Nicotiana spp. for the Expression and Purification of Functional IgG3 Antibodies Directed Against the Staphylococcus aureus Alpha Toxin

Immunoglobulin subclass IgG1 is bound and neutralized effectively by Staphylococcus aureus protein A, allowing the bacterium to evade the host’s adaptive immune response. In contrast, the IgG3 subclass is not bound by protein A and can be used to treat S. aureus infections, including drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). However, the yields of recombinant IgG3 are generally low because this subclass is prone to degradation, and recovery is hindered by the inability to use protein A as an affinity ligand for antibody purification. Here, we investigated plants (Nicotiana spp.) as an alternative to microbes and mammalian cell cultures for the production of an IgG3 antibody specific for the S. aureus alpha toxin. We targeted recombinant IgG3 to different subcellular compartments and tested different chromatography conditions to improve recovery and purification. Finally, we tested the antigen-binding capacity of the purified antibodies. The highest IgG3 levels in planta (>130 mg kg−1 wet biomass) were achieved by targeting the endoplasmic reticulum or apoplast. Although the purity of IgG3 exceeded 95% following protein G chromatography, product recovery requires further improvement. Importantly, the binding affinity of the purified antibodies was in the nanomolar range and thus comparable to previous studies using murine hybridoma cells as the production system.

Can Antinuclear Antibodies (ANA) be Monoclonal?

Background. Nuclear staining by immunofluorescence in a kidney biopsy is often seen in patients with positive antinuclear antibodies (ANA) in the serum. These ANA are usually polyclonal, but herein we report 9 cases with an unusual finding of monoclonal nuclear staining by immunofluorescence on kidney biopsy. Case Presentation. Nine cases with predominant stain for kappa or lambda light chain were identified by searching the renal pathology laboratory database for the past 10 years. All cases had positive stain for only kappa (six cases) or lambda (three cases) light chain in the nuclei. Eight out of nine cases had positive nuclear IgG stain, and one case had positive nuclear IgA stain. Among cases with positive nuclear IgG staining, six cases were positive for IgG1 subclass, one case was positive for IgG2 subclass, and one case was positive for IgG3 subclass. All patients with positive IgG nuclear stain, who had testing for ANA, had positive ANA. Patients with positive IgG1 subclass did not have monoclonal protein in the serum or urine, but the patient with positive IgG2 subclass and lambda light chain stain in the nuclei had IgG lambda monoclonal gammopathy. Conclusions. We identified a new unique pattern of nuclear stain by immunofluorescence in kidney biopsies that suggests the presence of monoclonal ANA. Workup for underlying monoclonal gammopathy is warranted in such patients.

Characterization of THSD7A-antibodies not binding to glomerular THSD7A in a patient with diabetes mellitus but no membranous nephropathy

Membranous nephropathy (MN) is an autoimmune disease caused by autoantibodies against the podocyte antigens phospholipase A2 receptor 1 (PLA2R1) and thrombospondin type 1 domain containing protein 7A (THSD7A) in 80% and 2–3% of patients, respectively. THSD7A antibodies are considered to be pathogenic and highly specific for MN patients. Using an indirect immunofluorescence test (IIFT) we detected THSD7A-antibodies (titre 1:10) in the serum of a patient with high proteinuria who, however, in the kidney biopsy was diagnosed with diabetic nephropathy and MN was excluded as a possible cause of proteinuria. Different immunofluorescence assays and Western blot techniques using recombinant THSD7A (rTHSD7A) or THSD7A from different human tissues revealed that the circulating THSD7A-autoantibodies were only of the IgG3 subclass. The patient serum reacted exclusively with rTHSD7A and only when the antigen was present in reducing Western blot conditions, or on formaldehyde-fixed cells for the IIFT. Our findings show for the first time the existence of circulating THSD7A-antibodies recognizing denatured/reduced rTHSD7A, which do not react with glomerular THSD7A in vivo and are thus presumptively non-pathogenic. As a consequence, kidney biopsy or Western blot analyses of THSD7A under non-reducing conditions should be performed to confirm the diagnosis of THSD7A-associated MN, especially in cases with low THSD7A-antibody levels in the IIFT.

The IgG3 subclass of β1-adrenergic receptor autoantibodies is an endogenous biaser of β1AR signaling

β1-Adrenergic receptor autoantibodies are associated with deleterious consequences; however, the IgG3 subclass provides beneficial outcomes in human heart failure. IgG3 β1AR autoantibody inhibits G-protein coupling, simultaneously activating ERK through G-protein–independent arrestin-dependent mechanisms.

Serological Analysis Reveals an Imbalanced IgG Subclass Composition Associated with COVID-19 Disease Severity

SummaryCOVID-19 is associated with a wide spectrum of disease severity, ranging from asymptomatic to acute respiratory distress syndrome (ARDS). Paradoxically, a direct relationship has been suggested between COVID-19 disease severity, and the levels of circulating SARS-CoV-2-specific antibodies, including virus neutralizing titers. Through a serological analysis of serum samples from 536 convalescent healthcare workers, we found that SARS-CoV-2-specific and virus-neutralizing antibody levels were indeed elevated in individuals that experienced severe disease. The severity-associated increase in SARS-CoV-2-specific antibody was dominated by IgG, with an IgG subclass ratio skewed towards elevated receptor binding domain (RBD)- and S1-specific IgG3. However, RBD- and S1-specific IgG1, rather than IgG3 were best correlated with virus-neutralizing titers. We propose that Spike-specific IgG3 subclass utilization contributes to COVID-19 disease severity through potent Fc-mediated effector functions. These results have significant implications for SARS-CoV-2 vaccine design, and convalescent plasma therapy.

The IgG3 Subclass of β1-adrenergic receptor autoantibody is an endogenous biaser of β1AR signaling

ABSTRACTAutoantibodies recognizing human β1ARs generated due to dysregulation in autoimmune response are generally associated with deleterious cardiac outcomes. However, cellular studies show that isolates of β1AR autoantibody from patients differentially modulate β1AR function. β1AR autoantibodies belong to the IgG class of immunoglobulins, however it is not known whether the IgG sub-classes mediate variability in β1AR responses. To determine whether the IgG3 subclass of β1AR autoantibodies uniquely modulate β1AR function, HEK293 cells stably expressing human β1ARs were utilized. Treatment of cells with IgG3(-) serum resulted in significant increase of cAMP compared to IgG3(+) serum. Pre-treatment of cells with IgG3(+) serum impaired dobutamine-mediated Adenylate Cyclase (AC) activity and cAMP generation whereas, it surprisingly increased AC activity and cAMP generation with β-blocker metoprolol. Consistently, purified IgG3(+) β1AR autoantibodies impaired dobutamine-mediated cAMP while elevating metoprolol-mediated AC activity and cAMP. Despite IgG3(+) autoantibodies reducing cAMP response to dobutamine, they mediate significant ERK activation upon dobutamine. IgG3(+) β1AR autoantibodies did not alter β2AR function, reflecting their specificity. The study shows that IgG3(+) β1AR autoantibody impairs agonist-mediated G-protein coupling while preferentially mediating G-protein-independent ERK activation. Furthermore, it uniquely biases β-blocker towards G-protein coupling. This unique biasing capabilities of IgG3(+) β1AR autoantibodies may underlie the beneficial outcomes in patients.

An MPER antibody neutralizes HIV-1 using germline features shared among donors

The membrane-proximal external region (MPER) of HIV-1 envelope glycoprotein (Env) can be targeted by neutralizing antibodies of exceptional breadth. MPER antibodies usually have long, hydrophobic CDRH3s, lack activity as inferred germline precursors, are often from the minor IgG3 subclass, and some are polyreactive, such as 4E10. Here we describe an MPER broadly neutralizing antibody from the major IgG1 subclass, PGZL1, which shares germline V/D-region genes with 4E10, has a shorter CDRH3, and is less polyreactive. A recombinant sublineage variant pan-neutralizes a 130-isolate panel at 1.4 μg/ml (IC50). Notably, a germline revertant with mature CDR3s neutralizes 12% of viruses and still binds MPER after DJ reversion. Crystal structures of lipid-bound PGZL1 variants and cryo-EM reconstruction of an Env-PGZL1 complex reveal how these antibodies recognize MPER and viral membrane. Discovery of common genetic and structural elements among MPER antibodies from different patients suggests that such antibodies could be elicited using carefully designed immunogens.