Receptor Binding Sites Research Articles

Distribution of [11C]-JNJ-42491293 in the marmoset brain: a positron emission tomography study.

JNJ-42491293 is a metabotropic glutamate 2 (mGlu2) positive allosteric modulator (PAM) that was radiolabelled with [11C]- to serve as a positron emission tomography (PET) ligand. Indeed, in vitro, the molecule displays high selectivity at mGlu2 receptors. However, PET experiments performed in rats, macaques and humans, have suggested that [11C]-JNJ-42491293 could interact with an unidentified, non-mGlu2 receptor binding site. The brain distribution of [11C]-JNJ-42491293 has not been determined in the brain of the common marmoset, a small non-human primate increasingly used in neuroscience research. Here, we investigated the distribution of [11C]-JNJ-42491293 in the marmoset brain. Three marmosets underwent brain magnetic resonance imaging (MRI) and 90-min dynamic PET scans with [11C]-JNJ-42491293 in combination with vehicle or the mGlu2 PAM AZD8529 (0.1, 1 and 10mg/kg). In the scans in which [11C]-JNJ-42491293 was co-administered with vehicle, the brain areas with the highest standardised uptake values (SUVs) were the midbrain, cerebellum and thalamus, while the lowest SUVs were found in the pons. The addition of AZD8529 (0.1, 1 and 10mg/kg) to [11C]-JNJ-42491293 did not modify the SUVs obtained with [11C]-JNJ-42491293 alone, and ex vivo blocking autoradiography with PAM AZD8529 (10, 100, 300µM) on marmoset brain sections showed increased signals in the blocking conditions compared to vehicle, suggesting that no competition occurred between the 2 ligands. The results we obtained here do not suggest that [11C]-JNJ-42491293 interacts selectively, or even at all, with mGlu2 receptors in the marmoset, in agreement with findings previously reported in macaque and human.

A Small Contribution to a Large System: The Leptin Receptor Complex.

Obesity is a classified epidemic, increasing the risk of secondary diseases such as diabetes, inflammation, cardiovascular disease, and cancer. The pleiotropic hormone leptin is the proposed link for the gut-brain axis controlling nutritional status and energy expenditure. Research into leptin signaling provides great promise toward discovering therapeutics for obesity and its related diseases targeting leptin and its cognate leptin receptor (LEP-R). The molecular basis underlying the human leptin receptor complex assembly remains obscure, due to the lack of structural information regarding the biologically active complex. In this work, we investigate the proposed receptor binding sites in human leptin utilizing designed antagonist proteins combined with AlphaFold predictions. Our results show that binding site I has a more intricate role in the active signaling complex than previously described. We hypothesize that the hydrophobic patch in this region engages a third receptor forming a higher-order complex, or a new LEP-R binding site inducing allosteric rearrangement.

Mechanisms that promote the evolution of cross-reactive antibodies upon vaccination with designed influenza immunogens

Immunogens that elicit broadly neutralizing antibodies targeting the conserved receptor-binding site (RBS) on influenza hemagglutinin may serve as candidates for a universal influenza vaccine. Here, we develop a computational model to interrogate antibody evolution by affinity maturation after immunization with two types of immunogens: a heterotrimeric "chimera" hemagglutinin that is enriched for the RBS epitope relative to other B cell epitopes and a cocktail composed of three non-epitope-enriched homotrimers of the monomers that comprise the chimera. Experiments in mice find that the chimera outperforms the cocktail for eliciting RBS-directed antibodies. We show that this result follows from an interplay between how B cells engage these antigens and interact with diverse helper Tcells and requires Tcell-mediated selection of germinal center B cells to be a stringent constraint. Our results shed light on antibody evolution and highlight how immunogen design and Tcells modulate vaccination outcomes.

Risk evaluation and mitigation strategies for newly detected SARS-CoV-2 Omicron BF.7 subvariant: A brief report.

Mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are always going on. The pathogenic characteristics of a virus are influenced by mutations in the viral genome. Therefore, the recently identified Omicron BF.7 subvariant might harm humans. Here we aimed to evaluate the potential risks of this newly detected variant and identify possible mitigation strategies. The frequent mutation associated with SARS-CoV-2 makes it more concerning compared to other viruses. The Omicron variant of SARS-CoV-2 has unique changes in the structural amino acid. Thus, Omicron subvariants are different from other coronavirus variants in terms of viral spread, disease severity, vaccine neutralization capacity, and immunity evade. Moreover, Omicron subvariant BF.7 is an offspring of BA.4 and BA.5. Similar S glycoprotein sequences are present among BF.7, BA.4, and BA.5. There is a change in the R346T gene in the receptor binding site of Omicron BF.7 than other Omicron subvariants. This BF.7 subvariant has created a limitation in current monoclonal antibody therapy. Omicron has mutated since it emerged, and the subvariants are improving in terms of transmission as well as antibody evasion. Therefore, the healthcare authorities should pay attention to the BF.7 subvariant of Omicron. The recent upsurge may create havoc all of a sudden. Scientists and researchers across the world should monitor the nature and mutations of SARS-CoV-2 variants. Also, they should find ways to fight the current circulatory variants and any future mutations.

Advanced investigation of the olfactory perception of semiochemical TMT on OR5K1 and Olfr175 by statistical physics approach

The adsorption of the trimethylthiazoline (TMT) on the human olfactory receptor OR5K1 and the mouse olfactory receptor Olfr175 was the object of the present paper. The main contribution of this work was to characterize stereographically and energetically OR5K1 and Olfr175 activated by trimethylthiazoline molecules docked on the human and the mouse olfactory binding pockets using the grand canonical ensemble in statistical physics. The experimental data and the advanced statistical physics models revealed that the adsorption of the trimethylthiazoline on the human olfactory receptor OR5K1 can be interpreted using the monolayer model with single energy, while the monolayer model with two energies described the interaction between the trimethylthiazoline molecules and the mouse olfactory receptor Olfr175. In fact, the investigated odorant was shown to be docked by a multi-docking process and non parallel orientation on OR5K1 and Olfr175 since the values of the number of TMT molecules per binding site n were superior to 1. The proposed models were applied to calculate the human and the mouse olfactory receptor binding site size distributions relative to TMT, which were spread out from 0.30 to 20 nm with a maximum at about 1.75 nm for OR5K1 and from 1 to 25 nm with a peak at about 4.25 nm for Olfr175. Furthermore, it was found from the calculated molar adsorption energies, which were lower than 11 kJ/mol, that physical adsorption process was occurred in the two olfactory systems. The adsorption energy distributions relative to TMT can be also calculated in order to understand of olfaction process in general through the determination of olfactory bands (i. e., adsorption energy distribution bands), which were situated between 0 and 10.50 kJ/mol and between 3 and 12.50 kJ/mol for OR5K1 and Olfr175, respectively. Referring to the investigation of thermodynamic functions governing the adsorption process such as the adsorption entropy, the Gibbs free enthalpy and the internal energy, it may be noted that the disorder peak of the two olfactory systems was reached when the equilibrium concentration was equal to the concentration at half saturation. In addition, the Gibbs free enthalpy and the internal energy were calculated and their negative values indicated that the adsorption process involved in the olfactory mechanism was exothermic and spontaneous nature.

Analysis of the conserved protective epitopes of hemagglutinin on influenza A viruses.

The conserved protective epitopes of hemagglutinin (HA) are essential to the design of a universal influenza vaccine and new targeted therapeutic agents. Over the last 15 years, numerous broadly neutralizing antibodies (bnAbs) targeting the HA of influenza A viruses have been isolated from B lymphocytes of human donors and mouse models, and their binding epitopes identified. This work has brought new perspectives for identifying conserved protective epitopes of HA. In this review, we succinctly analyzed and summarized the antigenic epitopes and functions of more than 70 kinds of bnAb. The highly conserved protective epitopes are concentrated on five regions of HA: the hydrophobic groove, the receptor-binding site, the occluded epitope region of the HA monomers interface, the fusion peptide region, and the vestigial esterase subdomain. Our analysis clarifies the distribution of the conserved protective epitope regions on HA and provides distinct targets for the design of novel vaccines and therapeutics to combat influenza A virus infection.

That H9N2 avian influenza viruses circulating in different regions gather in the same live-poultry market poses a potential threat to public health.

H9N2 avian influenza viruses are endemic and persistent in China, but those that are prevalent in different provinces are also causes of wide epidemics, related to the spread of wild birds and the cross-regional trade in live poultry. For the past 4 years, beginning in 2018, we have sampled a live-poultry market in Foshan, Guangdong, in this ongoing study. In addition to the prevalence of H9N2 avian influenza viruses in China during this period, we identified isolates from the same market belonging to clade A and clade B, which diverged in 2012-2013, and clade C, which diverged in 2014-2016, respectively. An analysis of population dynamics revealed that, after a critical divergence period from 2014 to 2016, the genetic diversity of H9N2 viruses peaked in 2017. Our spatiotemporal dynamics analysis found that clade A, B, and C, which maintain high rates of evolution, have different prevalence ranges and transmission paths. Clades A and B were mainly prevalent in East China in the early stage, and then spread to Southern China, becoming epidemic with clade C. Strains from different regions converge at the same live-poultry market to communicate, which may be one reasons the H9N2 viruses are difficult to eradicate and increasingly dominant throughout China. Selection pressure and molecular analysis have demonstrated that single amino acid polymorphisms at key receptor binding sites 156, 160, and 190 under positive selection pressure, suggesting that H9N2 viruses are undergoing mutations to adapt to new hosts. Live-poultry markets are important because people who visit them have frequent contact with poultry, H9N2 viruses from different regions converge at these markets and spread through contact between live birds and humans, generating increased risks of human exposure to these viruses and threatening public health safety. Thus, it is important to reducing the cross-regional trade of live poultry and strengthening the monitoring of avian influenza viruses in live-poultry markets to reduce the spread of avian influenza viruses.

Structure-Based Virtual Screening and Molecular Dynamics Simulation Assessments of Depsidones as Possible Selective Cannabinoid Receptor Type 2 Agonists.

The discovery of natural drug metabolites is a leading contributor to fulfilling the sustainable development goal of finding solutions to global health challenges. Depsidones are a class of polyketides that have been separated from lichens, fungi, sponges, and plants and possess various bioactivities, including cytotoxic, antimicrobial, antimalarial, antituberculosis, acetylcholinesterase and α-glucosidase inhibition, and anti-inflammatory effects. Endocannabinoid receptors (CB1 and CB2) are G-protein-coupled receptors (GPCRs), and their activation mediates many physiological processes. CB1 is the dominant subtype in the central nervous system, while CB2 is mainly expressed in the immune system. The two receptors exhibit high heterogeneity, making developing selective ligands a great challenge. Attempts to develop CB2 selective agonists for treating inflammatory diseases and neuropathic pain have not been successful due to the high homology of the binding sites of the CB receptors. In this work, 235 depsidones from various sources were investigated for the possibility of identifying CB2-selective agonists by performing multiple docking studies, including induced fit docking and Prime/molecular mechanics-generalized Born surface area (MM-GBSA) calculations to predict the binding mode and free energy. Simplicildone J (10), lobaric acid (110), mollicellin Q (101), garcinisidone E (215), mollicellin P (100), paucinervin Q (149), and boremexin C (161) had the highest binding scores (-12.134 kcal/mol, -11.944 kcal/mol, -11.479 kcal/mol, -11.394 kcal/mol, -11.322 kcal/mol, -11.305 kcal/mol, and -11.254 kcal/mol, respectively) when screened against the CB2 receptor (PDB ID: 6KPF). The molecular dynamic simulation was performed on the compounds with the highest binding scores. The computational outcomes show that garcinisidone E (215) and paucinervin Q (149) could be substantial candidates for CB2 receptor activation and warrant further in vivo and in vitro investigations.

Understanding the SARS-CoV-2 Virus Neutralizing Antibody Response: Lessons to Be Learned from HIV and Respiratory Syncytial Virus.

The SARS-CoV-2 pandemic commenced in 2019 and is still ongoing. Neither infection nor vaccination give long-lasting immunity and, here, in an attempt to understand why this might be, we have compared the neutralizing antibody responses to SARS-CoV-2 with those specific for human immunodeficiency virus type 1 (HIV-1) and respiratory syncytial virus (RSV). Currently, most of the antibodies specific for the SARS-CoV-2 S protein map to three broad antigenic sites, all at the distal end of the S trimer (receptor-binding site (RBD), sub-RBD and N-terminal domain), whereas the structurally similar HIV-1 and the RSV F envelope proteins have six antigenic sites. Thus, there may be several antigenic sites on the S trimer that have not yet been identified. The epitope mapping, quantitation and longevity of the SARS-CoV-2 S-protein-specific antibodies produced in response to infection and those elicited by vaccination are now being reported for specific groups of individuals, but much remains to be determined about these aspects of the host-virus interaction. Finally, there is a concern that the SARS-CoV-2 field may be reprising the HIV-1 experience, which, for many years, used a virus for neutralization studies that did not reflect the neutralizability of wild-type HIV-1. For example, the widely used VSV-SARS-CoV-2-S protein pseudotype has 10-fold more S trimers per virion and a different configuration of the trimers compared with the SARS-CoV-2 wild-type virus. Clarity in these areas would help in advancing understanding and aid countermeasures of the SARS-CoV-2 pandemic.

Advanced investigation of a putative adsorption process of nine non key food odorants (non-KFOs) on the broadly tuned human olfactory receptor OR2W1: Statistical physics modeling and molecular docking study

In the present paper, statistical physics formalism was used to understand the olfactory perception via the investigation of dose-olfactory response curves of a putative adsorption process of nine non key food odorants (non-KFOs) on the broadly tuned human olfactory receptor OR2W1, in order to quantitative characterize the interactions between the nine studied non-KFOs, i. e., furfuryl sulfide, furfuryl disulfide, benzyl methyl disulfide, furfuryl methyl disulfide, benzyl methyl sulfide, 1-phenylethanethiol, benzyl mercaptan, furfuryl methyl sulfide and 3-phenylpropanol molecules and OR2W1 binding sites at a molecular level. Two advanced adsorption models have been proposed: the advanced monolayer monoenergy model (monolayer model with identical and independent olfactory receptor binding sites) (Model 1) and the advanced monolayer model with two independent types of olfactory receptor binding sites (Model 2). It was concluded that the monolayer monoenergy model was selected as the most adequate model to fit the experimental dose-olfactory response curves tabulated in literature. Actually, the numerical values of the three fitted physico-chemical parameters (RM1, n and C1) were obtained by a non-linear regression. Indeed, modeling results suggested that the number of docked non-KFOs per OR2W1 binding site n values (1.24 < n < 1.94) was always superior to 1, which indicated the non-parallel orientation of the studied odorants on the olfactory receptor and the multi-molecular adsorption mechanism. The estimated molar adsorption energy ΔEa values (ranged from 6.07 to 12.16 kJ/mol) for the nine olfactory systems confirmed the physical the exothermic characters of the adsorption process since ΔEa values were lower than 40 kJ/mol and positive. Furthermore, these estimated parameters were applied to characterize stereographically and energetically the interaction between the nine non-KFOs and OR2W1 through the determination of the human receptor binding site size distributions (RSDs) and the adsorption energy distributions (AEDs), which were spread out from 0.25 to 6.50 nm and from 0 to 22.50 kJ/mol, respectively. The docking computation between these nine non-KFOs and OR2W1 proved that the estimated binding affinities were belonged to the adsorption energies spectrum in general and the specific adsorption energy band or the molecular vibration modes limited spectrum (between 2.50 kJ/mol and 17 kJ/mol) (approximate olfactory band).

Formation and characterization of BMP2/GDF5 and BMP4/GDF5 heterodimers

BackgroundProteins of the TGFβ family, which are largely studied as homodimers, are also known to form heterodimers with biological activity distinct from their component homodimers. For instance, heterodimers of bone morphogenetic proteins, including BMP2/BMP7, BMP2/BMP6, and BMP9/BMP10, among others, have illustrated the importance of these heterodimeric proteins within the context of TGFβ signaling.ResultsIn this study, we have determined that mature GDF5 can be combined with mature BMP2 or BMP4 to form BMP2/GDF5 and BMP4/GDF5 heterodimer. Intriguingly, this combination of a BMP2 or BMP4 monomer, which exhibit high affinity to heparan sulfate characteristic to the BMP class, with a GDF5 monomer with low heparan sulfate affinity produces a heterodimer with an intermediate affinity. Using heparin affinity chromatography to purify the heterodimeric proteins, we then determined that both the BMP2/GDF5 and BMP4/GDF5 heterodimers consistently signaled potently across an array of cellular and in vivo systems, while the activities of their homodimeric counterparts were more context dependent. These differences were likely driven by an increase in the combined affinities for the type 1 receptors, Alk3 and Alk6. Furthermore, the X-ray crystal structure of BMP2/GDF5 heterodimer was determined, highlighting the formation of two asymmetric type 1 receptor binding sites that are both unique relative to the homodimers.ConclusionsUltimately, this method of heterodimer production yielded a signaling molecule with unique properties relative to the homodimeric ligands, including high affinity to multiple type 1 and moderate heparan binding affinity.

Fluorescent, non-hydrolyzable nucleotide probes for ATP-activated ion channels.

In addition to its role as a ubiquitous, intracellular energy source and signaling molecule, adenosine 5’-triphosphate (ATP) also impacts extracellular signaling as both a neurotransmitter and a product of tissue damage. We previously developed a real-time nucleotide binding assay to study the gating of ATP-sensitive K+ (KATP) channels. Nucleotide binding was measured as Förster resonance energy transfer (FRET) between fluorescent, trinitrophenyl nucleotide derivatives and KATP channels tagged with the fluorescent, non-canonical amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropionic acid (ANAP). In order to further probe the shape and chemistry of the nucleotide binding sites of ATP activated channels, we have synthesized and purified trinitrophenyl analogs of the weakly hydrolyzable adenosine-5'-(γ-thio)-triphosphate (ATP- γ-S) and the non-hydrolyzable nucleotides adenosine-5'-[(β,γ)-methyleno]triphosphate (AMP-PCP) and adenosine-5'-[(β,γ)-imido]triphosphate. The trinitrophenyl group was added to the ribose moiety of these nucleotides through an aqueous reaction with 2,4,6-trinitrobenzenesulfonic acid under basic conditions. The reaction products were purified by precipitation in ethanol followed by reversed-phase high-performance liquid chromatography. Products were verified using 1H and 31P nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and UV/visible absorbance. These compounds will be useful for exploring the requirement (if any) for ATP hydrolysis to activate KATP channels and the unique chemistry of the ATP binding sites of purinergic P2X-family receptors.

Sigma conotoxin GVIIIA: Binding to 5HT3A with microsecond simulations and docking of diverse structures.

The 5HT3A receptor is an important neurological receptor that can be inhibited by conotoxins from cone snails. It is therefore important to understand the significance of the toxin in terms of the structure function relationship and potentially prove that a diverse set of structures can inhibit the receptor. Long time scale simulations were run for several microseconds to observe unbiased binding of the toxin to the receptor. An important factor to consider with the docking is that the binding was only observed to occur from the membrane to the receptor and not from the aqueous phase to the receptor. The final structure of the unbiased binding to the receptor was saved and used to test other predicted disulfide bond configurations of the GVIIIA toxin structure. It is important to sample varied possible structures because protein prediction software are not perfect and certain structures might cause inhibitory effects to 5HT3A, which can be tested with ClusPro clustering software. The PMF of the unbiased and relaxed docked structure will be compared with each other to show if similar results can be obtained. Certain positions are more likely to occur than others because if our unbiased simulations are valid, the peptide needs to start from the membrane and transition to the receptor binding site. The project seeks to probe how much tolerance there is in the structure of the natural toxin peptides, and this could be an important consideration for future therapeutic and toxin development for a variety of applications.

Structure and supramolecular organization of the canine distemper virus attachment glycoprotein

Canine distemper virus (CDV) is an enveloped RNA morbillivirus that triggers respiratory, enteric, and high incidence of severe neurological disorders. CDV induces devastating outbreaks in wild and endangered animals as well as in domestic dogs in countries associated with suboptimal vaccination programs. The receptor-binding tetrameric attachment (H)-protein is part of the morbilliviral cell entry machinery. Here, we present the cryo-electron microscopy (cryo-EM) structure and supramolecular organization of the tetrameric CDV H-protein ectodomain. The structure reveals that the morbilliviral H-protein is composed of three main domains: stalk, neck, and heads. The most unexpected feature was the inherent asymmetric architecture of the CDV H-tetramer being shaped by the neck, which folds into an almost 90° bent conformation with respect to the stalk. Consequently, two non-contacting receptor-binding H-head dimers, which are also tilted toward each other, are located on one side of an intertwined four helical bundle stalk domain. Positioning of the four protomer polypeptide chains within the neck domain is guided by a glycine residue (G158), which forms a hinge point exclusively in two protomer polypeptide chains. Molecular dynamics simulations validated the stability of the asymmetric structure under near physiological conditions and molecular docking showed that two receptor-binding sites are fully accessible. Thus, this spatial organization of the CDV H-tetramer would allow for concomitant protein interactions with the stalk and head domains without steric clashes. In summary, the structure of the CDV H-protein ectodomain provides new insights into the morbilliviral cell entry system and offers a blueprint for next-generation structure-based antiviral drug discovery.

In silico approach to identify novel allosteric intracellular antagonist for blocking the interleukin-8/CXCR2 receptor signaling pathway

The role of interleukin-8 (IL-8) and its receptor CXCR2 in inflammatory responses and tumor development and progression has been well documented. Our study aims to discover novel compounds as CXCR2 antagonists to block the IL-8 signaling pathway using an in silico drug design. Herein, a structure-based pharmacophore model was developed based on the crystal structure of inactive CXCR2 in a complex with an allosteric inhibitor. This model was validated and refined, followed by virtual screening with the ZINC15 database. Subsequent molecular docking allows for predicting the best pose of a ligand inside a receptor binding site. We found that the 35 top-ranked hits exhibited docking scores from −30.81 to −25.28 kJ/mol and better interaction potential comparing the reference inhibitor. Analysis of ADME and toxicity properties revealed the efficacy and safety of the selected seven compounds. To validate the stability of the protein-ligand complex structure MD simulations approach has also been performed and confirmed via the critical parameters. The MD results explained that the CXCR2 receptor bound with two best-proposed molecules, including ZINC77105530 and ZINC93176465, was quite stable states as observed from low RMSD, RMSF, Rg, SASA values, and high occupancy of the interaction types. Finally, our data identified that these compounds play as potential inhibitors of IL-8 signaling pathways with the MM/GBSA binding free energies of −41.77 ± 6.45 kcal/mol and −38.84 ± 6.16 kcal/mol, respectively. Communicated by Ramaswamy H. Sarma

Engineered soluble ACE2 receptor: Responding to change with change.

SARS coronavirus 2 (SARS-CoV-2) invades the human body by binding to major receptors such as ACE2 via its S-spike protein, so the interaction of receptor-binding sites has been a hot topic in the development of coronavirus drugs. At present, the clinical progress in monoclonal antibody therapy that occurred early in the pandemic is gradually showing signs of slowing. While recombinant soluble ACE2, as an alternative therapy, has been modified by many engineering methods, both the safety and functional aspects are approaching maturity, and this therapy shows great potential for broadly neutralizing coronaviruses, but its progress in clinical development remains stalled. Therefore, there are still several key problems to be considered and solved for recombinant soluble ACE2 to be approved as a clinical treatment as soon as possible.

Identification of B-Cell Epitopes on Capsid Protein Reveals Two Potential Neutralization Mechanisms in Red-Spotted Grouper Nervous Necrosis Virus.

Nervous necrosis virus (NNV), a formidable pathogen in marine and freshwater fish, has inflicted enormous financial tolls on the aquaculture industry worldwide. Although capsid protein (CP) is the sole structural protein with pathogenicity and antigenicity, public information on immunodominant regions remains extremely scarce. Here, we employed neutralizing monoclonal antibodies (MAbs) specific for red-spotted grouper NNV (RGNNV) CNPgg2018 in combination with partially overlapping truncated proteins and peptides to identify two minimal B-cell epitope clusters on CP, 122GYVAGFL128 and 227SLYNDSL233. Site-directed mutational analysis confirmed residues Y123, G126, and L128 and residues L228, Y229, N230, D231, and L233 as the critical residues responsible for the direct interaction with ligand, respectively. According to homologous modeling and bioinformatic evaluation, 122GYVAGFL128 is harbored at the groove of the CP junction with strict conservation among all NNV isolates, while 227SLYNDSL233 is localized in proximity to the tip of a viral protrusion having relatively high evolutionary dynamics in different genotypes. Additionally, 227SLYNDSL233 was shown to be a receptor-binding site, since the corresponding polypeptide could moderately suppress RGNNV multiplication by impeding virion entry. In contrast, 122GYVAGFL128 seemed dedicated only to stabilizing viral native conformation and not to assisting initial virus attachment. Altogether, these findings contribute to a novel understanding of the antigenic distribution pattern of NNV and the molecular basis for neutralization, thus advancing the development of biomedical products, especially epitope-based vaccines, against NNV. IMPORTANCE NNV is a common etiological agent associated with neurological virosis in multiple aquatic organisms, causing significant hazards to the host. However, licensed drugs or vaccines to combat NNV infection are very limited to date. Toward the advancement of broad-spectrum prophylaxis and therapeutics against NNV, elucidating the diversity of immunodominant regions within it is undoubtedly essential. Here, we identified two independent B-cell epitopes on NNV CP, followed by the confirmation of critical amino acid residues participating in direct interaction. These two sites were distributed on the shell and protrusion domains of the virion, respectively, and mediated the neutralization exerted by MAbs via drastically distinct mechanisms. Our work promotes new insights into NNV antigenicity as well as neutralization and, more importantly, offers promising targets for the development of antiviral countermeasures.

A TREM2-activating antibody with a blood–brain barrier transport vehicle enhances microglial metabolism in Alzheimer’s disease models

Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer’s disease (AD), suggesting that activation of this innate immune receptor may be a useful therapeutic strategy. Here we describe a high-affinity human TREM2-activating antibody engineered with a monovalent transferrin receptor (TfR) binding site, termed antibody transport vehicle (ATV), to facilitate blood–brain barrier transcytosis. Upon peripheral delivery in mice, ATV:TREM2 showed improved brain biodistribution and enhanced signaling compared to a standard anti-TREM2 antibody. In human induced pluripotent stem cell (iPSC)-derived microglia, ATV:TREM2 induced proliferation and improved mitochondrial metabolism. Single-cell RNA sequencing and morphometry revealed that ATV:TREM2 shifted microglia to metabolically responsive states, which were distinct from those induced by amyloid pathology. In an AD mouse model, ATV:TREM2 boosted brain microglial activity and glucose metabolism. Thus, ATV:TREM2 represents a promising approach to improve microglial function and treat brain hypometabolism found in patients with AD.

Structure and function of a novel lineage-specific neutralizing epitope on H protein of canine distemper virus.

Canine distemper virus (CDV) infects many sensitive species worldwide and its host range is expanding. The hemagglutinin (H) protein, the major neutralizing target, binds to cellular receptors and subsequently triggers fusion for initial viral infection. So it's necessary to clarify the precise neutralizing epitopes of H protein and extend the knowledge of mechanisms of virus neutralization. In this study, a neutralizing monoclonal antibody (mAb) 2D12 against CDV H protein, which had different reactivity with different CDV strains, was generated and characterized. A series of truncated H proteins were screened to define the minimal linear epitope 238DIEREFD244 recognized by 2D12. Further investigation revealed that the epitope was highly conserved in America-1 vaccine lineage of CDV strains, but different substitutions in the epitope appeared in CDV strains of the other lineages and two substitutions (D238Y and R241G) caused the change of antigenicity. Thus, the epitope represents a novel lineage-specific neutralizing target on H protein of CDV for differentiation of America-1 vaccine lineage and the other lineages of CDV strains. The epitope was identified to localize at the surface of H protein in two different positions in a three-dimensional (3D) structure, but not at the position of the receptor-binding site (RBS), so the mAb 2D12 that recognized the epitope did not inhibit binding of H protein to the receptor. But mAb 2D12 interfered with the H-F interaction for inhibiting membrane fusion, suggesting that the mAb plays key roles for formation of H-F protein oligomeric structure. Our data will contribute to the understanding of the structure, function, and antigenicity of CDV H protein and mechanisms of virus neutralization.

Hierarchical sequence-affinity landscapes shape the evolution of breadth in an anti-influenza receptor binding site antibody.

Broadly neutralizing antibodies (bnAbs) that neutralize diverse variants of a particular virus are of considerable therapeutic interest. Recent advances have enabled us to isolate and engineer these antibodies as therapeutics, but eliciting them through vaccination remains challenging, in part due to our limited understanding of how antibodies evolve breadth. Here, we analyze the landscape by which an anti-influenza receptor binding site (RBS) bnAb, CH65, evolved broad affinity to diverse H1 influenza strains. We do this by generating an antibody library of all possible evolutionary intermediates between the unmutated common ancestor (UCA) and the affinity-matured CH65 antibody and measure the affinity of each intermediate to three distinct H1 antigens. We find that affinity to each antigen requires a specific set of mutations - distributed across the variable light and heavy chains - that interact non-additively (i.e., epistatically). These sets of mutations form a hierarchical pattern across the antigens, with increasingly divergent antigens requiring additional epistatic mutations beyond those required to bind less divergent antigens. We investigate the underlying biochemical and structural basis for these hierarchical sets of epistatic mutations and find that epistasis between heavy chain mutations and a mutation in the light chain at the VH-VL interface is essential for binding a divergent H1. Collectively, this is the first work to comprehensively characterize epistasis between heavy and light chain mutations and shows that such interactions are both strong and widespread. Together with our previous study analyzing a different class of anti-influenza antibodies, our results implicate epistasis as a general feature of antibody sequence-affinity landscapes that can potentiate and constrain the evolution of breadth.