Receptor Binding Research Articles

Region-selective and site-specific glycation of influenza proteins surrounding the viral envelope membrane

Analysis of protein modifications is critical for quality control of therapeutic biologics. However, the identification and quantification of naturally occurring glycation of membrane proteins by mass spectrometry remain technically challenging. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. Straightforward sequencing was enhanced by MS/MS fragmentation of small peptides. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase. Topological analysis provides insights into the site-specific lysine glycation, localizing in the distinct structural regions of proteins surrounding the viral envelope membrane. Our finding highlights the proteome-wide discovery of lysine glycation of influenza membrane proteins and potential effects on the structural assembly, stability, receptor binding and enzyme activity, demonstrating that the impacts of accumulated glycation on the quality of products can be directly monitored by mass spectrometry-based structural proteomics analyses.

Factors associated with elevated SARS-CoV-2 immune response in children and adolescents.

Understanding the distinct immunologic responses to SARS-CoV-2 infection among pediatric populations is pivotal in navigating the COVID-19 pandemic and informing future public health strategies. This study aimed to identify factors associated with heightened antibody responses in children and adolescents to identify potential unique immune dynamics in this population. Data collected between July and December 2023 from the Texas Coronavirus Antibody REsponse Survey (Texas CARES), a statewide prospective population-based antibody survey among 1-to-19-year-old participants, were analyzed. Each participant had the following data available for analysis: (1) Roche Elecsys® Anti-SARS-CoV-2 Immunoassay for Nucleocapsid protein antibodies (Roche N-test), (2) qualitative and semi-quantitative detection of antibodies to the SARS CoV-2 spike protein receptor binding domain (Roche S-test), and (3) self-reported antigen/PCR COVID-19 test results, vaccination, and health status. Statistical analysis identified associations between participant characteristics and spike antibody quartile group. The analytical sample consisted of 411 participants (mean age 12.2 years, 50.6% female). Spike antibody values ranged from a low of 6.3 U/ml in the lowest quartile to a maximum of 203,132.0 U/ml in the highest quartile in the aggregate sample. Older age at test date (OR = 1.22, 95% CI: 1.12, 1.35, p < .001) and vaccination status (primary series/partially vaccinated, one or multiple boosters) showed significantly higher odds of being in the highest spike antibody quartile compared to younger age and unvaccinated status. Conversely, fewer days since the last immunity challenge showed decreased odds (OR = 0.98, 95% CI: 0.96, 0.99, p = 0.002) of being in the highest spike antibody quartile vs. more days since last immunity challenge. Additionally, one out of every three COVID-19 infections were asymptomatic. Older age, duration since the last immunity challenge (vaccine or infection), and vaccination status were associated with heightened spike antibody responses, highlighting the nuanced immune dynamics in the pediatric population. A significant proportion of children/adolescents continue to have asymptomatic infection, which has important public health implications.

Primary SARS-CoV-2 variant of concern infections elicit broad antibody Fc-mediated effector functions and memory B cell responses.

Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by human sera is a strong correlate of protection against symptomatic and severe Coronavirus Disease 2019 (COVID-19). The emergence of antigenically distinct SARS-CoV-2 variants of concern (VOCs) and the relatively rapid waning of serum antibody titers, however, raises questions about the sustainability of serum protection. In addition to serum neutralization, other antibody functionalities and the memory B cell (MBC) response are suggested to help maintaining this protection. In this study, we investigate the breadth of spike (S) protein-specific serum antibodies that mediate effector functions by interacting with Fc-gamma receptor IIa (FcγRIIa) and FcγRIIIa, and of the receptor binding domain (RBD)-specific MBCs, following a primary SARS-CoV-2 infection with the D614G, Alpha, Beta, Gamma, Delta, Omicron BA.1 or BA.2 variant. Irrespectively of the variant causing the infection, the breadth of S protein-specific serum antibodies that interact with FcγRIIa and FcγRIIIa and the RBD-specific MBC responses exceeded the breadth of serum neutralization, although the Alpha-induced B cell response seemed more strain-specific. Between VOC groups, both quantitative and qualitative differences in the immune responses were observed, suggesting differences in immunogenicity. Overall, this study contributes to the understanding of protective humoral and B cell responses in the light of emerging antigenically distinct VOCs, and highlights the need to study the immune system beyond serum neutralization to gain a better understanding of the protection against emerging variants.

The N and C-terminal deleted variant of the dengue virus NS1 protein is a potential candidate for dengue vaccine development

NS1 is an elusive dengue protein, involved in viral replication, assembly, pathogenesis, and immune evasion. Its levels in blood plasm are positively related to disease severity like thrombocytopenia, hemorrhage, and vascular leakage. Despite its pathogenic roles, NS1 is being used in various vaccine formulations due to its sequence conservancy, ability to produce protective antibodies and low risk for inducing antibody-dependent enhancement. In this study, we have used bioinformatics tools and reported literature to develop an NS1 variant (dNS1). Molecular docking studies were performed to evaluate the receptor-binding ability of the NS1 and dNS1 with TLR4. NS1 and dNS1 (153 to 312 amino acid region) genes were cloned, expressed and protein was purified followed by refolding. Docking studies showed the binding of NS1 and dNS1 with the TLR4 receptor which suggests that N and C-terminal sequences of NS1 are not critical for receptor binding. Antibodies against NS1 and dNS1 were raised in rabbits and binding affinity of anti-dNS1 anti-NS1 sera was evaluated against both NS1 and dNS1. Similar results were observed through western blotting which highlight that N and C-terminal deletion of NS1 does not compromise the immunogenic potential of dNS1 hence, supports its use in future vaccine formulations as a substitute for NS1.

Non-small cell lung cancer sensitisation to platinum chemotherapy via new thiazole-triazole hybrids acting as dual T-type CCB/MMP-9 inhibitors

Cisplatin remains the unchallenged standard therapy for NSCLC. However, it is not completely curative due to drug resistance and oxidative stress-induced toxicity. Drug resistance is linked to overexpression of matrix metalloproteinases (MMPs) and aberrant calcium signalling. We report synthesis of novel thiazole-triazole hybrids as MMP-9 inhibitors with T-type calcium channel blocking and antioxidant effects to sensitise NSCLC to cisplatin and ameliorate its toxicity. MTT and whole cell patch clamp assays revealed that 6d has a balanced profile of cytotoxicity (IC50 = 21 ± 1 nM, SI = 12.14) and T-type calcium channel blocking activity (⁓60% at 10 μM). It exhibited moderate ROS scavenging activity and nanomolar MMP-9 inhibition (IC50 = 90 ± 7 nM) surpassing NNGH with MMP-9 over −2 and MMP-10 over −13 selectivity. Docking and MDs simulated its receptor binding mode. Combination studies confirmed that 6d synergized with cisplatin (CI = 0.69 ± 0.05) lowering its IC50 by 6.89 folds. Overall, the study introduces potential lead adjuvants for NSCLC platinum-based therapy.

Countercurrent chromatography isolation of green propolis biomarkers: Potential blockers of SARS-COV-2 RBD and ACE2 interaction

Propolis is a natural resinous mixture produced by honeybees with numerous biological activities. Considering the recently reported potential of propolis as an adjuvant in COVID-19 treatment, a methodology for the fractionation of the hexane extract of Brazilian green propolis (HEGP) was developed for the obtention of prenylated biomarkers by countercurrent chromatography. The inhibition of the interaction between the receptor binding domain (RBD) of spike and ACE2 receptor was evaluated by the Lumitᵀᴹ immunoassay. Fractionation of HEGP was performed by both normal (CCC1 and CCC2, with extended elution) and reversed (CCC3) phase elution-extrusion modes with the solvent system hexane–ethanol–water 4:3:1. The normal elution mode of CCC1 (471 mg HEGP in a 80 mL column volume, 1.6 mm id) was scaled-up (CCC5, 1211 mg HEGP in a 112 mL column volume, 2.1 mm id), leading to the isolation of 89.9 mg of artepillin C, 1; 52.7 mg of baccharin, 2; and 26.6 mg of chromene, with purities of 93 %, 83 % and 88 %, respectively, by HPLC-PDA. Among the isolated compounds, artepillin C, 1, and baccharin, 2, presented the best results in the Lumitᵀᴹ immunoassay, showing 67% and 51% inhibition, respectively, at the concentration of 10 μM. This technique proved to be of low operational cost and excellent reproducibility.

Fully human single-domain antibody targeting a highly conserved cryptic epitope on the Nipah virus G protein

Nipah virus infection, one of the top priority diseases recognized by the World Health Organization, underscores the urgent need to develop effective countermeasures against potential epidemics and pandemics. Here, we identify a fully human single-domain antibody that targets a highly conserved cryptic epitope situated at the dimeric interface of the Nipah virus G protein (receptor binding protein, RBP), as elucidated through structures by high-resolution cryo-electron microscopy (cryo-EM). This unique binding mode disrupts the tetramerization of the G protein, consequently obstructing the activation of the F protein and inhibiting viral membrane fusion. Furthermore, our investigations reveal that this compact antibody displays enhanced permeability across the blood-brain barrier (BBB) and demonstrates superior efficacy in eliminating pseudovirus within the brain in a murine model of Nipah virus infection, particularly compared to the well-characterized antibody m102.4 in an IgG1 format. Consequently, this single-domain antibody holds promise as a therapeutic candidate to prevent Nipah virus infections and has potential implications for vaccine development.

Enhancing the cytotoxicity of immunotoxins by facilitating their dissociation from target receptors under the reducing conditions of the endocytic pathway

Immunotoxins (ITs) are recombinant chimeric proteins that combine a protein toxin with a targeting moiety to facilitate the selective delivery of the toxin to cancer cells. Here, we present a novel strategy to enhance the cytosolic access of ITs by promoting their dissociation from target receptors under the reducing conditions of the endocytic pathway. We engineered monobodySS, a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. MonobodySS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobodySS-based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobodySS-PE25. These ITs showed dose-dependent cytotoxicity against target receptor-expressing cancer cells and a wider therapeutic window due to higher efficacy at lower doses compared to controls with SS reduction inhibited. ERSS/28-PE25, with a KD of 28 nM for EGFR, demonstrated superior tumor-killing potency compared to ER/21-PE25, which lacks an SS bond, at equivalent and lower doses. In vivo, ERSS/28-PE25 outperformed ER/21-PE25 in suppressing tumor growth in EGFR-overexpressing xenograft mouse models. This study presents a strategy for developing solid tumor-targeting ITs using SS-containing paratopes to enhance cytosolic delivery and antitumor efficacy.

Bitter taste receptors as prognostic markers in adrenocortical carcinoma.

230 Background: Adrenocortical carcinoma (ACC) is a rare malignant neoplasm of the adrenal gland. Progressive forms of this cancer carry a dismal prognosis with 5-year survival rates &lt; 40%. As such, predictors of stage progression and prognostic biomarkers are a must. Taste 2 receptors (TAS2Rs) are localized on the surface of bitter receptor cells. Recent evidence has shown their role as prognostic biomarkers in multiple cancers, such as breast, prostate, and head and neck squamous cell carcinoma. The aim of this study was to elucidate the role of TAS2Rs in ACC. Methods: Differentially expressed genes (DEGs) based on high and low expression groups of TAS2R genes were identified through the 'DESeq2' package on R. Subsequent Gene Set Enrichment Analysis (GSEA) was performed using the 'TCGAbiolinks' package on R and Enrichr online tool. Immune infiltration analysis was conducted using TIMER2.0 and CIBERSORT. Survival analysis with an optimal expression cutoff for overall survival (OS) and a median cutoff for disease-free survival (DFS) based on The Cancer Genome Atlas Adrenocortical carcinoma (TCGA-ACC) data was executed using the 'survival' and 'survminer' R packages and GEPIA2 online tool. Results: Analysis of the TCGA-ACC cohort revealed OS estimates that indicated worse survival in patients with higher TAS2R14, TAS2R19, TAS2R20, and TAS2R30 expression (Table). Further analysis produced consistent results, demonstrating that elevated expressions of TAS2R14 and TAS2R19 are correlated with decreased DFS, with Hazard Ratios (HR) of 2.60 (p = 0.0046) and 2.20 (p = 0.022), respectively. Furthermore, the F-test and stage plots have shown consistent upregulation of selected TAS2R genes across stages I–IV (p &lt; 0.05). RNA-seq analysis revealed 1170 common (579 downregulated, 591 upregulated) DEGs between high and low TAS2R14 and TAS2R19 expression groups. GSEA showed upregulated genes in high expressers have relations to hedgehog signaling, epithelial to mesenchymal transition, TGF-B signaling, and organization of the extracellular matrix. On the other hand, down-regulated genes showed functions in metabolic reprogramming, chemokine receptor binding, MHC protein complex, and chemokine-mediated inflammation. GSEA on co-expressed genes showed pathways related to ferroptosis, autolysosome activity, and iron homeostasis and metabolism. Immune infiltration analysis revealed significantly increased CD4+, naïve and plasma B cell infiltration with higher TAS2R14 and TAS2R19 expression (Rho &gt; 0.25, p &lt; 0.05), in addition to increased M1 macrophage infiltration with higher TAS2R19 expression (Rho = 0.28, p &lt; 0.05). Conclusions: TAS2R genes are potentially involved in modulating carcinogenic processes, including stage progression, metastasis and invasion, immune regulation, and metabolic processes. This modulation may explain the poorer prognostic outcomes in ACC patients with elevated TAS2R expression. Further validation in external cohorts is necessary to confirm these results. [Table: see text]

5-Oxo-7,7-Dimethyl-5,6,7,8-Tetrahydro-4-H-Chromene Bearing N, N-Dimethylaniline as Turn-Off Fluorescent Chemosensor for Picric Acid in Real Water Sample.

We have synthesized a one-pot, three-component pyran-based fluorescence chemosensor using onion extract as a green catalyst. The confirmed structure of the 1:2 binding of receptor SPR-2-picric acid adduct revealed that the pyran-based receptor accommodated two guest picric acid molecules through non-covalent interactions. UV-Vis and fluorescence spectroscopy show high selectivity and sensitivity towards picric acid. The 1D/2D NMR and Job's plot analysis show the complexation and stoichiometric binding of the receptor SPR-2 with picric acid are 1:2. The 1H NMR spectral studies confirm that the formation of receptor SPR-2-picric acid adduct via weak hydrogen bonding. The cooperativity of the receptor SPR-2-picric acid adduct shows negative cooperativity due to the weak hydrogen bonding of receptor SPR-2 and picric acid. Further, the density functional theory (DFT) confirmed the molecular level interaction of the SPR-2 and receptor SPR-2-Picric acid adduct. The receptor was effectively used to assess picric acid concentrations in real water samples.

Characteristics of SARS-CoV-2 and Opisthorchis viverrini coinfections: insights into immune responses and clinical outcomes.

The effects of co-infections with SARS-CoV-2 and parasitic diseases have been little investigated in terms of immune response, disease dynamics, and clinical outcomes. This study aimed to explore the impact of co-infection with Opisthorchis viverrini and SARS-CoV-2 on the immune response concerning clinical symptoms and the severity of pulmonary abnormalities. A cross-sectional study was conducted, including healthy participants as controls, participants with opisthorchiasis, SARS-CoV-2 infection, and a co-infection group with both diseases. Characteristics of SARS-CoV-2 infection were assessed based on clinical parameters and severity of pulmonary abnormalities, whereas opisthorchiasis burden was evaluated by eggs-per-gram (EPG) counts. Immune responses were assessed by measuring levels of interferon-γ (IFN-γ), SARS-CoV-2 anti-spike receptor binding domain (RBD) IgG, and neutralizing antibody against SARS-CoV-2. In the co-infected group, clinical parameters and hospitalization rates were lower than in the SARS-CoV-2 group. Pulmonary abnormalities, such as bronchial fibrosis, were commonly observed in the SARS-CoV-2 group, leading to hospitalization in some cases. Participants with opisthorchiasis had higher IFN-γ levels than healthy individuals. IFN-γ levels were significantly lower in the co-infection group compared with the SARS-CoV-2 group (P = 0.002). There was a significant (P = 0.044) positive correlation between RBD-specific IgG and percent neutralization levels in the SARS-CoV-2 group. Levels of both were somewhat lower (not statistically significant) in the co-infection group. A negative correlation was observed between opisthorchiasis burden (EPG counts) and IFN-γ and RBD-specific IgG levels in the co-infected group. Following vaccination, the increase in IgG levels against the RBD protein was significantly lower in the co-infected group than in the SARS-CoV-2 group. These results suggest that O. viverrini infection suppresses immune responses and may lead to a reduction in severity in cases of SARS-CoV-2 co-infection.

Receptor interactions of protoxin and activated Vip3Aa structural conformations in Spodoptera exigua.

The Vip3Aa insecticidal protein, produced by Bacillus thuringiensis, has been effectively used in commercial Bt-crops to manage lepidopteran pests. Upon ingestion by larvae, the protoxin is processed by midgut proteases into the activated protein and binds specifically to its receptors in the midgut, leading to insect mortality. Cryo-EM resolution of the trypsin-processed Vip3Aa protein unveiled structural remodelling of the N-terminal region during the transition from protoxin to activated protein. This conformational change has been demonstrated to be crucial for toxicity against Spodoptera exigua larvae, a major global lepidopteran pest. In this study, we investigated the relevance of the structural remodelling for the specific binding to midgut receptors. We conducted in vitro binding assays with radiolabelled proteins and brush border membrane vesicles (BBMV) from S. exigua, employing structural mutants that lock the protein in either its protoxin or its activated conformation. Our results indicate that both structural stages of the protein share binding sites in the midgut epithelium. Moreover, in vivo competition assays revealed that Vip3Aa is able to bind to functional receptors in S. exigua larvae both as protoxin and as activated protein. Altogether, our findings point to both structural conformations contributing to receptor binding. In vivo, either spontaneous structural shift upon proteolytic cleavage or receptor-mediated remodelling could be occurring. However, the timing and context in which the conformational change occurs could influence membrane insertion and toxicity. Our results show the complex interplay between proteolytic processing, protein structure and receptor interactions in Vip3Aa's toxicity. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

SARS-CoV-2 JN.1 variant evasion of IGHV3-53/3-66 B cell germlines.

The severe acute respiratory syndrome coronavirus 2 variant JN.1 recently emerged as the dominant variant despite having only one amino acid change on the spike (S) protein receptor binding domain (RBD) compared with the ancestral BA.2.86, which never represented more than 5% of global variants. To define at the molecular level the JN.1 ability to spread globally, we interrogated a panel of 899 neutralizing human monoclonal antibodies. Our data show that the single leucine-455-to-serine mutation in the JN.1 spike protein RBD unleashed the global spread of JN.1, likely occurring by elimination of more than 70% of the neutralizing antibodies mediated by IGHV3-53/3-66 germlines. However, the resilience of class 3 antibodies with low neutralization potency but strong Fc functions may explain the absence of JN.1 severe disease.

SARS-CoV-2 seroprevalence in people living with HIV in South Sudan

ObjectivesThe burden of SARS-CoV-2 infection in people living with HIV (PLHIV) in South Sudan is unknown. MethodsWe conducted a cross-sectional seroprevalence survey of SARS-CoV-2 immunoglobulin (Ig) G antibodies and other diseases of public health importance (strongyloidiasis, toxoplasmosis) in PLHIV in South Sudan during April 1, 2020-April 30, 2022. We used a multiplex SARS-CoV-2 immunoassay to detect IgG antibodies targeting the SARS-CoV-2 spike, receptor binding domain, and nucelocapsid (N) proteins, and antigens for other pathogens (Strongyloides stercoralis and Toxoplasma gondii). ResultsAmong 3518 samples tested, seroprevalence of IgG antibodies to SARS-CoV-2 spike protein and receptor binding domain 591 and nucleocapsid ranged from 1.4% (95% confidence interval [CI]: 0.9-2.1%) in April-June 2020 to 53.3% (95% CI: 49.5-57.1%) in January-March 2022. The prevalence of S. stercoralis IgG ranged between 27.3% (95% CI: 23.4-31.5%) in October-December 2021 and 47.2% (95% CI: 37.8-56.8%) in July-September 2021, and, for T. gondii IgG, prevalence ranged from 15.5% (95% CI: 13.3-17.9%) in April-June 2020 to 36.2% (95% CI: 27.4-46.2%) July-September 2021. ConclusionsBy early 2022, PLHIV in South Sudan had high rates of SARS-CoV-2 seropositivity. Surveillance of diseases of global health concern in PLHIV is crucial to estimate population-level exposure and inform public health responses.

Sedum kamtschaticum Exerts Hypnotic Effects via the Adenosine A2A Receptor in Mice.

Insomnia is a common sleep disorder with significant societal and economic impacts. Current pharmacotherapies for insomnia are often accompanied by side effects, necessitating the development of new therapeutic drugs. In this study, the hypnotic effects and mechanisms of Sedum kamtschaticum 30% ethanol extract (ESK) and one of its active compounds, myricitrin, were investigated using pentobarbital-induced sleep experiments, immunohistochemistry (IHC), receptor binding assays, and enzyme-linked immunosorbent assay (ELISA). The pentobarbital-induced sleep experiments revealed that ESK and myricitrin reduced sleep latency and prolonged total sleep time in a dose-dependent manner. Based on c-Fos immunostaining, ESK, and myricitrin enhanced the GABAergic neural activity in sleep-promoting ventrolateral preoptic nucleus (VLPO) GABAergic. By measuring the level of GABA released from VLPO GABAergic neurons, ESK and myricitrin were found to increase GABA release in the hypothalamus. These effects were significantly inhibited by SCH. Moreover, ESK exhibited a concentration-dependent binding affinity for the adenosine A2A receptors (A2AR). In conclusion, ESK and myricitrin have hypnotic effects, and their underlying mechanisms may be related to the activation of A2AR.

CGAS Expression is Enhanced in Systemic Sclerosis Associated Interstitial Lung Disease and Stimulates Inflammatory Myofibroblast Activation.

The lungs of patients with Systemic Sclerosis Associated Interstitial Lung Disease (SSc-ILD) contain inflammatory myofibroblasts arising in association with fibrotic stimuli and perturbed innate immunity. The innate immune DNA binding receptor Cyclic GMP-AMP synthase (cGAS) is implicated in inflammation and fibrosis, but its involvement in SSc-ILD remains unknown. We examined cGAS expression, activity, and therapeutic potential in SSc-ILD using cultured fibroblasts, precision cut lung slices (PCLS), and a well-accepted animal model. Expression and localization of cGAS, cytokines, and type 1 interferons were evaluated in SSc-ILD lung tissues, bronchoalveolar lavage (BAL), and isolated lung fibroblasts. CGAS activation was assessed in a publicly available SSc-ILD single cell RNA sequencing dataset. Production of cytokines, type 1 interferons, and αSMA elicited by TGFβ1 or local substrate stiffness were measured in normal human lung fibroblasts (NHLFs) via qRT-PCR, ELISA, and immunofluorescence. Small molecule cGAS inhibition was tested in cultured fibroblasts, human PCLS, and the bleomycin pulmonary fibrosis model. SSc-ILD lung tissue and BAL are enriched for cGAS, cytokines, and type 1 interferons. The cGAS pathway shows constitutive activation in SSc-ILD fibroblasts and is inducible in NHLFs by TGFβ1 or mechanical stimuli. In these settings, and in human PCLS, cGAS expression is paralleled by the production of cytokines, type 1 interferons, and αSMA that are mitigated by a small molecule cGAS inhibitor. These findings are recapitulated in the bleomycin mouse model. cGAS signaling contributes to pathogenic inflammatory myofibroblast phenotypes in SSc-ILD. Inhibiting cGAS or its downstream effectors represents a novel therapeutic approach.

Structural Characterization of Human Bufavirus 1: Receptor Binding and Endosomal pH-Induced Changes.

Bufaviruses (BuV) are members of the Parvoviridae of the Protoparvovirus genus. They are non-enveloped, T = 1 icosahedral ssDNA viruses isolated from patients exhibiting acute diarrhea. The lack of treatment options and a limited understanding of their disease mechanisms require studying these viruses on a molecular and structural level. In the present study, we utilize glycan arrays and cell binding assays to demonstrate that BuV1 capsid binds terminal sialic acid (SIA) glycans. Furthermore, using cryo-electron microscopy (cryo-EM), SIA is shown to bind on the 2/5-fold wall of the capsid surface. Interestingly, the capsid residues stabilizing SIA binding are conserved in all human BuVs identified to date. Additionally, biophysical assays illustrate BuV1 capsid stabilization during endo-lysosomal (pH 7.4-pH 4) trafficking and capsid destabilization at pH 3 and less, which correspond to the pH of the stomach. Hence, we determined the cryo-EM structures of BuV1 capsids at pH 7.4, 4.0, and 2.6 to 2.8 Å, 3.2 Å, and 2.7 Å, respectively. These structures reveal capsid structural rearrangements during endo-lysosomal escape and provide a potential mechanism for this process. The structural insights gained from this study will add to the general knowledge of human pathogenic parvoviruses. Furthermore, the identification of the conserved SIA receptor binding site among BuVs provides a possible targetable surface-accessible pocket for the design of small molecules to be developed as anti-virals for these viruses.

Vaccination with ancestral SARS-CoV-2 spike adjuvanted with TLR agonists provides cross-protection against XBB.1

Many different platforms have been used to develop highly protective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans. However, protection has eroded over time due to the emergence of antigenically diverse viral variants, especially the Omicron subvariants. One successful platform for the generation of SARS-CoV-2 vaccines are recombinant spike protein vaccines, of which two are licensed in the United States and Europe. Typically, purified recombinant protein antigens are poorly immunogenic and adjuvants must be included in the formulation. Here, we adjuvanted recombinant ancestral SARS-CoV-2 Wuhan-Hu-1 spike proteins with an emulsion formulation combined with synthetic Toll-like receptor (TLR) 4 and 7/8 agonists. This combination led to the induction of a Th1-skewed immune response that included high titers of antibodies against Wuhan-Hu-1 spike. These serum antibodies included neutralizing and cross-reactive antibodies that recognized the spike from multiple SARS-CoV-2 variants, as well as the receptor binding domain (RBD) from SARS-CoV-1. Despite an absence of robust cross-neutralization, vaccination against Wuhan-Hu-1 spike in the context of TLR-containing emulsions provided complete cross-protection against disease from a lethal challenge with XBB.1 in a stringent K18-hACE2 mouse model. We believe that the combination of recombinant spike antigens with TLR agonist-based emulsion formulations could lead to the development of next-generation SARS-CoV-2 vaccines that provide significant protection from future emerging variants.

Determinants of immune responses predictive of protection against shigellosis in an endemic zone: a systems analysis of antibody profiles and function

Shigella is the third leading global cause of moderate or severe diarrhoea among children younger than 5years globally, and is the leading cause in children aged 24-59months. The mechanism of protection against Shigella infection and disease in endemic areas is uncertain. We aimed to compare the Shigella-specific antibody responses in individuals living in Shigella-endemic and non-endemic areas, and to identify correlates of protection in a Shigella-endemic location. We applied a systems approach to retrospectively analyse serological responses to Shigella across endemic and non-endemic populations. We profiled serum samples collected from 44individuals from the USA without previous exposure to Shigella and who were experimentally challenged with Shigella sonnei (non-endemic setting), and serum samples collected from 55Peruvian army recruits (endemic setting). In the endemic setting, a subset of 37samples collected from individuals infected with culture-confirmed Shigella flexneri 2a were divided into twogroups: susceptible, which included individuals infected within 90days of entering the camp (n=29); or resistant, which included individuals infected later than 90days after entering the camp (n=8). We analysed Shigella-specific antibody isotype, subclass, and Fc receptor binding profiles across IpaB, IpaC, IpaD, and lipopolysaccharide from Sflexneri 2a, 3a, and 6, and Ssonnei, and O-specific polysaccharide (OSP) from Sflexneri 2a and 3a and Ssonnei. We also evaluated antibody-mediated complement deposition and innate immune cell activation. The main outcome of interest was the detection of antibody markers and functionality associated with protection against shigellosis in a high-burden endemic setting. Adults with endemic exposure to Shigella possessed broad and functional antibody responses across polysaccharide, glycolipid, and protein antigens compared with individuals from non-endemic regions. In a setting with high Shigella burden, elevated levels of OSP-specific Fcα receptor (FcαR) binding antibodies were associated with resistance to shigellosis, whereas total OSP-specific IgA was not, suggesting a potentially unique functionality. OSP-specific FcαR binding IgA found in resistant individuals activated bactericidal neutrophil functions including phagocytosis, degranulation, and production of reactive oxygen species. Moreover, IgA depletion from resistant serum significantly reduced binding of OSP-specific antibodies to FcαR and antibody-mediated activation of neutrophils and monocytes. Our findings suggest that OSP-specific functional IgA responses contribute to protective immunity against Shigella infection in a high-burden setting. These findings will assist in the development and evaluation of Shigella vaccines. US National Institutes of Health.

Citrulline activates adenosine receptors: New insight into metabolic pathways interaction

Citrulline is a non-proteinogenic amino acid that forms as by-product in nitric oxide (NO) synthesis from arginine and may act in concert with NO as an independent signaling molecule that involves in the mechanism of vascular smooth muscle vasodilation.In this study we examined the effects of citrulline on pulmonary artery smooth muscles. Experimental design comprised outward potassium currents measurements in enzymatically isolated rat pulmonary artery smooth muscle (PASMc) cells using whole-cell patch clamp technique, isometric contractile force recordings on rat pulmonary artery rings and method of molecular docking simulation.Citrulline in a concentration 10−9-10−5 M relaxed phenylephrine (PHE)-preactivated SM of rat pulmonary artery in a dose-dependent manner (EC50 0,67 μM). This citrulline-induced relaxation was dependent on an intact endothelium. Bath application of citrulline (10-8-10-5 M) on isolated PASMc induced a significant increase in the amplitude of outward potassium current (Ik). The adenosine antagonist caffeine (10-6 M) effectively blocked both the citrulline-induced relaxation response and Ik increment. Molecular docking modeling suggests that caffeine blocking the potent activity of citrulline results from competitive interactions at the A2 adenosine receptor binding site.In summary, our data suggest that citrulline, released with NO at low concentrations, can effectively interact with adenosine receptors in smooth muscle cells, causing their relaxation, indicating surprising interaction between NO and adenosine pathways.