Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), primarily infects cells at mucosal surfaces. Serum neutralizing antibody ...responses are variable and generally low in individuals that suffer mild forms of COVID-19. Although potent immunoglobulin G (IgG) antibodies can neutralize the virus, less is known about secretory antibodies such as IgA that might affect the initial viral spread and transmissibility from the mucosa. Here, we characterize the IgA response to SARS-CoV-2 in a cohort of 149 convalescent individuals after diagnosis with COVID-19. IgA responses in plasma generally correlated with IgG responses. Furthermore, clones of IgM-, IgG-, and IgA-producing B cells were derived from common progenitor cells. Plasma IgA monomers specific to SARS-CoV-2 proteins were demonstrated to be twofold less potent than IgG equivalents. However, IgA dimers, the primary form of antibody in the nasopharynx, were, on average, 15 times more potent than IgA monomers against the same target. Thus, dimeric IgA responses may be particularly valuable for protection against SARS-CoV-2 and for vaccine efficacy.
Immunoglobulin G (IgG) antibodies contain a complex N-glycan embedded in the hydrophobic pocket between its heavy chain protomers. This glycan contributes to the structural organization of the Fc ...domain and determines its specificity for Fcγ receptors, thereby dictating distinct cellular responses. The variable construction of this glycan structure leads to highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported synthetic nanobodies that distinguish IgG glycoforms. Here, we present the structure of one such nanobody, X0, in complex with the Fc fragment of afucosylated IgG1. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N-glycan and acts as a 'glycan sensor', forming hydrogen bonds with the afucosylated IgG N-glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.
We discovered that
(
), a ubiquitous commensal bacterium, and its secreted peptidoglycan hydrolase (SagA) were sufficient to enhance intestinal barrier function and pathogen tolerance, but the ...precise biochemical mechanism was unknown. Here we show
has unique peptidoglycan composition and remodeling activity through SagA, which generates smaller muropeptides that more effectively activates nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mammalian cells. Our structural and biochemical studies show that SagA is a NlpC/p60-endopeptidase that preferentially hydrolyzes crosslinked Lys-type peptidoglycan fragments. SagA secretion and NlpC/p60-endopeptidase activity was required for enhancing probiotic bacteria activity against
pathogenesis
. Our results demonstrate that the peptidoglycan composition and hydrolase activity of specific microbiota species can activate host immune pathways and enhance tolerance to pathogens.
In the absence of an effective HIV-1 vaccine, passive immunization using broadly neutralizing Abs or Ab-like molecules could provide an alternative to the daily administration of oral antiretroviral ...agents that has recently shown promise as preexposure prophylaxis. Currently, no single broadly neutralizing Ab (bNAb) or combination of bNAbs neutralizes all HIV-1 strains at practically achievable concentrations in vivo . To address this problem, we created bispecific Abs that combine the HIV-1 inhibitory activity of ibalizumab (iMab), a humanized mAb directed to domain 2 of human CD4, with that of anti-gp120 bNAbs. These bispecific bNAbs (BibNAbs) exploit iMab’s potent anti–HIV-1 activity and demonstrated clinical efficacy and safety to anchor and thereby concentrate a second broadly neutralizing agent at the site of viral entry. Two BibNabs, PG9-iMab and PG16-iMab, exhibit exceptional breadth and potency, neutralizing 100% of the 118 viruses tested at low picomolar concentrations, including viruses resistant to both parental mAbs. The enhanced potency of these BibNAbs was entirely dependent on CD4 anchoring, not on membrane anchoring per se, and required optimal Ab geometry and linker length. We propose that iMab-based BibNAbs, such as PG9-iMab and PG16-iMab, are promising candidates for passive immunization to prevent HIV-1 infection.
The majority of patients with Alzheimer disease (AD) suffer from impaired cerebral circulation. Accumulating evidence suggests that fibrinogen, the main protein component of blood clots, plays an ...important role in this circulatory dysfunction in AD. Fibrinogen interacts with β-amyloid (Aβ), forming plasmin-resistant abnormal blood clots, and increased fibrin deposition is found in the brains of AD patients and mouse models. In this study, we investigated the biochemical and structural details of the Aβ-fibrinogen interaction. We identified the central region of Aβ42 as the most critical region for the interaction, which can be inhibited by specific antibodies against the central region of Aβ and by naturally occurring p3 peptides, Aβ17-40 and Aβ17-42. X-ray crystallographic analysis revealed that Aβ42 binding to fragment D of fibrinogen induced a structural change in the C-terminal region of the fibrinogen β-chain (β384-393). Furthermore, we identified an additional Aβ-binding site within the αC region of fibrinogen. Aβ binding to this αC region blocked plasmin-mediated fibrin cleavage at this site, resulting in the generation of increased levels of a plasmin-resistant fibrin degradation fragment. Overall, our study elucidates the Aβ-fibrinogen interaction and clarifies the mechanism by which Aβ-fibrinogen binding delays fibrinolysis by plasmin. These results may facilitate the development of effective therapeutics against the Aβ-fibrinogen interaction to treat cerebrovascular abnormalities in AD.
•Binding to fibrinogen is mediated by the central region of Aβ42 and is enhanced by its C-terminal residues.•Aβ42 binds the αC region of fibrinogen, delaying plasmin-mediated fibrin cleavage and generating a persistent αC degradation product.
GLP-1 and glucagon regulate glucose metabolism through a network of metabolic pathways initiated upon binding to their specific receptors that belong to class B G-protein coupled receptors (GPCRs). ...The therapeutic potential of glucagon is currently being evaluated, while GLP-1 is already used in the treatment of type 2 diabetes and obesity. Development of a second generation of GLP-1 based therapeutics depends on a molecular and structural understanding of the interactions between the GLP-1 receptor (GLP-1R) and its ligand GLP-1. There is considerable sequence conservation between GLP-1 and glucagon and between the hGLP-1R and human glucagon receptor (hGCGR), yet each receptor recognizes only its own specific ligand. Glucagon receptors in fish and frogs also exhibit ligand selectivity only towards glucagon and not GLP-1. Based on competitive binding experiments and assays of increase in intracellular cAMP, we demonstrate here that a GPCR in zebrafish (Danio rerio) exhibits dual ligand selectivity towards GLP-1 and glucagon, a characteristic not found in mammals. Further, many structural features found in hGLP-1R and hGCGR are also found in this zebrafish GPCR (zfGPCR). We show this by mapping of its sequence and structural features onto the hGLP-1R and hGCGR based on their partial and complementary crystal structures. Thus, we propose that zfGPCR represents a dual GLP-1R/GCGR. The main differences between the three receptors are in their stalk regions that connect their N-terminal extracellular domains (NECDs) with their transmembrane domains and the absence of loop 3 in the NECD in zfGLP-1R/GCGR. These observations suggest that the interactions between GLP-1 and glucagon with loop 3 and the stalk regions may induce different conformational changes in hGLP-1R and hGCGR upon ligand binding and activation that lead to selective recognition of their native ligands.
Cholestatic itch is a severe and debilitating symptom in liver diseases with limited treatment options. The class A G protein-coupled receptor (GPCR) Mas-related GPCR subtype X4 (MRGPRX4) has been ...identified as a receptor for bile acids, which are potential cholestatic pruritogens. An increasing number of GPCRs have been shown to interact with receptor activity–modifying proteins (RAMPs), which can modulate different aspects of GPCR biology. Using a combination of multiplexed immunoassay and proximity ligation assay, we show that MRGPRX4 interacts with RAMPs. The interaction of MRGPRX4 with RAMP2, but not RAMP1 or 3, causes attenuation of basal and agonist-dependent signaling, which correlates with a decrease of MRGPRX4 cell surface expression as measured using a quantitative NanoBRET pulse-chase assay. Finally, we use AlphaFold Multimer to predict the structure of the MRGPRX4–RAMP2 complex. The discovery that RAMP2 regulates MRGPRX4 may have direct implications for future drug development for cholestatic itch.
Although there is no effective cure for chronic hepatitis B virus (HBV) infection, antibodies are protective and correlate with recovery from infection. To examine the human antibody response to HBV, ...we screened 124 vaccinated and 20 infected, spontaneously recovered individuals. The selected individuals produced shared clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Single bNAbs protected humanized mice against infection but selected for resistance mutations in mice with prior established infection. In contrast, infection was controlled by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with an HBsAg peptide epitope revealed a stabilized hairpin loop. This structure, which contains residues frequently mutated in clinical immune escape variants, provides a molecular explanation for why immunotherapy for HBV infection may require combinations of complementary bNAbs.
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•Antibodies against HBsAg from different donors share sequence similarities•Broadly neutralizing antibodies (bNAbs) to HBsAg target 3 non-overlapping epitopes•Co-crystal structure reveals a hairpin loop that explains immune escape mutation•Combination of complementary bNAbs controlled HBV infection and prevented escape mutation
Human antibody immune response against HBV is poorly studied. Wang et al. cloned human anti-S antibodies from selected individuals and identified broadly neutralizing antibodies that can prevent or treat infection in humanized mice. Co-crystal structure explains the necessity of using complementary antibodies to suppress the emergence of escape mutations.
Ibalizumab is a humanized monoclonal antibody that binds human CD4--a key receptor for HIV--and blocks HIV-1 infection. However, HIV-1 strains with mutations resulting in loss of an N-linked glycan ...from the V5 loop of the envelope glycoprotein gp120 are resistant to ibalizumab. Previous structural analysis suggests that this glycan fills a void between the gp120 V5 loop and the ibalizumab light chain, perhaps causing steric hindrance that disrupts viral entry. If this void contributes to HIV-1 resistance to ibalizumab, we reasoned that 'refilling' it by engineering an N-linked glycan into the ibalizumab light chain at a position spatially proximal to gp120 V5 may restore susceptibility to ibalizumab. Indeed, one such ibalizumab variant neutralized 100% of 118 diverse HIV-1 strains tested in vitro, including 10 strains resistant to parental ibalizumab. These findings demonstrate that the strategic placement of a glycan in the variable region of a monoclonal antibody can substantially enhance its activity.
HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at ~2.5-3.0 Å resolution, are important for understanding enzyme function and ...mechanisms of drug resistance in addition to being helpful in the design of RT inhibitors. Despite hundreds of attempts, it was not possible to obtain the structure of a complex of HIV-1 RT with TMC278, a nonnucleoside RT inhibitor (NNRTI) in advanced clinical trials. A systematic and iterative protein crystal engineering approach was developed to optimize RT for obtaining crystals in complexes with TMC278 and other NNRTIs that diffract X-rays to 1.8 Å resolution. Another form of engineered RT was optimized to produce a high-resolution apo-RT crystal form, reported here at 1.85 Å resolution, with a distinct RT conformation. Engineered RTs were mutagenized using a new, flexible and cost effective method called methylated overlap-extension ligation independent cloning. Our analysis suggests that reducing the solvent content, increasing lattice contacts, and stabilizing the internal low-energy conformations of RT are critical for the growth of crystals that diffract to high resolution. The new RTs enable rapid crystallization and yield high-resolution structures that are useful in designing/developing new anti-AIDS drugs.
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