Multiplexed Epitope Mapping of HIV-1 Neutralizing Antibodies by Coupling Chemical Labeling of Cysteines to Deep Sequencing.

Identification of neutralizing antibody (NAb) binding sites or epitopes on an antigen is a prerequisite for epitope-focused vaccine design strategies. HIV-1 infection is associated with polyclonal antibody responses comprising NAbs that target multiple epitopes on the envelope glycoprotein (Env), the primary target of the immune response. Current epitope mapping methods, such as X-ray crystallography and cryo-EM microscopy that rely on purified antigen-antibody complexes, fail to reliably deconvolute epitope specificities of polyclonal HIV-1 antibodies.

Rapid Mapping of Protein Binding Sites and Conformational Epitopes by Coupling Yeast Surface Display to Chemical Labeling and Deep Sequencing.

Delineating the precise regions on an antigen that are targeted by antibodies is important for the development of vaccines and antibody therapeutics. X-ray crystallography and NMR are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, these are labor-intensive and require purified protein at high concentrations.

Recombinant quadrivalent influenza vaccine (RIV) induces robust cell-mediated and HA-specific B cell humoral immune responses among healthcare personnel.

Egg-free influenza vaccines, specifically cell culture-based inactivated influenza vaccine (ccIIV) and recombinant influenza vaccine (RIV), represent a significant advancement over traditional egg-based inactivated influenza vaccines (IIV), particularly for populations with extensive vaccination histories. This comprehensive immunological study investigated the comparative efficacy of ccIIV, IIV, and RIV in healthcare personnel (HCP) with repeated vaccination histories, examining both cellular and humoral immune responses through multiple analytical approaches. Our investigation employed a multi-faceted analytical framework, combining serological assessments via hemagglutination inhibition (HI) and microneutralization (MN) assays with detailed cellular immune response analysis.

Destabilising Effect of Class B CpG Adjuvants on Different Proteins and Vaccine Candidates.

Adjuvants function by enhancing the breadth, durability, and magnitude of the immune response, but little is known about their impact on vaccine stability. CpG is a widely used adjuvant that is included in several recently approved COVID-19 vaccines using Spike protein, RBD, or whole inactivated virus. : Here, we investigate the in vitro stability of the Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike protein, as well as a number of other proteins formulated with a class B CpG adjuvant.

Cryo-EM reveals conformational variability in the SARS-CoV-2 spike protein RBD induced by two broadly neutralizing monoclonal antibodies.

SARS-CoV-2 spike proteins play a critical role in infection by interacting with the ACE2 receptors. Their receptor-binding domains and N-terminal domains exhibit remarkable flexibility and can adopt various conformations that facilitate receptor engagement. Previous structural studies have reported the RBD of the spike protein in "up", "down", and various intermediate states, as well as its different conformational changes during ACE2 binding.

Stabilizing Prefusion SARS-CoV-2 Spike by Destabilizing the Postfusion Conformation.

As with many viral fusion proteins, the native conformation of SARS-CoV-2 Spike is metastable. Most COVID-19 vaccines utilize a stabilized Spike (Spike-2P) containing two proline substitutions, and subsequently, a further stabilized variant with four additional proline substitutions, Spike-6P, has been developed. In an alternative approach, we introduced two aspartic acid residues (2D) in the HR1 region of Spike at positions that are exposed and buried in the pre- and postfusion states, respectively, to destabilize the postfusion conformation.

Early 2022 breakthrough infection sera from India target the conserved cryptic class 5 epitope to counteract immune escape by SARS-CoV-2 variants.

Unlabelled: During the coronavirus disease 2019 (COVID-19) pandemic, the vast majority of epitope mapping studies have focused on sera from mRNA-vaccinated populations from high-income countries. In contrast, here, we report an analysis of 164 serum samples isolated from patients with breakthrough infection in India during early 2022 who received two doses of the ChAdOx viral vector vaccine. Sera were screened for neutralization breadth against wild-type (WT), Kappa, Delta, and Omicron BA.

Understanding the physiological role and cross-interaction network of VapBC35 toxin-antitoxin system from Mycobacterium tuberculosis.

The VapBC toxin-antitoxin (TA) system, composed of VapC toxin and VapB antitoxin, has gained attention due to its relative abundance in members of the M. tuberculosis complex. Here, we have functionally characterised VapBC35 TA system from M.

Ex Vivo Delivery of mRNA to Immune Cells via a Nonendosomal Route Obviates the Need for Nucleoside Modification.

Base modification and the use of lipid nanoparticles are thought to be essential for efficient in vivo delivery and expression of mRNA. However, for ex vivo immune cell engineering, the need for either of the two is unclear. Previous reports have suggested that nucleic acids may be efficiently delivered to immune cells ex vivo, through a nonendosomal delivery route, but the need for base modification has not been determined.

Deciphering the role of VapBC13 and VapBC26 toxin antitoxin systems in the pathophysiology of Mycobacterium tuberculosis.

The expansion of VapBC TA systems in M. tuberculosis has been linked with its fitness and survival upon exposure to stress conditions. Here, we have functionally characterized VapBC13 and VapBC26 TA modules of M.

Backbone assignment of CcdB_G100T toxin from E.coli in complex with the toxin binding C-terminal domain of its cognate antitoxin CcdA.

The CcdAB system expressed in the E.coli cells is a prototypical example of the bacterial toxin-antitoxin (TA) systems that ensure the survival of the bacterial population under adverse environmental conditions. The solution and crystal structures of CcdA, CcdB and of CcdB in complex with the toxin-binding C-terminal domain of CcdA have been reported.

Improved Prediction of Stabilizing Mutations in Proteins by Incorporation of Mutational Effects on Ligand Binding.

While many computational methods accurately predict destabilizing mutations, identifying stabilizing mutations has remained a challenge, because of their relative rarity. We tested ΔΔG predictions from computational predictors such as Rosetta, ThermoMPNN, RaSP, and DeepDDG, using 82 mutants of the bacterial toxin CcdB as a test case. On this dataset, the best computational predictor is ThermoMPNN, which identifies stabilizing mutations with a precision of 68%.

A(H2N2) and A(H3N2) influenza pandemics elicited durable cross-reactive and protective antibodies against avian N2 neuraminidases.

Human cases of avian influenza virus (AIV) infections are associated with an age-specific disease burden. As the influenza virus N2 neuraminidase (NA) gene was introduced from avian sources during the 1957 pandemic, we investigate the reactivity of N2 antibodies against A(H9N2) AIVs. Serosurvey of healthy individuals reveal the highest rates of AIV N2 antibodies in individuals aged ≥65 years.

Enhancing Immunogenicity of a Thermostable, Efficacious SARS-CoV-2 Vaccine Formulation through Oligomerization and Adjuvant Choice.

Currently deployed SARS-CoV-2 vaccines all require storage at refrigerated or sub-zero temperatures. We demonstrate that after month-long incubation at 37 °C, solubilization, and formulation with squalene-in-water emulsion adjuvant, a stabilized receptor binding domain retains immunogenicity and protective efficacy. We also examine the effects of trimerization of the stabilized RBD, as well as of additional adjuvants, on both B and T-cell responses.

Molecular bases for strong phenotypic effects of single synonymous codon substitutions in the E. coli ccdB toxin gene.

Background: Single synonymous codon mutations typically have only minor or no effects on gene function. Here, we estimate the effects on cell growth of ~ 200 single synonymous codon mutations in an operonic context by mutating almost all positions of ccdB, the 101-residue long cytotoxin of the ccdAB Toxin-Antitoxin (TA) operon to most degenerate codons. Phenotypes were assayed by transforming the mutant library into CcdB sensitive and resistant E.

Enhanced protective efficacy of a thermostable RBD-S2 vaccine formulation against SARS-CoV-2 and its variants.

With the rapid emergence of variants of concern (VOC), the efficacy of currently licensed vaccines has reduced drastically. VOC mutations largely occur in the S1 subunit of Spike. The S2 subunit of SARS-CoV-2 is conserved and thus more likely to elicit broadly reactive immune responses that could improve protection.

Mutations in S2 subunit of SARS-CoV-2 Omicron spike strongly influence its conformation, fusogenicity, and neutralization sensitivity.

The Omicron subvariants have substantially evaded host-neutralizing antibodies and adopted an endosomal route of entry. The virus has acquired several mutations in the receptor binding domain and N-terminal domain of S1 subunit, but remarkably, also incorporated mutations in S2 which are fixed in Omicron sub-lineage. Here, we found that the mutations in the S2 subunit affect the structural and biological properties such as neutralization escape, entry route, fusogenicity, and protease requirement.

A CcdB toxin-derived peptide acts as a broad-spectrum antibacterial therapeutic in infected mice.

The bacterial toxin CcdB (Controller of Cell death or division B) targets DNA Gyrase, an essential bacterial topoisomerase, which is also the molecular target for fluoroquinolones. Here, we present a short cell-penetrating 24-mer peptide, CP1-WT, derived from the Gyrase-binding region of CcdB and examine its effect on growth of Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and a carbapenem- and tigecycline-resistant strain of Acinetobacter baumannii in both axenic cultures and mouse models of infection. The CP1-WT peptide shows significant improvement over ciprofloxacin in terms of its in vivo therapeutic efficacy in treating established infections of S.

Identification of diphenylurea derivatives as novel endocytosis inhibitors that demonstrate broad-spectrum activity against SARS-CoV-2 and influenza A virus both in vitro and in vivo.

Rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) poses enormous challenge in the development of broad-spectrum antivirals that are effective against the existing and emerging viral strains. Virus entry through endocytosis represents an attractive target for drug development, as inhibition of this early infection step should block downstream infection processes, and potentially inhibit viruses sharing the same entry route. In this study, we report the identification of 1,3-diphenylurea (DPU) derivatives (DPUDs) as a new class of endocytosis inhibitors, which broadly restricted entry and replication of several SARS-CoV-2 and IAV strains.

Neutralizing Efficacy of Encapsulin Nanoparticles against SARS-CoV2 Variants of Concern.

Rapid emergence of the SARS-CoV-2 variants has dampened the protective efficacy of existing authorized vaccines. Nanoparticle platforms offer a means to improve vaccine immunogenicity by presenting multiple copies of desired antigens in a repetitive manner which closely mimics natural infection. We have applied nanoparticle display combined with the SpyTag-SpyCatcher system to design encapsulin-mRBD, a nanoparticle vaccine displaying 180 copies of the monomeric SARS-CoV-2 spike receptor-binding domain (RBD).

Biophysical Correlates of Enhanced Immunogenicity of a Stabilized Variant of the Receptor Binding Domain of SARS-CoV-2.

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We have previously reported the design and characterization of a mammalian cell expressed RBD derivative, mRBD1-3.2, that has higher thermal stability and greatly enhanced immunogenicity relative to the wild type mRBD.

Mutational scan inferred binding energetics and structure in intrinsically disordered protein CcdA.

Unlike globular proteins, mutational effects on the function of Intrinsically Disordered Proteins (IDPs) are not well-studied. Deep Mutational Scanning of a yeast surface displayed mutant library yields insights into sequence-function relationships in the CcdA IDP. The approach enables facile prediction of interface residues and local structural signatures of the bound conformation.

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics.

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase.