The Fab region of IgG impairs the internalization pathway of FcRn upon Fc engagement.

Binding to the neonatal Fc receptor (FcRn) extends serum half-life of IgG, and antagonizing this interaction is a promising therapeutic approach in IgG-mediated autoimmune diseases. Fc-MST-HN, designed for enhanced FcRn binding capacity, has not been evaluated in the context of a full-length antibody, and the structural properties of the attached Fab regions might affect the FcRn-mediated intracellular trafficking pathway. Here we present a comprehensive comparative analysis of the IgG salvage pathway between two full-size IgG1 variants, containing wild type and MST-HN Fc fragments, and their Fc-only counterparts. We find no evidence of Fab-regions affecting FcRn binding in cell-free assays, however, cellular assays show impaired binding of full-size IgG to FcRn, which translates into improved intracellular FcRn occupancy and intracellular accumulation of Fc-MST-HN compared to full size IgG1-MST-HN. The crystal structure of Fc-MST-HN in complex with FcRn provides a plausible explanation why the Fab disrupts the interaction only in the context of membrane-associated FcRn. Importantly, we find that Fc-MST-HN outperforms full-size IgG1-MST-HN in reducing IgG levels in cynomolgus monkeys. Collectively, our findings identify the cellular membrane context as a critical factor in FcRn biology and therapeutic targeting.

Effect of FcRn antagonism on protective antibodies and to vaccines in IgG-mediated autoimmune diseases pemphigus and generalised myasthenia gravis.

Antagonism of the neonatal Fc receptor (FcRn) by efgartigimod has been studied in several autoimmune diseases mediated by immunoglobulin G (IgG) as a therapeutic approach to remove pathogenic IgGs. Whereas reduction of pathogenic titres has demonstrated efficacy in multiple autoimmune diseases, reducing total IgG could potentially increase infection risk in patients receiving FcRn antagonists. The objective of this study was to analyse the effect of FcRn antagonism with efgartigimod on existing protective antibody titres and the ability to mount an immune response after vaccine challenge. Serum levels of total IgG and protective antibodies against tetanus toxoid (TT), varicella zoster virus (VZV), and pneumococcal capsular polysaccharide (PCP) were measured in all patients enrolled in an open-label trial of efgartigimod for the treatment of pemphigus. Vaccine specific-responses were assessed by measuring changes in IgG titres in patients with generalised myasthenia gravis (gMG) who were treated with efgartigimod and who received influenza, pneumococcal, or coronavirus disease 2019 (COVID-19) vaccines during participation in the double-blind trial ADAPT or open-label extension, ADAPT+ (n = 17). FcRn antagonism reduced levels of protective anti-TT, anti-VZV, and anti-PCP antibodies and total IgG to a similar extent; anti-TT and anti-VZV titres remained above minimally protective thresholds for the majority of patients, (10/12) 83% and (14/15) 93% respectively. Protective antibodies returned to baseline values upon treatment cessation. Antigen-specific IgG responses to influenza, pneumococcal, and COVID-19 immunisation were detected in patients with gMG who received these vaccines while undergoing therapy with efgartigimod. In conclusion, FcRn antagonism with efgartigimod did not hamper generation of IgG responses but did transiently reduce IgG titres of all specificities.

FcRn Antagonism Leads to a Decrease of Desmoglein-Specific B Cells: Secondary Analysis of a Phase 2 Study of Efgartigimod in Pemphigus Vulgaris and Pemphigus Foliaceus.

Background: Immunoglobulin G (IgG) levels are maintained by the IgG-recycling neonatal Fc-receptor (FcRn). Pemphigus vulgaris and pemphigus foliaceus are debilitating autoimmune disorders triggered by IgG autoantibodies against mucosal and epidermal desmogleins. Recently, a phase 2 clinical trial (NCT03334058; https://clinicaltrials.gov/NCT03334058) was completed in participants with pemphigus using efgartigimod, an FcRn inhibitor, in combination with prednisone. Efgartigimod demonstrated an early effect on diease activity and was well tolerated. In addition to the safety and efficacy assessment, clinical trials present an opportunity to gain more insights into the mechanism of disease, the mode of action of treatment, and potential for corticosteroid-sparing activity. Objective: The aim of our study was to assess the impact of FcRn antagonism by efgartigimod on immunological parameters known to be directly involved in pemphigus pathology, such as cellular and serological responses. Methods: We investigated total and antigen-specific IgG subclass level kinetics during and after treatment, assessed antigen-specific B-cell responses, followed T- and B-cell immunophenotypes, and analyzed how different immunophenotypes link to clinical response. Results: Treatment resulted in reduction of total IgG as well as autoreactive IgG antibody levels. Surprisingly, unlike total IgG and vaccine- or natural-infection-elicited IgG, which returned to baseline levels after stopping efgartigimod treatment, autoreactive antibody levels remained low in several study participants. Efgartigimod showed no effect on total leukocytes, neutrophils, monocytes, or lymphocytes in patients treated with extended efgartigimod therapy. Intriguingly, antigen-specific analyses revealed a loss of desmoglein-specific B cells in several participants responding to efgartigimod, in line with prolonged reduction of pathogenic IgG levels. Conclusions: Efgartigimod treatment of participants with pemphigus improved their conditions and exerted an immunomodulatory effect beyond the blockade of IgG recycling. Further studies in larger populations with an appropriate placebo control are needed to confirm these potentially important observations to establish long-term clinical responses in autoimmune diseases.

Distinct immune profiles of HIV-infected subjects are linked to specific lipid mediator signature.

INTRODUCTION: To date, with no prophylactic human immunodeficiency virus (HIV) vaccine available, HIV incidence rates remain undefeated. Despite full virological suppression, HIV+ individuals exhibit a higher rate of cardiovascular disorders and cancers what is attributed to the residual, persistent levels of immune activation. METHODS: We have established the Virological and Immunological Monitoring (VIM) platform and forty VIM samples that included treated immunological responders (IRs) or nonresponders (INRs), viremic untreated subjects and uninfected controls, were phenotyped by flow cytometry and plasma was used to quantify proinflammatory eicosanoids and the specialized proresolving mediators by liquid chromatography tandem mass spectrometry. RESULTS: While HIV infection profoundly altered lipid mediator (LM) profile, differences were also seen in patients on viral suppressive therapy. IRs exhibited higher levels of proresolving mediators as compared to INRs and notable differences in plasma LM were also seen in early and late treated individuals. CONCLUSIONS: This study demonstrated distortions in proinflammatory/proresolution processes in infected patients including those with controlled viremia.

Route of immunization defines multiple mechanisms of vaccine-mediated protection against SIV.

Antibodies are the primary correlate of protection for most licensed vaccines; however, their mechanisms of protection may vary, ranging from physical blockade to clearance via the recruitment of innate immunity. Here, we uncover striking functional diversity in vaccine-induced antibodies that is driven by immunization site and is associated with reduced risk of SIV infection in nonhuman primates. While equivalent levels of protection were observed following intramuscular (IM) and aerosol (AE) immunization with an otherwise identical DNA prime-Ad5 boost regimen, reduced risk of infection was associated with IgG-driven antibody-dependent monocyte-mediated phagocytosis in the IM vaccinees, but with vaccine-elicited IgA-driven neutrophil-mediated phagocytosis in AE-immunized animals. Thus, although route-independent correlates indicate a critical role for phagocytic Fc-effector activity in protection from SIV, the site of immunization may drive this Fc activity via distinct innate effector cells and antibody isotypes. Moreover, the same correlates predicted protection from SHIV infection in a second nonhuman primate vaccine trial using a disparate IM canarypox prime-protein boost strategy, analogous to that used in the first moderately protective human HIV vaccine trial. These data identify orthogonal functional humoral mechanisms, initiated by distinct vaccination routes and immunization strategies, pointing to multiple, potentially complementary correlates of immunity that may support the rational design of a protective vaccine against HIV.

Highlights from the HIV Cure and Reservoir Symposium, 11-12 September 2017, Ghent, Belgium.

For the second time, the HIV Cure Research Center (HCRC) at Ghent University organised the HIV Cure and Reservoir Symposium, in Ghent, Belgium, where in this two-day conference, virologists, molecular biologists, immunologists and clinicians presented the most recent achievements in the field of HIV cure, including data on therapeutic vaccines, HIV remission strategies such as 'shock and kill' or 'block and lock', benefits of early ART and potential of haematopoietic stem cell transplant in achieving cure. Furthermore, methods to characterise and quantify the HIV reservoir were discussed along with HIV reservoir characterisation in different body parts, including the central nervous system. An HIV activist and representative of a patients' agency also presented the patients' perspective on HIV cure. This report is a summary of all topics discussed during this symposium.

HIV Reservoir Characterization Symposium: 19 September 2016, Ghent, Belgium.

The HIV Cure Research Center (HCRC) in Ghent organised the first HIV Reservoir Characterization Symposium, and brought together virologists, molecular biologists, immunologists and clinicians to discuss the most recent developments in HIV reservoir characterisation with a view to achieving an HIV cure. The one-day symposium covered new developments in the field of HIV reservoir and HIV cure research, with the latest news on the European HIV cure trials. This report summarises the major themes discussed during the symposium.

A Functional Role for Antibodies in Tuberculosis.

While a third of the world carries the burden of tuberculosis, disease control has been hindered by a lack of tools, including a rapid, point-of-care diagnostic and a protective vaccine. In many infectious diseases, antibodies (Abs) are powerful biomarkers and important immune mediators. However, in Mycobacterium tuberculosis (Mtb) infection, a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach, we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses, such that Ltb infection is associated with unique Ab Fc functional profiles, selective binding to FcγRIII, and distinct Ab glycosylation patterns. Moreover, compared to Abs from Atb, Abs from Ltb drove enhanced phagolysosomal maturation, inflammasome activation, and, most importantly, macrophage killing of intracellular Mtb. Combined, these data point to a potential role for Fc-mediated Ab effector functions, tuned via differential glycosylation, in Mtb control.

HLA-C levels impact natural killer cell subset distribution and function.

Differences in HLA-C expression are inversely correlated with HIV viral load set-point and slower progression to AIDS, linked to enhanced cytotoxic T cell immunity. Yet, beyond T cells, HLA-C serves as a dominant ligand for natural killer (NK) cell killer immunoglobulin-like receptors (KIR). Thus, we speculated that HLA-C expression levels may also impact NK activity, thereby modulating HIV antiviral control. Phenotypic and functional profiling was performed on freshly isolated PBMCs. HLA-C expression was linked to changes in NK subset distribution and licensing, particularly in HLA-C1/C1, KIR2DL3+2DL2-individuals. Moreover, high levels of HLA-C, were associated with reduced frequencies of anergic CD56neg NKs and lower frequencies of KIR2DL1/2/3+ NK cells, pointing to an HLA-C induced influence on the NK cell development in the absence of disease. In HIV infection, several spontaneous controllers, that expressed higher levels of HLA-C demonstrated robust NK-IFN-γ secretion in response to target cells, highlighting a second disease induced licensing phenotype. Thus this population study points to a potential role for HLA-C levels both in NK cell education and development.

TLR2 activation causes no morbidity or cardiovascular failure, despite excessive systemic nitric oxide production.

AIMS: Septic shock is the leading cause of death in intensive care units worldwide, resulting from a progressive systemic inflammatory reaction causing cardiovascular and organ failure. Nitric oxide (NO) is a potent vasodilator and inhibition of NO synthases (NOS) can increase blood pressure in septic shock. However, NOS inhibition does not improve outcome, on the contrary, and certain NO donors may even provide protection. In addition, NOS produce superoxide in case of substrate or cofactor deficiency or oxidation. We hypothesized that excessive systemic iNOS-derived NO production is insufficient to trigger cardiovascular failure and shock. METHODS AND RESULTS: We found that the systemic injection with various synthetic Toll-like receptor-2 (TLR2), TLR3, or TLR9 agonists triggered systemic NO production identical to that of lipopolysaccharide (LPS) or tumour necrosis factor. In contrast to the latter, however, these agonists did not cause hypothermia or any other signs of discomfort or morbidity, and inflammatory cytokine production was low. TLR2 stimulation with the triacylated lipopeptide Pam3CSK4 not only caused identical NO levels in circulation, but also identical iNOS expression patterns as LPS. Nevertheless, Pam3CSK4 did not cause hypotension, bradycardia, reduced blood flow, or inadequate tissue perfusion in the kidney or the liver. CONCLUSION: We demonstrate that excessive iNOS-derived NO in circulation is not necessarily linked to concomitant cardiovascular collapse, morbidity, or mortality. As such, our data indicate that the central role of iNOS-derived NO in inflammation-associated cardiovascular failure may be overestimated.

Single-cell analysis of the dynamics and functional outcomes of interactions between human natural killer cells and target cells.

Natural killer (NK) cells are a subset of innate immune lymphocytes that interrogate potential target cells and rapidly respond by lysing them or secreting inflammatory immunomodulators. Productive interactions between NK cells and targets such as tumor cells or virally infected cells are critical for immunological control of malignancies and infections. For individual NK cells, however, the relationship between the characteristics of these cell-cell interactions, cytolysis, and secretory activity is not well understood. Here, we used arrays of subnanoliter wells (nanowells) to monitor individual NK cell-target cell interactions and quantify the resulting cytolytic and secretory responses. We show that NK cells operate independently when lysing a single target cell and that lysis is most probable during an NK cell's first encounter with a target. Furthermore, we demonstrate that the secretion of interferon-γ (IFN-γ) occurs most often among NK cells that become the least motile upon contacting a target cell but is largely independent of cytolysis. Our findings demonstrate that integrated analysis of the cell-cell interaction parameters, cytolytic activity, and secretory activity of single NK cells can reveal new insights into how these complex functions are related within individual cells.

Decreased Fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically HIV-1 infected individuals.

In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection.