RESUMEN
Interest in measuring immunoglobulin G Subclasses (IgG Subclasses) is increasing as more information is gathered and understanding regarding conditions associated with deficiencies of each IgG Subclass grows. Different methodologies are available for the measurement of IgG Subclasses, but their specificities vary. As a result, laboratories choose the methodology that better suits their routine, but which may not necessarily align with the needs of their population. In addition, the lack of standardization for the quantification of IgG Subclasses causes diagnostic gaps when comparing results provided by different methodologies. Thus, the purpose of our research is to compare the analytical performance of The Binding Site's (TBS) Optilite® human Immunoglobulin G (IgG) and IgG Subclasses Immunoturbidimetry assay, with the Nephelometry method routinely used in our clinical laboratory, Siemens BNII®. Our results show that the Immunoturbidimetry assay appears to be the most reliable to evaluate IgG Subclasses: the sum of IgG Subclasses and Total IgG correlate better than by Nephelometry. Although these methodologies share a similar principle, the comparison of results appears to be compromised. Therefore, prior to switching methodologies, further studies should be conducted to assess which methodology could be better applied to specific populations. It is also essential to standardise IgG Subclasses assays to reduce discrepancies that arise from comparing results.
Asunto(s)
Inmunoglobulina G , Inmunoturbidimetría , Nefelometría y Turbidimetría , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Inmunoglobulina G/inmunología , Nefelometría y Turbidimetría/métodos , Inmunoturbidimetría/métodos , Reproducibilidad de los Resultados , Femenino , Masculino , Adulto , Persona de Mediana Edad , AncianoRESUMEN
Shigella is the second leading cause of diarrheal diseases, accounting for >200,000 infections and >50,000 deaths in children under 5 years of age annually worldwide. The incidence of Shigella-induced diarrhea is relatively low during the first year of life and increases substantially, reaching its peak between 11 to 24 months of age. This epidemiological trend hints at an early protective immunity of maternal origin and an increase in disease incidence when maternally acquired immunity wanes. The magnitude, type, antigenic diversity, and antimicrobial activity of maternal antibodies transferred via placenta that can prevent shigellosis during early infancy are not known. To address this knowledge gap, Shigella-specific antibodies directed against the lipopolysaccharide (LPS) and virulence factors (IpaB, IpaC, IpaD, IpaH, and VirG), and antibody-mediated serum bactericidal (SBA) and opsonophagocytic killing antibody (OPKA) activity were measured in maternal and cord blood sera from a longitudinal cohort of mother-infant pairs living in rural Malawi. Protein-specific (very high levels) and Shigella LPS IgG were detected in maternal and cord blood sera; efficiency of placental transfer was 100% and 60%, respectively, and had preferential IgG subclass distribution (protein-specific IgG1 > LPS-specific IgG2). In contrast, SBA and OPKA activity in cord blood was substantially lower as compared to maternal serum and varied among Shigella serotypes. LPS was identified as the primary target of SBA and OPKA activity. Maternal sera had remarkably elevated Shigella flexneri 2a LPS IgM, indicative of recent exposure. Our study revealed a broad repertoire of maternally acquired antibodies in infants living in a Shigella-endemic region and highlights the abundance of protein-specific antibodies and their likely contribution to disease prevention during the first months of life. These results contribute new knowledge on maternal infant immunity and target antigens that can inform the development of vaccines or therapeutics that can extend protection after maternally transferred immunity wanes.
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Anticuerpos Antibacterianos/sangre , Disentería Bacilar/inmunología , Disentería Bacilar/prevención & control , Inmunoglobulina G/sangre , Vacunas contra la Shigella/inmunología , Adolescente , Adulto , Estudios de Cohortes , Femenino , Humanos , Inmunidad Materno-Adquirida , Inmunoglobulina G/clasificación , Lactante , Recién Nacido , Malaui , Masculino , Embarazo , Shigella flexneri/inmunología , Adulto JovenRESUMEN
Characterizing the serologic features of asymptomatic SARS-CoV-2 infection is imperative to improve diagnostics and control of SARS-CoV-2 transmission. In this study, we evaluated the antibody profiles in 272 plasma samples collected from 59 COVID-19 patients, consisting of 18 asymptomatic patients, 33 mildly ill patients and 8 severely ill patients. We measured the IgG against five viral structural proteins, different isotypes of immunoglobulins against the Receptor Binding Domain (RBD) protein, and neutralizing antibodies. The results showed that the overall antibody response was lower in asymptomatic infections than in symptomatic infections throughout the disease course. In contrast to symptomatic patients, asymptomatic patients showed a dominant IgG-response towards the RBD protein, but not IgM and IgA. Neutralizing antibody titers had linear correlations with IgA/IgM/IgG levels against SARS-CoV-2-RBD, as well as with IgG levels against multiple SARS-CoV-2 structural proteins, especially with anti-RBD or anti-S2 IgG. In addition, the sensitivity of anti-S2-IgG is better in identifying asymptomatic infections at early time post infection compared to anti-RBD-IgG. These data suggest that asymptomatic infections elicit weaker antibody responses, and primarily induce IgG antibody responses rather than IgA or IgM antibody responses. Detection of IgG against the S2 protein could supplement nucleic acid testing to identify asymptomatic patients. This study provides an antibody detection scheme for asymptomatic infections, which may contribute to epidemic prevention and control.
Asunto(s)
Anticuerpos Antivirales/sangre , Infecciones Asintomáticas , Inmunoglobulina G/sangre , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Proteínas Estructurales Virales/inmunología , Adolescente , Adulto , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/fisiología , Sitios de Unión de Anticuerpos , Femenino , Humanos , Inmunoglobulina G/clasificación , Inmunoglobulina M/inmunología , Cinética , Masculino , Persona de Mediana Edad , Pruebas de Neutralización/estadística & datos numéricos , SARS-CoV-2/química , Adulto JovenRESUMEN
Background: Patients with primary humoral immunodeficiency are more prone to invasive as well as recurrent pneumococcal infections. Therefore, anti-pneumococcal vaccination including the 13-valent conjugate vaccine is recommended. Nevertheless, to date, no data is available on immunogenicity of this vaccine in this population. Objective: To assess the immunogenicity and the persistence of protection up to one year after a 13-valent pneumococcal conjugate vaccine in patients with primary humoral immunodeficiency. Methods: Twenty-nine patients with common variable immunodeficiency or IgG subclass deficiency were vaccinated. Immune response and immune protection at baseline as well as at one, six and twelve months after vaccination were evaluated by measuring specific IgG serum concentrations (ELISA), and opsonophagocytic activities directed against selected pneumococcal (MOPA). Results: By ELISA, half of the patients had protective IgG concentrations before vaccination, 35.7% showed an immune response one month after vaccination, 71.4%, 66.7% and 56.0% of the patients were protected at one, six and twelve months respectively. Conversely, by MOPA, 3.4% of the patients were protected at baseline, 10.7% showed an immune response and 28.6%, 48.2% and 33.3% were protected at one, six and twelve months respectively. IgG subclass deficiency, Ig replacement therapy and higher IgG2 concentrations at diagnosis were associated with long-term protection. Conclusion: Pneumococcal conjugate vaccine improves immune protection and antibodies' functionality in a subset of patients with primary immunodeficiency. Prime-boost vaccine strategy needs to be better and individually adapted.
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Inmunodeficiencia Variable Común/inmunología , Inmunodeficiencia Variable Común/terapia , Deficiencia de IgG/inmunología , Deficiencia de IgG/terapia , Vacunas Neumococicas/uso terapéutico , Adulto , Anciano , Anticuerpos Antibacterianos/sangre , Especificidad de Anticuerpos , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Masculino , Persona de Mediana Edad , Fagocitosis/inmunología , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/prevención & control , Serogrupo , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/inmunología , Factores de Tiempo , Vacunas Conjugadas/uso terapéutico , Adulto JovenRESUMEN
Increasing evidence suggests infants develop unique neutralizing antibody (nAb) responses to HIV compared to adults. Here, we dissected the nAb response of an infant whose virus is in clinical trials as a vaccine immunogen, with a goal of characterizing the broad responses in the infant to this antigen. We isolated 73 nAbs from infant BG505 and identified a large number of clonal families. Twenty-six antibodies neutralized tier 2 viruses-in some cases, viruses from the same clade as BG505, and in others, a different clade, although none showed notable breadth. Several nAbs demonstrated antibody-dependent cellular cytotoxicity activity and targeted the V3 loop. These findings suggest an impressive polyclonal response to HIV infection in infant BG505, adding to the growing evidence that the nAb response to HIV in infants is polyclonal-a desirable vaccine response to a rapidly evolving virus like HIV.
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Anticuerpos Neutralizantes/biosíntesis , Linfocitos B/inmunología , Anticuerpos Anti-VIH/biosíntesis , Infecciones por VIH/prevención & control , VIH-1/inmunología , Inmunoglobulina G/biosíntesis , Adulto , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/clasificación , Citotoxicidad Celular Dependiente de Anticuerpos , Linfocitos B/virología , Preescolar , Células Clonales , Epítopos/química , Anticuerpos Anti-VIH/clasificación , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Inmunoglobulina G/clasificación , MasculinoRESUMEN
Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for cancer immunotherapeutic drug discovery while also revealing limitations in knowledge of structure-activity relationships. The current understanding of renal filtration originates largely from data reported for dextrans, IgG, albumin, and selected globular proteins. For a one-armed IgG-based T-cell imaging agent, we observed higher renal signal than typically observed for bivalent IgGs, prompting us to explore the factors governing renal filtration of biologics. We constructed a small representative library of IgG-like formats with varied shapes and hinge flexibilities falling broadly into two categories: branched molecules including bivalent IgG and (scFv)2Fc, and nonbranched molecules including one-armed IgG, one-armed IgG with stacked Fab, and one-armed IgG with a rigid IgA2 hinge. Transmission electron microscopy revealed Y-shaped structures for the branched molecules and pseudo-linear structures for the nonbranched molecules. Single-photon emission CT imaging, autoradiography, and tissue harvest studies demonstrated higher renal uptake and catabolism for nonbranched molecules relative to branched molecules. Among the nonbranched molecules, the one-armed IgG with rigid IgA2 hinge molecule demonstrated higher kidney uptake and decreased systemic exposure relative to molecules with a more flexible hinge. Our results show that differences in shape and hinge flexibility drive the increased glomerular filtration of one-armed relative to bivalent antibodies and highlight the practical advantages of using imaging to assess renal filtration properties. These findings are particularly relevant for T-cell-dependent bispecific molecules, many of which have nonstandard antibody structures.
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Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Barrera de Filtración Glomerular/metabolismo , Inmunoglobulina G/inmunología , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único/métodos , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Biespecíficos/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Femenino , Barrera de Filtración Glomerular/efectos de los fármacos , Humanos , Inmunoglobulina G/clasificación , Ratones SCIDRESUMEN
Affinity chromatography is the linchpin of antibody downstream processing and typically relies on bacterial immunoglobulin (Ig)-binding proteins, epitomized by staphylococcal protein A-based ligands. However, such affinity ligands are fairly costly and suffer from chemical instability, leading to ligand denaturation and leaching from chromatographic support. Innovations in this area are aimed at developing robust and highly selective antibody ligands capable of withstanding harsh column sanitization conditions. We report the development and first-stage characterization of a selective short linear peptide ligand of the IgG Fc region capable of capturing all four IgG subclasses. The ligand was discovered through in vitro directed evolution. A focused phage-display library based on a previously identified peptide lead was subjected to a single-round screen against a pool of human IgG. The hits were identified with next-generation sequencing and ranked according to the enrichment ratio relative to their frequency in the pre-screened library. The top enriched peptide GSYWYNVWF displaying highest affinity for IgG was coupled to bromohydrin-activated agarose beads via a branched linker. The resulting affinity matrix was characterized with a dynamic binding capacity of approx. 43 mg/mL, on par with commercially employed protein A-based resin.
Asunto(s)
Cromatografía de Afinidad/métodos , Evolución Molecular Dirigida/métodos , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/aislamiento & purificación , Biblioteca de Péptidos , Alcoholes/química , Secuencia de Aminoácidos , Secuenciación de Nucleótidos de Alto Rendimiento , Ensayos Analíticos de Alto Rendimiento , Humanos , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/clasificación , Inmunoglobulina G/metabolismo , Ligandos , Unión Proteica , Sefarosa/química , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismoRESUMEN
BACKGROUND: Human preformed antibodies (Abs), anti-galactose-alpha-1,3-galactose (Gal) and anti-N-glycolylneuraminic acid (Neu5Gc), can react with porcine antigens of wild-type pigs. To provide basic population data of the Abs for potential application in clinical xenotransplantation, we developed enzyme-linked immunosorbent assay methods and investigated the serum titers of anti-Gal and anti-Neu5Gc Abs, including immunoglobulin (Ig) M and IgG along with its subclasses, in humans. METHODS: Anti-Gal and anti-Neu5Gc Abs serum titers were measured in 380 healthy Korean adults using the in-house enzyme-linked immunosorbent assays. The frequency and median values of anti-Gal and anti-Neu5Gc were measured, and their class and subclass distribution were evaluated. RESULTS: The detection frequencies of anti-Gal were 99.2%, 95.0%, 23.2%, 94.5%, 12.4%, and 3.4% for IgM, IgG, IgG1, IgG2, IgG3, and IgG4, respectively. The detection frequencies of anti-Neu5Gc Abs were 87.4%, 96.6%, 1.6%, 46.3%, 0.0%, and 0.0% for IgM, IgG, IgG1, IgG2, IgG3, and IgG4, respectively. The median values of anti-Gal IgM (1001.6 ng/mL) and IgG (1198.3 ng/mL) were significantly higher than those of anti-Neu5Gc Abs (IgM, 328.4 ng/mL; IgG, 194.7 ng/mL; P < .001). IgG2 titers of both anti-Gal and anti-Neu5Gc Abs correlated better with the IgG class than the titers of other IgG subclasses. CONCLUSIONS: The titers of anti-Gal Abs were higher than those of anti-Neu5Gc Abs. IgG2 was the main IgG subclass in both anti-Gal and anti-Neu5Gc Abs. Variation in the titers of anti-Gal or anti-Neu5Gc Abs may partly explain the biological and immunologic changes that occur in recipients of xenotransplants.
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Disacáridos/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Ácidos Neuramínicos/inmunología , Adulto , Animales , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G/clasificación , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Masculino , Persona de Mediana Edad , PorcinosRESUMEN
We analyzed antibody response patterns according to the level of disease severity in patients with novel coronavirus disease 2019 (COVID-19) in Japan. We analyzed 611 serum specimens from 231 patients with COVID-19 (mild, 170; severe, 31; critical, 30). Immunoglobulin M (IgM) and IgG antibodies against nucleocapsid protein (N) and spike 1 protein (S1) were detected by enzyme-linked immunosorbent assays. The peaks of fitting curves for the optical density (OD) values of IgM and IgG antibodies against N appeared simultaneously, while those against S1 were delayed compared with N. The OD values of IgM against N and IgG against both N and S1 were significantly higher in the severe and critical cases than in the mild cases at 11 days after symptom onset. The seroconversion rates of IgG were higher than those of IgM against both N and S1 during the clinical course based on the optimal cut-off values defined in this study. The seroconversion rates of IgG and IgM against N and S1 were higher in the severe and critical cases than in the mild cases. Our findings show that a stronger antibody response occurred in COVID-19 patients with greater disease severity and there were low seroconversion rates of antibodies against N and S1 in the mild cases.
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Anticuerpos Antivirales/sangre , COVID-19/epidemiología , COVID-19/inmunología , SARS-CoV-2/inmunología , Anticuerpos Antivirales/clasificación , COVID-19/patología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Inmunoglobulina M/sangre , Inmunoglobulina M/clasificación , Japón/epidemiologíaRESUMEN
BACKGROUND: Intravenous Immune Globulin (IVIG) is used to treat numerous immune-mediated and inflammatory conditions. There is growing awareness of hemolysis, occasionally severe, as a side-effect of this therapy. While most cases are associated with anti-A and/or anti-B isoagglutinins, the frequency and mechanism of hemolysis remain poorly characterized. STUDY DESIGN AND METHODS: A prospective observational study was conducted to determine incidence, natural history and risk factors for IVIG-mediated hemolysis. A total of 99 infusions of high-dose IVIG (2 g/kg or higher) administered to 78 non-group O patients were monitored and graded according to Canadian IVIG Hemolysis Pharmacovigilance Group. Serum ferritin and C3/C4 levels were monitored as indicators of macrophage activation and complement consumption, respectively. Supplementary investigations included assessment for ABO zygosity, Secretor status, FcR polymorphisms, eluate IgG subclass, monocyte monolayer assay, and a panel of cytokines. RESULTS: Hemolysis was observed in 32 of 99 (32%) of infusions, with 19 of 99 (19%) grade 2 or higher. Hemolysis was only apparent 5-10 days after a completed IVIG infusion in 84% of cases and was associated with increases in serum ferritin without complement-consumption. In univariate analysis, increased risk was observed in group AB patients, first-time IVIG recipients, those not taking immuosuppressive medications, or patients treated with a specific IVIG brand; however, in multivariate analysis, product association was no longer observed. No other patient- or practice-related risk factors were identified. CONCLUSION: IVIG-mediated hemolysis is common and frequently severe. Monitoring for 5-10 days following an infusion should be considered in non-O patients receiving high-dose IVIG with known risk factors.
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Ferritinas/sangre , Hemólisis/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulinas Intravenosas/efectos adversos , Sistema del Grupo Sanguíneo ABO/inmunología , Adulto , Anciano , Canadá/epidemiología , Complemento C3/inmunología , Complemento C4/inmunología , Citocinas/sangre , Femenino , Hemaglutininas/sangre , Humanos , Inmunoglobulina G/clasificación , Inmunoglobulinas Intravenosas/administración & dosificación , Inmunoglobulinas Intravenosas/uso terapéutico , Incidencia , Infusiones Intravenosas , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Farmacovigilancia , Estudios Prospectivos , Factores de RiesgoAsunto(s)
Cromatografía Liquida/métodos , Inmunoglobulina G/sangre , Inmunoturbidimetría/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Reacciones Falso Positivas , Humanos , Inmunoglobulina G/clasificación , Inmunoglobulina G/genética , Paraproteinemias/sangre , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: CD83 is known to regulate lymphocyte maturation, activation, homeostasis, and antibody response to immunization and infection. While CD83 has a major part in B cell function, its role in influenza A virus infection has not yet been investigated. METHODS: We investigated the role of CD83 using C57BL/6J wild type mice and CD83 knockout (KO) mice after intraperitoneal administration of the influenza A/WSN/1933 virus. We analyzed cells of the peritoneal cavity, splenocytes, and cells of the bone marrow with FACS to investigate CD83 expression and cell population change in response to the virus infection. ELISA was performed with sera and peritoneal cavity fluids to detect A/WSN/1933 virus-specific IgG and the subclasses of IgG. RESULTS: FACS analysis data showed a transient but distinct induction of CD83 expression in the peritoneal B cells of wild type mice. CD83 KO mice exhibited a delayed recovery of B cells in the bone marrow after influenza virus infection and overall, a smaller T cell population compared to wild type mice. The peritoneal cavity and serum of the wild type mice contained a high titer of IgG within 14 days after infection, whereas the CD83 KO mice had a very low titer of IgG. CONCLUSIONS: These results show the importance of CD83 in lymphocytes homeostasis and antibody production during influenza A virus infection.
Asunto(s)
Antígenos CD/genética , Antígenos CD/inmunología , Regulación de la Expresión Génica/inmunología , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Virus de la Influenza A/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Infecciones por Orthomyxoviridae/inmunología , Animales , Anticuerpos Antivirales/sangre , Formación de Anticuerpos , Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cavidad Peritoneal/citología , Bazo/citología , Antígeno CD83RESUMEN
In recent years, advanced HPLC-MS strategies based on intact protein ("top-down") or protein subunit ("middle-up/middle-down") analysis have been implemented for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up/middle-down analysis for polyclonal IgGs exhibiting extensive sequence variability. Specifically, we addressed IgGs from mouse, representing an important model system in immunological investigations. To obtain Fc/2 portions as conserved subunits of IgGs, we made use of the bacterial protease SpeB. For this purpose, we initially determined SpeB cleavage sites in murine IgGs. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry. This enabled simultaneous relative quantification of IgG subclasses and their N-glycosylation variants, both of which influence IgG effector functions. To assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. The study revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts, respectively. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine, and may thus be implemented for quality control of functional antibodies.
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Anticuerpos/inmunología , Antígenos de Plantas/inmunología , Cromatografía Líquida de Alta Presión/métodos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/clasificación , Inmunoglobulina G/inmunología , Espectrometría de Masas/métodos , Alérgenos/inmunología , Animales , Femenino , Glicosilación , Fragmentos Fc de Inmunoglobulinas/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Procesamiento Proteico-Postraduccional , VacunaciónRESUMEN
Transplacental antibody transfer from mother to fetus provides protection from infection in the first weeks of life, and the four different subclasses of IgG (IgG1, IgG2, IgG3, and IgG4) have diverse roles in protection against infection. In this study, we evaluated concentrations and transplacental transfer ratios of the IgG subclasses in a healthy UK-based cohort of mother-cord pairs, and investigated associations with maternal, obstetric, and fetal factors. In agreement with previous studies, we found a strong association between maternal and cord IgG for all subclasses. We report a transfer efficiency hierarchy of IgG1>IgG3>IgG4=IgG2 in our study population, and our review of the literature demonstrates that there is no consensus in the hierarchy of subclass transfer, despite the commonly made statement that the order is IgG1>IgG4>IgG3>IgG2. We report additional data regarding negative associations between elevated maternal IgG concentrations and maternal/cord transfer ratios, finding an effect on IgG1, IgG2, and IgG3 subclasses. Levels of IgG subclasses were the same between venous and arterial blood samples from the umbilical cord, but there was a significantly higher level of total IgG in arterial blood. We found no correlation between placental FcRn protein levels and IgG transfer in our cohort, suggesting that IgG is the main determinant of observed differences in transplacental transfer ratios at term. Neonatal IgG1 and IgG4 levels were increased with later gestation at delivery, independent of any increase in transplacental transfer, indicating that the benefit of later gestation is through accumulation of these subclasses in the fetus. Neonatal IgG2 levels and transfer ratios were reduced in rhesus-negative pregnancies, suggesting that administered anti-D antibodies may compete for transplacental transfer of this subclass. Maternal influenza vaccination resulted in elevated maternal and neonatal levels of IgG4, whereas maternal Tdap vaccination had no impact on neonatal levels of the subclasses, nor transfer. However, within Tdap vaccinated pregnancies, later gestation at Tdap vaccination was associated with higher transplacental transfer. Our study provides information regarding levels and transfer of IgG subclasses in healthy term pregnancies and demonstrates the importance of recording detailed clinical information in studies of antibody transfer, including parity, ethnicity, and timing of maternal vaccine delivery.
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Sangre Fetal/inmunología , Inmunidad Materno-Adquirida , Inmunoglobulina G/sangre , Intercambio Materno-Fetal , Circulación Placentaria/inmunología , Adulto , Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/administración & dosificación , Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/inmunología , Femenino , Humanos , Inmunogenicidad Vacunal , Inmunoglobulina G/clasificación , Recién Nacido , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/inmunología , Londres , Masculino , Persona de Mediana Edad , Embarazo , Vacunación , Adulto JovenRESUMEN
Early postnatal life is characterized by a critical time period in which the developing neonatal immune system transitions from passive immunity, induced by protective maternal antibodies, to the competence of a fully functioning immune system. The inflammatory capability of both maternal and neonatal antibodies is governed by N-linked glycosylation of the Fc region, and though this has been examined extensively in adults, there is currently little information regarding antibody glycosylation patterns during early postnatal life. To characterize the murine IgG Fc glycosylation profile during early life, we used nano-LC-ESI-Qq-TOF mass spectrometry analysis to assess subclass specific Asn-297 glycosylation patterns in the serum of BALB/c mice from 5-60 days of age. From birth to adulthood, we observed a decline in proinflammatory Fc glycosylation in all IgG subclasses. This was shown by significantly reduced agalactosylated and monogalactosylated structures combined with increased sialylation after weaning at 45 and 60 days of age. This information indicates that the transition between neonatal life and adulthood in mice is accompanied by reduction of inflammatory IgG antibodies. Our study contributes to a growing body of literature indicating the importance of IgG Fc glycosylation and its association with inflammation during different life stages.
Asunto(s)
Sistema Inmunológico/crecimiento & desarrollo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Femenino , Glicosilación , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Fragmentos Fc de Inmunoglobulinas/clasificación , Inmunoglobulina G/clasificación , Masculino , Ratones , Ratones Endogámicos BALB C , Embarazo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
OBJECTIVE: To clinically characterize patients with anti-metabotropic glutamate receptor (mGluR) 1 encephalitis, to identify prognostic factors, and to study the immunoglobulin G (IgG) subclasses and effects of antibodies on neuronal mGluR1 clusters. METHODS: Clinical information on new and previously reported patients was reviewed. Antibodies to mGluR1 and IgG subclasses were determined with brain immunohistochemistry and cell-based assays, and their effects on mGluR1 clusters were studied on rat hippocampal neurons. RESULTS: Eleven new patients were identified (10 adults, 1 child);4 were female. In these and 19 previously reported cases (n = 30, median age 55 years), the main clinical manifestation was a subacute cerebellar syndrome that in 25 (86%) patients was associated with behavioral/cognitive changes or other neurologic symptoms. A tumor was found in 3 of 26 (11%). Brain MRI was abnormal in 7 of 19 (37%) at onset and showed cerebellar atrophy in 10 of 12 (83%) at follow-up. Twenty-five of 30 (83%) patients received immunotherapy. Follow-up was available for 25: 13 (52%) had clinical stabilization; 10 (40%) showed significant improvement; and 2 died. At the peak of the disease, patients with bad outcome at 2 years (modified Rankin Scale score > 2, n = 7) were more likely to have higher degree of initial disability, as reflected by a worse Scale for Assessment and Rating of Ataxia score, and more frequent need of assistance to walk. Antibodies to mGluR1 were mainly IgG1 and caused a significant decrease of mGluR1 clusters in cultured neurons. CONCLUSIONS: Anti-mGluR1 encephalitis manifests as a severe cerebellar syndrome, often resulting in long-term disability and cerebellar atrophy. The antibodies are pathogenic and cause significant decrease of mGluR1 clusters in cultured neurons.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/diagnóstico , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Cerebelosas/diagnóstico , Enfermedades Cerebelosas/inmunología , Encefalitis/diagnóstico , Encefalitis/inmunología , Receptores de Glutamato Metabotrópico/inmunología , Adulto , Anciano , Animales , Atrofia/patología , Enfermedades Autoinmunes del Sistema Nervioso/complicaciones , Células Cultivadas , Enfermedades Cerebelosas/etiología , Enfermedades Cerebelosas/patología , Niño , Embrión de Mamíferos , Encefalitis/complicaciones , Femenino , Estudios de Seguimiento , Hipocampo/citología , Humanos , Inmunoglobulina G/clasificación , Inmunoterapia , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Neuronas , Pronóstico , RatasRESUMEN
The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8+ T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8+ T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8+ T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8+ T-cell populations in vivo, which may serve prognostic and diagnostic roles.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/virología , Papillomaviridae/inmunología , Tomografía de Emisión de Positrones/métodos , Animales , Antígenos , Clonación Molecular , Epítopos/genética , Epítopos/metabolismo , Femenino , Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/clasificación , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunoglobulina G/clasificación , Inmunoglobulina G/inmunología , Pulmón/virología , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/inmunologíaRESUMEN
Monoclonal antibodies (MAbs) have the potential to assist in the battle against multidrug-resistant bacteria such as carbapenem-resistant Klebsiella pneumoniae (CR-Kp). However, the characteristics by which these antibodies (Abs) function, such as the role of antibody subclass, must be determined before such modalities can be carried from the bench to the bedside. We performed a subclass switch on anticapsular monoclonal murine IgG3 (mIgG3) hybridomas and identified and purified a murine IgG1 (mIgG1) hybridoma line through sib selection. We then compared the ability of the mIgG1 and mIgG3 antibodies to control CR-Kp sequence type 258 (ST258) infection both in vitro and in vivo We found by enzyme-limited immunosorbent assay (ELISA) and flow cytometry that mIgG3 has superior binding to the CR-Kp capsular polysaccharide (CPS) and superior agglutinating ability compared to mIgG1 The mIgG3 also, predictably, had better complement-mediated serum bactericidal activity than the mIgG1 and also promoted neutrophil-mediated killing at concentrations lower than that of the mIgG1 In contrast, the mIgG1 had marginally better activity in improving macrophage-mediated phagocytosis. Comparing their activities in a pulmonary infection model with wild-type as well as neutropenic mice, both antibodies reduced organ burden in a nonlethal challenge, regardless of neutrophil status, with mIgG1 having the highest overall burden reduction in both scenarios. However, at a lethal inoculum, both antibodies showed reduced efficacy in neutropenic mice, with mIgG3 retaining the most activity. These findings suggest the viability of monoclonal Ab adjunctive therapy in neutropenic patients that cannot mount their own immune response, while also providing some insight into the relative contributions of immune mediators in CR-Kp protection.IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is an urgent public health threat that causes life-threatening infections in immunocompromised hosts. Its resistance to nearly all antibiotics necessitates novel strategies to treat it, including the use of monoclonal antibodies. Monoclonal antibodies are emerging as important adjuncts to traditional pharmaceuticals, and studying how they protect against specific bacteria such as Klebsiella pneumoniae is crucial to their development as effective therapies. Antibody subclass is often overlooked but is a major factor in how an antibody interacts with other mediators of immunity. This paper is the first to examine how the subclass of anticapsular monoclonal antibodies can affect efficacy against CR-Kp Additionally, this work sheds light on the viability of monoclonal antibody therapy in neutropenic patients, who are most vulnerable to CR-Kp infection.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Enterobacteriaceae Resistentes a los Carbapenémicos/inmunología , Inmunoglobulina G/clasificación , Inmunoglobulina G/inmunología , Klebsiella pneumoniae/inmunología , Infecciones del Sistema Respiratorio/prevención & control , Animales , Antibacterianos/farmacología , Anticuerpos Antibacterianos/clasificación , Anticuerpos Monoclonales/clasificación , Anticuerpos Monoclonales/genética , Sitios de Unión de Anticuerpos , Carbapenémicos/farmacología , Klebsiella pneumoniae/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Neutropenia , Fagocitosis , Infecciones del Sistema Respiratorio/inmunologíaRESUMEN
The screening for IgG subclass donor-specific antibodies (DSAs) in allograft recipients uses IgG1-4 subclass-specific monoclonal antibodies (mAbs) that should be mono-specific. The cross-reactivity discrepancies reported for IgG subclass-specific mAbs warranted a critical cross-reactivity pattern analysis of the IgG subclass-specific mAbs most commonly used to detect DSAs. We tested the reactivity of 2 anti-IgG1-, 3 anti-IgG2-, 1 anti-IgG3-, and 2 anti-IgG4-specific PE-conjugated mAbs against microbeads coated with IgG1-4 proteins separately. Each IgG subclass protein was coated at three densities on the beads (0.5, 1, and 2 µg of protein per 106 beads), and the PE-conjugated mAbs were titrated from 0.04 µg/mL to 5 µg/mL. The IgG subclass reactivity of the sample was acquired on the Luminex multiplex platform. Among the IgG subclass-specific mAbs, only the anti-IgG3 (clone: HP6050) mAb was mono-specific. All other mAbs tested were binding to IgG subclass proteins other than their respective immunogen, thereby being cross-reactive. IgG subclass cross-reactivity patterns were dependent on the concentration of both IgG subclass-specific mAbs and IgG1-4 protein targets coated onto the beads. With the current IgG subclass mAbs available, 3 of the 15 possible combinations of IgG1-4 subclass protein could be identified. While the remaining 12 unique combinations cannot be distinguished clearly, 6 groups that corresponded to two different unique combinations of IgG1-4 subclass protein could be identified. The dilution of serum samples and IgG subclass-specific mAbs, other than the anti-IgG3 (clone: HP6050), must be further optimized before their implementation in IgG subclass DSA screening in allograft recipients.
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Anticuerpos Monoclonales/inmunología , Inmunoensayo , Inmunoglobulina G/sangre , Isoanticuerpos/sangre , Trasplante de Órganos , Receptores de Trasplantes , Especificidad de Anticuerpos , Biomarcadores/sangre , Reacciones Cruzadas , Humanos , Inmunoglobulina G/clasificación , Isoanticuerpos/clasificación , Valor Predictivo de las Pruebas , Reproducibilidad de los ResultadosRESUMEN
Evaluation of the murine isotype antibodies is essential in subunit vaccine development because inbred mouse strains with diverse genetic backgrounds respond different to recombinant proteins. In this regard, the main goal of this study was to measuring and comparing the profile of IgG isotype responses in C57BL/6 mice. For this purpose, the extracellular region of plasmodium vivax thrombospondin-related adhesive protein (PvTRAP) gene was expressed in Escherichia coli Rosetta (DE3)-pET23a. Then, the recombinant PvTRAP alone or emulsified with Freund's complete adjuvant were applied for immunization of the C57BL/6 mice. The role of antibodies and cellular immune responses induced by recombinant PvTRAP were evaluated. The results showed the level of anti-rPvTRAP IgG2c was significantly higher than IgG2a in the groups that received rPvTRAP alone (mean OD490 = 0.798 ± 0.12 and 0.39 ± 0.1, respectively) and emulsified with CFA/IFA (mean OD490 = 1.48 ± 0.07 and 0.605 ± 0.13, respectively; P < 0.05, independent sample t-test). Additionally, the immunized mice with rPvTRAP and rPvTRAP + CFA/IFA had an intermediate-avidity IgG2a antibody but high-avidity IgG2c antibody as well as the mean of serum antibody titers results exhibited that in both rPvTRAP and rPvTRAP + CFA/IFA mouse groups, IgG2a end-point titer (1:3200 and 1:25,600, respectively) was noteworthy lower than IgG2c (1:25,600 and 1:102,400, respectively). Moreover, the results revealed the eliciting significant levels of IFN-γ (P < 0.05, independent sample t-test) and no detectable level of IL-4 in the mouse groups received rPvTRAP alone and emulsified with CFA/IFA as compared to the mouse control groups. In general, our results showed that for correctly interpreting of Th1 immune responses in C57BL/6 mouse strain it is critical to measure IgG2c instead of IgG2a along with IFN-γ.