Antibody Binding Sites Research Articles

Antibody landscape of C57BL/6 mice cured of B78 melanoma via a combined radiation and immunocytokine immunotherapy regimen.

Sera of immune mice that were previously cured of their melanoma through a combined radiation and immunocytokine immunotherapy regimen consisting of 12 Gy of external beam radiation and the intratumoral administration of an immunocytokine (anti-GD2 mAb coupled to IL-2) with long-term immunological memory showed strong antibody-binding against melanoma tumor cell lines via flow cytometric analysis. Using a high-density whole-proteome peptide array (of 6.090.593 unique peptides), we assessed potential protein-targets for antibodies found in immune sera. Sera from 6 of these cured mice were analyzed with this high-density, whole-proteome peptide array to determine specific antibody-binding sites and their linear peptide sequence. We identified thousands of peptides that were targeted by these 6 mice and exhibited strong antibody binding only by immune (after successful cure and rechallenge), not naïve (before tumor implantation) sera and developed a robust method to detect these differentially targeted peptides. Confirmatory studies were done to validate these results using 2 separate systems, a peptide ELISA and a smaller scale peptide array utilizing a slightly different technology. To the best of our knowledge, this is the first study of the full set of germline encoded linear peptide-based proteome epitopes that are recognized by immune sera from mice cured of cancer via radio-immunotherapy. We furthermore found that although the generation of B-cell repertoire in immune development is vastly variable, and numerous epitopes are identified uniquely by immune serum from each of these 6 immune mice evaluated, there are still several epitopes and proteins that are commonly recognized by at least half of the mice studied. This suggests that every mouse has a unique set of antibodies produced in response to the curative therapy, creating an individual "fingerprint." Additionally, certain epitopes and proteins stand out as more immunogenic, as they are recognized by multiple mice in the immune group.

Structural Insights of the SARS-CoV-2 Nucleocapsid Protein: Implications for the Inner-workings of Rapid Antigen Tests.

The nucleocapsid (N) protein is an abundant component of SARS-CoV-2 and a key analyte for lateral-flow rapid antigen tests. Here, we present new structural insights for the SARS-CoV-2 N protein using cryo-electron microscopy (EM) and molecular modeling tools. Epitope mapping based on structural data supported host-immune interactions in the C-terminal portion of the protein, while other regions revealed protein-protein interaction sites. Complementary modeling results suggested that N protein structures from known variants of concern (VOC) are nearly 100% conserved at specific antibody-binding sites. Collectively, these results suggest that rapid tests that target the nucleocapsid C-terminal domain should have similar accuracy across all VOCs. In addition, our combined structural modeling workflow may guide the design of immune therapies to counter viral processes as we plan for future variants and pandemics.

Imprinted SARS-CoV-2 humoral immunity induces convergent Omicron RBD evolution

Continuous evolution of Omicron has led to a rapid and simultaneous emergence of numerous variants that display growth advantages over BA.5 (ref. 1). Despite their divergent evolutionary courses, mutations on their receptor-binding domain (RBD) converge on several hotspots. The driving force and destination of such sudden convergent evolution and its effect on humoral immunity remain unclear. Here we demonstrate that these convergent mutations can cause evasion of neutralizing antibody drugs and convalescent plasma, including those from BA.5 breakthrough infection, while maintaining sufficient ACE2-binding capability. BQ.1.1.10 (BQ.1.1 + Y144del), BA.4.6.3, XBB and CH.1.1 are the most antibody-evasive strains tested. To delineate the origin of the convergent evolution, we determined the escape mutation profiles and neutralization activity of monoclonal antibodies isolated from individuals who had BA.2 and BA.5 breakthrough infections2,3. Owing to humoral immune imprinting, BA.2 and especially BA.5 breakthrough infection reduced the diversity of the neutralizing antibody binding sites and increased proportions of non-neutralizing antibody clones, which, in turn, focused humoral immune pressure and promoted convergent evolution in the RBD. Moreover, we show that the convergent RBD mutations could be accurately inferred by deep mutational scanning profiles4,5, and the evolution trends of BA.2.75 and BA.5 subvariants could be well foreseen through constructed convergent pseudovirus mutants. These results suggest that current herd immunity and BA.5 vaccine boosters may not efficiently prevent the infection of Omicron convergent variants.

Development of an anti-CAR antibody response in SIV-infected rhesus macaques treated with CD4-MBL CAR/CXCR5 T cells.

T cells expressing a simian immunodeficiency (SIV)-specific chimeric antigen receptor (CAR) and the follicular homing molecule, CXCR5, were infused into antiretroviral therapy (ART) suppressed, SIV-infected rhesus macaques to assess their ability to localize to the lymphoid follicle and control the virus upon ART interruption. While the cells showed evidence of functionality, they failed to persist in the animals beyond 28 days. Development of anti-CAR antibodies could be responsible for the lack of persistence. Potential antigenic sites on the anti-SIV CAR used in these studies included domains 1 and 2 of CD4, the carbohydrate recognition domain (CRD) of mannose-binding lectin (MBL), and an extracellular domain of the costimulatory molecule, CD28, along with short linker sequences. Using a flow cytometry based assay and target cells expressing the CAR/CXCR5 construct, we examined the serum of the CD4-MBL CAR/CXCR5-T cell treated animals to determine that the animals had developed an anti-CAR antibody response after infusion. Binding sites for the anti-CAR antibodies were identified by using alternative CARs transduced into target cells and by preincubation of the target cells with a CD4 blocking antibody. All of the treated animals developed antibodies in their serum that bound to CD4-MBL CAR/CXCR5 T cells and the majority were capable of inducing an ADCC response. The CD4 antibody-blocking assay suggests that the dominant immunogenic components of this CAR are the CD4 domains with a possible additional site of the CD28 domain with its linker. This study shows that an anti-drug antibody (ADA) response can occur even when using self-proteins, likely due to novel epitopes created by abridged self-proteins and/or the self-domain of the CAR connection to a small non-self linker. While in our study, there was no statistically significant correlation between the ADA response and the persistence of the CD4-MBL CAR/CXCR5-T cells in rhesus macaques, these findings suggest that the development of an ADA response could impact the long-term persistence of self-based CAR immunotherapies.

Characterization of a Novel CD4 Mimetic Compound YIR-821 against HIV-1 Clinical Isolates.

Small CD4-mimetic compound (CD4mc), which inhibits the interaction between gp120 with CD4, acts as an entry inhibitor and induces structural changes in the HIV-1 envelope glycoprotein trimer (Env) through its insertion within the Phe43 cavity of gp120. We recently developed YIR-821, a novel CD4mc, that has potent antiviral activity and lower toxicity than the prototype NBD-556. To assess the possibility of clinical application of YIR-821, we tested its antiviral activity using a panel of HIV-1 pseudoviruses from different subtypes. YIR-821 displayed entry inhibitor activity against 53.5% (21/40) of the pseudoviruses tested and enhanced neutralization mediated by coreceptor binding site (CoRBS) antibodies in 50% (16/32) of these. Furthermore, when we assessed the antiviral effects using a panel of pseudoviruses and autologous plasma IgG, enhancement of antibody-mediated neutralization activity was observed for 48% (15/31) of subtype B strains and 51% (28/55) of non-B strains. The direct antiviral activity of YIR-821 as an entry inhibitor was observed in 53% of both subtype B (27/51) and non-B subtype (40/75) pseudoviruses. Enhancement of antibody-dependent cellular cytotoxicity was also observed with YIR-821 for all six selected clinical isolates, as well as for the transmitted/founder (T/F) CH58 virus-infected cells. The sequence diversity in the CD4 binding site as well as other regions, such as the gp120 inner domain layers or gp41, may be involved in the multiple mechanisms related to the sensitive/resistant phenotype of the virus to YIR-821. Our findings may facilitate the clinical application of YIR-821. IMPORTANCE Small CD4-mimetic compound (CD4mc) interacts with the Phe43 cavity and triggers conformational changes, enhancing antibody-mediated neutralization and antibody-dependent cellular cytotoxicity (ADCC). Here, we evaluated the effect of YIR-821, a novel CD4mc, against clinical isolates, including both subtype B and non-B subtype viruses. Our results confirm the desirable properties of YIR-821, which include entry inhibition, enhancement of IgG-neutralization, binding, and ADCC, in addition to low toxicity and long half-life in a rhesus macaque model, that might facilitate the clinical application of this novel CD4mc. Our observation of primary viruses that are resistant to YIR-821 suggests that further development of CD4mcs with different structural properties is required.

Turning antibodies off and on again using a covalently tethered blocking peptide

In their natural form, antibodies are always in an "on-state" and are capable of binding to their targets. This leads to undesirable interactions in a wide range of therapeutic, analytical, and synthetic applications. Modulating binding kinetics of antibodies to turn them from an "off-state" to an "on-state" with temporal and spatial control can address this. Here we demonstrate a method to modulate binding activity of antibodies in a predictable and reproducible way. We designed a blocking construct that uses both covalent and non-covalent interactions with the antibody. The construct consisted of a Protein L protein attached to a flexible linker ending in a blocking-peptide designed to interact with the antibody binding site. A mutant Protein L was developed to enable photo-triggered covalent crosslinking to the antibody at a specific location. The covalent bond anchored the linker and blocking peptide to the antibodylight chain keeping the blocking peptide close to the antibody binding site. This effectively put the antibody into an "off-state". We demonstrate that protease-cleavable and photocleavable moieties in the tether enable controlled antibody activation to the "on-state" for anti-FLAG and cetuximab antibodies. Protein L can bind a range of antibodies used therapeutically and in research for wide applicability.

A group of infection-enhancing and focus size-reducing monoclonal antibodies recognized an ‘a and c’ strands epitope in the pr domain of Dengue Virus prM

Partial cleavage of a dengue virus envelope protein, prM, by furin results in a mixture of extracellular particles with variable levels of maturation and infectivity. Partially mature particles can infect leukocytes via interaction between the prM-anti-prM antibody complex with Fcγ receptors. Known prM epitopes involved in antibody-mediated infection are localized to the pr domain. In this study, a group of murine anti-prM monoclonal antibodies with strong infection-enhancing activity was found to reduce the focus size of subsets of multiple dengue serotypes that they could enhance. By employing sets of overlapping peptides, four antibodies recognizing 2-mercaptoethanol-insensitive epitopes were mapped to a common tetrapeptide located distantly in the b-c loop and furin binding site. Substitution mutations of each, or both, of the tetrapeptides in virus-like particles, however, failed to reduce binding. Further mapping experiments were performed using immature virus-like particles with abolished furin binding site to minimize the differential influence of various pr substitutions on pr-M cleavage. Reduction of antibody binding was detected when single alanine substitutions were introduced into the ‘a’ strand and ‘c' strand of pr domain. These findings suggest that the pr ‘a and c' strands region is the major binding site of these unusual focus size-reducing anti-prM antibodies.

Longitudinal Characteristics of Anti-Platelet Factor 4 Immunoglobulin G in Vaccine-Induced Immune Thrombotic Thrombocytopenia

Introduction: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenoviral vector-based vaccines against the SARS-CoV-2 virus. VITT typically presents as moderate to severe thrombocytopenia often accompanied by arterial and/or venous thrombi occurring in unusual locations. The thrombocytopenia and thrombosis seen in VITT are triggered by antibodies against platelet factor 4 (PF4, CXCL4), leading to the activation of platelets and inducing a hypercoagulable state, similar to what is observed in heparin-induced thrombocytopenia (HIT). In a previous study, we identified a unique VITT antibody binding site. involving amino acids located at the heparin binding region of PF4. In this study, we describe the change in VITT anti-PF4 IgG binding regions and binding response, as well as the ability of longitudinal patient samples to activate platelets at initial presentation and up to 109 days after vaccination with Vaxzevria AZD1222 (AstraZeneca ChAdOx1 nCoV-19). Methods: We longitudinally studied sera from VITT patients (n = 7) sent for confirmatory testing to the McMaster Platelet Immunology Laboratory. These VITT patient samples were received at time of diagnosis and follow up samples up to 109 days after vaccination (range 8 - 109 days). Confirmed VITT patients presented with clinical features including thrombocytopenia (platelet count < 150 x 109/L) with thrombotic manifestations and tested positive in both the anti-PF4 IgG/A/M immunoassay (positive optical density (OD) > 0.4) and in the PF4-serotonin release assay (PF4-SRA, positive release > 20%). Alanine scanning mutagenesis was used to identify the changes in antibody binding sites on PF4 from VITT patients at diagnosis and one follow-up time point. We also determined the change in binding responses and dissociation rates at time of diagnosis and follow-up anti-PF4 antibodies using biolayer interferometry (BLI). Results: Anti-PF4 immunoglobulin G antibody levels are positive and high at diagnosis (OD median 2.35, range 1.895 - 3.347) and decreased but remain relatively high and positive (OD >1.0, median 2.09, range 1.178 - 2.631) in all 7 VITT patients, longitudinally. In addition, most follow-up samples were weaker at activating platelets with or without exogenous PF4 compared to their initial sample. However, follow-up sera beyond 45 days still strongly activated platelets with PF4 in 2/7 (28.5%) VITT patients in the PF4-SRA. In 4/7 (57.1%) VITT patients, the number of amino acids on PF4 that continued to be important for VITT antibody binding decreased from 7 on average at time of diagnosis to 2 amino acids in the defined VITT binding site at follow-up. Binding responses to PF4 and PF4/heparin measured by BLI also decreased from initial diagnosis (PF4, median 1.872 nm, range 0.587 - 2.443 nm; PF4/heparin, median 1.362 nm, range 0.450 - 2.072 nm) when compared to follow-up (PF4, median 0.356 nm, range 0.052 - 1.770 nm; PF4/heparin, median 0.159 nm, range 0.006 - 1.206 nm) in all 7 VITT patient samples. Binding responses are a measure of abundance of antigen-specific antibodies present in a given sample. Lastly, we also measured dissociation rates of the antibodies with BLI, which reflects the avidity of an antibody for a given antigen. In similar trend as the binding responses, the dissociation rates of the anti-PF4 and anti-PF4/heparin antibodies decreased in 4/7 (57.1%) VITT patients at follow-up when compared to initial diagnosis. Conclusion: Despite persistent anti-PF4 antibody levels in VITT patients over time, there is reduced platelet activation possibly due to the loss of antibodies to the VITT binding region necessary for platelet activation, or due to a decrease in binding response and avidity. This study suggests that VITT is similar to HIT because over time, there is a decline in platelet activating anti-PF4 antibodies. Clinical implications of persistent anti-PF4 antibodies in VITT require further study.

Paragraph-antibody paratope prediction using graph neural networks with minimal feature vectors.

The development of new vaccines and antibody therapeutics typically takes several years and requires over $1bn in investment. Accurate knowledge of the paratope (antibody binding site) can speed up and reduce the cost of this process by improving our understanding of antibody-antigen binding. We present Paragraph, a structure-based paratope prediction tool that outperforms current state-of-the-art tools using simpler feature vectors and no antigen information. Source code is freely available at www.github.com/oxpig/Paragraph. Supplementary data are available at Bioinformatics online.

PH-Regulated Strategy and Mechanism of Antibody Orientation on Magnetic Beads for Improving Capture Performance of Staphylococcus Species.

Immunomagnetic beads (IMBs) have been widely used to capture and isolate target pathogens from complex food samples. The orientation of the antibody immobilized on the surface of magnetic beads (MBs) is closely related to the effective recognition with an antigen. We put forward an available strategy to orient the antibody on the surface of MBs by changing the charged amino group ratio of the reactive amino groups at optimal pH value. Quantum dots labeling antigen assay, antigen-binding fragment (Fab) accessibility assay and lysine mimicking were used for the first time to skillfully illustrate the antibody orientation mechanism. This revealed that the positively charged ε-NH2 group of lysine on the Fc relative to the uncharged amino terminus on Fab was preferentially adsorbed on the surface of MBs with a negatively charged group at pH 8.0, resulting in antigen binding sites of antibody fully exposed. This study contributes to the understanding of the antibody orientation on the surface of MBs and the potential application of IMBs in the separation and detection of pathogenic bacteria in food samples.

In Silico Genome Analysis Reveals the Evolution and Potential Impact of SARS-CoV-2 Omicron Structural Changes on Host Immune Evasion and Antiviral Therapeutics.

New variants of SARS-CoV-2 continue to evolve. The novel SARS-CoV-2 variant of concern (VOC) B.1.1.529 (Omicron) was particularly menacing due to the presence of numerous consequential mutations. In this study, we reviewed about 12 million SARS-CoV-2 genomic and associated metadata using extensive bioinformatic approaches to understand how evolutionary and mutational changes affect Omicron variant properties. Subsampled global data based analysis of molecular clock in the phylogenetic tree showed 29.56 substitutions per year as the evolutionary rate of five VOCs. We observed extensive mutational changes in the spike structural protein of the Omicron variant. A total of 20% of 7230 amino acid and structural changes exclusive to Omicron's spike protein were detected in the receptor binding domain (RBD), suggesting differential selection pressures exerted during evolution. Analyzing key drug targets revealed mutation-derived differential binding affinities between Delta and Omicron variants. Nine single-RBD substitutions were detected within the binding site of approved therapeutic monoclonal antibodies. T-cell epitope prediction revealed eight immunologically important functional hotspots in three conserved non-structural proteins. A universal vaccine based on these regions may likely protect against all these SARS-CoV-2 variants. We observed key structural changes in the spike protein, which decreased binding affinities, indicating that these changes may help the virus escape host cellular immunity. These findings emphasize the need for continuous genomic surveillance of SARS-CoV-2 to better understand how novel mutations may impact viral spread and disease outcome.

Detailed Molecular Interactions between Respiratory Syncytial Virus Fusion Protein and the TLR4/MD-2 Complex In Silico.

Molecular interactions between respiratory syncytial virus (RSV) fusion protein (F protein) and the cellular receptor Toll-like receptor 4 (TLR4) and myeloid differentiation factor-2 (MD-2) protein complex are unknown. Thus, to reveal the detailed molecular interactions between them, in silico analyses were performed using various bioinformatics techniques. The present simulation data showed that the neutralizing antibody (NT-Ab) binding sites in both prefusion and postfusion proteins at sites II and IV were involved in the interactions between them and the TLR4 molecule. Moreover, the binding affinity between postfusion proteins and the TLR4/MD-2 complex was higher than that between prefusion proteins and the TLR4/MD-2 complex. This increased binding affinity due to conformational changes in the F protein may be able to form syncytium in RSV-infected cells. These results may contribute to better understand the infectivity and pathogenicity (syncytium formation) of RSV.

Solving the MCM paradox by visualizing the scaffold of CMG helicase at active replisomes

Genome duplication is safeguarded by constantly adjusting the activity of the replicative CMG (CDC45-MCM2-7-GINS) helicase. However, minichromosome maintenance proteins (MCMs)—the structural core of the CMG helicase—have never been visualized at sites of DNA synthesis inside a cell (the so-called MCM paradox). Here, we solve this conundrum by showing that anti-MCM antibodies primarily detect inactive MCMs. Upon conversion of inactive MCMs to CMGs, factors that are required for replisome activity bind to the MCM scaffold and block MCM antibody binding sites. Tagging of endogenous MCMs by CRISPR-Cas9 bypasses this steric hindrance and enables MCM visualization at active replisomes. Thus, by defining conditions for detecting the structural core of the replicative CMG helicase, our results explain the MCM paradox, provide visual proof that MCMs are an integral part of active replisomes in vivo, and enable the investigation of replication dynamics in living cells exposed to a constantly changing environment.

AML-327 Novel Anti–B7-H3 Blocking Antibody Enhances NK Cell–Mediated Cytotoxicity and Improves Outcomes in AML-Bearing Mice

B7-H3 is an immune checkpoint molecule that is overexpressed in various human malignancies. Several monoclonal antibodies (mAbs) targeting B7-H3 have shown promising results against solid tumors. However, B7-H3's role in AML remains unexplored. i) To analyze the prognostic significance of B7-H3 in AML. ii) To investigate the immunomodulatory role of B7-H3 in AML in vitro and in vivo. We analyzed B7-H3 expression in 100 AML patients and 20 healthy donors by flow cytometry and tested for associations with clinical features. To investigate the role of B7-H3 in immunomodulation, B7-H3 was knocked down in AML cell lines using shRNA or using anti-B7-H3 blocking mAbs (T1-A5, HEK1B3A3, and 58B1A2). A human-mouse chimeric (chT-1A5) antibody was generated, and its binding site on B7-H3 protein was characterized by epitope mapping. Finally, we treated mice bearing AML cells with anti-B7-H3 antibodies and measured human AML engraftment with flow cytometry and bioluminescence imaging. Expression of B7-H3 was significantly higher in AML patients than in healthy donors (p<0.01) and in CD34+ AML cells than in CD34- cells (p<0.01). Clinically, we observed that high B7-H3 expression was associated with a poor prognosis. Furthermore, we observed that inhibition of B7-H3 expression or blocking of its activity in AML cells using a novel monoclonal antibody (T-1A5) significantly enhanced natural killer (NK) cell-mediated cytotoxicity in vitro and in vivo. Moreover, we observed that the human-mouse chimera of this antibody (ChT-1A5) induced dose-dependent cell-mediated cytotoxicity (ADCC) in B7-H3+ primary AML cells but not in normal hematopoietic cells, suggesting the specify of this antibody for AML cells. Epitope mapping using peptides derived from the B7-H3 protein identified the FG loop region as the binding site for the T1-A5 antibody. Finally, treatment with T-1A5 or chT-1A5 in combination with human NK cells significantly reduced leukemia burden and extended survival in AML xenograft and patient-derived xenograft models. Our data indicate that B7-H3 is overexpressed in AML and that an anti-B7-H3 antibody (T1-A5) not only blocks B7-H3's immunomodulatory function but also induces ADCC in AML cells in vitro and in vivo.

Ascorbylation of a Reactive Cysteine in the Major Apple Allergen Mal d 1.

The protein Mal d 1 is responsible for most allergic reactions to apples (Malus domestica) in the northern hemisphere. Mal d 1 contains a cysteine residue on its surface, with its reactive side chain thiol exposed to the surrounding food matrix. We show that, in vitro, this cysteine residue is prone to spontaneous chemical modification by ascorbic acid (vitamin C). Using NMR spectroscopy and mass spectrometry, we characterize the chemical structure of the cysteine adduct and provide a three-dimensional structural model of the modified apple allergen. The S-ascorbylated cysteine partially masks a major IgE antibody binding site on the surface of Mal d 1, which attenuates IgE binding in sera of apple-allergic patients. Our results illustrate, from a structural perspective, the role that chemical modifications of allergens with components of the natural food matrix can play.

SEMA: Antigen B-cell conformational epitope prediction using deep transfer learning.

One of the primary tasks in vaccine design and development of immunotherapeutic drugs is to predict conformational B-cell epitopes corresponding to primary antibody binding sites within the antigen tertiary structure. To date, multiple approaches have been developed to address this issue. However, for a wide range of antigens their accuracy is limited. In this paper, we applied the transfer learning approach using pretrained deep learning models to develop a model that predicts conformational B-cell epitopes based on the primary antigen sequence and tertiary structure. A pretrained protein language model, ESM-1v, and an inverse folding model, ESM-IF1, were fine-tuned to quantitatively predict antibody-antigen interaction features and distinguish between epitope and non-epitope residues. The resulting model called SEMA demonstrated the best performance on an independent test set with ROC AUC of 0.76 compared to peer-reviewed tools. We show that SEMA can quantitatively rank the immunodominant regions within the SARS-CoV-2 RBD domain. SEMA is available at https://github.com/AIRI-Institute/SEMAi and the web-interface http://sema.airi.net.

Omicron variant in COVID-19 current pandemic: a reason for apprehension.

COVID-19 emerged in Wuhan, China, but was caused by the original coronavirus, severe acute respiratory syndrome associated coronavirus-2 (SARS-CoV2). In early 2020, there was a widespread breakout of cases well over world, resulting in an epidemic that rapidly escalated to become a pandemic. This abruptly shook the global healthcare system. The emergence of the alpha, beta, and delta SARS-CoV-2 were associated with new waves of infections, sometimes across the entire world but until this month i.e.,between Nov-Dec, 2021, Delta variant reigned supreme until the emergence of a newer variant i.e., Omicron(B.1.1.529) of SARS-CoV-2. Delta had 13 mutations. Of these, nine are in the spike protein, the protrusion on thesurface ofthe virus that helps it latch onto human cells.Specifically, two are in a molecular hook, called the "receptor-bindingdomain". Omicron, a creation caused bymonstrous mutations. At least 32 mutations are in the spike protein and 10 in the receptor-binding domain. was designated a COVID-19 variant of concern (VoC) by the WorldHealth Organization (WHO) on 26th November 2021. Structurally, the omicron variant has shown too mutated at antibody binding sites which would leverage them for escaping the possible immune response by the body. We don't yet know much about the other alterations and how they might affect the virus's behavior. Omicron COVID-19 strain after identifying individuals with symptoms that were not the same as those seen in the Delta form. People with night sweats have also been reported. The new omicron variant has more mutations than the prevailing rampant delta virus. This makes the newer variant more transmissible, better able to evade itself from various vaccines readily available in the current scenario. These overall increases in the percentage changes in a single day cases of COVID-19 reported cases can be attributed to the beginning of third wave or can be speculated as newer surge of omicron variantcases. Yet another new variant has been detected in Francewith 46 mutations and 37 deletions in its genetic code, many affecting the spike protein. 'B.1.640.2' is the current nomenclature for this variation.

Modeling of pneumococcal serogroup 10 capsular polysaccharide molecular conformations provides insight into epitopes and observed cross-reactivity.

Streptococcus pneumoniae is an encapsulated gram-negative bacterium and a significant human pathogen. The capsular polysaccharide (CPS) is essential for virulence and a target antigen for vaccines. Although widespread introduction of pneumococcal conjugate vaccines (PCVs) has significantly reduced disease, the prevalence of non-vaccine serotypes has increased. On the basis of the CPS, S. pneumoniae serogroup 10 comprises four main serotypes 10A, 10B, 10C, and 10F; as well as the recently identified 10D. As it is the most prevalent, serotype 10A CPS has been included as a vaccine antigen in the next generation PCVs. Here we use molecular modeling to provide conformational rationales for the complex cross-reactivity reported between serotypes 10A, 10B, 10C, and 10F anti-sera. Although the highly mobile phosphodiester linkages produce very flexible CPS, shorter segments are conformationally defined, with exposed -D-galactofuranose ( DGalf) side chains that are potential antibody binding sites. We identify four distinct conformational epitopes for the immunodominant DGalf that assist in rationalizing the complex asymmetric cross-reactivity relationships. In particular, we find that strongly cross-reactive serotypes share common epitopes. Further, we show that human intelectin-1 has the potential to bind the exposed exocyclic 1,2-diol of the terminal DGalf in each serotype; the relative accessibility of three- or six-linked DGalf may play a role in the strength of the innate immune response and hence serotype disease prevalence. In conclusion, our modeling study and relevant serological studies support the inclusion of serotype 10A in a vaccine to best protect against serogroup 10 disease.

SARS-CoV-2 spike N-terminal domain modulates TMPRSS2-dependent viral entry and fusogenicity.

SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) remains poorly characterized despite enrichment of mutations in this region across variants of concern (VOCs). Here, we examine the contribution of the NTD to infection and cell-cell fusion by constructing chimeric spikes bearing B.1.617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus. We find that the Delta NTD on a Kappa or wild-type (WT) background increases S1/S2 cleavage efficiency and virus entry, specifically in lung cells and airway organoids, through use of TMPRSS2. Delta exhibits increased cell-cell fusogenicity that could be conferred to WT and Kappa spikes by Delta NTD transfer. However, chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD do not show more efficient TMPRSS2 use or fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions in a spike context-dependent manner, and allosteric interactions may be lost when combining regions from more distantly related VOCs.

Comparison of lateral flow immunoassays based on oriented and nonoriented immobilization of antibodies for the detection of aflatoxin B1

In recent years, some studies have found that oriented immobilization of antibodies to microspheres can fully expose the antigen binding sites of antibodies, which can improve the sensitivity of sandwich immunoassays for the detection of proteins. Can this antibody immobilization strategy also improve the sensitivity of competitive immunoassays for the detection of small molecules? To answer this question, the conjugate MS-SPG-Ab (oriented immobilization of aflatoxin B1 antibody to time-resolved fluorescent microspheres via streptococcal protein G) and the conjugate MS-Ab (nonoriented immobilization of aflatoxin B1 antibody to time-resolved fluorescent microspheres) were prepared, and a lateral flow immunoassay (LFIA) for the detection of aflatoxin B1 (AFB1) was established. The detection performance of the two methods was compared. The results showed that under the condition that the number of “effective” antibodies immobilized on TRF-MS was similar, compared with the nonoriented immobilization strategy (IC50 = 0.21 ng mL−1), the LFIA method established by the oriented immobilization strategy reduced the sensitivity of AFB1 detection (IC50 = 0.37 ng mL−1). However, this method can obtain higher detection precision for AFB1, the CV values were all below 8%. And it has stronger tolerance to the matrix of maize and peanut samples. The bias of LFIAs based on oriented immobilization technology (−14.93%–7.92%) was lower than nonoriented immobilization technology (28.16%–34.19%) for AFB1 detection in the two sample extracts. This study suggests that the LFIA method based on the oriented immobilization of antibodies can improve the accuracy of the detection results when performing rapid screening of small molecules.