IgG Subclasses Research Articles

The Importance of Monitoring Antigen-Specific Memory B Cells, and How ImmunoSpot Assays Are Suitable for This Task

Determining an individual’s humoral immune reactivity to a pathogen, autoantigen, or environmental agent is traditionally accomplished through the assessment of specific antibody levels in blood. However, in many instances, titers of specific antibodies decline over time and thus do not faithfully reveal prior antigen exposure or establishment of immunological memory. To estimate an individual’s humoral immune competence, it is therefore necessary to assess functional B cell memory. Here, we describe novel B cell ELISPOT and FluoroSpot assays (collectively referred to as ImmunoSpot) that can be rapidly developed and validated to characterize the memory B cell (Bmem) repertoire specific for any desired antigen ex vivo and at single-cell resolution. Moreover, multiplexed variants of the B cell FluoroSpot assay enable high-throughput testing of antigen-specific B cells secreting distinct antibody classes and/or IgG subclasses, with minimal cell material requirements. B cell ImmunoSpot assays also enable measurement of affinity distributions within the antigen-specific Bmem compartment and permit cross-reactivity measurements that can provide insights into Bmem established against future pathogen variants. Collectively, the ImmunoSpot® system presented here is highly reproducible, and can be readily validated for regulated tests. The newly gained ability to monitor the antigen-specific Bmem compartment should catalyze a more comprehensive understanding of humoral immunity in health and disease.

Six Injections of Modified Adjuvanted PQ Grass Is Effective and Well-Tolerated in a Pivotal Phase III Trial.

PQ Grass 27600 SU (PQ Grass) cumulative dose is a pre-seasonal, six-injection, aluminium-free, modified subcutaneous immunotherapy product under development for the treatment of allergic rhinitis (AR). A pivotal Phase III randomised double-blind, placebo-controlled clinical trial was performed to evaluate the efficacy and safety of PQ Grass in subjects with seasonal AR. An adaptive group sequential trial PQGrass306 (G306) with one pre-defined interim analysis was designed, using 2 parallel groups applying a 1:1 active versus placebo randomisation of patients aged 18-65. The primary efficacy endpoint was the EAACI (European Academy of Allergy and Clinical Immunology) Combined Symptom and Medication Score (EAACI-CSMS0-6) averaged over the peak grass pollen season (GPS). 858 subjects were screened and 555 subjects were randomised. Based on the results of the pre-defined interim analysis, the trial was stopped for success showing superiority in favour of PQ Grass. The primary endpoint EAACI-CSMS0-6 (peak GPS) demonstrated a highly significant and clinically meaningful point difference of PQ Grass over placebo of -0.27 points (95% CI: -0.42 to -0.12), corresponding to a relative difference of -20.3% (p = 0.0005). Highly consistent and beneficial results were obtained for PQ Grass for all key secondary endpoints. Significant induction of blocking IgG4 and IgA antibody subclasses occurred. PQ Grass was well tolerated, and no unexpected safety signals occurred. This pivotal Phase III trial demonstrated a significant and clinically meaningful effect on the primary endpoint as well as highly consistent secondary endpoint results and a supportive safety profile.

A third COVID-19 vaccine dose in kidney transplant recipients induces antibody response to vaccine and Omicron variants but shows limited Ig subclass switching.

Solid organ transplant recipients (SOTRs) suffer more frequent and more severe infections due to their compromised immune responses resulting from immunosuppressive treatments designed to prevent organ rejection. Pharmacological immunosuppression can adversely affect immune responses to vaccination. A cohort of kidney transplant recipients (KTRs) received their third dose of ancestral, monovalent COVID-19 vaccine in the context of a clinical trial and antibody responses to the vaccine strain, as well as two Omicron variants BA.1 and BA.5 were investigated and compared with healthy controls who also received a third dose of mRNA vaccine (HCs). Total IgG and live virus neutralizing antibody titers were reduced in KTRs compared with controls for all variants. KTRs displayed altered IgG subclass switching, with significantly lower IgG3 antibodies. Responses in KTRs were also very heterogeneous, with some individuals showing strong responses but a significant number showing no Omicron-specific neutralizing antibodies. Taken together, immune responses after COVID-19 vaccination in KTRs were not only lower than HCs but highly variable, indicating that simply increasing the number of vaccine doses alone may not be sufficient to provide greater protection in this population. These findings underscore the need for tailored vaccination strategies for immunosuppressed populations, such as KTRs. Alternative formulations and doses of COVID-19 vaccines should be considered for people with severely compromised immune systems, as more frequent vaccinations may not significantly improve the response, especially regarding neutralizing antibodies.IMPORTANCEThis study addresses the challenges faced by kidney transplant recipients (KTRs) in mounting effective immune responses against COVID-19. By evaluating the antibody responses to a third dose of monovalent mRNA COVID-19 vaccine and its effectiveness against Omicron subvariants (BA.1 and BA.5), this study reveals significant reductions in both binding and neutralizing antibodies in KTRs compared with healthy controls. The research highlights altered IgG subclass switching and heterogeneous responses within the KTR population. Reduced recognition of variants, coupled with differences in IgG subclasses, decreases both the quality and quantity of protective antibodies after vaccination in KTRs. These findings underscore the need for tailored vaccination strategies for immunosuppressed populations, such as KTRs. Alternative formulations and doses of COVID-19 vaccines should be considered for people with severely compromised immune systems, as more frequent vaccinations may not significantly improve the response, especially regarding neutralizing antibodies.

P-1988. COVID-19 vaccine induced antibodies in human milk: exploring form and function

Abstract Background Following SARS-CoV-2 vaccination/infection of pregnant/lactating women, human milk contains SARS-CoV-2 antibodies (Ab). Limited data exists on whether those Ab induce protection for infants against COVID-19. Among 45 lactating women who received primary and booster SARS-CoV-2 vaccines from 2021 to 2022, we previously showed that anti-receptor binding domain (RBD) IgG and IgA significantly increased in serum and milk following booster SARS-CoV-2 vaccine but did not differ by maternal nucleoprotein (NP) status. In this exploratory analysis, we aimed to further characterize the Ab present in human milk following maternal booster SARS-CoV-2 vaccination. SARS-CoV-2 Neutralization Antibodies in Human Milk by Maternal Nucleoprotein Status before and after SARS-CoV-2 Booster Vaccine. Neutralization titers in human milk significantly increased among women with hybrid immunity (NP+) after booster vaccine (P=0.04) but not for women with vaccine only immunity (NP-). Methods Using a subset of paired longitudinal milk and blood samples from a cohort of 45 lactating women who received primary and booster SARS-CoV-2 vaccines from 2021 to 2022, we characterized neutralizing Ab, RBD IgG subclasses (IgG1-IgG4), and Ab-dependent complement activation (ADCA). Antibody-Dependent Complement Activation (ADCA) using Human Milk from Lactating Women before and after SARS-CoV-2 Booster Vaccine. Two women with hybrid immunity (NP+) had significantly positive ADCA after SARS-CoV-2 booster vaccine, human milk from all others resulted in low or no activation of complement. Results For 7 NP+ (hybrid immune) and 7 NP- (vaccine immune) women, SARS-CoV-2 neutralization titers increased in serum and milk after booster vaccine among NP+ women (p=0.04) but not for NP- women (Fig1). After booster vaccine, ADCA was positive but variable in maternal serum and milk with only 2/12 (16%) women demonstrating high-levels of ADCA in human milk (Fig2). These same two women demonstrated different IgG subclass profiles in milk after booster vaccine with moderate to high levels of IgG1 and IgG3, and low levels of IgG4 (Fig3). This was inverse for all other women who demonstrated moderate to low levels of IgG1 and high titers of IgG4 in human milk. Notably, both women with high ADCA response were NP+ and vaccinated with primary SARS-CoV-2 vaccine more than 4 months post-partum, while all others received primary SARS-CoV-2 vaccine while pregnant or less than a month post-partum. IgG Subclasses in Human Milk before and after SARS-CoV-2 booster vaccine by Complement Activation Status Human milk with high complement activation demonstrated higher levels of IgG1 and IgG3 after booster vaccine compared to women with low complement activation. Conclusion The different Ab profile may indicate a tolerizing response that could be related to pregnancy at time of initial antigen exposure or from repeat SARS-CoV-2 vaccination and warrants future investigation (additional samples are under analysis). Nonetheless, human milk demonstrates robust binding and neutralizing Ab following SARS-CoV-2 vaccination, offering other potential mechanisms for protection of infants against COVID-19 infection. Disclosures Anne-Marie Rick, MD, MPH, PhD, Pfizer: Advisor/Consultant

Immunosuppressive Therapy Modifies Anti-Spike IgG Subclasses Distribution After Four Doses of mRNA Vaccination in a Cohort of Kidney Transplant Recipients

Background: IgG4 is the least immunogenic subclass of IgG. Immunization with mRNA vaccines against SARS-CoV-2, unlike other vaccines, induces an increase in IgG4 against the spike protein in healthy populations. This study investigated whether immunosuppressive therapy affects the immune response, focusing on IgG subclass changes, to four doses of mRNA vaccine in kidney transplant recipients (KTRs). Methods: This study includes 146 KTRs and 23 dialysis patients (DPs) who received three mRNA-1273 vaccine doses and a BNT162b2 booster. We evaluated anti-spike IgG titers and subclasses, T-CD4+ and T-CD8+ cellular responses, and serum neutralizing activity (SNA). Results: At the fourth dose, 75.8% of COVID-19 naïve KTRs developed humoral and cellular responses (vs. 95.7% in DPs). There was a correlation between anti-spike IgG titers/subclasses and SNA (p < 0.001). IgG subclass kinetics after the third/fourth dose differed between COVID-19 naïve KTRs and DPs. Immunosuppressive therapy influenced IgG subclasses: mTOR inhibitors (mTORi) positively influenced IgG1 and IgG3 (p < 0.05), while mycophenolic acid negatively affected IgG1, IgG3, and IgG4 (p < 0.05). SNA is correlated with breakthrough infections after four doses of vaccine in KTRs. mTORi was the only factor associated with SNA > 65% in naïve KTRs [4.29 (1.21–15.17), p = 0.024]. Conclusions: KTRs show weaker cellular and humoral immune responses to mRNA vaccines and a class shift towards non-inflammatory anti-S IgG4 upon booster doses. IgG subclasses show a positive correlation with SNA and are influenced by immunosuppression. Increased SNA after four doses of vaccine is protective against infection. mTORi may benefit non-responding KTRs.

An engineered Palivizumab IgG2 subclass for synthetic gp130 and fas mediated signaling.

Recently, we phenocopied Interleukin (IL-)6 signaling using the dimerized single-chain variable fragment (scFv) derived from the respiratory syncytial virus (RSV) IgG1-antibody Palivizumab (PscFvLHFc) to activate a Palivizumab anti-idiotypic nanobody (AIPVHH)-gp130 receptor fusion protein. Palivizumab was unable to activate STAT3 signaling, so we aimed to create a similar ligand capable of triggering this pathway. Here, we created three variants of the ligand called PscFvLH0Fc, PscFvLH4Fc and PscFvLH8Fc by shortening the spacer region connecting PscFvLH and Fc from 23 amino acids in PscFvLHFc to 0 amino acids or expanding it by rigid linkers of 4 or 8 alpha helical loops, respectively. The rigid-linker ligands had completely altered cellular activation patterns via AIPVHHgp130 fusion proteins. Deleting the extracellular stalk region between transmembrane and AIPVHH in the synthetic receptors AIP2VHHgp130Δstalk and AIP3VHHgp130Δstalk to increase rigidity and enhanced the biological activity of the short spacer PscFvFc ligands. Since scFv constructs are less stable than antibodies and have not been FDA approved, we looked for different antibody backbones. Transferring Palivizumab's variable region to a more rigid and hence more agonistic IgG2 backbone (PIgG2) maintained affinity while improving agonistic properties activating cells expressing AIP2VHHgp130Δstalk and AIP3VHHgp130Δstalk but not their full-length counterparts. Furthermore, we engineered a tetravalent Palivizumab variant (PscFvPIgG2) capable of inducing higher-order receptor clustering, activating Fas-induced apoptosis. In summary, we engineered a fully-synthetic cytokine/cytokine receptor pair based on the IgG2-variant of Palivizumab and the AIPVHHgp130Δstalk variants opening avenues for therapeutic applications utilizing non-physiological targets in immunotherapy.

Immune Profile Differences between IgG4-Related Diseases and Primary Sjögren's Syndrome.

Immunoglobulin G4-related disease (IgG4-RD) share clinical features with primary Sjögren's syndrome (pSS). This study aimed to identify altered serological parameters and potential biomarkers of IgG4-RD and pSS. Forty IgG4-RD patients, 40 pSS patients, and 40healthy controls (HC) were enrolled in this study. Routine serological parameters and clinical manifestations were assessed. IgG subclasses (IgGSc) were detected using a Siemens BN P, and lymphocyte subsets were analyzed using flow cytometry. Cytokines assays were performed using cytometric bead array. Compared to pSS, IgG4-RD patients had higher IgG4 (p <0.001) and lower IgG1 (p =0.014). The natural killer (NK) cells (p = 0.004), CD4+ T cells (p = 0.028), and TBNK cells (p = 0.040) were increased in IgG4-RD compared to pSS. IgG4 used to differentiate IgG4-RD from pSS produced an area under the curve (AUC) of up to 0.952. In addition, we compared serum parameters, immune cells, and cytokines of IgG4-RDwith mouth dryness or eye dryness with those of pSS with the same symptoms, and similar serological changes were observed. IgG4-RD patients with mouth dryness had higher IgG4 (p <0.001) and Th cells (p = 0.016) but lower IgG1 (p = 0.009) compared to pSS with dry mouth. IgG4-RD patients with eye dryness had higher levels of IgG4 (p <0.001), Treg cells (p = 0.037), and NK cells (p = 0.017) than pSS patients with eye dryness. Moreover, IgG4-RD patients with mouth and eye dryness had higher levels of B (p = 0.006), Th (p = 0.026), Th2 (p = 0.007), and Treg cells (p = 0.028) than IgG4-RD patients without mouth and eye dryness. Immune system disorder is an outstanding feature of IgG4-RD, and its feature differ from pSS. Assessment of immune status is important in the diagnosis and differential diagnosis of IgG4-RD.

The Discovery of Autoimmune Nodopathies and the Impact of IgG4 Antibodies in Autoimmune Neurology.

In the past decade, significant progress has been made on the understanding of IgG4-mediated autoimmune diseases, of both the central and the peripheral CNS. In addition to the description of diverse antigenic targets, the description of IgG subclasses associated with specific pathogenic autoantibodies has provided useful insights into the pathophysiology and, more importantly, into the therapeutic implications of the autoantibody subclasses. This understanding has affected how myasthenia gravis, autoimmune encephalitis, and autoimmune neuropathies are treated. In the case of autoimmune neuropathies, the discovery of antigenic targets located at the node of Ranvier has led to the definition of a new diagnostic category, the autoimmune nodopathies, which differentiate them from the classical forms of Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy. These neuropathies including those caused by autoantibodies targeting contactin-1, contactin-associated protein 1, and neurofascin are mainly, though not always exclusively, mediated by IgG4 antibodies, and respond to therapies similarly to other IgG4-mediated neurologic and non-neurologic diseases, providing evidence that not only the antigenic target but also the autoantibody subclass play a role in understanding both the disease pathophysiology and response to therapies. In this article, we describe the history and main findings on autoimmune nodopathies; highlight the particularities and similarities of IgG4-mediated neurologic diseases, including autoimmune nodopathies and neuromuscular junction and certain CNS disorders; elaborate on the unique functional properties of IgG4 in influencing their specific response to immunotherapies stressing the rationale of the most suitable present and future targeted therapies; and discuss how best to apply and monitor maintenance therapies for inducing disease stability in all IgG4 neurologic autoimmunities including the need for potential future biomarkers.

Pancreatitis in children: practical management from the BSPGHAN Pancreatitis Working Group

Pancreatitis, a condition characterised by inflammation of the pancreas, has multiple aetiologies. Improving clinical proficiency in prompt diagnosis and effective management leads to better outcomes for children with acute pancreatitis, acute recurrent pancreatitis and chronic pancreatitis. Establishing consensus guidance via the British Society of Paediatric Gastroenterology Hepatology and Nutrition Pancreatitis Working Group has ensured further focus on these patients who are often cared for in a multidisciplinary framework and may prompt future research in this area. Initial assessment includes serum amylase/lipase, triglyceride levels, full blood count, C reactive protein, renal and liver function profile, glucose, calcium and capillary blood gas. Fasted transabdominal ultrasound for all children and young people with suspected pancreatitis is recommended to identify pancreatic parenchyma and pancreatobiliary ductal changes, and complications. For fluid resuscitation, use crystalloids or Ringer’s lactate: initial bolus of 10 to 20 mL/kg, 1.5–2 times maintenance volume, with hourly monitoring of urine output over the initial 24–48 hours. Initiate oral intake within the first 24 hours after fluid resuscitation; fat restriction is not recommended. For suspected autoimmune pancreatitis, workup includes immunoglobulin levels (IgG, IgM, IgA, IgG subclasses), complement components and autoantibody profile to confirm diagnosis. Significant interventional management for pancreatitis and related complications is performed via endoscopic retrograde cholangiopancreatography or endoscopic ultrasound; referral to a specialised paediatric hepatobiliary surgical team is highly recommended. Close collaboration with a specialist centre can improve diagnostic and management pathways and outcomes for children.

Induction of Fc-dependent functional antibodies against different variants of SARS-CoV-2 varies by vaccine type and prior infection

BackgroundSARS-CoV-2 transmission and COVID-19 disease severity is influenced by immunity from natural infection and/or vaccination. Population-level immunity is complicated by the emergence of viral variants. Antibody Fc-dependent effector functions are as important mediators in immunity. However, their induction in populations with diverse infection and/or vaccination histories and against variants remains poorly defined.MethodsWe evaluated Fc-dependent functional antibodies following vaccination with two widely used vaccines, AstraZeneca (AZ) and Sinovac (SV), including antibody binding of Fcγ-receptors and complement-fixation in vaccinated Brazilian adults (n = 222), some of who were previously infected with SARS-CoV-2, as well as adults with natural infection only (n = 200). IgG, IgM, IgA, and IgG subclasses were also quantified.ResultsAZ induces greater Fcγ-receptor-binding (types I, IIa, and IIIa/b) antibodies than SV or natural infection. Previously infected individuals have significantly greater vaccine-induced responses compared to naïve counterparts. Fcγ-receptor-binding is highest among AZ vaccinated individuals with a prior infection, for all receptor types, and substantial complement-fixing activity is only seen among this group. SV induces higher IgM than AZ, but this does not drive better complement-fixing activity. Some SV responses are associated with subject age, whereas AZ responses are not. Importantly, functional antibody responses are well retained against the Omicron BA.1 S protein, being best retained for Fcγ-receptor-1 binding, and are higher for AZ than SV.ConclusionsHybrid immunity, from combined natural exposure and vaccination, generates strong Fc-mediated antibody functions which may contribute to immunity against evolving SARS-CoV-2 variants. Understanding determinants of Fc-mediated functions may enable future vaccines with greater efficacy against different variants.

Unveiling the link: anti-protein disulfide isomerase A3 autoantibody expression and polycystic ovary syndrome risk in euthyroid autoimmune thyroiditis women

BackgroundPolycystic ovary syndrome (PCOS) is a common complication of autoimmune thyroiditis (AIT) in women, but the underlying mechanism remains unclear. Protein disulfide isomerase A3 (PDIA3) is a ubiquitous protein. We have reported that PDIA3 autoantibody (PDIA3Ab) production results from autoimmune responses against thyrocytes, resulting in its high expression in euthyroid AIT patients. This study aimed to explore potential correlations between PDIA3Ab expression and concurrent PCOS in euthyroid AIT women.MethodsThis is a single-center cross-sectional study. All participants, who visited the First Hospital of China Medical University from April 2023 to May 2024, were assigned to four groups according to AIT and PCOS diagnostic criteria. The PDIA3Ab levels of total IgG and IgG subclasses were detected using ELISA.ResultsFrom highest to lowest, PDIA3Ab total serum IgG levels were categorized as follows: AIT-PCOS group > AIT-non-PCOS group > non-AIT-PCOS group > non-AIT-non-PCOS group Significant differences were observed between each pair of groups, except for the non-AIT-PCOS and non-AIT-non-PCOS groups. Further analysis of the subclasses of PDIA3Ab revealed that serum IgG1 levels in the AIT-PCOS and AIT-non-PCOS groups were significantly higher than those in the non-AIT-PCOS and non-AIT-non-PCOS groups. In addition, the AIT-PCOS group had significantly higher serum IgG3 levels than the other three groups. Binary logistic regression analysis revealed that the PDIA3Ab total IgG level was an independent risk factor for concurrent PCOS in euthyroid AIT women (Q4 vs. Q1: OR, 95%CI = 5.082, 1.348–19.16). Furthermore, a trend test demonstrated a titer-dependent increase in PCOS prevalence among AIT women as the PDIA3Ab total IgG level increased.ConclusionsThe expression of serum PDIA3Ab may indicate an increased risk of PCOS in euthyroid AIT women and could potentially serve as new targets for markers or immune intervention.

Simultaneous isotyping and semi-quantitation of anti-drug antibodies to an IgG1 biotherapeutic using hybrid LBA-LC-MS/MS.

Commonly, ligand-binding platforms are being used for immunogenicity assessment, but with the recent advent of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for protein quantification, this technology has become an alternative for the measurement of anti-drug antibodies (ADAs), when combined with an immunocapture step to extract them out of the biological sample. The monoclonal antibody adalimumab was immobilized on magnetic beads to isolate ADAs against this drug from serum samples. Multiple repetitions of immunopurification were used to minimize nonspecific binding and improve drug tolerance while maintaining sufficient recovery. A subsequent tryptic digestion released peptides, from which unique peptide sequences, originating from the constant region of seven ADA subclasses, were selected. These were then analyzed by LC-MS/MS against (unextracted) subclass-specific reference standards for semi-quantification. With two immunocapture and two immunopurification steps, the method simultaneously measures the ADA subclasses IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA within their relevant ranges, with good repeatability and drug tolerance, and limited interference of endogenous immunoglobulins. The method was successfully applied for the analysis of serum samples of subjects dosed with adalimumab. Hybrid LBA-LC-MS/MS is a viable platform for measuring ADAs and adds value, especially when ADA isotyping is needed.

PET Foils Functionalized with Reactive Copolymers as Adaptable Microvolume ELISA Spot Array Platforms for Multiplex Serological Analysis of SARS-CoV-2 Infections.

Microvolume ELISA platforms have become vital in diagnostics for their high-throughput capabilities and minimal sample requirements. High-quality substrates with advanced surface properties are essential for these applications. They enable both efficient biomolecule immobilization and antifouling properties, which are critical for assay sensitivity and specificity. This study presents PET-based microvolume ELISA spot arrays coated with amine- and DBCO-reactive copolymers MCP-2 and Copoly Azide. The platforms were designed for the sensitive and specific detection of specific antibodies such as COVID-19 biomarkers. Supporting robust attachment of the SARS-CoV-2 nucleoprotein (NP), these arrays outperform traditional approaches. It was demonstrated that covalent attachment methods proved more efficient than passive adsorption, together with the reduction of non-specific binding. Analytical performance was verified with classical ELISA and real-time Surface Plasmon Resonance (SPR) analysis. It enables sensitive detection of IgG and IgA antibodies, including IgG subclasses, in human serum. Clinically, the platform achieved 100.0% sensitivity and 92.9% specificity for anti-NP antibody detection in COVID-19-positive and negative samples. Additionally, DNA-directed immobilization extended the platform's utility to multiplex serological measurements. These findings underscore the potential of PET-based microvolume ELISA arrays as scalable, high-throughput diagnostic tools suitable for detecting multiple biomarkers in a single assay and easily integrated into microfluidic devices.

Platelet aggregation responses to Salmonella Typhimurium are determined by host anti-Salmonella antibody levels.

Invasive non-typhoidal Salmonella infections are responsible for >75 000 deaths/year and >500 000 cases/year globally. Seventy-five percent of these cases occur in Sub-Saharan Africa, an increasing number of which are from multi-drug resistant strains. Interactions between bacteria and platelets can lead to thrombus formation, which can be beneficial for control of infection (immunothrombosis), or harmful through uncontrolled inflammation and organ damage (thromboinflammation). It is unknown whether Salmonella Typhimurium can activate human platelets. To assess this, light transmission aggregometry was used to measure platelet activation by two different Salmonella Typhimurium strains in 26healthy donors in platelet-rich plasma and washed platelets. In platelet-rich plasma, but not in washed platelets, Salmonella Typhimurium activated platelets in a donor- and strain-dependent manner mediated through the low affinity immune receptor FcγRIIA and the feedback agonists, ADP and thromboxane A2. Plasma swap studies between strong and weak responders demonstrated a plasma component was responsible for the variation between donors. Depletion of anti-Salmonella antibodies from plasma abolished Salmonella-induced platelet aggregation responses, and addition of polyclonal anti-Salmonella antibody allowed aggregation in washed platelets. Correlating levels of anti-Salmonella total IgG or the IgG1, IgG2, IgG3 and IgG4 subclasses to platelet responses revealed total IgG levels, rather than levels of individual subclasses, positively correlated with maximum platelet aggregation results, and negatively with lag times. Overall, we show that anti-Salmonella IgG antibodies are responsible for donor variation in platelet aggregation responses to Salmonella and mediate this activity through FcγRIIA.

Multiorgan involvement and circulating IgG1 predict hypocomplementaemia in IgG4-related disease

ObjectivesHypocomplementaemia is common in patients with IgG4-related disease (IgG4-RD). We aimed to determine the IgG4-RD features associated with hypocomplementaemia and investigate mechanisms of complement activation in this disease.MethodsWe performed a...

Interrogating post-transplant donor HLA-specific antibody characteristics and effector functions using clinical bead assays

Single antigen bead (SAB) assays are the most common and sensitive method used to detect and monitor post-transplant donor specific HLA antibodies (DSA). However, a direct comparison across traditional and modified SAB assays to improve routine DSA monitoring using pre-treated IgG sera to eliminate interference has not been performed. We performed a technical comparison of 251 post-transplant DSA from n = 91 serum samples tested neat (pre-treated, undiluted), at a single 1:16 dilution, in the C1q bead assay, and for IgG subclasses (IgG1, IgG2, IgG3, IgG4) with IgG-enriched sera. We found that DSAs that are detectable by 1:16 dilution and/or C1q are associated with higher IgG MFI values and results could be predicted by testing neat sera. DSA detected at 1:16 dilution correlated with >7000 IgG MFI in neat sera and identified DSA that exceeded the SAB linear range for semiquantitative measurements. C1q positive DSA correlated with >15,000 IgG MFI in neat sera. C1q binding correlated most strongly with total IgG MFI (Spearman r = 0.82, p = 0.002) and not specific subclasses, demonstrating that DSA C1q binding capacity in this cohort is driven by HLA-specific IgG concentration. Evaluation of engineered pan-HLA class I-specific human IgG1 and IgG2 subclass monoclonal antibodies by SAB C1q and C3d assays revealed that IgG2 antibodies can bind complement at higher concentrations. The strengths and limitations of modified SAB assays must be considered to optimize efficient testing and accurate clinical interpretation.

Complement activation by IgG subclasses is governed by their ability to oligomerize upon antigen binding.

Complement activation through antibody-antigen complexes is crucial in various pathophysiological processes and utilized in immunotherapies to eliminate infectious agents, regulatory immune cells, or cancer cells. The tertiary structures of the four IgG antibody subclasses are largely comparable, with the most prominent difference being the hinge regions connecting the Fab and Fc domains, providing them with unique structural flexibility. Complement recruitment and activation depend strongly on IgG subclass, which is commonly rationalized by differences in hinge flexibility and the respective affinities for C1, the first component of the classical complement pathway. However, a unifying mechanism of how these different IgG subclass properties combine to modulate C1 activation has not yet been proposed. We here demonstrate that complement activation is determined by their varying ability to form IgG oligomers on antigenic surfaces large enough to multivalently bind and activate C1. We directly visualize the resulting IgG oligomer structures and characterize their distribution by means of high-speed atomic force microscopy, quantify their complement recruitment efficiency from quartz crystal microbalance experiments, and characterize their ability to activate complement on tumor cell lines as well as in vesicle-based complement lysis assays. We present a mechanistic model of the multivalent interactions that govern C1 binding to IgG oligomers and use it to extract kinetic rate constants from real-time interaction data from which we further calculate equilibrium dissociation constants. Together, we provide a comprehensive view on the parameters that govern complement activation by the different IgG subclasses, which may inform the design of future antibody therapies.

IgG4-Related Disease Involving the Ear: A Case Report.

IgG4-related disease is a chronic inflammatory disease with widespread clinical presentation. It mimics various malignant, infectious, and inflammatory conditions, leading to confusion in diagnosis and management. Otological manifestations, though relatively rare, can lead to significant complications. A 45-year-old male with a recent history of ventilation tube placement in the right ear presented with a sensation of imbalance associated with profound hearing loss. He was managed in line of acute otitis media with labyrinthitis with steroids and antibiotics and removal of the ventilation tube. He returned in one week and presented with right-sided lower motor neuron-type facial paresis. Computed tomography images of the temporal bone showed a soft tissue density lesion in the right middle ear cavity extending to the mastoid antrum. He underwent a right cortical mastoidectomy with decompression of the facial nerve. Histopathology and immunohistochemistry of granulation tissues from the middle ear and the mastoid revealed evidence suggestive of probable IgG4 disease. IgG4-related disease is a relatively new entity, and its pathogenesis has not been properly understood. IgG4 subclass has been involved in this disease resulting in fibro-inflammatory conditions leading to tumor-like masses or fibrosis of the affected organs. Treatment includes glucocorticoids and immunosuppressant medications.

Development of bovine IgG3-specific assays using a novel recombinant single-domain binding reagent

Cattle express three subclasses of IgG antibody - IgG1, IgG2 and IgG3. Unlike IgG1 and IgG2, IgG3 was described relatively recently and the role of this subclass in immunity is unknown. Using recombinant bovine IgG1, IgG2 and IgG3 monoclonal antibodies (mAbs), we demonstrated that only one of the commercially available anti-bovine IgG mAbs tested was able to recognize IgG3, and no mAb exclusively bound IgG3. Here, we evaluated a small ankyrin repeat protein, called Ankyron™ AZS40101, that was generated to bind to bovine IgG3. Ankyron™ AZS40101 bound specifically to IgG3 with minimal reactivity to IgG1 and IgG2. Ankyron™ AZS40101 was shown to be useful in ELISA assays as either a capture or detection reagent. Utilisation of Ankyron™ AZS40101 alongside IgG1 and IgG2 specific mAbs to detect antigen-specific IgG subclasses in the serum of cattle sequentially vaccinated with heterologous foot-and-mouth disease virus capsid antigens revealed a low-level antigen-specific IgG3 response, in addition to IgG1 and IgG2 responses. Assessment of the total IgG1, IgG2 and IgG3 levels in healthy cattle plasma samples showed that IgG3 was measurable at a mean concentration of 0.19 mg/mL, although this was significantly lower than those of IgG1 (mean 3.28 mg/mL) and IgG2 (mean 3.41 mg/mL). Thus, Ankyron™ AZS40101 is a new reagent that provides utility for measurement of bovine IgG3 responses to infection and vaccination.

High-Coverage Disulfide Mapping Enabled by Programmable Disulfide-Ene Reaction Integrated onto a Bottom-Up Protein Analysis Workflow.

Mapping disulfide linkages is crucial for characterizing pharmaceutical proteins during drug development and quality control. Traditional bottom-up protein analysis workflows often suffer from incomplete mapping for tryptic peptides consisting of multiple disulfide bonds. Although the employment of a partial reduction of disulfide bonds can improve disulfide mapping, it becomes a bottleneck of analysis because individual tuning is often needed. Herein, we have developed an online disulfide-ene reaction system in which the composition of the reaction solvent can be programmed to achieve optimal partial reduction of tryptic disulfide peptides after liquid chromatography separation. By coupling this system onto a bottom-up protein analysis workflow, high coverage for sequencing (71-83%) and disulfide mapping (84-100%) was achieved for standard proteins consisting of 4-19 disulfide bonds. The analytical capability was further demonstrated by mapping 13 scrambled disulfide bonds in lysozyme and achieving compositional analysis of IgG isotypes (κ and λ) and subclasses (IgG1-IgG4) from human plasma.