Abstract Objectives Tuberculosis (TB) remains a substantial cause of morbidity and mortality among people living with human immunodeficiency virus (HIV) worldwide. However, the immunological mechanisms associated with the enhanced susceptibility among HIV‐positive individuals remain largely unknown. Methods Here, we used a simian immunodeficiency virus (SIV)/TB‐coinfection Mauritian cynomolgus macaque (MCM) model to examine humoral responses from the plasma of SIV‐negative (n = 8) and SIV‐positive (n = 7) MCM 8‐week postinfection with Mycobacterium tuberculosis (Mtb). Results Antibody responses to Mtb were impaired during SIV coinfection. Elevated inflammatory bulk IgG antibody glycosylation patterns were observed in coinfected macaques early at 8‐week post‐Mtb infection, including increased agalactosylation (G0) and reduced di‐galactosylation (G2), which correlated with endpoint Mtb bacterial burden and gross pathology scores, as well as the time‐to‐necropsy. Conclusion These studies suggest that humoral immunity may contribute to control of TB disease and support growing literature that highlights antibody Fc glycosylation as a biomarker of TB disease progression.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern such as Omicron hampered efforts in controlling the ongoing coronavirus disease 2019 pandemic due to their ability to escape neutralizing antibodies induced by vaccination or prior infection, highlighting the need to develop broad-spectrum vaccines and therapeutics. Most human monoclonal antibodies (mAbs) reported to date have not demonstrated true pan-sarbecovirus neutralizing breadth especially against animal sarbecoviruses. Here, we report the isolation and characterization of highly potent mAbs targeting the receptor binding domain (RBD) of huACE2-dependent sarbecovirus from a SARS-CoV survivor vaccinated with BNT162b2. Among the six mAbs identified, one (E7) showed better huACE2-dependent sarbecovirus neutralizing potency and breadth than any other mAbs reported to date. Mutagenesis and cryo-electron microscopy studies indicate that these mAbs have a unique RBD contact footprint and that E7 binds to a quaternary structure-dependent epitope.

Emerging SARS-CoV-2 variants, notably Omicron, continue to remain a formidable challenge to worldwide public health. The SARS-CoV-2 receptor-binding domain (RBD) is a hotspot for mutations, reflecting its critical role at the ACE2 interface during viral entry. Here, we comprehensively investigated the impact of RBD mutations, including 5 variants of concern (VOC) or interest—including Omicron (BA.2)—and 33 common point mutations, both on IgG recognition and ACE2-binding inhibition, as well as FcγRIIa- and FcγRIIIa-binding antibodies, in plasma from two-dose BNT162b2-vaccine recipients and mild-COVID-19 convalescent subjects obtained during the first wave using a custom-designed bead-based 39-plex array. IgG-recognition and FcγR-binding antibodies were decreased against the RBD of Beta and Omicron, as well as point mutation G446S, found in several Omicron sub-variants as compared to wild type. Notably, while there was a profound decrease in ACE2 inhibition against Omicron, FcγR-binding antibodies were less affected, suggesting that Fc functional antibody responses may be better retained against the RBD of Omicron in comparison to neutralization. Furthermore, while measurement of RBD–ACE2-binding affinity via biolayer interferometry showed that all VOC RBDs have enhanced affinity to human ACE2, we demonstrate that human ACE2 polymorphisms, E35K (rs1348114695) has reduced affinity to VOCs, while K26R (rs4646116) and S19P (rs73635825) have increased binding kinetics to the RBD of VOCs, potentially affecting virus–host interaction and, thereby, host susceptibility. Collectively, our findings provide in-depth coverage of the impact of RBD mutations on key facets of host–virus interactions.

Background The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines.Methods We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid alpha-Galactosylceramide, or MF59 (R) squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine.Findings Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59 (R) adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59 (R) increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5.Interpretation These results demonstrated that an RBD-Fc protein subunit/MF59 (R) adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial.Copyright (c) 2023 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains a formidable challenge to worldwide public health. The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is a hotspot for mutations, reflecting its critical role at the ACE2 interface during viral entry. We comprehensively investigated the impact of RBD mutations, including 6 variants of concern (VOC) or interest (Alpha, Beta, Gamma, Delta, Kappa and Omicron) and 33 common point mutations, on IgG recognition, FcγR-engagement, and ACE2-binding inhibition in plasma from BNT162b2-vaccine recipients (two-weeks following second dose) and mild-to-moderate COVID-19 convalescent subjects using our custom bead-based 39-plex array. We observed that IgG-recognition and FcγR-binding antibodies were most profoundly decreased against Beta and Omicron RBDs, as well as point mutations G446S, found in Omicron, and N501T, a key mutation found in animal adapted SARS-CoV-2 viruses. Measurement of RBD-ACE2 binding affinity via Biolayer Interferometry showed all VOC RBDs have enhanced affinity to human ACE2. Furthermore we demonstrate that human ACE2 polymorphisms, E35K (rs1348114695), K26R (rs4646116) and S19P (rs73635825), have altered binding kinetics to the RBD of VOCs potentially affecting virus-host interaction and thereby host susceptibility.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis study was supported by the Victorian Government and Medical Research Future Fund (MRFF) GNT2002073 (to A.K.W, P.M.H., W.H.T., D.I.G., S.J.K., and A.W.C.), GNT2005544 (to A.K.W., J.A.J., D.I.G., S.J.K., and A.W.C), the Paul Ramsay Foundation (A.K.W., D.I.G., S.J.K. and A.W.C.). A.K.W, J.A.J., D.I.G., W.H.T., S.J.K. and A.W.C. are supported by NHMRC fellowships. W-H.T. is a Howard Hughes Medical Institute Wellcome Trust International Research Scholar (208693/Z/17/Z). N.A.G. is supported by an ARC DECRA fellowship.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Study protocols were approved by the University of Melbourne Human Research Ethics Committee (#2056689).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present work are contained in the manuscript

Abstract Objectives Following infection with SARS‐CoV‐2, virus‐specific antibodies are generated, which can both neutralise virions and clear infection via Fc effector functions. The importance of IgG antibodies for protection and control of SARS‐CoV‐2 has been extensively reported. By comparison, other antibody isotypes including IgA have been poorly characterised. Methods Here, we characterised plasma IgA from 41 early convalescent COVID‐19 subjects for neutralisation and Fc effector functions. Results Convalescent plasma IgA from > 60% of the cohort had the capacity to inhibit the interaction between wild‐type RBD and ACE2. Furthermore, a third of the cohort induced stronger IgA‐mediated ACE2 inhibition than matched IgG when tested at equivalent concentrations. Plasma IgA and IgG from this cohort broadly recognised similar RBD epitopes and had similar capacities to inhibit ACE2 from binding to 22 of the 23 prevalent RBD mutations assessed. However, plasma IgA was largely incapable of mediating antibody‐dependent phagocytosis in comparison with plasma IgG. Conclusion Overall, convalescent plasma IgA contributed to the neutralising antibody response of wild‐type SARS‐CoV‐2 RBD and various RBD mutations. However, this response displayed large heterogeneity and was less potent than IgG.

Joining a function-enhanced Fc-portion of human IgG to the SARS-CoV-2 entry receptor ACE2 produces an antiviral decoy with strain transcending virus neutralizing activity. SARS-CoV-2 neutralization and Fc-effector functions of ACE2-Fc decoy proteins, formatted with or without the ACE2 collectrin domain, were optimized by Fc-modification. The different Fc-modifications resulted in distinct effects on neutralization and effector functions. H429Y, a point mutation outside the binding sites for FcγRs or complement caused non-covalent oligomerization of the ACE2-Fc decoy proteins, abrogated FcγR interaction and enhanced SARS-CoV-2 neutralization. Another Fc mutation, H429F did not improve virus neutralization but resulted in increased C5b-C9 fixation and transformed ACE2-Fc to a potent mediator of complement-dependent cytotoxicity (CDC) against SARS-CoV-2 spike (S) expressing cells. Furthermore, modification of the Fc-glycan enhanced cell activation via FcγRIIIa. These different immune profiles demonstrate the capacity of Fc-based agents to be engineered to optimize different mechanisms of protection for SARS-CoV-2 and potentially other viral pathogens.

The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralize virus, have potential for the treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralize the virus. We show that combining two clones with distinct binding epitopes within the RBD into a single protein construct to generate biparatopic reagents dramatically enhances their neutralizing capacity. Furthermore, the biparatopic nanobodies exhibit enhanced control over clinically relevant RBD variants that escaped recognition by the individual nanobodies. Structural analysis of biparatopic binding to spike (S) protein revealed a unique binding mode whereby the two nanobody paratopes bridge RBDs encoded by distinct S trimers. Accordingly, biparatopic nanobodies offer a way to rapidly generate powerful viral neutralizers with enhanced ability to control viral escape mutants.

Allelic variants are found amongst all human IgG subclasses and can influence a range of Fc functions. IgG allotypes have been associated with reduced risk of clinical malaria, an ancient disease that has co-evolved with humanity. However, how they contribute to malaria control is not fully understood. Here, we identified a novel IgG3 allotype in pregnant women from Papua New Guinea who were highly exposed to malaria. The new G3m29 (IGHG3*30) allotype was prominent in women of Sepik ethnicity. Plasma from women with G3m29 induced enhanced antibody-dependent phagocytosis of Plasmodium falciparum CS2-infected erythrocytes. A human monoclonal antibody to P. falciparum VAR2CSA, recombinantly generated as IGHG3*30, showed enhanced affinity to FcγRIIa and induced increased antibody-dependent phagocytosis in comparison to other IgG3 allotypes. This study provides clues to the role of allotypic variants of IgG3 in the control of malaria.

Fc-mediated immune functions have been correlated with protection in the RV144 HIV vaccine trial and are important for immunity to a range of pathogens. IgG antibodies (Abs) that form complexes with Fc receptors (FcRs) on innate immune cells can activate Fc-mediated immune functions. Genetic variation in both IgGs and FcRs have the capacity to alter IgG-FcR complex formation via changes in binding affinity and concentration. A growing challenge lies in unraveling the importance of multiple variations, especially in the context of vaccine trials that are conducted in homogenous genetic populations. Here we use an ordinary differential equation model to quantitatively assess how IgG1 allotypes and FcγR polymorphisms influence IgG-FcγRIIIa complex formation in vaccine-relevant settings. Using data from the RV144 HIV vaccine trial, we map the landscape of IgG-FcγRIIIa complex formation predicted post-vaccination for three different IgG1 allotypes and two different FcγRIIIa polymorphisms. Overall, the model illustrates how specific vaccine interventions could be applied to maximize IgG-FcγRIIIa complex formation in different genetic backgrounds. Individuals with the G1m1,17 and G1m1,3 allotypes were predicted to be more responsive to vaccine adjuvant strategies that increase antibody FcγRIIIa affinity (e.g. glycosylation modifications), compared to the G1m-1,3 allotype which was predicted to be more responsive to vaccine boosting regimens that increase IgG1 antibody titers (concentration). Finally, simulations in mixed-allotype populations suggest that the benefit of boosting IgG1 concentration versus IgG1 affinity may be dependent upon the presence of the G1m-1,3 allotype. Overall this work provides a quantitative tool for rationally improving Fc-mediated functions after vaccination that may be important for assessing vaccine trial results in the context of under-represented genetic populations.

Immune effector responses against Plasmodium falciparum include antibody-mediated activation of innate immune cells, which can induce Fc effector functions, including antibody-dependent cellular cytotoxicity, and the secretion of cytokines and chemokines. These effector functions are regulated by the composition of immunoglobulin G (IgG) Fc N-linked glycans. However, a role for antibody-mediated natural killer (NK) cells activation or Fc N-linked glycans in pregnant women with malaria has not yet been established. Herein, we studied the capacity of IgG antibodies from pregnant women, with placental malaria or non-placental malaria, to induce NK cell activation in response to placental malaria-associated antigens DBL2 and DBL3. Antibody-mediated NK cell activation was observed in pregnant women with malaria, but no differences were associated with susceptibility to placental malaria. Elevated anti-inflammatory glycosylation patterns of IgG antibodies were observed in pregnant women with or without malaria infection, which were not seen in healthy non-pregnant controls. This suggests that pregnancy-associated anti-inflammatory Fc N-linked glycans may dampen the antibody-mediated activation of NK cells in pregnant women with malaria infection. Overall, although anti-inflammatory glycans and antibody-dependent NK cell activation were detected in pregnant women with malaria, a definitive role for these antibody features in protecting against placental malaria remains to be proven.

Abstract Objectives SARS‐CoV‐2 can be transmitted by aerosols, and the ocular surface may be an important route of transmission. Little is known about protective antibody responses to SARS‐CoV‐2 in tears after infection or vaccination. We analysed the SARS‐CoV‐2‐specific IgG and IgA responses in human tears after either COVID‐19 infection or vaccination. Methods We measured the antibody responses in 16 subjects with COVID‐19 infection for an average of 7 months before, and 15 subjects before and 2 weeks post‐Comirnaty (Pfizer‐BioNtech) vaccination. Plasma, saliva and basal tears were collected. Eleven pre‐pandemic individuals were included as healthy controls. Results IgG antibodies to spike and nucleoprotein were detected in tears, saliva and plasma from subjects with prior SARS‐CoV‐2 infection in comparison with uninfected controls. While receptor‐binding domain (RBD)‐specific antibodies were detected in plasma, minimal RBD‐specific antibodies were detected in tears and saliva. By contrast, high levels of IgG antibodies to spike and RBD, but not nucleoprotein, were induced in tears, saliva and plasma of subjects receiving 2 doses of the Comirnaty vaccine. Increased levels of IgA1 and IgA2 antibodies to SARS‐CoV‐2 antigens were detected in plasma following infection or vaccination but were unchanged in tears and saliva. Comirnaty vaccination induced high neutralising Abs in the plasma, but limited neutralising antibodies were detected in saliva or tears. Conclusion Both infection and vaccination induce SARS‐CoV‐2‐specific IgG antibodies in tears. RBD‐specific IgG antibodies in tears were induced by vaccination but were not present 7 months post‐infection. This suggests the neutralising antibodies may be low in the tears late following infection.

The SARS-CoV-2 receptor binding domain (RBD) is both the principal target of neutralizing antibodies and one of the most rapidly evolving domains, which can result in the emergence of immune escape mutations, limiting the effectiveness of vaccines and antibody therapeutics. To facilitate surveillance, we developed a rapid, high-throughput, multiplex assay able to assess the inhibitory response of antibodies to 24 RBD natural variants simultaneously. We demonstrate how this assay can be implemented as a rapid surrogate assay for functional cell-based serological methods to measure the SARS-CoV-2 neutralizing capacity of antibodies at the angiotensinconverting enzyme 2-RBD (ACE2-RBD) interface. We describe the enhanced affinity of RBD variants N439K, S477N, Q493L, S494P, and N501Y to the ACE2 receptor and demonstrate the ability of this assay to bridge a major gap for SARS-CoV-2 research, informing selection of complementary monoclonal antibody candidates and the rapid identification of immune escape to emerging RBD variants following vaccination or natural infection.

Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics.

Immunoglobulin G (IgG) antibodies that activate Fc-mediated immune functions have been correlated with vaccine efficacy, but it is difficult to unravel the relative roles of multiple IgG and Fc receptor (FcR) features that have the capacity to influence IgG-FcR complex formation but vary on a personalized basis. Here, we develop an ordinary differential-equation model to determine how personalized variability in IgG subclass concentrations and binding affinities influence IgG-FcγRIIIa complex formation and validate it with samples from the HIV RV144 vaccine trial. The model identifies individuals who are sensitive, insensitive, or negatively affected by increases in HIV-specific IgG1, which is validated with the addition of HIV-specific IgG1 monoclonal antibodies to vaccine samples. IgG1 affinity to FcγRIIIa is also prioritized as the most influential parameter for dictating activation broadly across a population. Overall, this work presents a quantitative tool for evaluating personalized differences underlying FcR activation, which is relevant to ongoing efforts to improve vaccine efficacy.

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

Abstract Objectives Tuberculosis comorbidity with chronic diseases including diabetes, HIV and chronic kidney disease is of rising concern. In particular, latent tuberculosis infection (LTBI) comorbidity with end‐stage kidney disease (ESKD) is associated with up to 52.5‐fold increased risk of TB reactivation to active tuberculosis infection (ATBI). The immunological mechanisms driving this significant rise in TB reactivation are poorly understood. To contribute to this understanding, we performed a comprehensive assessment of soluble and cellular immune features amongst a unique cohort of patients comorbid with ESKD and LTBI. Methods We assessed the plasma and cellular immune profiles from patients with and without ESKD and/or LTBI (N = 40). We characterised antibody glycosylation, serum complement and cytokine levels. We also assessed classical and non‐classical monocytes and T cells with flow cytometry. Using a systems‐based approach, we identified key immunological features that discriminate between the different disease states. Results Individuals with ESKD exhibited a highly inflammatory plasma profile and an activated cellular state compared with those without ESKD, including higher levels of inflammatory antibody Fc glycosylation structures and activated CX3CR1+ monocytes that correlate with increased inflammatory plasma cytokines. Similar elevated inflammatory signatures were also observed in ESKD+/LTBI+ compared with ESKD−/LTBI+, suggesting that ESKD induces an overwhelming inflammatory immune state. In contrast, no significant inflammatory differences were observed when comparing LTBI+ and LTBI− individuals. Conclusion Our study highlights the highly inflammatory state induced by ESKD. We hypothesise that this inflammatory state could contribute to the increased risk of TB reactivation in ESKD patients.

AIM:To characterize anterior eye health and tear film characteristics in individuals with human immunodeficiency virus (HIV) undergoing anti-retroviral therapy. METHODS:This cross-sectional study involved 35 adults, categorized as healthy controls (n = 18) or as HIV-positive patients (n = 17), with no history of opportunistic infection or current ocular fundus abnormalities. Participants underwent a comprehensive anterior eye assessment. Primary outcome measures were dry eye symptoms (Ocular Surface Disease Index survey), tear film osmolarity, and extent of meibomian gland dropout. Secondary outcomes measures were ocular redness, tear film stability, and ocular surface staining. Levels of 36 cytokines were assayed from basal tears using a multiplex bead array. RESULTS:The HIV-positive group showed more extensive meibomian gland dropout relative to controls (mean ± SD, controls: 29.6 ± 5.8 versus 37.0 ± 13.9%, p = 0.045). The extent of meibomian gland dropout was negatively correlated with blood CD4 T-cell count (a marker of immunodeficiency) at diagnosis (r = -0.69, p = 0.006). All other tests of anterior ocular health, including dry eye symptom levels, were not significantly different between the groups. There were no significant inter-group differences for the 36 cytokines assayed in the tear film. CONCLUSIONS:We find greater meibomian gland dropout in HIV-positive individuals that is related to disease severity at diagnosis. Given this feature predisposes to dry eye disease, it suggests the need for long-term studies of anterior eye health in people with HIV.

Evaluating the biophysical and functional nature of IgG is key to defining correlates of protection in infectious disease, and autoimmunity research cohorts, as well as vaccine efficacy trials. These studies often require small quantities of IgG to be purified from plasma for downstream analysis with high throughput immunoaffinity formats which elute IgG at low-pH, such as Protein G and Protein A. Herein we sought to compare Protein G purification of IgG with an immunoaffinity method which elutes at physiological pH (Melon Gel). Critical factors impacting Fc functionality with the potential to significantly influence FcγR binding, such as IgG subclass distribution, N-glycosylation, aggregation, and IgG conformational changes were investigated and compared. We observed that transient exposure of IgG to the low-pH elution buffer, used during the Protein G purification process, artificially enhanced recognition of Fcγ Receptors (FcγRs) as demonstrated by Surface Plasmon Resonance (SPR), FcγR dimer ELISA, and a functional cell-based assay. Furthermore, low-pH exposed IgG caused conformational changes resulting in increased aggregation and hydrophobicity; factors likely to contribute to the observed enhanced interaction with FcγRs. These results highlight that methods employed to purify IgG can significantly alter FcγR-binding behavior and biological activity and suggest that the IgG purification approach selected may be a previously overlooked factor contributing to the poor reproducibility across current assays employed to evaluate Fc-mediated antibody effector functions.