Immunoglobulin C-gene expression. I. The commitment to IgG subclass of secretory cells is determined by the quality of the nonspecific stimuli

Polyclonal stimulation of normal splenic B lymphocytes with either lipopolysac-charide (LPS) or helper T lymphocytes specific for B cell surface antigens results in the selective expression of IgG subclasses by the secretory cells: in addition to IgM-secreting plaque-forming cells (PFC), thymus-independent stimulation leads to the development of IgG₂ and IgG₃ PFC, while helper cell-dependent activation leads to IgG₁ and IgG₂ PFC. This cannot be solely explained by selective stimulation of distinct B cell subpopulations, because purified LPS-reactive blasts if restimulated by helper cells switch to IgG₁ while if maintained with LPS switch to IgG₃. The simultaneous stimulation of splenic B cells with LPS and helper cells results in additive IgM and IgG₂ responses, but in the selective suppression of IgG₃ PFC with a concomitant synergic enhancement of IgG₁ responses. These results are interpreted to indicate that the expression of IgG C genes in proliferating B lymphocytes is directed by the quality of nonspecific stimuli.     

Differential capacities of outer membrane proteins from Neisseria meningitidis B to prime the murine immune system after vaccination

Understanding the specificity of antibody response to Neisseria meningitidis serogroup B (Men B) is a key requirement for the development of an effective vaccine. This study was designed to investigate the antigen specificity of murine IgG1 and IgG2b antibodies induced by different primary immunization schedules and the booster dose with the Cuban Men B vaccine. Immunoblotting analyses were performed using outer membrane vesicles (OMV) from the vaccine strain (B:4,7:P1.19,15). IgG subclasses binding to PorA, PorB and RmpM were determined by digital scanning of the immunoreactive bands. Bactericidal antibody response after vaccination was also evaluated. The results indicated that IgG2b anti-PorA was the main antibody response induced by two doses of the vaccine. A primary series of three doses was found important for increasing IgG2b as well as IgG1 to PorB and RmpM. The fourth dose favoured the recognition of RmpM as detected by the increase of specific IgG1 and IgG2b. IgG subclasses anti-PorA did not change significantly if animals received two, three or four doses of the vaccine during the primary immunization or after the booster dose for all vaccine groups. The booster response to PorB and RmpM of groups BC2 and BC3 showed a significant increase in IgG2b levels compared with the primary response. However, the recall and the primary response of group BC4 were similar, suggesting a saturated dose-effect response after four doses of vaccine. The same was seen for bactericidal antibody response when human complement source was used in the assay.     

Adjuvants Influence the Immunoglobin Subclass Distribution of Immune Responses in Vivo

Mice were immunized against fluorescein isothiocyanate (FITC)-labelled human gamma globulin (HGG) in the absence or presence of different adjuvants. The immune response was assayed every other day with regard to both total Ig-secreting cells and FITC-specific plaque-forming cells (PFC). The adjuvants influenced the type of immune response induced to the same antigenic determinant. Thus, addition of Freund's complete (FCA) or incomplete (FIA) adjuvant preferentially led to the secretion of IgG1 PFC of an average high affinity. Most newly appearing IgG-secreting cells were also detected as FITC-specific PFC. The use of lipopolysaccharide (LPS) as an adjuvant resulted in the induction of both IgM and IgG, particularly of the IgG3 and IgG2b subclasses. However, these antibodies had relatively low affinity, and a large number of total IgG-secreting cells induced by LPS had no detectable FITC specificity. The FCA/FIA- and LPS-induced responses to FITC-HGG were additive when injected together, indicating that they act on distinct subpopulations of B lymphoid cells. The adjuvant response to LPS, but not the response to FCA/FIA, was totally absent in mice of the C3H/Hej strain, which are non-responders to the polyclonal activating properties of LPS. Finally, the response induced by FCA or FIA was T-cell-dependent and the LPS response T-cell-independent as assayed in nude mice.     

Regulation of Isotype Immunoglobulin Production by Adjuvants in Vivo

Mice were immunized against fluorescein isothiocyanate (FITC)-labelled human gamma globulin (HGG) or dextran sulphate (DXS) in the absence or presence of different adjuvants. The immune response was assayed as the total Ig-secreting cells and FITC-specific plaque-forming cells (PFC) found in various lymphoid organs. The adjuvants influenced the isotype of antibodies produced to the same antigenic determinant. The PFC of different IgG subclasses were favoured by different adjuvants. The IgG3 isotype was produced mainly after immunization with either antigen and lipopolysaccharide (LPS) or Li salt as adjuvant; IgG1 was produced with incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), alum, poly I:C, Quil A, Be salt, and poly A:U. Some of the above adjuvants (Be salt and poly A:U) favoured the production of IgG2b, and others (CFA, alum, Quil A, and poly I:C) favoured the IgG2a isotype besides the main isotype. Attempts were made to correlate the activation by the various adjuvants of certain TH subtypes with the isotypes produced.     

Agonists of Toll-like receptors 3, 4, 7, and 9 are candidates for use as adjuvants in an outer membrane vaccine against Neisseria meningitidis serogroup B

The bacterium Neisseria meningitidis is the causative agent of meningitis and sepsis. A generally effective vaccine against N. meningitidis serogroup B is not yet available, but outer membrane vesicle vaccines are in development. These vaccines contain lipopolysaccharide (LPS). The inclusion of N. meningitidis wild-type LPS in a vaccine is controversial because of its high toxicity. Therefore, the adjuvant activity of a panel of different Toll-like receptor (TLR) agonists in combination with LPS-deficient meningococcal outer membrane complexes was compared after immunization of mice. The results demonstrate that TLR3, TLR4, TLR7, and TLR9 agonists enhance immune responses against LPS-deficient outer membrane complexes. Their adjuvant activity was characterized by higher levels of antigen-specific immunoglobulin G (IgG), IgG2a, and IgG2b; a higher IgG2a/IgG1 ratio; lower total IgE levels; and most importantly, higher serum bactericidal antibody titers compared to LPS-deficient outer membrane complexes alone.     

IgG subclass antibodies to serogroup B meningococcal outer membrane antigens following infection and vaccination

IgG and IgG subclass antibodies to the outer membrane antigens from Neisseria meningitidis (serogroup B, serotype 15:P1.16) were quantitated by an enzyme-linked immunosorbent assay (ELISA) in sera from 40 patients with group B:15:P1.16 meningococcal disease and 24 volunteers immunized with a serotype 15:P1.16 outer membrane vesicle vaccine. A second injection was given 6 weeks after the first immunization. Patient sera obtained two and six weeks after onset of the disease had significantly higher levels of total IgG, IgG1, IgG2, and IgG3 antibodies to the outer membrane antigens than acute sera, convalescent sera from patients with systemic non-meningococcal bacterial infections and sera from healthy controls. The levels of total IgG and IgG1 remained high one and three years later. Sera from the vaccinees showed high levels of total IgG and IgG1 6, 12 and 26 weeks after the first immunization and high levels of IgG3 6 weeks after the second immunization. No increase of IgG2 or IgG4 levels was observed in the postimmunization sera. Immunoblotting of three convalescent sera demonstrated individual patterns of IgG subclass binding to various outer membrane antigens with most distinct binding of IgG1 and IgG3 antibodies to the class I protein, the H.8 lipoprotein and the lipopolysaccharide. Since IgG1 and IgG3 are the most effective antibodies for complement activation and phagocytosis, group B meningococcal disease and immunization with the serotype 15:P1.16 outer membrane vesicle vaccine stimulate production of those IgG subclasses which have the strongest opsonic and bactericidal activity.     

Synovial Fluid from Rheumatoid Arthritis Patients Induces Polyclonal Antibody Formation in Vivo

Our previous studies demonstrated the presence of a T-cell replacing factor in the synovial fluid (SF) of patients with rheumatoid arthritis (RA) and that RA-SF can activate, selectively, the induction of IgG2b antibody secreting cells in lipopolysaccharide (LPS)-pretreated mouse spleen cell cultures. In the present study the effect of RA-SF was tested in vivo in mice. Injection of the polyclonal activator LPS induced the production of IgM and IgG3 secreting cells in normal mice. However, the addition of RA-SF led to a selective increase in the production of IgG2b with a peak response on day 5 and IgG1 plaque-forming cells (PFC) with a peak on day 7. Neither the IgG2b nor IgG1 responses were caused by specific immunity against heterologous proteins present in RA-SF, as injection of in vitro inactive RA-SF samples did not induce PFC. The effect on B cells of RA-SF was further evaluated by injection of RA-SF in combination with LPS to the Xid B-cell deficient CBA/N mice. RA-SF had identical effects in CBA/N as in normal mice. The biological implication of these findings is discussed. Our earlier results support the idea that B cells are endogenously activated in RA patients. We have speculated that this activation is caused by the B-cell differentiation factor which is present in SF. Therefore, we also tested whether RA-SF could influence antibody-forming cells in mice that spontaneously develop autoimmunity. We found that injection of RA-SF alone, in the absence of any other activating substance, induced a very marked increase of IgG producing cells in (NZW x NZB) F1 hybrid mice. From a relatively high background level the RA-SF could still induce an up to 100-fold increase in the numbers of PFC in spleens of such mice.     

Lipopolysaccharide- and cholera toxin-specific subclass distribution of B-cell responses in cholera.

The immunoglobulin subclass responses to homologous lipopolysaccharide (LPS) and to cholera toxin (CT) in adult patients infected with Vibrio cholerae O1 and V. cholerae O139 were studied. LPS-specific antibody-secreting cells (ASC) of both the immunoglobulin A1 (IgA1) and IgA2 subclasses were seen, with the IgA1 ASC response predominating in both V. cholerae O1- and O139-infected patients. For antibodies in plasma, by day 11 after onset of disease, all V. cholerae O1- infected patients responded to homologous LPS with the IgA1 subclass (P = 0.001), whereas fewer (68%) responded with the IgA2 subclass (P = 0.007). About 89% of V. cholerae O139-infected patients responded with the IgA1 subclass (P = 0.003), and only 21% responded with the IgA2 subclass (not significant [NS]). Both groups of cholera patients showed significant increases in LPS-specific IgG1, IgG2, and IgG3 antibodies in plasma. In feces, the response to homologous LPS occurred in both groups of patients with the IgA1 and IgA2 subclasses, with 55 to 67% of patients showing a positive response. V. cholerae O1- and O139-infected patients showed CT-specific ASC responses of the different IgG and IgA subclasses in the circulation, and the pattern followed the order IgG1 > IgA1 > IgG2 > IgA2, with low levels of IgG3 and IgG4 ASC. Plasma anti-CT antibody responses in all subclasses were seen by day 11 after onset of disease. Although there were no increases in CT-specific ASC of the IgG3 (NS) and IgG4 (NS) subtypes, there were significant increases of these two subclasses in plasma (P 相似文献   

Correlation between Specific Immunosuppression and Polyclonal B Cell Activation Induced by a Protein Secreted by Streptococcus mutans

The relationship between polyclonal B cell activation and immunosuppressor effects induced by F5'EP-Sm, a non-cytotoxic protein secreted by Streptococcus mutans , was studied in C57BL/6 mice. Mice created with F5'EP-Sm exhibited a considerable increase in splenic nonspecific Ig plaque-forming cells (PFC) compared with untreated mice. The isotypic pattern of non-specific PFC responses favours IgG2a∼IgG2b>IgG3>IgG1∼IgM, when taken as a ratio between treated and untreated animals. When F5'EP-Sm was administered 2 days before immunization with sheep red blood cells (SRBC), the non-specific PFC production was accompanied by an ephemeral increase in specific PFC against SRBC 1 day after immunization, which was quickly replaced by a strong immunosuppression. In contrast, when F5'EP-Sm was injected after priming, there was little or no demonstrable suppression of specific PFC, and the increase of non-specific PFC was much less evident. The kinetic curves representing increase or decrease in relation to controls of specific and non-specific PFC are almost mirror images in each of the isotypes. The in vivo suppressor effect was abrogated in thymectomized mice, although the involvement of the T cell compartment is probably secondary to the B cell mitogen effect, since T-depleted spleen cells proliferate and synthesize non-specific Ig when stimulated in vitro with F5'EP-Sm.     

Human antibody responses to bacteriophage phi X 174: sequential induction of IgM and IgG subclass antibody

We studied the appearance of antigen-specific immunoglobulin classes and IgG subclasses in normal adult human subjects in response to primary, secondary, and tertiary immunization with the T-cell-dependent neo-antigen bacteriophage phi X 174. To complete the study we developed a sensitive, specific, and reproducible ELISA assay which was closely comparable to the widely used neutralization assay for total antibody (r = +0.97) and for IgG antibody (r = +0.93), and reasonably comparable for IgM antibody (r = +0.76). We confirmed that the initial response to primary immunization was predominantly, but not exclusively, IgM antibody. The secondary and tertiary responses demonstrated memory, amplification, and switch from IgM to IgG antibody. There was an orderly appearance of phage-specific IgG subclasses. IgG3 and IgG1 antibodies appeared 2 to 6 weeks after primary immunization. In all subjects there was a marked increase in IgG1 and IgG3 antibody after secondary immunization, and IgG2 antibody followed closely; IgG4 antibody appeared in some subjects. IgM antibody persisted in significant amounts (approx 50%) throughout the secondary response period. Following tertiary immunization, IgG1, IgG2, and IgG3 antibody consistently increased, and IgG4 antibody appeared in all subjects; IgG1 antibody predominated. Low levels of IgM antibody (approx 1% of total) persisted during the tertiary response. The persisting antibody on long-term follow-up (median 4 years after immunization) was virtually all (greater than 90%) IgG1.     

Analysis of IgG subclass production in cell cultures from IgA deficient patients and in normal controls as a function of age

IgA deficient individuals may also have low serum levels of IgG subclasses, especially IgG2. In the present study we examined the development of plasma cells producing IgM, IgA or IgG, and the IgG1 and IgG2 subclasses, following lipopolysaccharide (LPS) and pokeweed mitogen (PWM) stimulation of mononuclear cells (MNC) from normal and IgA deficient individuals as a function of age. Studies of blood MNC from 38 normal donors (age range 2-44 years) revealed an age-related distribution pattern of mu, gamma, alpha, gamma 1 and gamma 2 plasma cells produced in mitogen-stimulated and control cultures. Decreased IgA responses to both LPS and PWM were consistently observed in cultures of MNC from all of the nine children with IgA deficiency. When compared with age-matched controls the IgG response was also diminished in PWM stimulated cultures, whereas the IgM responses were normal. The IgG deficit was due to reduced responses for the gamma 1 and gamma 2 subclasses, and was most pronounced for IgG2; IgG2 plasma cell differentiation was particularly depressed in LPS cultures. In contrast to normal adult cells, blood MNC from the nine children with IgA deficiency and age-matched controls (2-17 years) yielded more IgG1 than IgG2 plasma cells in both control and LPS cultures, while the pattern of response to PWM was similar in all groups (gamma 1 greater than gamma 2). A good concordance was found between the level of secreted Ig in the culture supernatants and the relative number of IgM or, IgG and IgA plasma cells identified by immunofluorescence staining of cytoplasmic immunoglobulins.     

IgG2b-dependent down regulation of the LPS-induced PFC-response and its blockade by Fc gamma 2bR protein

Fc gamma receptor (Fc gamma R)-dependent immunoregulation by murine heat-aggregated (HAgg) IgG subclasses on the bacterial lipopolysaccharide (LPS)-induced plaque forming cell (PFC) response to trinitrophenylated sheep red blood cell (TNP-SRBC) antigen and the competitive effect by Fc gamma 2bR-protein on the down regulation by HAgg-IgG2b were studied in murine T-cell-deprived spleen cell cultures. HAgg-IgG1 and HAgg-IgG3 enhanced the PFC response, but HAgg-IgG2b strongly suppressed the LPS-induced PFC response. HAgg-IgG1 could not compete with the suppressive effect of HAgg-IgG2b. The HAgg-IgG2b seemed to act on both macrophages (M phi) and B-cells, because the cell cultures that had been reconstituted with HAgg-IgG2b-pretreated M phi and untreated B-cells and vice versa showed poor PFC responses. The suppression induced by HAgg-IgG2b on the LPS-induced PFC response in the T-cell-deprived cultures was abolished by the addition of phospholipase C (PLC)-treated Fc gamma 2bR protein at the early stage of the culture. The mechanisms by which HAgg-IgG2b suppress the LPS-induced PFC response and PLC-treated Fc gamma 2bR protein restores this response were discussed.     

Human immunoglobulin G subclass immune response to outer membrane antigens in meningococcal group B vaccine

The immunoglobulin G (IgG) subclass distribution of antibodies against the major outer membrane proteins from serotype 2a Neisseria meningitidis in human vaccinees was studied by immunoblotting. The volunteers received two doses of a noncovalent complex of group B polysaccharide and outer membrane material from the same meningococcal strain. Six weeks after the first vaccination the antibodies mounted against the class 1 and 5 proteins belonged mainly to the IgG1 and IgG3 subclasses. However, the binding of IgG3 to the class 5 proteins decreased markedly in serum samples taken after 25 weeks. Antibody binding to the serotype-specific class 2 protein was dependent on renaturation of the antigen by a dipolar ionic detergent (R. E. Mandrell and W. D. Zollinger, J. Immunol. Methods 67:1-11, 1984). The immune response against this protein showed more individual variation and consisted of IgG1 or IgG3 or both, often combined with IgG4.     

Enhanced Th2 immunity after DNA prime-protein boost immunization with amyloid beta (1-42) plus CpG oligodeoxynucleotides in aged rats

Generation and accumulation of fibrillar amyloid beta (Abeta) is widely considered as the pathogenic basis of neurodegeneration in Alzheimer's disease (AD). Both active immunization with fibrillar Abeta and passive immunization with anti-Abeta antibodies in transgenic mouse models of AD result in prevention/dissociation of Abeta plaque formation and restoration of cognitive functions. However, similar immunization studies in humans had to be halted because 6% of the AD patients developed acute meningoencephalitis, likely due to anti-Abeta specific autoimmune Th1 cells. Hence, making Abeta immunotherapy successful requires production of strong antibody responses without Th1-type immunity. In an attempt to develop safer vaccines, we examined the influence of oligodeoxynucleotides as adjuvant on the Th1 and Th2 immune response to Abeta in aged rats. We further investigated whether a DNA prime-protein boost strategy could elicit a more robust Th2 response. The results of the present study showed that all the animals injected with either Abeta peptide alone or Abeta encoding plasmid alone or plasmid DNA prime followed by peptide boost have elicited specific anti-Abeta antibodies. When co-administered, synthetic oligodeoxynucleotides (ODN) further enhanced the anti-Abeta titres. More importantly, the IgG subclasses of the antibodies generated by DNA prime-peptide boost regimen with ODN as adjuvant were primarily of IgG2b and IgG1 isotypes, suggesting that heterologous immunization strategy along with ODN would be advantageous in eliciting more beneficial Th2-type humoral immune response.     

Changes of tetanus specific IgG, IgM and IgG subclasses after DPT vaccination

We evaluated tetanus specific IgG, IgM, IgG subclasses after DPT vaccination in infants and children. Tetanus toxoid specific IgG, IgM IgG subclasses were measured to characterize the isotope profile of antibody against tetanus toxoid. The values of the tetanus specific IgG in the positive group were significantly increased compared to those of the control group, and were significantly increased after two inoculation. Tetanus specific IgG was very low in adults and neonates. In our tetanus specific IgG subclasses study, forty-five of 56 cases (80%) showed predominantly IgG1 antibody responses to tetanus toxoid, while twenty-five of 56 cases (45%) showed IgG4 responses. Both IgG1 and IgG4 responses were demonstrated in 17 cases (30%). So we suggest that IgG was mainly involved in humoral immune response after DPT vaccination, and IgG1 may play an important role among IgG subclasses. IgG4, alone or together with IgG1, can also play a role in immune response to tetanus toxoid.     

Prior exposure to subimmunogenic amounts of some bacterial lipopolysaccharides induces specific immunological unresponsiveness.

Pretreatment (priming) of BALB/c mice with a low (subimmunogenic) dose of Escherichia coli O113 lipopolysaccharide (LPS) generates immunological memory 7 to 30 days later; the direct (immunoglobulin M) plaque-forming cell (PFC) responses produced after subsequent immunization with an optimal dose are 4 to 20 times greater than those of unprimed mice. By contrast, priming with a low dose of E. coli O55 LPS, followed by immunization with an optimally immunogenic dose 2 to 30 days later, resulted in a significantly reduced antibody response. Similar results were obtained with Serratia marcescens LPS. Dose-response studies indicated that such unresponsiveness is antigen specific and could be induced with subimmunogenic amounts of LPS. Priming reduced the magnitude of the PFC response to all immunizing doses of LPS tested. Unresponsiveness is not due to (i) an alteration in the time course of the PFC response or to (ii) a change in the isotype of the anti-LPS antibody produced after priming and immunization.     

Physical linkage of naturally complexed bacterial outer membrane proteins enhances immunogenicity

The outer membrane proteins (OMPs) of bacterial pathogens are essential for their growth and survival and especially for attachment and invasion of host cells. Since the outer membrane is the interface between the bacterium and the host cell, outer membranes and individual OMPs are targeted for development of vaccines against many bacterial diseases. Whole outer membrane fractions often protect against disease, and this protection cannot be fully reproduced by using individual OMPs. Exactly how the interactions among individual OMPs influence immunity is not well understood. We hypothesized that one OMP rich in T-cell epitopes can act as a carrier for an associated OMP which is poor in T-cell epitopes to generate T-dependent antibody responses, similar to the hapten-carrier effect. Major surface protein 1a (MSP1a) and MSP1b1 occur as naturally complexed OMPs in the Anaplasma marginale outer membrane. Previous studies demonstrated that immunization with the native MSP1 heteromer induced strong immunoglobulin G (IgG) responses to both proteins, but only MSP1a stimulated strong CD4⁺ T-cell responses. Therefore, to test our hypothesis, constructs of CD4⁺ T-cell epitopes from MSP1a linked to MSP1b1 were compared with individually administered MSP1a and MSP1b1 for induction of MSP1b-specific IgG. By linking the T-cell epitopes from MSP1a to MSP1b1, significantly higher IgG titers against MSP1b1 were induced. Understanding how the naturally occurring intermolecular interactions between OMPs influence the immune response may lead to more effective vaccine design.     

Local and systemic antibody responses in mice immunized intranasally with native and detergent-extracted outer membrane vesicles from Neisseria meningitidis

The mouse humoral immune response toward native or detergent-extracted outer membrane vesicles (NOMVs and DOMVs, respectively) from Neisseria meningitidis was determined after intranasal immunization. Both preparations elicited high frequencies of NOMV-specific antibody-forming cells (AFCs) locally in the nasal associated lymphoid tissue (NALT) after three or four weekly doses. The diffuse NALT (D-NALT) contained ca. 10-fold more NOMV-specific AFCs than those observed in the mediastinal lymph node, spleen, and bone marrow. AFCs observed in the D-NALT were primarily immunoglobulin A positive (IgA(+)) and were maintained for at least 1 month. In contrast, the organized NALT (O-NALT) contained low numbers of AFCs, and the response was relatively short-lived. In other lymphoid tissues, AFCs producing various IgG subclasses and IgM were present with IgG2b-producing AFCs being dominant or codominant with IgA or IgG2a. In serum and in all of the tissues examined, with the exception of the NALT, NOMVs clearly induced a stronger antibody response and a broader range of antibody isotypes than DOMVs. The development of NOMV-specific AFCs in spleen and bone marrow after intranasal immunization was slow compared to intravenous immunization but, once established, the intranasally elicited responses increased steadily for at least 75 days. NOMV-specific antibodies induced via several routes of immunization had high bactericidal activities in serum. Our results indicated that intranasally administered OMVs induced strong local and systemic antibody responses in mice that were relatively long-lived.     

Stimulation of secretory antibodies against Bordetella pertussis antigens in the lungs of mice after oral or intranasal administration of liposome-incorporated cell-surface antigens

Lipopolysaccharide (LPS) and outer membrane protein (OMP) preparations of Bordetella pertussis were incorporated into multilamellar liposomes composed of soya bean-derived phospholipids which were then used for oral and intranasal immunization of mice. Specific antibody responses of animals immunized by either route were measured in lung washes. A specific IgA response to LPS was detected after immunization with the OMP-containing liposomes but not with the LPS-containing liposomes, indicating adjuvant activity of the proteins. The OMP-containing liposomes were significantly more effective in inducing immune responses than the OMP preparation alone. Responses were highest when mice were given a booster 30 days after primary immunization. Maximum responses occurred 20 days after the booster but specific antibody was still detected 75 days after the secondary immunization. These results suggest that this liposome antigen delivery system has potential in stimulating secretory antibody responses which may be helpful in protecting against infection from B. pertussis.