (LF))-specific antibody responses of humans receiving the UK licensed anthrax vaccine were determined. The PA-specific IgG response peaked two weeks post immunization and fell back to pre-boost levels by week 12. The heterogeneity of the host population modulated the extent of the PA-specific antibody response. Significantly lower levels of LF-specific antibodies were also detected. Vaccinated individuals recognized the same PA epitope as the protective mouse lethal toxin neutralizing monoclonal 2D3 suggesting that this may also be a target for human protection.     

Analysis of a common inheritable idiotype in IgA-deficient sera using monoclonal antibodies

In these studies we describe the production of three mAb raised to an idiotype on an IgG anticasein antibody isolated from the serum of one IgA-deficient blood donor. These are IgM kappa and block the binding of casein Ag to anticasein antibody. Sera of unrelated IgA-deficient donors were tested for the presence of the idiotype; 15 of 56 IgA-deficient sera (25%) contain the anticasein idiotype, whereas 1 of 45 normal sera was positive. Anticasein antibodies as a whole were predominantly of the IgG1 and IgG3 subclass; idiotype-positive anticaseins are predominantly of the IgG1 subclass. For IgA-deficient donors, the relative amount of idiotype-positive anticasein antibody was correlated with the level of anticasein present in the serum. Studies were done to investigate the potential inheritance of the idiotype in families; in three of four families the idiotype was inherited in an apparent autosomal dominant pattern. Our data show that a common cross-reactive idiotype can be detected in the sera of IgA-deficient individuals and their family members. This suggests that V region markers may be conserved in this humoral immunodeficiency disease.     

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.     

Mass Spectrometry Detection of G3m and IGHG3 Alleles and Follow-Up of Differential Mother and Neonate IgG3

Mass spectrometry (MS) analysis for detection of immunoglobulins (IG) of the human IgG3 subclass is described that relies on polymorphic amino acids of the heavy gamma3 chains. IgG3 is the most polymorphic human IgG subclass with thirteen G3m allotypes located on the constant CH2 and CH3 domains of the gamma3 chain, the combination of which leads to six major G3m alleles. Amino acid changes resulting of extensive sequencing previously led to the definition of 19 IGHG3 alleles that have been correlated to the G3m alleles. As a proof of concept, MS proteotypic peptides were defined which encompass discriminatory amino acids for the identification of the G3m and IGHG3 alleles. Plasma samples originating from ten individuals either homozygous or heterozygous for different G3m alleles, and including one mother and her baby (drawn sequentially from birth to 9 months of age), were analyzed. Total IgG3 were purified using affinity chromatography and then digested by a combination of AspN and trypsin proteases, and peptides of interest were detected by mass spectrometry. The sensitivity of the method was assessed by mixing variable amounts of two plasma samples bearing distinct G3m allotypes. A label-free approach using the high-performance liquid chromatography (HPLC) retention time of peptides and their MS mass analyzer peak intensity gave semi-quantitative information. Quantification was realized by selected reaction monitoring (SRM) using synthetic peptides as internal standards. The possibility offered by this new methodology to detect and quantify neo-synthesized IgG in newborns will improve knowledge on the first acquisition of antibodies in infants and constitutes a promising diagnostic tool for vertically-transmitted diseases.