Mechanism of antibody-specific deglycosylation and immune evasion by Streptococcal IgG-specific endoglycosidases
Beatriz TrastoyJonathan J. DuJavier O. CifuenteLorena RudolphMikel García-AlijaErik H. KlontzDaniel DeredgeNazneen SultanaChau G. HuynhMaria W. FlowersChao LiDiego E. SastreLai‐Xi WangFrancisco CorzanaÁlvaro MallagarayEric J. SundbergMarcelo E. Guerin
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Abstract:
Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-β-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications.Keywords:
Evasion (ethics)
Immunoglobulin (Ig)M and IgG antibodies, prepared in the rabbit against the protective antigen of Pseudomonas aeruginosa P4, were compared as to their biological activities in vitro and in vivo. In vitro biological activities of these antibodies were determined by passive hemagglutination, bactericidal, and opsonophagocytic tests. Increased effectiveness of IgM over IgG on a molar basis was demonstrated in all of these tests. However, in mouse protection tests, in which the purified globulins were injected intraperitoneally 4 hr prior to challenge with P. aeruginosa suspended in hog gastric mucin, IgM anticapsular antibody was found to be less effective than IgG antibody. The exact mechanism whereby IgG antibody exerts more protective ability than IgM antibody is still unknown. We present evidence to suggest that the difference in activity between the two classes of antibody is due to the ability of the IgG antibody to enter the bloodstream more rapidly than the IgM antibody and also to the ability of IgG to diffuse rapidly through the tissues of the organs.
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The neonatal Fc receptor (FcRn) plays an important role in regulating the serum half-lives of IgG antibodies. A correlation has been established between the pH-dependent binding affinity of IgG antibodies to FcRn and their serum half-lives in mice. In this study, molecular modeling was used to identify Fc positions near the FcRn binding site in a human IgG antibody that, when mutated, might alter the binding affinity of IgG to FcRn. Following mutagenesis, several IgG2 mutants with increased binding affinity to human FcRn at pH 6.0 were identified at Fc positions 250 and 428. These mutants do not bind to human FcRn at pH 7.5. A pharmacokinetics study of two mutant IgG2 antibodies with increased FcRn binding affinity indicated that they had serum half-lives in rhesus monkeys ∼2-fold longer than the wild-type antibody.
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The objective of this study was to quantitively evaluate SARS-CoV-2 antibody levels in currently availably immunoglobulin products.This study examined 142 unique lots of 11 different immunoglobulin products (both intravenous [IV] and subcutaneous formulations).The immunoglobulin lots were assessed for Ig-binding activities against severe acute respiratory syndrome coronavirus disease 2 (SARS-CoV-2) receptor binding domain, spike, and nucleocapsid proteins by ELISA assays. Additionally, functionality of 48 lots were assessed for their ability to inhibit several SARS-CoV-2 variants spike binding to angiotensin-converting enzyme 2 (ACE2).Of the 11 immunoglobulin products evaluated, 8 of them (72%) contained detectable anti-SARS-CoV-2 antibodies. Immunoglobulin products manufactured after the year 2020 (with expiration dates 2023–2024) had significantly higher antibody levels compared with products manufactured prepandemic (before 2020). Sixty percent of the products with expiration dates of 2023 and 85% of the products with expiration dates of 2024 contained antibodies to SARS-CoV-2 proteins. Immunoglobulin products with later dates of expiration were strongly associated with inhibition of ACE2-binding activity. The 10% products used for IV administration had greater inhibition of ACE2-binding activity than the 20% products used for SC administration.Immunoglobulin products with later expiration dates (2023–2025) had significantly higher antibody levels, binding, and inhibition activities against SARS-CoV-2 proteins when compared with prepandemic lots. These more recent immunoglobulin products may be therapeutically beneficial to patients who do not have a robust SARS-CoV-2 vaccine response.This is a large study of 11 different immunoglobulin products commonly used in patients with predominantly antibody deficiency. The more recent immunoglobulin products (expiration dates 2023–2025) not only contained detectable higher anti-SARS-CoV-2 antibody levels but also had higher binding and inhibition activities against SARS-CoV-2 proteins when compared with prepandemic product. Interestingly, the 10% IV immunoglobulin products had greater inhibition of ACE2-binding activity than the 20% SC immunoglobulins, which clinicians may want to consider when starting immunoglobulin replacement therapy in a patient with recurrent COVID-19 infection or poor vaccine response. This study was limited by a lack of consistency in number of lots of each product evaluated. Clinicians need to be aware that the more recent immunoglobulin products (expiration dates 2023–2025) may offer greater therapeutic effect and be especially beneficial to patients who lack adequate SARS-CoV-2 vaccine response.
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The porcine immunoglobulins M (IgM), A (IgA), and G (IgG) were isolated and purified and some of the properties of the porcine milk IgA were examined. Monospecific antisera which were prepared against these immunoglobulins in rabbits were then used to absorb a particular class of immunoglobulin from sow serum, colostrum, and milk in an attempt to identify the immunoglobulin classes of neutralizing antibodies to the porcine enteric virus, transmissible gastroenteritis (TGE). The results of these absorption studies suggest that in colostrum and milk from sows experimentally (orally) or naturally infected with live virulent TGE virus, IgA is the predominant immunoglobulin class of TGE antibodies. Both IgA and IgG TGE antibodies appeared to be present in the serum from these sows, but with IgG TGE antibodies predominating. In contrast, in the serum, colostrum and milk from sows vaccinated intramuscularly or intramammarily with live attenuated TGE virus, the TGE antibody activity was associated mainly with the IgG class of immunoglobulins. These results provide additional data indicating that the route of infection or vaccination markedly influences the immunoglobulin class of antibodies in colostrum and milk. Secondly, IgA antibodies in mammary secretions are probably essential for providing optimal passive immunity of nursing pigs against infection with TGE virus.
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Abstract Anti‐GM1 immunoglobulin G (IgG) antibodies are frequently present in sera from patients with Guillain–Barré syndrome (GBS). A previous report on a patient who had a neuropathy with immunoglobulin M (IgM) M‐protein binding to a conformational epitope formed by phosphatidic acid (PA) and gangliosides prompted us to investigate the binding of IgG antibodies in GBS sera to a mixture of GM1 and PA (GM1/PA). Of 121 GBS patients, 32 had anti‐GM1 IgG antibodies. All 32 also had antibody activity against GM1/PA. Twenty‐five (78%) of 32 patients had greater activity against GM1/PA than against GM1 alone. Twelve patients who had no anti‐GM1 IgG antibodies had IgG antibody activity against GM1/PA. No GBS patient had IgG antibody against PA alone. In contrast, two rabbit anti‐GM1 antisera had greater activity against GM1 alone than against GM1/PA. IgG antibody with greater binding activity against a mixture of GM1 and a phospholipid than against GM1 alone may have an important role in the pathogenesis of GBS and has implications for diagnosis. Muscle Nerve 27: 302–306, 2003
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Multifocal motor neuropathy
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An enzyme-linked immunosorbent assay (ELISA) for total antibodies to Toxoplasma gondii was modified to measure specific immunoglobulin M (IgM) antibodies. The assay requires three incubation periods totaling 2 h and enzyme-labeled-heavy-chain-specific antibodies to human IgM. The objective read-out in absorbance was normalized to percent of a standardized positive control for interpretations. No difference was observed between the assay results with or without previous absorption of the samples by Staphylococcus aureus protein A to remove most of the IgG antibodies. Addition of serum containing very high levels of IgG antibodies to another containing both IgG and IgM antibodies did not change the IgM assay values for the latter. None of the 22 sera containing high levels of IgM rheumatoid factor (RF) gave positive ELISA IgM results, even though 8 of them also had high levels of IgG toxoplasma antibodies. Mixtures of sera containing high concentrations of RF with sera having high levels of IgG toxoplasma antibodies also failed to show any false-positive reactions in the IgM toxoplasma assay. Thus, this ELISA for T. gondii IgM antibodies was not affected by IgG toxoplasma antibodies and RF.
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Serum obtained from normal human subjects contains antibodies reactive in an enzyme-linked immunosorbent assay with the glucuronoxylomannan (GXM) of Cryptococcus neoformans. The frequency of occurrence of class-specific antibodies among normal subjects was 28% for immunoglobulin G (IgG), 98% for IgM, and 3% for IgA. Anti-GXM antibodies with kappa light chains occurred in 98% of normal subjects, while the occurrence of lambda light chains was 28%. Each of five subjects with high levels of anti-GXM IgG antibodies had readily detectable antibodies of the IgG2 isotype; two of the five subjects had readily detectable IgG1 antibody. An examination of sera from human immunodeficiency virus-infected patients showed that human immunodeficiency virus infection was accompanied by a significant decrease in the occurrence of IgM antibodies and anti-GXM antibodies with kappa light chains; these decreases occurred early in infection when CD4 counts were still > or = 500 cells per microliter. A slight but not statistically significant decrease in the occurrence of anti-GXM IgG antibodies was seen only in patients with CD4 levels of < 200 cells per microliter. Sera from normal subjects with high levels of anti-GXM IgG antibodies were examined to identify any contribution of the antibodies to complement activation or to opsonization of the yeast cells. An analysis of the kinetics for activation and binding of C3 to the yeast cell showed no pattern of quantitative or qualitative differences between sera with high or low levels of anti-GXM IgG antibodies. Phagocytosis studies showed that the naturally occurring IgG antibodies did not contribute to opsonization of the yeast cells.
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The development of total immunoglobulin G (IgG) antibodies and antibodies of the four IgG subclasses in serum against Haemophilus influenzae type b capsular polysaccharide (CPS) was studied in 24 children and 11 adults with invasive Haemophilus influenzae type b infections, by using an enzyme-linked immunosorbent assay. None of the 8 children aged 10 months or younger had increases in the IgG class or in any of the IgG subclasses. In contrast, 14 of 16 children between 10 months and 6 years of age and 10 of 11 adults had significant increases in total IgG, IgG1, or IgG2 antibodies in various combinations, but none of them had increases in IgG3 or IgG4 antibodies. The increases in IgG1 and IgG2 antibodies in the children were of similar magnitudes. Of 11 adult patients, 9 had significant increases in IgG2 antibodies, while only 4 had increases in IgG1 antibodies. In conclusion, this study shows that children younger than approximately 1 year have no IgG response to H. influenzae type b CPS, while individuals above this age have a mixed IgG1 and IgG2 response.
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The development of immunoglobulin G (IgG) subclass-specific anti-Treponema pallidum antibodies during the course of syphilis in humans was studied with sera from 50 untreated male patients. The patients were divided into five diagnosis groups. In the fluorescent treponemal antibody test, which delineates the presence of cross-reacting antibodies, as well as specific antitreponema antibodies, IgG1, IgG2, and IgG3 subclass antibodies were already present during the seronegative primary stage. Specific antibodies, which were detected by the fluorescent treponemal antibody absorption test, were first present during the serotype-variable primary stage. These antibodies were almost exclusively of the IgG1 and IgG3 subclasses. In later stages, antibodies of other subclasses were detectable. Titration of IgG1 antitreponema antibodies in three electrophoretically different IgG fractions revealed an asymmetric distribution in these fractions during primary syphilis. The antibodies were largely confined to the most basic fraction during primary syphilis. A sudden change in the distribution was noted between the end of the primary stage and the secondary stage; an even distribution of IgG1 antitreponema antibodies existed in the late latent stage. These findings confirm and extend previous results from our laboratory. The development of antibodies detected by both tests is discussed in terms of a sequential stimulation of the immune system due to the presence of an extracellular layer covering the treponemas or, alternatively, in terms of a suppression of the immune response during early syphilis.
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To determine the effect of anti-immunoglobulin antibodies on the measurement of the humoral immune response in hepatitis C virus (HCV) infected patients. Anti-immunoglobulin antibodies were defined using sheep immunoglobulins as a target to characterize distinct changes in patterns of immunoglobulin levels. Serum immunoglobulin A, G and M concentrations were measured by ELISA in 45 patients with recent-onset HCV infection and 45 matched normal individuals. It was found that normal individuals had mean IgA, IgG and IgM levels of 2.67 mg/ml, 9.39 mg/ml and 1.77 mg/ml, respectively while HCV infected patients had mean levels of 3.19 mg/ml, 10.76 mg/ml and 1.94 mg/ml. These represented significant increases in immunoglobulin levels in the sera of HCV patients compared to normal individuals (p < 0.0001, p < 0.00004 and p < 0.0004). Anti-immunoglobulin antibodies lead to an overestimation of serum immunoglobulin levels in HCV patients. Interestingly, the mean levels of immunoglobulins A, G and M in HCV infected sera, determined after purification from anti-sheep immunoglobulins, was 2.73 mg/ml, 9.55 mg/ml and 1.79 mg/ml. Therefore, there was no significant difference in HCV patients compared to normal individuals (p < 0.42, p < 0.36 and p < 0.44). The presence of circulating immune complex in serum during the early phase of infection may contribute to immunopathological effects in the infected host and provide some new insights into antibody response to HCV.
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