Binding Inhibition Assay Research Articles

A-235 Systemic Inflammation is associated with worse outcomes from SARS-CoV-2 infection but not neutralizing antibody

Abstract Background Numerous studies have demonstrated an association between systemic inflammation and mortality in patients with COVID-19. However, it is unclear how neutralizing antibody to severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2) associates with outcomes. Further, how systemic inflammation impacts neutralizing antibody production is relatively poorly understood. The aim of this study was to determine the association between the neutralizing antibody response to SARS-CoV-2, inflammatory markers, and clinical outcomes in a cohort of hospitalized patients without prior exposure/vaccination. Methods This prospective observational study approved by the Washington University IRB included adults admitted through the ED at Barnes Jewish Hospital between 10/2020 and 10/2021 with symptomatic RT-PCR confirmed SARS-CoV-2 infection. A total of 395 serial samples were assessed from 208 patients without previous vaccination or documented exposure to SARS-CoV-2. Neutralization of SARS-CoV-2 was assessed using the Architect ACE2 binding inhibition assay (Abbott, Research Use only) which measures the % inhibition of interactions between antibodies and the SARS-COV-2 viral Spike Receptor Binding Domain. While 12.5% inhibition of ACE2 was considered positive >80% neutralization is the expected standard. CRP was assessed using the Architect Multigent CRP assay with 9.6mg/dL as the categorical cutoff for risk and interleukin 6 (IL-6) was assessed using the Alinity IL-6 assay (Abbott, Research Use Only) using 30 pg/mL as the threshold for risk in categorical analyses. The electronic medical record (EPIC) was interrogated for comorbidities, age, gender, 30-day mortality, 10-day intubation, and pharmacological interventions. Correlation between biomarkers was assessed using Spearman r. Kaplan-Meier curves and Cox proportional hazards models were constructed for 30-day mortality and 10-day intubation. Results 37 of 208 (18%) of patients died within thirty days of ED presentation and 59 (28%) required intubation within 10-days. There was a significant correlation between IL-6 and CRP (r=0.34) but not with ACE-2 binding (Spearman r <0.06 for both IL-6 and CRP). There was significantly higher CRP in those that died (median = 14 mg/dL, IQR = 8-21) than those that survived (5 mg/dL, 2-11). IL-6 was higher in patients that died (344 pg/mL, 0-870) than those that survived (65 pg/mL, 28-140). Median ACE-2 inhibition trended higher in those that survived (18%, 0-65%) than those that died (3%, 0-48%). Survival curves demonstrated that patients with IL-6 > 30 pg/mL, CRP > 9.6 mg/dL, and ACE2 binding <12.5% at baseline were more likely to die within 30-days. Multivariate modeling found that only IL-6 (Hazard Ratio, 1.28, 95%CI 1.08-1.52 for each doubling) and age (1.04, 1.01-1.08) were significantly associated with 30-day mortality. Conclusions Systemic inflammation measured by IL-6 and CRP upon admission is highly associated with 30-day mortality from SARS-CoV-2 infection. While ACE-2 inhibition was lower in those that died from COVID-19 at admission, it was not independently associated with mortality or correlated with inflammatory markers. This implies an importance of other aspects of the immune response for mediating inflammation and reducing risk of mortality from SARS-CoV-2.

Structure-Based De Novo Design for the Discovery of Miniprotein Inhibitors Targeting Oncogenic Mutant BRAF.

Being a component of the Ras/Raf/MEK/ERK signaling pathway crucial for cellular responses, the VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has emerged as a promising target for anticancer drug discovery due to oncogenic mutations that lead to pathway hyperactivation. Despite the discovery of several small-molecule BRAF kinase inhibitors targeting oncogenic mutants, their clinical utility has been limited by challenges such as off-target effects and suboptimal pharmacological properties. This study focuses on identifying miniprotein inhibitors for the oncogenic V600E mutant BRAF, leveraging their potential as versatile drug candidates. Using a structure-based de novo design approach based on binding affinity to V600E mutant BRAF and hydration energy, 39 candidate miniprotein inhibitors comprising three helices and 69 amino acids were generated from the substructure of the endogenous ligand protein (14-3-3). Through in vitro binding and kinase inhibition assays, two miniproteins (63 and 76) were discovered as novel inhibitors of V600E mutant BRAF with low-micromolar activity, with miniprotein 76 demonstrating a specific impediment to MEK1 phosphorylation in mammalian cells. These findings highlight miniprotein 76 as a potential lead compound for developing new cancer therapeutics, and the structural features contributing to its biochemical potency against V600E mutant BRAF are discussed in detail.

Identification of moonlighting adhesins of highly-adhesive Lactobacillus plantarum PO23 isolated from the intestine of Paralichthys olivaceus

At present, feeding probiotics has been an effective method to prevent and control enterogenous diseases of cultured fish. It is very important and urgent to study the molecular mechanism of the adhesion and colonization of probiotics to improve the use efficiency accurately. In our previous research, highly-adhesive Lactobacillus plantarum PO23 (LP-PO23) was isolated from the intestine of olive flounder (Paralichthys olivaceus). To clarify the specific adhesion of LP-PO23 to intestinal epithelial cells (IECs), six adhesins, d-lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor Tu (EF-Tu), enolase (ENO), 60 kDa chaperonin (Cpn60) and chaperone protein DnaK (DnaK) were identified as moonlighting proteins by far-Western blot and liquid chromatography-mass spectrometry in this research. The recombinant proteins rLDH, rGAPDH, rEF-Tu, rENO, rCpn60 and rDnak containing FLAG tag were expressed and employed as antigens to produce mouse polyclonal antibodies (pAbs). Using pAbs, Western blot, immunofluorescence (IF) and immunoelectron microscopy (IEM) were carried out to confirm the surface distribution of six adhesins on LP-PO23 cells respectively. Finally, the specific binding of rLDH, rGAPDH, rEF-Tu, rENO, rCpn60 and rDnak to IECs was verified by IF. In binding inhibition assay, the mouse anti-GAPDH pAb displayed the strongest inhibition activity among six pAbs. These results confirmed that six moonlighting adhesins participated in the specific adhesion of LP-PO23 to IECs and would facilitate understanding the adhesion mechanism of lactobacilli.

A universal recombinant adenovirus type 5 vector-based COVID-19 vaccine.

A universal recombinant adenovirus type-5 (Ad5) vaccine against COVID19 (Ad-US) was constructed, and immunogenicity and broad-spectrum of Ad5-US were evaluated with both intranasal and intramuscular immunization routes. The humoral immune response of Ad5-US in serum and bronchoalveolar lavage fluid were evaluated by the enzyme-linked immunosorbent assay (ELISA), recombinant vesicular stomatitis virus based pseudovirus neutralization assay, and angiotensin-converting enzyme-2 (ACE2) -binding inhibition assay. The cellular immune response and Th1/Th2 biased immune response of Ad5-US were evaluated by the IFN-γ ELISpot assay, intracellular cytokine staining, and Meso Scale Discovery (MSD) profiling of Th1/Th2 cytokines. Intramuscular priming followed by an intranasal booster with Ad5-US elicited the broad-spectrum and high levels of IgG, IgA, pseudovirus neutralizing antibody (PNAb), and Th1-skewing of the T-cell response. Overall, the adenovirus type-5 vectored universal SARS-CoV-2 vaccine Ad5-US was successfully constructed, and Ad5-US was highly immunogenic and broad spectrum. Intramuscular priming followed by an intranasal booster with Ad5-US induced the high and broad spectrum systemic immune responses and local mucosal immune responses.

Functional dissection of the spike glycoprotein S1 subunit and identification of cellular cofactors for regulation of swine acute diarrhea syndrome coronavirus entry.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel porcine enteric coronavirus, and the broad interspecies infection of SADS-CoV poses a potential threat to human health. This study provides experimental evidence to dissect the roles of distinct domains within the SADS-CoV spike S1 subunit in cellular entry. Specifically, we expressed the S1 and its subdomains, S1A and S1B. Cell binding and invasion inhibition assays revealed a preference for the S1B subdomain in binding to the receptors on the cell surface, and this unknown receptor is not utilized by the porcine epidemic diarrhea virus. Nanoparticle display demonstrated hemagglutination of erythrocytes from pigs, humans, and mice, linking the S1A subdomain to the binding of sialic acid (Sia) involved in virus attachment. We successfully rescued GFP-labeled SADS-CoV (rSADS-GFP) from a recombinant cDNA clone to track viral infection. Antisera raised against S1, S1A, or S1B contained highly potent neutralizing antibodies, with anti-S1B showing better efficiency in neutralizing rSADS-GFP infection compared to anti-S1A. Furthermore, depletion of heparan sulfate (HS) by heparinase treatment or pre-incubation of rSADS-GFP with HS or constituent monosaccharides could inhibit SADS-CoV entry. Finally, we demonstrated that active furin cleavage of S glycoprotein and the presence of type II transmembrane serine protease (TMPRSS2) are essential for SADS-CoV infection. These combined observations suggest that the wide cell tropism of SADS-CoV may be related to the distribution of Sia or HS on the cell surface, whereas the S1B contains the main protein receptor binding site. Specific host proteases also play important roles in facilitating SADS-CoV entry.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel pathogen infecting piglet, and its unique genetic evolution characteristics and broad species tropism suggest the potential for cross-species transmission. The virus enters cells through its spike (S) glycoprotein. In this study, we identify the receptor binding domain on the C-terminal part of the S1 subunit (S1B) of SADS-CoV, whereas the sugar-binding domain located at the S1 N-terminal part of S1 (S1A). Sialic acid, heparan sulfate, and specific host proteases play essential roles in viral attachment and entry. The dissection of SADS-CoV S1 subunit's functional domains and identification of cellular entry cofactors will help to explore the receptors used by SADS-CoV, which may contribute to exploring the mechanisms behind cross-species transmission and host tropism.

Biochemical and structural characterization of chlorhexidine as an ATP-assisted inhibitor against type 1 methionyl-tRNA synthetase from Gram-positive bacteria

Methionyl-tRNA synthetase (MetRS) catalyzes the attachment of l-methionine (l-Met) to tRNAMet to generate methionyl-tRNAMet, an essential substrate for protein translation within ribosome. Owing to its indispensable biological function and the structural discrepancies with human counterpart, bacterial MetRS is considered an ideal target for developing antibacterials. Herein, chlorhexidine (CHX) was identified as a potent binder of Staphylococcus aureus MetRS (SaMetRS) through an ATP-aided affinity screening. The co-crystal structure showed that CHX simultaneously occupies the enlarged l-Met pocket (EMP) and the auxiliary pocket (AP) of SaMetRS with its two chlorophenyl groups, while its central hexyl linker swings upwards to interact with some conserved hydrophobic residues. ATP adopts alternative conformations in the active site cavity, and forms ionic bonds and water-mediated hydrogen bonds with CHX. Consistent with this synergistic binding mode, ATP concentration-dependently enhanced the binding affinity of CHX to SaMetRS from 10.2 μM (no ATP) to 0.45 μM (1 mM ATP). While it selectively inhibited two representative type 1 MetRSs from S. aureus and Enterococcus faecalis, CHX did not show significant interactions with three tested type 2 MetRSs, including human cytoplasmic MetRS, in the enzyme inhibition and biophysical binding assays, probably due to the conformational differences between two types of MetRSs at their EMP and AP. Our findings on CHX may inspire the design of MetRS-directed antimicrobials in future.

Diphenyl-tetrazol-propanamide Derivatives Act as Dual-Specific Antagonists of Platelet CLEC-2 and Glycoprotein VI.

Platelet C-type lectin-like receptor 2 (CLEC-2) induces platelet activation and aggregation after clustering by its ligand podoplanin (PDPN). PDPN, which is not normally expressed in cells in contact with blood flow, is induced in inflammatory immune cells and some malignant tumor cells, thereby increasing the risk of venous thromboembolism (VTE) and tumor metastasis. Therefore, small-molecule compounds that can interfere with the PDPN-CLEC-2 axis have the potential to become selective antiplatelet agents. Using molecular docking analysis of CLEC-2 and a PDPN-CLEC-2 binding-inhibition assay, we identified a group of diphenyl-tetrazol-propanamide derivatives as novel CLEC-2 inhibitors. A total of 12 hit compounds also inhibited PDPN-induced platelet aggregation in humans and mice. Unexpectedly, these compounds also fit the collagen-binding pocket of the glycoprotein VI molecule, thereby inhibiting collagen interaction. These compounds also inhibited collagen-induced platelet aggregation, and one compound ameliorated collagen-induced thrombocytopenia in mice. For clinical use, these compounds will require a degree of chemical modification to decrease albumin binding. Nonetheless, as dual activation of platelets by collagen and PDPN-positive cells is expected to occur after the rupture of atherosclerotic plaques, these dual antagonists could represent a promising pharmacophore, particularly for arterial thrombosis, in addition to VTE and metastasis.

Venomous gland transcriptome and venom proteomic analysis of the scorpion Androctonus amoreuxi reveal new peptides with anti-SARS-CoV-2 activity

The recent COVID-19 pandemic shows the critical need for novel broad spectrum antiviral agents. Scorpion venoms are known to contain highly bioactive peptides, several of which have demonstrated strong antiviral activity against a range of viruses. We have generated the first annotated reference transcriptome for the Androctonus amoreuxi venom gland and used high performance liquid chromatography, transcriptome mining, circular dichroism and mass spectrometric analysis to purify and characterize twelve previously undescribed venom peptides. Selected peptides were tested for binding to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and inhibition of the spike RBD – human angiotensin-converting enzyme 2 (hACE2) interaction using surface plasmon resonance-based assays. Seven peptides showed dose-dependent inhibitory effects, albeit with IC50 in the high micromolar range (117–1202 µM). The most active peptide was synthesized using solid phase peptide synthesis and tested for its antiviral activity against SARS-CoV-2 (Lineage B.1.1.7). On exposure to the synthetic peptide of a human lung cell line infected with replication-competent SARS-CoV-2, we observed an IC50 of 200 nM, which was nearly 600-fold lower than that observed in the RBD – hACE2 binding inhibition assay. Our results show that scorpion venom peptides can inhibit the SARS-CoV-2 replication although unlikely through inhibition of spike RBD – hACE2 interaction as the primary mode of action. Scorpion venom peptides represent excellent scaffolds for design of novel anti-SARS-CoV-2 constrained peptides. Future studies should fully explore their antiviral mode of action as well as the structural dynamics of inhibition of target virus-host interactions.

Development and validation of a pentaplex assay for the identification of antibodies against common viral diseases in cattle.

Animal welfare and economic implications of infectious diseases in cattle demand an efficient surveillance as the foundation for control and eradication programmes. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic bovine leukosis virus (EBLV) cause common and often underdiagnosed diseases in cattle that are endemic in most countries [1]. A hallmark of individual exposure to a viral pathogen is the presence of antibodies directed towards that virus. The aim of this study was to develop and validate a pentaplex assay to simultaneously detect and quantify antibodies against BRSV, PI3V, BoHV-1, BVDV and EBLV in serum, as an efficient tool to yield epidemiological data. Monoplex assays were initially developed using either complete BRSV or BoHV-1 viral lysates, or recombinant proteins for BVDV, EBLV or PI3V as capture antigens. In addition, 125 serum samples from unvaccinated cattle, which were classified as positive or negative for each of the viruses by commercial ELISA kits, were used for validation. Conditions established for the Luminex monoplex assays were adopted for the pentaplex assay. The accuracy, determined by the area under the ROC curve, was greater than 0.97, and assay diagnostic sensitivities and specificities were over 95 and 90%, respectively, for all antigens. Intra (r) and interassay (R) coefficients of variation were under 10 and 20 %, respectively. Selectivity towards target viruses was shown by binding inhibition assays where unbound viruses reduced fluorescence intensities. Diagnostic agreement for samples analysed simultaneously in the monoplex and multiplex assays was almost perfect. In conclusion, a highly sensitive pentaplex assay was validated for the simultaneous identification of antibodies directed against BVDV, BoHV-1, PI3V, BRSV and EBLV in serum. The developed pentaplex assay complies with performance characteristics established by international guidelines for diagnostic tests and may be used as a tool for the implementation of epidemiological surveillance.

Primary Exposure to SARS-CoV-2 via Infection or Vaccination Determines Mucosal Antibody-Dependent ACE2 Binding Inhibition.

Mucosal antibodies play a critical role in preventing SARS-CoV-2 infections or reinfections by blocking the interaction of the receptor-binding domain (RBD) with the angiotensin-converting enzyme 2 (ACE2) receptor on the cell surface. In this study, we investigated the difference between the mucosal antibody response after primary infection and vaccination. We assessed longitudinal changes in the quantity and capacity of nasal antibodies to neutralize the interaction of RBD with the ACE2 receptor using the spike protein and RBD from ancestral SARS-CoV-2 (Wuhan-Hu-1), as well as the RBD from the Delta and Omicron variants. Significantly higher mucosal IgA concentrations were detected postinfection vs postvaccination, while vaccination induced higher IgG concentrations. However, ACE2-inhibiting activity did not differ between the cohorts. Regarding whether IgA or IgG drove ACE2 inhibition, infection-induced binding inhibition was driven by both isotypes, while postvaccination binding inhibition was mainly driven by IgG. Our study provides new insights into the relationship between antibody isotypes and neutralization by using a sensitive and high-throughput ACE2 binding inhibition assay. Key differences are highlighted between vaccination and infection at the mucosal level, showing that despite differences in the response quantity, postinfection and postvaccination ACE2 binding inhibition capacity did not differ.

(R)evolution of the Standard Addition Procedure for Immunoassays.

A new method to transfer the standard addition procedure for concentration determination to immunoassays with non-linear calibration curves was developed. The new method was successfully applied to simulated data and benchmarked against a state-of-the-art algorithm, showing a significantly improved performance with improvement factors between 2 and 192. The logit function was used to transform the immunoassay signal response of test samples spiked with known analyte concentrations. The relationship between logit(signal) and log-transformed estimated total analyte concentration is linear if the estimated total analyte concentration is correct. Finally, the new method was validated experimentally using different assays in varying, relevant complex matrices, such as serum, saliva, and milk. Different concentrations of testosterone and amitriptyline between 0.05 and 3.0 µg L-1 were quantified using a binding inhibition assay in combination with reflectometric interference spectroscopy (RIfS) as the transduction principle. The sample concentration was calculated using a numerical method. Samples could be quantified with recoveries between 70 and 118%. The standard addition method accounts for individual matrix interference on the immunoassay by spiking the test sample itself. Although the experiments were carried out using RIfS, the method can be applied to any immunoassay that meets the analytical requirements.

A Severe Acute Respiratory Syndrome Coronavirus 2 Anti-Spike Immunoglobulin G Assay: A Robust Method for Evaluation of Vaccine Immunogenicity Using an Established Correlate of Protection.

As the COVID-19 pandemic continues, variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge. Immunogenicity evaluation of vaccines and identification of correlates of protection for vaccine effectiveness is critical to aid the development of vaccines against emerging variants. Anti-recombinant spike (rS) protein immunoglobulin G (IgG) quantitation in the systemic circulation (serum/plasma) is shown to correlate with vaccine efficacy. Thus, an enzyme-linked immunosorbent assay (ELISA)-based binding assay to detect SARS-CoV-2 (ancestral and variant strains) anti-rS IgG in human serum samples was developed and validated. This assay successfully met acceptance criteria for inter/intra-assay precision, specificity, selectivity, linearity, lower/upper limits of quantitation, matrix effects, and assay robustness. The analyte in serum was stable for up to 8 freeze/thaw cycles and 2 years in -80 °C storage. Similar results were observed for the Beta, Delta, and Omicron BA.1/BA.5/XBB.1.5 variant-adapted assays. Anti-rS IgG assay results correlated significantly with neutralization and receptor binding inhibition assays. In addition, usage of international reference standards allows data extrapolation to WHO international units (BAU/mL), facilitating comparison of results with other IgG assays. This anti-rS IgG assay is a robust, high-throughput method to evaluate binding IgG responses to S protein in serum, enabling rapid development of effective vaccines against emerging COVID-19 variants.

The Combination of Binding Avidity of Ovomucoid-Specific IgE Antibody and Specific IgG4 Antibody Can Predict Positive Outcomes of Oral Food Challenges during Stepwise Slow Oral Immunotherapy in Children with Hen's Egg Allergy.

To increase the prediction accuracy of positive oral food challenge (OFC) outcomes during stepwise slow oral immunotherapy (SS-OIT) in children with a hen's egg (HE) allergy, we evaluated the predictive value of the combination of antigen-specific IgE (sIgE) with antigen binding avidity and sIgG4 values. Sixty-three children with HE allergy undergoing SS-OIT were subjected to repeated OFCs with HE. We measured the ovomucoid (OVM)-sIgE by ImmunoCAP or densely carboxylated protein (DCP) microarray, sIgG4 by DCP microarray, and the binding avidity of OVM-sIgE defined as the level of 1/IC50 (nM) measured by competitive binding inhibition assays. The OFC was positive in 37 (59%) patients undergoing SS-OIT. Significant differences in DCP-OVM-sIgE, CAP-OVM-sIgE, I/IC50, DCP-OVM-sIgG4, the multiplication products of DCP-OVM-sIgE, and the binding avidity of DCP-OVM-sIgE (DCP-OVM-sIgE/IC50) and DCP-OVM-sIgE/sIgG4 were compared between the negative and positive groups (p < 0.01). Among them, the variable with the greatest area under the receiver operating characteristic curve was DCP-OVM-sIgE/IC50 (0.84), followed by DCP-OVM-sIgE/sIgG4 (0.81). DCP-OVM-sIgE/IC50 and DCP-OVM-sIgE/sIgG4 are potentially useful markers for the prediction of positive OFCs during HE-SS-OIT and may allow proper evaluation of the current allergic status in the healing process during HE-SS-OIT.

Isolation of Anti-SARS-CoV-2 Natural Products Extracted from Mentha canadensis and the Semi-synthesis of Antiviral Derivatives.

Traditional herbal medicine offers opportunities to discover novel therapeutics against SARS-CoV-2 mutation. The dried aerial part of mint (Mentha canadensis L.) was chosen for bioactivity-guided extraction. Seven constituents were isolated and characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Syringic acid and methyl rosmarinate were evaluated in drug combination treatment. Ten amide derivatives of methyl rosmarinate were synthesized, and the dodecyl (13) and 3-ethylphenyl (19) derivatives demonstrated significant improvement in the anti-SARS-CoV-2 plaque reduction assay, achieving IC50 of 0.77 and 2.70 μM, respectively, against Omicron BA.1 as compared to methyl rosmarinate's IC50 of 57.0 μM. Spike protein binding and 3CLpro inhibition assays were performed to explore the viral inhibition mechanism. Molecular docking of compounds 13 and 19 to 3CLpro was performed to reveal potential interaction. In summary, natural products with anti-Omicron BA.1 activity were isolated from Mentha canadensis and derivatives of methyl rosmarinate were synthesized, showing 21- to 74-fold improvement in antiviral activity against Omicron BA.1.

Buffalo sperm membrane glycan-binding proteins reveal precise and preferential binding signatures with specific glycans targets on oviduct epithelium and zona pellucida-an implication in fertilization

Sperm membrane glycan-binding proteins (lectins) interact with the counterpart glycans in the oviduct, oocytes, and vice-versa. It has already been well known that specific glycans are present on oviductal epithelium and zona pellucida (ZP) in different mammalian species. Some of these glycans are necessary for oviductal sperm reservoir formation and gamete recognition. The specific binding phenomenon of lectin-glycans is one of the vital factors for successful fertilization in mammals. We hypothesized that buffalo sperm membrane glycan-binding proteins have specific glycan targets in the oviduct and ZP supporting the fertilization event. In the present investigation, sperm membrane proteins were extracted and assessed for their binding capacity with glycans using a high-throughput glycan microarray. The most promising glycan binding signals were evaluated to confirm the sperm putative receptors for glycan targets in the oviductal epithelial cells (OEC) and on ZP using an in-vitro competitive binding inhibition assay. Based on an array of 100 glycans, we found that N-acetyllactosamine (LacNAc), Lewis-a trisaccharide, 3′-sialyllactosamine and LacdiNAc were the most promising glycans and selected for further in-vitro validation. We established an inhibitory concentration of 12 mM Lewis-a trisaccharide and 10 μg/ml Lotus tetragonolobus (LTL) lectin for the sperm-OEC binding interaction, indicating its specificity and sensitivity. We observed that 3 mM 3′-sialyllactosamine, and LacdiNAc were the most competitive inhibitory concentration in sperm-ZP binding, suggesting a specific and abundance-dependent binding affinity. The competitive binding affinity of Maackia amurensis (MAA) lectin with Neu5Ac(α2-3)Gal(β1-4)GlcNAc further supports the abundance of 3′-sialyllactosamine on ZP responsible for sperm binding. Our findings develop the strong evidence on buffalo sperm putative receptors underlying their locking specificities with Lewis-a trisaccharide in oviduct and 3′-sialyllactosamine on ZP. The functional interaction of buffalo sperm lectins with the target glycans in OEC and ZP appears to be accomplished in an abundance-dependent manner, facilitating the fertilization event in buffaloes.

Human salivary protein-derived peptides specific-salivary SIgA antibodies enhanced by nasal double DNA adjuvant in mice play an essential role in preventing Porphyromonas gingivalis colonization: an in-vitro study

BackgroundWe previously showed that fimbriae-bore from Poryphyromonas gingivalis (Pg), one of the putative periodontopathogenic bacteria specifically bound to a peptide domain (stat23, prp21) shared on statherin or acidic proline-rich protein 1 (PRP1) molecule of human salivary proteins (HSPs). Here, we investigated whether the nasal administration of DNA plasmid expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotide 1826 as double DNA adjuvant (dDA) with stat23 and prpr21 induces antigen (Ag)-specific salivary secretory IgA (SIgA) antibodies (Abs) in mice. Further, we examined that stat23- and prpr21-specific salivary SIgA Abs induced by dDA have an impact on Pg-binding to human whole saliva-coated hydroxyapatite beads (wsHAPs).Material and methodsC57BL/6N mice were nasally immunized with dDA plus sta23 or/and prp21 peptide as Ag four times at weekly intervals. Saliva was collected one week after the final immunization and was subjected to Ag-specific ELISA. To examine the functional applicability of Ag-specific SIgA Abs, SIgA-enriched saliva samples were subjected to Pg binding inhibition assay to wsHAPs.ResultsSignificantly elevated levels of salivary SIgA Ab to stat23 or prp21 were seen in mice given nasal stat23 or prp21 with dDA compared to those in mice given Ag alone. Of interest, mice nasally given the mixture of stat23 and prp21 as double Ags plus dDA, resulted in both stat23- and prp21-specific salivary SIgA Ab responses, which are mediated through significantly increased numbers of CD11c+ dendritic cell populations and markedly elevated Th1 and Th2 cytokines production by CD4+ T cells in the mucosal inductive and effector tissues. The SIgA Ab-enriched saliva showed significantly reduced numbers of live Pg cells binding to wsHAPs as compared with those in mice given double Ags without dDA or naïve mice. Additionally, saliva from IgA-deficient mice given nasal double Ags plus dDA indicated no decrease of live Pg binding to wsHAPs.ConclusionThese findings show that HSP-derived peptides-specific salivary SIgA Abs induced by nasal administration of stat23 and prp21 peptides plus dDA, play an essential role in preventing Pg attachment and colonization on the surface of teeth, suggesting a potency that the SIgA may interrupt and mask fimbriae-binding domains in HSPs on the teeth.

Severe Acute Respiratory Syndrome Coronavirus 2 Receptor (Human Angiotensin-Converting Enzyme 2) Binding Inhibition Assay: A Rapid, High-Throughput Assay Useful for Vaccine Immunogenicity Evaluation

Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) show immune evasion of vaccine-derived immunity, highlighting the need for better clinical immunogenicity biomarkers. To address this need, an enzyme-linked immunosorbent assay-based, human angiotensin-converting enzyme 2 (hACE2) binding inhibition assay was developed to measure antibodies against the ancestral strain of SARS-CoV-2 and was validated for precision, specificity, linearity, and other parameters. This assay measures the inhibition of SARS-CoV-2 spike (S) protein binding to the receptor, hACE2, by serum from vaccine clinical trials. Inter- and intra-assay precision, specificity, linearity, lower limit of quantitation, and assay robustness parameters successfully met the acceptance criteria. Heme and lipid matrix effects showed minimal interference on the assay. Samples were stable for testing in the assay even with 8 freeze/thaws and up to 24 months in -80 °C storage. The assay was also adapted for variants (Delta and Omicron BA.1/BA.5), with similar validation results. The hACE2 assay showed significant correlation with anti-recombinant S immunoglobulin G levels and neutralizing antibody titers. This assay provides a rapid, high-throughput option to evaluate vaccine immunogenicity. Along with other clinical biomarkers, it can provide valuable insights into immune evasion and correlates of protection and enable vaccine development against emerging COVID-19 variants.

Age-adjusted impact of prior COVID-19 on SARS-CoV-2 mRNA vaccine response.

More people with a history of prior infection are receiving SARS-CoV-2 vaccines. Understanding the level of protection granted by 'hybrid immunity', the combined response of infection- and vaccine-induced immunity, may impact vaccination strategies through tailored dosing. A total of 36 infected ('prior infection') and 33 SARS-CoV-2 'naïve' individuals participated. Participants provided sera six months after completing a round of BNT162b2 vaccination, to be processed for anti-spike antibody measurements and the receptor binding domain-ACE2 binding inhibition assays. The relationships between antibody titer, groups and age were explored. Anti-spike antibody titers at 6 months post-vaccination were significantly higher, reaching 13- to 17-fold, in the 'prior infection' group. Semi-log regression models showed that participants with 'prior infection' demonstrated higher antibody titer compared with the 'naïve' even after adjusting for age. The enhancement in antibody titer attributable to positive infection history increased from 8.9- to 9.4- fold at age 30 to 19- to 32-fold at age 60. Sera from the 'prior infection' group showed higher inhibition capacity against all six analyzed strains, including the Omicron variant. Prior COVID-19 led to establishing enhanced humoral immunity at 6 months after vaccination. Antibody fold-difference attributed to positive COVID-19 history increased with age, possibly because older individuals are prone to symptomatic infection accompanied by potentiated immune responses. While still pending any modifications of dosing recommendations (i.e. reduced doses for individuals with prior infection), our observation adds to the series of real-world data demonstrating the enhanced and more durable immune response evoked by booster vaccinations following prior infection.

SARS-CoV-2 Omicron escapes mRNA vaccine booster-induced antibody neutralisation in patients with autoimmune rheumatic diseases: an observational cohort study

ObjectivesThis study investigates whether COVID-19 vaccines can elicit cross-reactive antibody responses against the Omicron variant in patients with autoimmune rheumatic diseases (ARDs).MethodsThis observational cohort study comprised 149 patients with ARDs...

Protein-protein conjugation enhances the immunogenicity of SARS-CoV-2 receptor-binding domain (RBD) vaccines

SummarySeveral effective SARS-CoV-2 vaccines have been developed using different technologies. Although these vaccines target the isolates collected early in the pandemic, many have protected against serious illness from newer variants. Nevertheless, efficacy has diminished against successive variants and the need for effective and affordable vaccines persists especially in the developing world. Here, we adapted our protein-protein conjugate vaccine technology to generate a vaccine based on receptor-binding domain (RBD) antigen. RBD was conjugated to a carrier protein, EcoCRM®, to generate two types of conjugates: crosslinked and radial conjugates. In the crosslinked conjugate, antigen and carrier are chemically crosslinked; in the radial conjugate, the antigen is conjugated to the carrier by site-specific conjugation. With AS01 adjuvant, both conjugates showed enhanced immunogenicity in mice compared to RBD, with a Th1 bias. In hACE2 binding inhibition and pseudovirus neutralization assays, sera from mice vaccinated with the radial conjugate demonstrated strong functional activity.