Mouse antibody response to group A streptococcal carbohydrate

In an attempt to more fully understand the generation of antibody diversity to carbohydrate (CHO) Ag, we produced and characterized a panel of hybridoma cell lines specific for group A streptococcal CHO from mice injected with the intact bacteria (minus the hyaluronic acid capsule and cell wall protein Ag). We have analyzed the use of H and L chain V region genes in the early (day 7) and late response (hyperimmune) and have sequenced the dominant VH gene used in several of our hybridomas. Our data allowed us to assess the extent to which the recombination of various V, D, and J gene segments and somatic mutation contribute to antibody diversification in this system. In this report we confirm that a minimum of two VH and four VK gene segments are used to encode this response. We extend this analysis to show that multiple D and J gene segments are used and that a significant amount of junctional variability is tolerated in CDR 3. Our results indicate that the level of somatic mutation in the hyperimmune response is generally low in comparison with the response to haptens and protein Ag. These data also suggest that there is a positive selection for mutation in CDR 1 during the hyperimmune response to group A streptococcal CHO.     

Genetic influences on the immune response of rats to streptococcal A carbohydrate

Selective breeding of Sprague-Dawley rats immunized with group A streptococcal vaccine (GASV) revealed genetic influences on the magnitude of the precipitin response to streptococcal group A carbohydrate (SACHO). After four brother-sister inbreedings, a family of low-precipitin responders (LPR) was segregated from a family of high-precipitin responders (HPR). Precipitin analysis of all four generations of rats bred for low-precipitin responses revealed an earlier influence of inbreeding on the secondary as compared to the primary precipitin response. Prolonged immunization and/or variation in the dose of antigen failed to enhance the magnitude of the precipitin response of LPR. Examination of anti-SACHO antisera for cross-specificity revealed A-variant carbohydrate precipitins in only 1%. This system may offer an opportunity to examine the clinical relevance of anti-SACHO antibodies to rheumatic heart disease.     

Human immune response to group A streptococcal carbohydrate (A-CHO). II. Antigen-independent stimulation of IgM anti-A-CHO production in purified B cells by a monoclonal anti-idiotopic antibody

The paper describes the induction by a monoclonal anti-idiotopic antibody (anti-Id mAb) of specific antibody production to group A streptococcal carbohydrate (A-CHO) in purified human B cells of several unrelated individuals. The anti-Id mAb, designated 16F498 (anti-Id498), recognizes a recurrent idiotope (Id 498) associated with the combining site of human antibodies to N-acetyl-D-glucosamine (GlcNAc), the immunodominant group of A-CHO. Id498 is expressed on IgM anti-GlcNAc antibodies but does not occur on IgG antibodies with the same specificity. It occurs also on a minor population of IgM antibodies without specificity for A-CHO. Id498 was found in 19 of 27 sera from unselected healthy donors and thus seems to be frequently expressed within the adult B cell repertoire. The in vitro induction of anti-A-CHO antibodies was analyzed in human B cells extensively depleted for T cells. Specific antibody secretion required cross-linked anti-Id which was achieved by coupling the mAb to agarose beads. No antibody secretion could be induced by soluble anti-Id (1 and 10 micrograms/ml). An optimal response required soluble T cell-derived factors which were added as a mixture of recombinant interleukin 2 with a T cell hybridoma supernatant that augments B cell growth and differentiation. Under these conditions an antigen-independent specific increase of IgM anti-A-CHO production (2.6- to 10-fold, or up to 2000 ng/ml respectively) could be induced in blood B cell populations of four of six normal individuals expressing the Id498 at serum level.     

Preparation of monoclonal antibodies to nuclear DNA of mammalian cells by immunization with group A streptococcal polysaccharide conjugated with synthetic polyelectrolytes

Monoclonal antibodies (MAb) were obtained by hybridization of spleen cells from BALB/c mice immunized with streptococcal group A polysaccharide (A-PS) conjugated with synthetic polyelectrolytes (PEL). These MAb reacted with nuclei from human and mouse cells. MAb reacting with nuclei were obtained after prolonged immunization with conjugates and were not formed by hybridization of spleen cells from non-immunized mice or by the immunization with PEL. The investigation of Mab (B1/2 and A5/2) reacting with nuclei has shown that these Mab are directed against DNA and do not react with other tissue substances. No cross-reactions of Mab with A-PS used for immunization have been revealed. Mab B1/2 and A5/2 belong to autoantibodies.     

Modulation of lymphocyte functions by group A streptococcal membrane

Protoplast membranes isolated from group A streptococci suppress functions of mouse B cells in vivo and in vitro. Intraperitoneal injection 24 or 72 hr (but not 12 hr) before collection of lymphoid cells results in a selective decrease in the mitogenic response of bone marrow cells to dextran sulfate (DS). The response of bone marrow cells to lipopolysaccharide (LPS), and spleen cells to both DS and LPS, is unaltered. In vitro exposure of lymphocytes to membranes concomitantly with mitogen reduces the response to both DS and LPS, however, the DS response is more susceptible to low doses of membrane. Suppression of the response to DS in vitro is not mediated by cells bearing Thy 1.2 antigen. Neither the phytohemagglutinin (PHA)-responsive cells nor the adherent cells participate in suppression of the LPS response in vitro. In contrast to the suppression of B-cell functions neither the PHA nor concanavalin A (Con A) response of mouse bone marrow, spleen, or thymus cells is altered by streptococcal protoplast membranes injected 24 hr before collection of cells. In vitro exposure of spleen cells to a limited range of concentrations of membrane results in an enhanced proliferative response of spleen cells stimulated by suboptimal doses of PHA. This synergism is not mediated by the adherent cells. Addition of membranes to spleen cell cultures in vitro has no effect upon the response of spleen cells to suboptimal doses of Con A or to optimal doses of either Con A or PHA. Higher concentrations of membranes reduce the proliferative response of both control and mitogen-stimulated cells. This nonselective suppression by high doses of membranes is not due to toxicity. Delayed hypersensitivity to sheep erythrocytes is potentiated by injection of membranes. These studies suggest that streptococcal membranes preferentially suppress the immature B cells and enhance certain T-cell functions.     

Human immune response to multiple injections of murine monoclonal IgG

Murine monoclonal antibody infusions in humans should induce a human anti-mouse immunoglobulin (mIgG) immune response, especially if multiple infusions over an extended period of time are necessary for therapeutic efficacy. We have administered multiple infusions of the murine monoclonal antibody T101 to patients with cutaneous T cell lymphoma (CTCL) or chronic lymphocytic leukemia (CLL). Five of 10 CTCL patients, compared with zero of six CLL patients, developed antibodies to mIgG. In those CTCL patients who did not demonstrate anti-mIgG antibodies, we were unable to correlate the lack of response to any of a large number of clinical parameters. Anti-mIgG antibodies were of both the mu and gamma isotypes and were detectable 14 days after the first infusion. Multiple infusions were associated with elevated titers. The anti-idiotypic portion of the anti-mIgG titer steadily increased with each infusion until eventually, in one patient receiving eight weekly infusions, well over one-half the serum anti-mIgG recognized only T101 and not four other murine IgG2AK antibodies tested. To increase our confidence in these findings, four separate assay systems were used to make these determinations. The identification of anti-idiotype antibodies as the dominant species of the immune response to multiple infusions of murine monoclonal antibody has major implications for future work with monoclonal antibodies. Although it has been suggested that human monoclonal antibodies would obviate an immune response, our work implies that such antibodies might still induce anti-idiotype antibodies if multiple infusions are administered.     

Serologic and topographic characterization of idiotopes on murine monoclonal anti-streptococcal group A carbohydrate antibodies

We have employed five spectrotypically distinct monoclonal anti-variable region antibodies in the definition and characterization of a set of idiotopes expressed on murine monoclonal antibodies specific for streptococcal group A carbohydrate (GAC). By evaluating which of a panel of monoclonal anti-GAC antibodies were bound by the various anti-idiotopes, we observed four distinct reactivity profiles for the five anti-idiotopes ranging from highly restricted (binding of the homologous anti-GAC monoclonal antibody only) to broadly cross-reactive (binding of 18 of the 38 IgG3 anti-GAC antibodies). With N-acetyl-D-glucosamine and soluble GAC used as haptens, this spectrum of reactivity profiles was paralleled by a gradient of susceptibility to hapten inhibition of anti-idiotope binding to idiotope. The degree of cross-reactivity exhibited by a given anti-idiotope was found to be inversely related to its susceptibility to hapten inhibition. The topographic relationships among the idiotopes, defined by the results of competitive binding assays, were suggestive of a linear idiotope map spanning the variable region from the antigen-binding site to the vicinity of the constant region. Additional data from competitive inhibition assays with isolated and recombined H and L chains from a prototype monoclonal anti-GAC antibody (HGAC 39), and from isoelectric focusing of whole or reduced and alkylated HGAC 39, suggested that one of the idiotopes was located, at least primarily, on the VL domain.     

Monoclonal autoantibodies to different epithelial structures of the thymus gland obtained by the immunization with streptococcal group A antigens

By the indirect immunofluorescence method it was shown to which epithelial thymus structures monoclonal antibodies (mAT) reacting with the different epidermal structures are directed. These mAT related to the autoantibodies were obtained earlier, as a result of lymphoid cells polyclonal activation, by the immunization of BALB/c mice with streptococcal group A nonspecific protein antigens of the cell wall. It was shown that mAT A6/1, reacting with the basal layer of the skin epithelium are directed to the epithelium of the cortical and medullar thymus zones, which is regarded as the so called endocrinal epithelium. These mAT, by the study with immunoblotting method, react with the protein of mV SOkD, B5/1 mAT to the skin epithelium, on the thymus sections react with the single cells around the Hassel bodies.     

Production of hybridomas synthesizing monoclonal antibodies to streptococcal group A polysaccharide

Nine stable hybridomas synthetizing monoclonal antibodies to the antigenic determinants of polysaccharide A of group A streptococcus were obtained. Three monoclonal antibodies possessed precipitating properties. The formation of hybridomas was found to be influenced by the presence of immune splenocytes and the standard conditions of cell fusion. The highest yield of hybridomas was observed under the conditions ensuring the growth of cell in 80-100% of the wells. Rapid and specific screening was found to be an important stage in obtaining hybridomas.     

Directing the immune response to carbohydrate antigens

Peptide mimetics may substitute for carbohydrate antigens in vaccine design applications. At present, the structural and immunological aspects of antigenic mimicry, which translate into immunologic mimicry, as well as the functional correlates of each, are unknown. In contrast to screening peptide display libraries, we demonstrate the feasibility of a structure-assisted vaccine design approach to identify functional mimeotopes. By using concanavalin A (ConA), as a recognition template, peptide mimetics reactive with ConA were identified. Designed peptides were observed to compete with synthetic carbohydrate probes for ConA binding, as demonstrated by enzyme-linked immunosorbent assay and isothermal titration calorimetry (ITC) analysis. ITC measurements indicate that a multivalent form of one particular mimetic binds to ConA with similar affinity as does trimannoside. Splenocytes from mimeotope-immunized mice display a peptide-specific cellular response, confirming a T-cell-dependent nature for the mimetic. As ConA binds to the Envelope protein of the human immunodeficiency virus, type 1 (HIV-1), we observed that mimeotope-induced serum also binds to HIV-1-infected cells, as assessed by flow cytometry, and could neutralize T-cell line adapted HIV-1 isolates in vitro, albeit at low titers. These studies emphasize that mimicry is based more upon functional rather than structural determinants that regulate mimeotope-induced T-dependent antibody responses to polysaccharide and emphasize that rational approaches can be employed to develop further vaccine candidates.     

Modulation of the immune response to the severe acute respiratory syndrome spike glycoprotein by gene-based and inactivated virus immunization

Although the initial isolates of the severe acute respiratory syndrome (SARS) coronavirus (CoV) are sensitive to neutralization by antibodies through their spike (S) glycoprotein, variants of S have since been identified that are resistant to such inhibition. Optimal vaccine strategies would therefore make use of additional determinants of immune recognition, either through cellular or expanded, cross-reactive humoral immunity. Here, the cellular and humoral immune responses elicited by different combinations of gene-based and inactivated viral particles with various adjuvants have been assessed. The T-cell response was altered by different prime-boost immunizations, with the optimal CD8 immunity induced by DNA priming and replication-defective adenoviral vector boosting. The humoral immune response was enhanced most effectively through the use of inactivated virus with adjuvants, either MF59 or alum, and was associated with stimulation of the CD4 but not the CD8 response. The use of inactivated SARS virus with MF59 enhanced the CD4 and antibody response even after gene-based vaccination. Because both cellular and humoral immune responses are generated by gene-based vaccination and inactivated viral boosting, this strategy may prove useful in the generation of SARS-CoV vaccines.     

Immune response to superoxide dismutase in group A streptococcal infection

Extracellular localisation of manganese-dependent superoxide dismutase (SodA) by group A streptococcus (GAS) may have a role in protection of this pathogenic bacterium from exogenously produced reactive oxygen species. In this study we show that SodA is found both in surface protein extracts and in culture supernatants of GAS. To investigate whether SodA is a possible vaccine candidate outbred Quackenbush mice were subcutaneously vaccinated with recombinant SodA. Strong antibody responses which were moderately opsonic were elicited. These antibodies were unable to protect mice from intraperitoneal challenge with M1 GAS. We also show that SodA and p145 (a conserved peptide from the M-protein) antibodies are present at significantly higher levels amongst patients with rheumatic heart disease than in control subjects from the same endemic region. The higher SodA antibody levels in patients may be indicative of a role for this protein in pathogenesis of rheumatic heart disease but are more likely to be a marker of recent or recurrent streptococcal infection.     

A role for carbohydrate moieties in the immune response to malaria

Treatment of antigen prepared from asexual blood stages of the human malarial parasite Plasmodium falciparum with a mixture of glycosidases resulted in a reduction in the ability of the antigen to bind antibodies from immune human and monkey sera in an ELISA assay. Some of the epitopes in the parasite material were heat stable, protease resistant, and sensitive to glycosidases. Proteins of Mr 110,000 and 65,000 from parasitized RBC were shown to have reduced antigenicity in Western blots after glycosidase treatment. The carbohydrate side chains of parasite glycoproteins therefore make a contribution to the total antigenicity of the parasite.     

Role of group A streptococcal IgG Fc-receptor in induction of anti-IgG by immunization in the rabbit

Previous work has demonstrated that streptococcal IgG Fc-receptors (FcR) may trigger production of anti-IgG after immunization of rabbits with group A streptococci. This effect seemed dependent on in vitro binding of IgG, derived from the growth medium, to the vaccine strains. In the experiments presented here, IgG was eluted from streptococcal strains to be used for immunization of rabbits by 1 M KSCN and washing, a treatment which did not affect the capacity of the strains to bind newly added IgG. Using two IgG FcR-positive group A streptococcal strains (M-types 1 and 22) for intravenous immunization, anti-IgG was found in the sera of 26 out of 28 rabbits, examined 8 weeks after immunization. In contrast, anti-IgG was not induced in 16 rabbits receiving either group A, type T27 or group B, type Ia streptococci both of which lack surface FcR activity. Finally, immunization with purified streptococcal IgG FcR (0.35 mg, given subcutaneously combined with Freund's complete adjuvant and two weeks later intraconjunctivally without adjuvant) also induced anti-IgG. In all rabbits, anti-human rather than anti-rabbit IgG was detected. It is proposed that in vivo interaction between the bacterial FcR and rabbit IgG, resulting in conformation changes in IgG, is a prerequisite for the induction of anti-IgG. Thus, streptococcal triggering of anti-IgG, ascribable to IgG Fc-receptor activity and not requiring presence of foreign IgG, has been demonstrated in the rabbit.     

Role of group A streptococcal IgG Fc-receptor in induction of anti-IgG by immunization in the rabbit

Abstract Previous work has demonstrated that streptococcal IgG Fc-receptors (FcR) may trigger production of anti-IgG after immunization of rabbits with group A streptococci. This effect seemed dependent on in vitro binding of IgG, derived from the growth medium, to the vaccine strains. In the experiments presented here, IgG was eluted from streptococcal strains to be used for immunization of rabbits by 1 M KSCN and washing, a treatment which did not affect the capacity of the strains to bind newly added IgG. Using two IgG FcR-positive group A streptococcal strains (M-types 1 and 22) for intravenous immunization, anti-IgG was found in the sera of 26 out of 28 rabbits, examined 8 weeks after immunization. In contrast, anti-IgG was not induced in 16 rabbits receiving either group A, type T27 or group B, type Ia streptococci both of which lack surface FcR activity. Finally, immunization with purified streptococcal IgG FcR (0.35 mg, given subcutaneously combined with Freund's complete adjuvant and two weeks later intraconjunctivally without adjuvant) also induced anti-IgG. In all rabbits, anti-human rather than anti-rabbit IgG was detected. It is proposed that in vivo interaction between the bacterial FcR and rabbit IgG, resulting in conformation changes in IgG, is a prerequisite for the induction of anti-IgG. Thus, streptococcal triggering of anti-IgG, ascribable to IgG Fc-receptor activity and not requiring presence of foreign IgG, has been demonstrated in the rabbit.