Receptor Binding Research Articles

A SARS-CoV-2 vaccine on an NIR-II/SWIR emitting nanoparticle platform.

The COVID-19 pandemic caused a global health crisis that resulted in millions of deaths. Effective vaccines have played central roles in curtailing the pandemic. Here, we developed a down-converting near-infrared IIb (NIR-IIb; 1500 to 1700 nanometers) luminescent, pure NaErF4@NaYF4 rare-earth nanoparticle (pEr) as vaccine carriers. The pEr nanoparticles were coated with three layers of cross-linked biocompatible polymers (pEr-P3; ~55 nanometers) and conjugated to the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Upon subcutaneous injection of the pEr-P3-RBD nanovaccine in mice, in vivo NIR-IIb imaging revealed active vaccine trafficking and migration to lymph nodes through lymphatic vessels. Two doses of the adjuvant-free vaccine elicited long-lasting (>7 months) high titers of serum viral neutralization antibody and anti-RBD immunoglobulin G, along with robust RBD-specific germinal center B cells and T follicular helper cells. We devised in vivo NIR-II molecular imaging of RBD-specific cells in lymph nodes, opening noninvasive assessments of vaccine-elicited immune responses longitudinally.

First detection and molecular characterization of porcine epidemic diarrhea virus (PEDV) in India: evidence of a new variant in Karnataka

BackgroundPorcine epidemic diarrhea (PED) is a significant pig disease causing high mortality in suckling pigs and high morbidity across all age groups. It is highly prevalent in Southeast Asia, posing a threat of transboundary transmission to India. Although antibodies were detected as early as 2003 in Assam, there was no evidence of viral detection or molecular characterization until this study. This study reports the first clinical outbreak of PED in India, followed by the detection and genetic characterization of PED virus (PEDV) during 2022-23.MethodsThe outbreak was characterized, and fecal samples (n = 21) were collected from affected pigs. These samples were screened for PEDV using RT-PCR, targeting the N, S, and M genes. Serosurveillance was conducted in eight districts, and serum samples (n = 339) were tested for PEDV antibodies using ELISA. Partial N, S, and M gene sequencing, followed by phylogenetic analysis using MEGA v11.0.13, was performed to identify the prevailing genotype and variations in the coding region.ResultsThis study identified the first clinical outbreak of PEDV in India, with morbidity rates of 55-57.1% and symptoms including yellow watery diarrhea, vomiting, and abdominal pain. PEDV was confirmed in 17 of 21 fecal samples by amplifying the N, S, and M genes. Serosurveys showed seropositivity in Mandya (2.8%), Bengaluru Rural (6.6%), and Kolar (21.6%), districts indicating PEDV circulation in the state of Karnataka, India. The phylogenetic analysis of the S and M genes placed our study sequences within the Genotype 2a (G2a) clade, aligning with other known G2a strains. In contrast, the phylogenetic tree of the N gene clustered our sequences within the Genotype 1a (G1a) clade suggesting potential recombination. The Indian PEDV strains clustered with strains of China, with unique amino acid substitutions in the S gene, particularly in the receptor binding region.ConclusionThis study reports the first clinical outbreak of PED in India and identifies the circulating genotype of PEDV. The study emphasizes the need for large-scale surveillance studies to understand the disease’s status. Understanding PEDV’s genetic diversity and evolution is essential to develop area-specific vaccines to mitigate the disease impact on India’s pig population.

Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity

Background Botulinum neurotoxin type A (BoNT/A) represents a major threat to global public health because of its most potent toxicity with the longest persistence. Several camelid single-domain antibodies (or VHHs) have been reported to exhibit high neutralizing activity against the receptor binding domain (HC) of the BoNT/A subtype used to generate them. However, it remains unclear if these VHHs can neutralize effectively HC of other BoNT/A subtypes. This study aimed to generate HC domains of all eight BoNT/A subtypes and to screen for VHHs with broad reactivity against these domains. Methods HC domains of BoNT/A1-A8 were recombinantly produced in Escherichia coli. The bont/HCA1 fragment was amplified from sludge sample and cloned into pET45b vector by Gibson assembly. Expression vectors for HC domains of BoNT/A2-A8 were derived from pET45b-HCA1 by site-directed mutagenesis and/or in-house gene synthesis. Similarly, VHHs were synthesized and cloned into pET22b vector. Recombinant protein were purified by Ni-NTA spin columns and analyzed by SDS-PAGE. ELISA was used to confirm the antigenicity of HC domains and to evaluate the reactivity of VHHs to these domains. Results SDS-PAGE analysis and ELISA results with commercial polyclonal antibody demonstrated the HC domains of all eight BoNT/A subtypes were correctly produced. ELISA results using a VHH panel indicated that, apart from ciA-C2, a well-characterized VHH specific for HC of BoNT/A1, two new VHHs were found to recognize the HC domains of all BoNT/A subtypes, of which VHH-A3 displayed EC50 values for these domains close to those of ciA-C2. Conclusion This study provided a resource to comprehensively identify antitoxins conferring broad protection against BoNT/A.

Research on Neuroimmune Gastrointestinal Diseases Based on Artificial Intelligence: Molecular Dynamics Analysis of Caffeine and DRD3 Protein.

The aim of this study was to develop a clinical application model for the rational use of caffeine. Caffeine is related to the incidence of neuro immune gastrointestinal diseases. Coffee consumption needs to be optimized in order to reduce the incidence rate. By using KEEG analysis to explore potential molecular signaling pathways involved in the progression of neurological immune gastrointestinal diseases, and analyzing the details of this signaling Pathway using molecular simulation results, which can support AI system for doctor. The research team designed a controlled experiment to analyze the differences in reward and reinforcement of Brain pleasure/addiction and dopamine related signaling pathways function between multiple groups of people with different coffee drinking habits and a blank control group. The study team used molecular dynamics methods to investigate the signaling route that links coffee with the binding of dopamine receptor D3.AI is used to predict the prevalence of gastric reflux disease. Human experiments have shown a correlation between caffeine intake and gastroesophageal reflux disease. AI algorithm results can provide clinical support, and molecular simulation results are consistent with human experimental results. Caffeine and DRD3 protein have a stable interaction system. The research team elucidated the intermolecular interaction between caffeine and DRD3, and AI algorithms can predict the likelihood of disease occurrence, providing a new strategy for clinical practice. This study has passed ethical approval at Chifeng Cancer Hospital, and the ethical documents for this study have been submitted to the World Health Organization for filing.

Pathology and Therapeutic Significance of Fibroblast Growth Factors

The fibroblast growth factor (FGF) family includes 22 proteins in humans. Based on their mode of action, there are three families of FGFs: paracrine FGFs (FGF 1–10, 16, 17, 18, 20, and 22), intracrine FGFs (FGF 11–14), and endocrine FGFs (FGF 19, 21, and 23). FGF signaling plays critical roles in embryonic development, tissue repair, regeneration, angiogenesis, and metabolic regulation. They exert their cellular functions by binding, dimerization, and activation of transmembrane FGF receptors (FGFRs). Aberrant FGF signaling is associated with various human diseases. Thus, understanding the unique properties of FGF signaling will help to explore new therapeutic interventions against FGF-mediated pathological conditions. This review will discuss the differential expression and regulation of each FGF under normal human physiological and pathological conditions. Moreover, we will outline current therapeutics and treatment strategies that have been developed against FGF-related pathology.

Cortical α4β2-nicotinic acetylcholine receptors and cognitive decline in Parkinson's disease

Background Autopsy and in vivo molecular imaging studies suggest altered binding of the α4β2-nicotinic cholinergic receptor (α4β2-nAChR) with cognitive dysfunction in Parkinson's disease (PD). Objective To determine the relationship between cortical and hippocampal binding of the α4β2-nAChR with [18F]XTRA PET, a high-affinity radiotracer that enables quantification of α4β2-nAChR in these regions, and cognitive function in individuals with PD. Methods Individuals with PD (N = 32) and age-similar, controls without PD or dementia (N = 10) completed a cognitive assessment and one 90-min, [18F]XTRA PET scan. Metabolite-corrected arterial input function radioactivity time-activity curves were generated to obtain total distribution volume (VT) across 12 regions of interest (ROIs). [18F]XTRA binding was compared 1) between controls and people with PD and 2) between controls, persons with PD with normal cognition (PD-NC), and persons with PD with MCI (PD-MCI). Results [18F]XTRA binding was higher in the occipital cortex of the combined group of PD participants compared to age-similar controls. No regions showed lower binding in PD. VT with, but not without, partial volume correction was different between controls, PD-NC, and PD-MCI groups, and this was driven by higher binding in PD-MCI compared to controls. Regression of regional VT on cognitive domain T-scores, adjusting for age, showed that worse performance in visual-spatial memory tasks was associated with higher VT in the precuneus and the entire parietal cortex. Conclusions Higher α4β2-nAChR binding in posterior cortical regions is found in PD and associated with worse visual perception and memory, possibly due to lower receptor occupancy by endogenous acetylcholine.

Salivary Proteome Is Altered in Children With Small Area Thermal Burns.

Saliva is a child appropriate biofluid, but it has not previously been used to evaluate the systemic response to burn injury in children. The aim of this study was to investigate the salivary proteome of children with small area thermal skin burns relative to different burn characteristics (mechanism, time to re-epithelialization and risk of emotional distress). SWATH Mass Spectrometry was used to quantify the abundance of 742 proteins in the saliva of children with burns (n=22) and healthy controls (n=37). Eight proteins were differentially abundant in the saliva of children with burns compared to healthy children, and these were associated with immune processes, epidermal cell differentiation and transferrin receptor binding. Eleven proteins were differentially abundant in patients with burns of different mechanisms. Scald burns had an over-representation of immune/inflammatory response processes, and contact burns had an over-representation of cornification, intermediate filament assembly and cell death cellular processes. Four proteins were elevated in patients who were at high risk for emotional distress and 15 proteins were correlated with time to wound re-epithelialization. This pilot study proves that saliva can be used for paediatric biomarker discovery and can be used as a diagnostic and prognostic sample to investigate systemic changes in a paediatric burn cohort.

MERS-CoV spike vaccine-induced N-terminal domain-specific antibodies are more protective than receptor binding domain-specific antibodies.

The COVID-19 pandemic underscores the need to prepare for future emerging coronavriuses (CoVs) by understanding the principles behind effective CoV vaccine design such as protective immunity and antibody responses. To study which epitopes and subdomains contribute to invivo protection, we utilized the prefusion-stabilized spike protein of MERS-CoV, MERS S-2P, as a vaccine immunogen. Vaccination with MERS S-2P elicited both receptor-binding domain (RBD)- and non-RBD-specific antibodies, including N-terminal domain (NTD)-specific G2-and CDC2-A2-like antibodies. Intriguingly, the immunogen MERS S-2P_ΔRBD, MERS S-2P with the RBDs removed, protects comparably to S1 and S-2P immunogens against MERS-CoV challenge. Moreover, passive transfer studies of polyclonal IgG from MERS S-2P immunized mice depleted of subdomain-specific antibodies demonstrated that non-RBD antibodies protected more thannon-NTD antibodies. Altogether, these findings illustrate that in-vivo protection is not solely driven byRBD-specific antibodies and highlights the importance of targeting non-RBD sites in future CoV vaccine designs.

In silico Evaluation of Anacyclus pyrethrum Composition for Inhibition of Spike RBD-ACE-2 Interaction to Treat COVID-19

Background: The spike glycoprotein of SARS-CoV-2, via its S1-subunit, binds with host angiotensin-converting enzyme 2 (ACE-2) receptors, and its S2-subunit mediates the fusion of the virus to the host cell. The entry of SARS-CoV-2 inside the host cell can be prevented by inhibition of the receptor binding domain (RBD) of S1-subunit of the spike. Anacyclus pyrethrum, a native herb of Algeria, Spain and Morocco has antidepressant, analgesic, antimicrobial, anesthetic, antioxidant, anti-inflammatory, aphrodisiac, antidiabetic and immunostimulant effects. Still, its antiviral effect has not been established yet. Methodology: The present study deals with ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity), molecular docking and molecular dynamic simulation based investigation to evaluate the potential of Anacyclus pyrethrum constituents for effective spike RBD inhibition. Results: ADMET analysis revealed that 10 out of 12 significant constituents belongs to toxicity class 4 to 6 proving least toxicity of the plant extract with high LD50 values. Molecular docking analysis of 10 considered compounds revealed that morphinan-6-one, 4,5.alpha.-epoxy-3-hydroxy-17-methyl, a derivative of morphine (well-known analgesic and anti-inflammatory compound) gave the maximum negative binding energy of -6.9Kcal/mol in best-docked conformation with spike RBD having 2 hydrogen bonds. Molecular dynamic simulation disclosed effective RMSD, RMSF, and Rg values over the simulation trajectory with significant hydrogen bonding proving stable interaction of the compound with that of the spike RBD. Conclusion: Hence, all these outcomes revealed the outstanding potential of the Anacyclus pyrethrum extract to inhibit the spike RBD of SARS-CoV-2. Therefore, further in-vitro investigation can develop natural and effective treatments against COVID-19 disease.

TOLLIP inhibits the replication of PEDV by autophagic degradation of Nsp9.

Selective autophagy plays a crucial role in innate antiviral immunity by targeting essential viral components and host factors necessary for virus propagation. Among these factors, the nonstructural protein 9 (Nsp9) of Porcine Epidemic Diarrhea Virus (PEDV) is required for viral replication. However, the host factors regulating Nsp9 have remained elusive. In our study, we discovered that Nsp9 undergoes degradation through selective autophagy. Using coimmunoprecipitation combined with mass spectrometry analysis, we identified Toll-interacting protein (TOLLIP) as an autophagy cargo receptor binding to Nsp9 and facilitating its autophagic degradation. Additionally, we found that TOLLIP interacts with LC3A, LC3C, and GABARAPL1. Further investigations revealed that Nsp9 specifically enhances the binding of TOLLIP to LC3A, rather than LC3C or GABARAPL1. Importantly, TOLLIP promotes the engulfment of Nsp9 by LC3A-coated autophagosomes and mediates Nsp9 trafficking to lysosomes, ultimately leading to LC3A-dependent degradation of Nsp9. Consequently, TOLLIP suppresses PEDV replication. Overall, our findings highlight the role of TOLLIP in connecting viral proteins to LC3A-dependent autophagosome formation, emphasizing its significance in combating viruses through selective autophagy.

A Decrease of Antibodies Against SARS-CoV-2 Antigens Does Not Reflect a Decrease of Neutralization Rate: A Prospective Study to Evaluate Kinetic and Dynamic Humoral Immune Response After Vaccination During Pregnancy.

Despite being at increased risk for severe COVID-19, pregnant women were initially excluded from vaccine clinical trials, which limited data regarding vaccine effectiveness and protection in this group. Aiming to better understand the immune response to vaccination during pregnancy, we compared the kinetics and titers of neutralizing and IgG antibodies generated against SARS-CoV-2 during vaccination with BNT162b2 (Pfizer-BioNTech) or CoronaVac (Sinovac Biotech) in a cohort of pregnant women. We evaluated participants before vaccination and 30 days after each vaccine dose, using a multiplex bead assay to measure IgG antibodies against SARS-CoV-2 antigens (total spike, spike-1, spike-2, receptor binding domain, and nucleocapsid) and a live virus fluorescence reduction neutralization assay (FRNA) to quantify neutralizing antibodies. Our data showed that vaccination resulted in a robust humoral response, with a considerable increase in the levels of IgG and neutralizing antibodies after the first vaccine dose and a sustained neutralizing response after the vaccine boost. In addition, antispike IgG assays presented a slight decrease after the second dose, while the neutralization rate remained stable. Immune response to the SARS-CoV-2 vaccine in pregnant women demonstrated an important increase in neutralizing antibodies.

Isolation and molecular characterization of an enteric isolate of the Genotype-Ia Bovine coronavirus with notable mutations in the receptor binding domain of the spike glycoprotein.

BCoV new isolate was plaque purified, isolated, and propagated in vitro using MDBK and HRT-18. The full-length genome sequencing of this new BCoV isolate (31Kbs) was drafted and deported in the GenBank. The genome organization is (5'-UTR-Gene-1-32kDa-HE-S-4.9 kDa-4.8kDa-12.7kDa-E-M-N-UTR-3'). Phylogenetic analysis based on the sequences of (the full-length genome, S, HE, and N) showed that the BCoV-13 clustered with other North American BCoV genotype I members. The sequence analysis shows several synonymous mutations among various domains of the S glycoprotein, especially the receptor binding domain. We found nine notable nucleotide deletions immediately downstream of the RNA binding domain of the nucleocapsid gene. Further gene function studies are encouraged to study the function of these mutations on the BCoV molecular pathogenesis and immune regulation. This research enhances our understanding of BCoV genomics and contributes to improved diagnostic and control measures for BCoV infections in cattle.

Dataset of SARS-CoV-2 spike protein receptor binding domain variants in complex with antigen-binding fragments targeting COVID-19 vaccine-referenced variants.

In this paper, we present a dataset of homology-modeled structures of SARS-CoV-2 Spike protein Receptor Binding Domain (RBD) variants complexed with antigen-binding fragments (Fab) derived from the PDB structures 8GPY (Omicron BA.4/5 RBD in complex with a neutralizing antibody scFv), 8H7Z (BA.2 RBD in complex with BA7535 Fab), and 8XE9 (XBB.1.5 RBD in complex with BD55-1205). The dataset consists of six sets of complex structures generated by combining 14 RBD variants with three different Fab pairs. The SARS-CoV-2 Spike protein sequence variants included in the dataset are Wild Type, BETA, EPSILON, IOTA, ETA, GAMMA, LAMBDA, KAPPA, BA.2, BA.4, XBB.1.5, EG.5.1, and KP.2. Notably, the KP.2 variant, which has gained attention due to its inclusion in recent vaccine updates (https://www.ema.europa.eu/), is also part of this dataset. The models were refined using the Schrödinger Bioluminate suite of software. For each variant, the homology-modeled RBDs were complexed with the Fab fragments from 8GPY, 8H7Z, and 8XE9 by grafting them onto the RBDs, followed by energy minimization, both individually and in combination. This process resulted in a comprehensive dataset of RBD-Fab complexes, suitable for comparative analysis and further investigations. Binding affinities between the RBD-Fab pairs were calculated using the Prime MM-GBSA tool (VSGB solvation model and OPLS4 force field), enabling the ranking of antigen-antibody interactions. Structural minimizations were performed to accurately estimate interaction energies, providing insights into the efficacy of antibody binding across different variants. This dataset provides high-quality structural data that can be reused for in-depth studies of antibody-antigen interactions, particularly in the context of vaccine efficacy and viral immune evasion strategies. The inclusion of the KP.2 variant, which is central to the latest COVID-19 vaccine updates, makes these homology models especially relevant. Supplementary files include the homology-modeled structures, computed binding affinities, and quality assessments (QMEANDisCo) for each model, ensuring reproducibility and reliability for further analyses.

Coronil effectively inhibits the interaction of clinically relevant Omicron mutants of SARS-CoV-2 Spike Proteins with human ACE2 receptor

BackgroundAccumulating evidence suggests that the receptor binding domain (RBD) of the SARS-CoV-2 Omicron variant has several times more binding affinity to the human angiotensin-converting enzyme 2 (ACE2) receptor compared to the RBD of the original covid-19 strain. This increased binding affinity of the Omicron variant is responsible for its increased internalization and infectivity. PurposeIn the present study, the impact of Coronil, a tri-herbal formulation of extracts from Withania somnifera, Tinospora cordifolia and Ocimum sanctum on the binding properties of Omicron SARS-CoV-2 variant spike proteins (S proteins) was investigated. MethodsChemical composition of Coronil was determined by the Prominence-XR UHPLC system. The ELISA-based ACE2 binding inhibition assay was performed to delineate the effect of Coronil on the interaction between human ACE2 receptor and different Omicron variant S-proteins such as BA.4/BA5, XBB, BA.2.75.2, BA4.6/BF.7, BA.2.75.2, BQ.1.1 and a recently found spike protein variant JN.1 which is thought to emerge from BA.2.86. ResultsCoronil showed a dose-dependent inhibitory effect on the interactions between ACE2 and receptor binding domains (RBD) of all variants of S-proteins evaluated in this study including the recently emerged, highly transmissible variant spike protein JN.1. Although, Coronil significantly reduced the binding percentage in almost all the variant spike proteins, the maximum inhibition was achieved against BA.4/BA.5 where it inhibited the S protein – ACE2 interaction even at a low concentration of 3 µg/ml (16.6 %). This binding inhibition was further increased to 60.3 and 84.6 % at 100 and 300 µg/ml respectively. ConclusionThis capability of Coronil to inhibit the binding of S-protein variants with ACE2 receptor may interfere with viral binding and internalization resulting in reduced infectivity of these Omicron spike protein variants. Overall, our data underscores the potential of Coronil in combating the various newly emerged Omicron spike protein variants. These findings may provide a basis for further studies of Coronil for its clinical effectiveness against these Omicron variants.

Phase 1/2 study of XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 monoclonal antibody, in combination with atezolizumab in patients with advanced solid tumors and in MSS CRC.

206 Background: XTX101 is an investigational tumor-activated, Fc-enhanced, high affinity binding aCTLA-4 designed to block CTLA-4 and deplete regulatory T cells upon activation in the tumor by proteases. XTX101 also contains mutations designed to enhance Fcγ receptor binding. Fc enhancement augments FcγR co-engagement on antigen presenting cells and has been linked with efficacy of aCTLA-4 combinations in patients (pts) with “cold tumors”, including microsatellite stable (MSS) colorectal cancer (CRC). Parts 1A/1B of the study evaluated XTX101 monotherapy in pts with advanced solid tumors. XTX101 was generally well tolerated with limited peripheral XTX101 activation (~13%) compared to evidence of XTX101 activation in the tumor microenvironment (73-96%, in n=2 on-treatment tumor biopsies). A durable partial response (PR) and resolution of hepatic metastases was observed in a pt with PD-L1 negative NSCLC at the monotherapy recommended phase 2 dose (RP2D) of 150 mg once every six weeks (Q6W) [1]. Methods: Part 1C evaluated the safety and feasibility of XTX101 in combination with atezolizumab in pts with advanced solid tumors using 3+3 dose escalation to establish the RP2D. Phase 2 is enrolling pts with MSS CRC with at least 1 prior regimen for metastatic CRC. Initial safety and anti-tumor activity data from Phase 2 in ~20 pts will be presented. Results: As of July 15, 2024, 15 pts were enrolled in Part 1C: MSS CRC (n=12), esophageal cancer, NSCLC, and ampullary carcinoma (n=1 each). Median age 69 and 3 prior lines of therapy (1-12). Across all XTX101 dose levels (75 mg to 150 mg), treatment-related adverse events (TRAEs) of any grade with >10% incidence included infusion reactions (n=4), fatigue and diarrhea (n=2 each), no G4 or G5 TRAEs were reported. In Part 1C, 2 pts experienced a dose limiting toxicity: 1 pt had G3 colitis, and 1 pt had G3 liver enzyme elevation (both were at the XTX101 150 mg dose level and resolved with immunosuppression). Two unconfirmed PRs were reported: in a pt with MSS CRC, including resolution of a liver target lesion, and in a pt with ampullary carcinoma (accompanied by a Ca 19-9 decrease from 700 to 41). In addition, a decrease in intra-tumoral Tregs was observed in paired tumor biopsies from a pt. The combination RP2D was established as XTX101 100 mg Q6W and atezolizumab 1200 mg once every three weeks (Q3W), and enrollment in the Phase 2 part of the study for pts with MSS CRC is ongoing at the RP2D. Conclusions: In Part 1C dose escalation, the combination of XTX101, a tumor-activated, Fc-enhanced aCTLA-4, and atezolizumab was generally well tolerated in pts with advanced solid tumors and demonstrated initial evidence of anti-tumor activity in cold tumors. These data support the initiation of an ongoing Phase 2 study evaluating the combination in pts with MSS CRC with and without liver metastases. 1. Davar ESMO IO 2023. Clinical trial information: NCT04896697 .

Childhood family environment and μ-opioid receptor availability in vivo in adulthood.

Animal studies have reported associations of early maternal separation with altered μ-opioid receptor function but data on humans are scarce. We now investigated whether childhood family environment is related to μ-opioid receptor availability in the human brain in adulthood. Healthy participants (n = 37-39 in the analyses) were recruited from the prospective population-based Young Finns Study (YFS) that started in 1980. Childhood family environment was evaluated in 1980, including scores for stress-prone life events, disadvantageous emotional family atmosphere, and adverse socioeconomic environment. We used positron emission tomography (PET) with radioligand [11C]carfentanil to measure μ-opioid receptor availability in adulthood. Age- and sex-adjusted analyses showed that exposure to stress-prone life events in childhood was related to lower μ-opioid receptor binding in the orbitofrontal cortex, hippocampus, putamen, amygdala, insula, thalamus, anterior cingulate cortex, and dorsal caudate in adulthood (when compared to participants not exposed to stress-prone life events). Unfavorable socioeconomic family environment or disadvantageous emotional family atmosphere was not associated with μ-opioid receptor availability in adulthood. In conclusion, exposure to environmental instability (i.e., to stress-prone life events below traumatic threshold) during early development is associated with dysregulation of the u-opioid receptor transmission in adulthood. The findings increase understanding of the neurobiological mechanisms involved in the associations between childhood adversities and adulthood mental disorders.

Estrogenic activity of alkylcyclohexanones and alkylcyclohexanols in rainbow trout estrogen receptor binding and liver slices Vtg mRNA expression assays.

Alkylcyclohexanones and alkylcyclohexanols are industrial chemicals produced in large volumes. Little is known regarding estrogenic activity of these chemicals in aquatic species. A series of para-alkyl subsituted hexanones and hexanols with varying chain length and branching were tested in a suite of in vitro rainbow trout binding assays and in an ex vivo rainbow trout liver slice/vitellogenin (Vtg) expression assay to assess estrogenic potential. Furthermore, the extent of biotransformation of these chemicals under test conditions was monitored. Cyclohexane, cyclohexanol, and 4-ethylcyclohexanol had no detectable estrogenic activity in these assays. Para substituted alkylcyclohexanones and alkylcyclohexanols with side chains containing three to six carbons were active in both assays. Alkylcyclohexanones and alkylcyclohexanols with other than para substitution were also estrogenic when tested in these assays. The binding affinity relative to estradiol in cytosol of active chemicals was low, ranging from 0.0016 to 0.000029%. Biotransformation of alkylcyclohexanones to alkylcyclohexanols occurred in the cytosol binding assay and in the liver slice assay, thus making those exposures a mixture of alcohol and ketone forms. Biotransformation did not occur in rainbow trout nuclear extract and recombinant binding assays, thus the exposure in these assays was to only the dosed chemical. Both alkylcyclohexanones and alkylcyclohexanols bound to the estrogen receptor in these metabolically limited assays. Significant induction of Vtg mRNA expression was seen in slice exposed to chemicals with measurable binding affinities. Often the induction levels were below maximum efficacy and occurred at concentrations slightly lower than toxic concentrations. Therefore, while these chemicals produced an estrogenic effect, it is considerably weaker than that of the endogenous hormone and occurred at concentrations close to those which produce toxicity.

Putative mapping of α-subunits in the human brain: A PET study of GABAA receptor binding

Abstract The benzodiazepines (BZ) bind to the GABAA receptor (GABAAR) at the interface of its α-/γ-subunits and exert pharmacological activity as allosteric modulators. However, the distribution of the six distinct α-subunits (α1–α6) in the human brain has not been mapped in detail, primarily due to lack of α-subunit selective radioligands. AZD7325 and AZD6280 were two drug candidates with partial α-subunit selectivity in vitro, in development for the treatment of anxiety. GABAAR occupancy of both drugs was examined in the human brain using [11C]flumazenil PET imaging, which visualizes GABAARs containing the α1-, α2-, α3-, or α5-subunits with similar sensitivity. Importantly, the pattern of occupancy was heterogeneous across brain regions and different between the two drugs. This observation encouraged us to extend the analysis in an attempt to generate tentative maps of α-subunits in the human brain. Parametric images of [11C]flumazenil binding in 12 subjects, obtained at baseline and following administration of different doses of AZD7325 or AZD6280, were entered into a comprehensive analysis to identify GABAAR occupancy components of the two drugs. The major outcome parameters of the fitted models were maps of the contributions of these components to the overall occupancy and binding. The maps were then explored in terms of gross anatomy and were correlated with gene expression data for the relevant α-subunits to speculate on possible α-subunit identity of the derived components. The overall occupancy was disentangled into three distinct components (C1 to C3) by the preferred model. C1 was occupied by both drugs, C2 was only occupied by AZD7325, and C3 was not occupied by either drug. The patterns of component-specific contributions were diverse and complex, dissimilar to each other and to the overall [11C]flumazenil binding. Of the three components, C1 had the highest contribution throughout most of the brain except some cerebral nuclei, such as amygdala. The contribution of C2 was notable in cortex and basal ganglia, and very low in thalamus and brain stem. Within the cortex, the contribution of C3 was localized with highest values in sharply demarcated areas of the limbic, cingulate, and insular cortex. Otherwise, it had the highest contribution among components in some subcortical nuclei, was behind C1 in thalamus, and was negligible in brain stem. All three components had a high-degree, statistically significant positive correlation with GABAAR α-subunit gene (GABRA) expression: C1 foremost with GABRA1, C2 foremost with GABRA2, and C3 foremost with GABRA5. The correlations suggest that C1 might correspond to the distribution of α1- (and possibly α3-), C2 to that of α2-, and C3 to that of α5-subunit-containing GABAARs, respectively. The components identified by the present analysis of occupancy patterns at the [11C]flumazenil binding site provided putative in vivo maps of α-subunit-specific GABAAR distribution in the human brain. The findings demonstrate the feasibility of developing small molecules having preference for certain α-subunits, even if full selectivity was not yet achieved. Accordingly, the results should encourage and support the development of optimized, fully selective compounds to the benefit of basic research and drug development for the treatment of neurological and psychiatric conditions.

Extracellular vesicle surface engineering with integrins (ITGAL & ITGB2) to specifically target ICAM-1-expressing endothelial cells.

Extracellular vesicles (EVs) are taken up by most cells, however specific or preferential cell targeting remains a hurdle. This study aims to develop an EV that targets cells involved in inflammation, specifically those expressing intercellular adhesion molecule-1 (ICAM-1). To target these cells, we overexpress the ICAM-1 binding receptor "lymphocyte function-associated antigen-1" (LFA-1) in HEK293F cells, by sequential transfection of plasmids of the two LFA-1 subunits, ITGAL and ITGB2 (CD11a and CD18). The LFA-1 receptor was strongly overexpressed on the EVs released by the transfected cells. We further loaded these EVs with a therapeutic peptide, targeting myeloid differentiation primary response 88 (Myd88; EVMyd88), through a developed EV open-and-close procedure. Myd88 is a downstream common intracellular messenger for most TLR-receptors. EV expression of LFA-1 increases EV binding to ICAM-1-expressing cells, an effect that was dose-dependently inhibited by a specific neutralizing ICAM-1 antibody. Further, activated human endothelial cells treated with LFA-1 EVMyd88 had increased uptake of these EVs, resulting in dose-dependent inhibition of induced release of IL-8, presumably by targeting Myd88. We conclude that LFA-1-expressing EVMyd88 may be a candidate suitable for delivering therapeutic peptides in inflammatory diseases associated with TLR-activation.

P-2023. A ‘Variant-Proof’ Cocktail Of Two Heavy Chain-Only Antibodies Targets 3 Epitopes In The Spike Protein S1 And S2 Subunits And Broadly Neutralizes SARS-Cov-2 With Exceptional Potency

Abstract Background More than 7 million people have died from COVID-19 to date and SARS-CoV-2 continues to cause substantial disease around the world. High-risk patients like immunocompromised and elderly are often not able to elicit adequate immune responses to vaccination and strongly benefit from an additional layer of protection in the form of complementary antibody therapy. Monoclonal antibodies act immediately and can provide immune support for months. However, all previously authorized antibodies lost their neutralization potency against the currently circulating and constantly mutating SARS-CoV-2 variants, leaving a vast unmet medical need. The combination XVR012 consists of the molecules XVR013m and XVR014. Methods The combination XVR012 consists of the molecules XVR013m and XVR014 (Figure 1). XVR013m is a mono-specific VHH-Fc antibody that targets a unique epitope in the S2 subunit, XVR014 is a bi-specific bivalent VHHx-Fc-VHHy antibody construct, targeting 2 non-competing epitopes in the Receptor-Binding Domain of the spike (Figure 2). Both antibodies contain a human IgG1 Fc with LS mutations for half-life extension. Structural analysis of the XVR013m epitope Left panel: Structural analysis of the VHH R3DC23 (green) bound to the HR2 domain of the S2 unit of the SARS-CoV-2 spike protein, between the terminal N1194 glycan (orange) and the viral membrane. Right panel: One R3DC23 (green) binds the interface of two HR2 coils and each HR2 coil is bound by two R3DC23 VHHs. Results XVR012 delivers a triple mode of action in preventing infection of host cells, i.e., (1) sterical hinderance of ACE2 receptor binding, (2) induction of S1 subunit shedding thereby preventing viral attachment and (3) inhibition of the fusion process between the viral and host cell membrane. XVR012 broadly neutralizes SARS-CoV-1 and SARS-CoV-2 viruses in vitro and demonstrates an IC50 ranging from 4.8 to 8,7 ng/mL against all SARS-CoV-2 variants tested so far in a pseudovirus neutralization assay, including the currently circulating BA.2.86.1, HK.3, EG.5.1 and HV.1 (Table 1). In vivo, efficacy was demonstrated in the Syrian Golden Hamster model in a therapeutic setting, showing complete reduction of lung viremia for all XVR012 dose levels tested, even with a very low dose of 0,5 mg/kg for the XVR013m component. (Figure 3). PK parameters have been determined in a Tg32 SCID mouse model and support an envisioned duration of protection up to 6 months. Neutralization potency of VSV pseudo-typed with spike proteins of recent SARS-CoV-2 variants. Data are presented as mean IC50 based on 3 independent experiments, except for (*) 1 independent experiment. Conclusion In summary, this second-generation cocktail targeting three unique and highly conserved epitopes in the S1 and S2 subunits of the spike is ready to proceed to clinical testing and may provide a long-term solution to the populations at highest risk. Lung infectious viral titers in Syrian golden hamster post-infection challenge model (Wuhan strain) on day 4 post infection. Mean values + standard error of the mean (SEM) are reported. * Two animals in 2 mg/kg group of XVR013m and one animal in the 20 mg/kg group of XVR014 were experimentally confirmed in PK assays to not have been exposed to the drug. Dotted lines represent the lower limit of detection (LLOD) range. Disclosures Florence M. Herschke, PhD, ExeVir Bio: Patent inventor|ExeVir Bio: Employee Bert Schepens, PhD, Exevir: Bert Schepens is Inventor on patents that describe the nanobodies that are mentioned in the abstract and presentation Loes van Schie, PhD, Exevir: Grant/Research Support|Exevir: I am a co-inventor on the patents protecting the biologicals described in the abstract and presentation Xavier Saelens, PhD, ExeVir Bio: I am an inventor on patent applications WO2022/167666 A1 and WO2023/222825 A1 which incorporate discoveries and inventions described here.|ExeVir Bio: I am a scientific co-founder of ExeVir Bio and in receipt of ExeVir Bio share options