Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) is characterized by the abrupt onset of significant obsessive-compulsive symptoms (OCS) and/or severe food restriction, together with other neuropsychiatric manifestations. An autoimmune pathogenesis triggered by infection has been proposed for at least a subset of PANS. The older diagnosis of Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus (PANDAS) describes rapid onset of OCD and/or tics associated with infection with Group A Streptococcus. The pathophysiology of PANS and PANDAS remains incompletely understood. We recently found serum antibodies from children with rigorously defined PANDAS to selectively bind to cholinergic interneurons (CINs) in the striatum. Here we examine this binding in children with relapsing and remitting PANS, a more heterogeneous condition, collected in a distinct clinical context from those examined in our previous work, from children with a clinical history of Streptococcus infection. IgG from PANS cases showed elevated binding to striatal CINs in both mouse and human brain. Patient plasma collected during symptom flare decreased a molecular marker of CIN activity, phospho-riboprotein S6, in ex vivo brain slices; control plasma did not. Neither elevated antibody binding to CINs nor diminished CIN activity was seen with plasma collected from the same children during remission. These findings replicate what we have seen previously in PANDAS and support the hypothesis that at least a subset of PANS cases have a neuroimmune pathogenesis. Given the critical role of CINs in modulating basal ganglia function, these findings confirm striatal CINs as a locus of interest in the pathophysiology of both PANS and PANDAS.

In this study, we performed a computational study of binding mechanisms for the SARS-CoV-2 spike Omicron XBB lineages with the host cell receptor ACE2 and a panel of diverse class one antibodies. The central objective of this investigation was to examine the molecular factors underlying epistatic couplings among convergent evolution hotspots that enable optimal balancing of ACE2 binding and antibody evasion for Omicron variants BA.1, BA2, BA.3, BA.4/BA.5, BQ.1.1, XBB.1, XBB.1.5, and XBB.1.5 + L455F/F456L. By combining evolutionary analysis, molecular dynamics simulations, and ensemble-based mutational scanning of spike protein residues in complexes with ACE2, we identified structural stability and binding affinity hotspots that are consistent with the results of biochemical studies. In agreement with the results of deep mutational scanning experiments, our quantitative analysis correctly reproduced strong and variant-specific epistatic effects in the XBB.1.5 and BA.2 variants. It was shown that Y453W and F456L mutations can enhance ACE2 binding when coupled with Q493 in XBB.1.5, while these mutations become destabilized when coupled with the R493 position in the BA.2 variant. The results provided a molecular rationale of the epistatic mechanism in Omicron variants, showing a central role of the Q493/R493 hotspot in modulating epistatic couplings between convergent mutational sites L455F and F456L in XBB lineages. The results of mutational scanning and binding analysis of the Omicron XBB spike variants with ACE2 receptors and a panel of class one antibodies provide a quantitative rationale for the experimental evidence that epistatic interactions of the physically proximal binding hotspots Y501, R498, Q493, L455F, and F456L can determine strong ACE2 binding, while convergent mutational sites F456L and F486P are instrumental in mediating broad antibody resistance. The study supports a mechanism in which the impact on ACE2 binding affinity is mediated through a small group of universal binding hotspots, while the effect of immune evasion could be more variant-dependent and modulated by convergent mutational sites in the conformationally adaptable spike regions.

Clostridioides difficile infection (CDI) is a significant concern caused by widespread antibiotic use, resulting in diarrhea and inflammation from the gram-positive anaerobic bacterium C. difficile. Although bezlotoxumab (Bez), a monoclonal antibody (mAb), was developed to address CDI recurrences, the recurrence rate remains high, partly due to reduced neutralization efficiency against toxin B2. In this study, we aimed to enhance the binding of Bez to C. difficile toxin B2 by combining computational simulations and mutational analyses. We identified specific mutations in Bez, including S28R, S31W/K, Y32R, S56W and G103D/S in the heavy chain (Hc), and S32F/H/R/W/Y in the light chain (Lc), which significantly improved binding to toxin B2 and formed critical protein-protein interactions. Through molecular dynamics simulations, several single mutations, such as HcS28R, LcS32H, LcS32R, LcS32W and LcS32Y, exhibited superior binding affinities to toxin B2 compared to Bez wild-type (WT), primarily attributed to Coulombic interactions. Combining the HcS28R mutation with four different mutations at residue LcS32 led to even greater binding affinities in double mutants (MTs), particularly HcS28R/LcS32H, HcS28R/LcS32R and HcS28R/LcS32Y, reinforcing protein-protein binding. Analysis of per-residue decomposition free energy highlighted key residues contributing significantly to enhanced binding interactions, emphasizing the role of electrostatic interactions. These findings offer insights into rational Bez MT design for improved toxin B2 binding, providing a foundation for developing more effective antibodies to neutralize toxin B2 and combat-related infections.Communicated by Ramaswamy H. Sarma.

Antibodies have increasingly been developed as drugs with over 100 now licensed in the US or EU. During development, it is often necessary to increase or reduce the affinity of an antibody and rational attempts to do so rely on having a structure of the antibody-antigen complex often obtained by modeling. The antigen-binding site consists primarily of six loops known as complementarity-determining regions (CDRs), and an open question has been whether these loops change their conformation when they bind to an antigen. Existing surveys of antibody-antigen complex structures have only examined CDR conformational change in case studies or small-scale surveys. With an increasing number of antibodies where both free and complexed structures have been deposited in the Protein Data Bank, a large-scale survey of CDR conformational change during binding is now possible. To this end, we built a dataset, AbAgDb, that currently includes 177 antibodies with high-quality CDRs, each of which has at least one bound and one unbound structure. We analyzed the conformational change of the Cα backbone of each CDR upon binding and found that, in most cases, the CDRs (other than CDR-H3) show minimal movement, while 70.6% and 87% of CDR-H3s showed global Cα RMSD ≤ 1.0Å and ≤ 2.0Å, respectively. We also compared bound CDR conformations with the conformational space of unbound CDRs and found most of the bound conformations are included in the unbound conformational space. In future, our results will contribute to developing insights into antibodies and new methods for modeling and docking.

High mutation and replication rates of HIV-1 result in the continuous generation of variants, allowing it to adapt to changing host environments. Mutations often have deleterious effects, but variants carrying them are rapidly purged. Surprisingly, a particular variant incapable of entering host cells was found to be rescued by host antibodies targeting HIV-1. Understanding the molecular mechanism of this rescue is important to develop and improve antibody-based therapies. To unravel the underlying mechanisms, we performed fully atomistic molecular dynamics simulations of the HIV-1 gp41 trimer responsible for viral entry into host cells, its entry-deficient variant, and its complex with the rescuing antibody. We find that the Q563R mutation, which the entry-deficient variant carries, prevents the native conformation of the gp41 6-helix bundle required for entry and stabilizes an alternative conformation instead. This is the consequence of substantial changes in the secondary structure and interactions between the domains of gp41. Binding of the antibody F240 to gp41 reverses these changes and re-establishes the native conformation, resulting in rescue. To test the generality of this mechanism, we performed simulations with the entry-deficient L565A variant and antibody 3D6. We find that 3D6 binding was able to reverse structural and interaction changes introduced by the mutation and restore the native gp41 conformation. Viral variants may not only escape antibodies but be aided by them in their survival, potentially compromising antibody-based therapies, including vaccination and passive immunization. Our simulation framework could serve as a tool to assess the likelihood of such resistance against specific antibodies.Communicated by Ramaswamy H. SarmaCommunicated by Ramaswamy H. Sarma.

Label-free techniques including Surface Plasmon Resonance (SPR) and Biolayer Interferometry (BLI) are biophysical tools widely used to collect binding kinetics data of bimolecular interactions. To efficiently analyze SPR and BLI binding kinetics data, we have built a new high throughput analysis tool named the TitrationAnalysis. It can be used as a package in the Mathematica scripting environment and ultilize the non-linear curve-fitting module of Mathematica for its core function. This tool can fit the binding time course data and estimate association and dissociation rate constants ( k a and k d respectively) for determining apparent dissociation constant ( K D ) values. The high throughput fitting process is automatic, requires minimal knowledge on Mathematica scripting and can be applied to data from multiple label-free platforms. We demonstrate that the TitrationAnalysis is optimal to analyze antibody-antigen binding data acquired on Biacore T200 (SPR), Carterra LSA (SPR imaging) and ForteBio Octet Red384 (BLI) platforms. The k a , k d and K D values derived using TitrationAnalysis very closely matched the results from the commercial analysis software provided specifically for these instruments. Additionally, the TitrationAnalysis tool generates user-directed customizable results output that can be readily used in downstream Data Quality Control associated with Good Clinical Laboratory Practice operations. With the versatility in source of data input source and options of analysis result output, the TitrationAnalysis high throughput analysis tool offers investigators a powerful alternative in biomolecular interaction characterization.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a +sense single-strand RNA virus. The virus has four major surface proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), respectively. The constitutive proteins present a high grade of symmetry. Identifying a binding site is difficult. The virion is approximately 50-200 nm in diameter. Angiotensin-converting enzyme 2 (ACE2) acts as the cell receptor for the virus. SARS-CoV-2 has an increased affinity to human ACE2 compared with the original SAR strain. Topological space, and its symmetry, is a critical component in molecular interactions. By exploring this space, a suitable ligand space can be characterized accordingly. A spike protein (S) computational model in a complex with ACE 2 was generated using silica methods. Topological spaces were probed using high computational throughput screening techniques to identify and characterize the topological space of both SARS and SARS-CoV-2 spike protein and its ligand space. In order to identify the symmetry clusters, computational analysis techniques, together with statistical analysis, were utilized. The computations are based on crystallographic protein data bank PDB-based models of constitutive proteins. Cartesian coordinates of component atoms and some cluster maps were generated and analyzed. Dihedral angles were used in order to compute a topological receptor space. This computational study uses a multimodal representation of spike protein interactions with some fragment proteins. The chemical space of the receptors (a dimensional volume) suggests the relevance of the receptor as a drug target. The spike protein S of SARS and SARS-CoV-2 is analyzed and compared. The results suggest a mirror symmetry of SARS and SARS-CoV-2 spike proteins. The results show thatSARS-CoV-2 space is variable and has a distinct topology. In conclusion, surface proteins grant virion variability and symmetry in interactions with a potential complementary target (protein, antibody, ligand). The mirror symmetry of dihedral angle clusters determines a high specificity of the receptor space.

The detection of site-specific phosphorylation in the microtubule-associated protein tau is emerging as a means to diagnose and monitor the progression of Alzheimer\'s Disease and other neurodegenerative diseases. However, there is a lack of phospho-specific monoclonal antibodies and limited validation of their binding specificity. Here, we report a novel approach using yeast biopanning against synthetic peptides containing site-specific phosphorylations. Using yeast cells displaying a previously validated phospho-tau (p-tau) single-chain variable region fragment (scFv), we show selective yeast cell binding based on single amino acid phosphorylation on the antigen. We identify conditions that allow phospho-specific biopanning using scFvs with a wide range of affinities (KD = 0.2 to 60 nM). Finally, we demonstrate the capability of screening large libraries by performing biopanning in 6-well plates. These results show that biopanning can effectively select yeast cells based on phospho-site specific antibody binding, opening doors for the facile identification of high-quality monoclonal antibodies.

Rabbit hemorrhagic disease virus (RHDV) typically causes a fatal disease in rabbits. In Australia, RHDV was imported to control the feral rabbit population, while it poses a severe threat to native rabbits in other countries. RHDV variants are genetically diverse and serological studies using antibodies isolated from infected rabbits or raised against RHDV virus-like particles (VLPs) have found RHDV variants antigenically distinct. In this study, we determined the X-ray crystal structure of an RHDV GI.2 (N11 strain) protruding (P) domain in complex with a diagnostic monoclonal antibody (2D9) Fab. We showed that 2D9 interacted with conserved and variable residues on top of the P domain with nanomolar affinity. To better illustrate 2D9 specificity, we determined the X-ray crystal structure of an RHDV GI.1b (Ast89 strain) that was a 2D9 non-binder. Structural analysis indicated that amino acid substitutions on the GI.1b P domain likely restricted 2D9 binding. Interestingly, a model of the GI.2 P domain-Fab complex superimposed onto a cryo-EM structure of an RHDV VLP revealed that 2D9 Fab molecules clashed with neighboring Fabs and indicated that there was a reduced antibody binding occupancy. Moreover, the RHDV GI.2 histo-blood group antigen (HBGA) co-factor binding site appeared obstructed when 2D9 was modeled on the VLP and suggested that 2D9 might also function by blocking HBGA attachment. Overall, this new data provides the first structural basis of RHDV antibody specificity and explains how amino acid variation at the binding site likely restricts 2D9 cross-reactivity. IMPORTANCE Isolated RHDV antibodies have been used for decades to distinguish between antigenic variants, monitor temporal capsid evolution, and examine neutralizing capacities. In this study, we provided the structural basis for an RHDV GI.2 specific diagnostic antibody (2D9) binding and reveal that a small number of amino acid substitutions at the binding site could differentiate between RHDV GI.2 and GI.1b. This novel structural information provides a framework for understanding how RHDV displays a specific antigenic epitope and engages an antibody at the atomic level. Importantly, part of the 2D9 binding region was earlier reported to contain a neutralizing epitope and our structural modeling as well as recent human norovirus antibody-mediated neutralization studies, suggest that the 2D9 antibody has the potential to block HBGA attachment. These new findings should aid in characterizing antigenic variants and advance the development of novel monoclonal antibodies for diagnostics and therapeutics.

Protein display technologies link proteins to distinct nucleic acid sequences (barcodes), enabling multiplexed protein assays via DNA sequencing. Here, we develop Cas9 display (CasPlay) to interrogate customized peptide libraries fused to catalytically inactive Cas9 (dCas9) by sequencing the guide RNA (gRNA) barcodes associated with each peptide. We first confirm the ability of CasPlay to characterize antibody epitopes by recovering a known binding motif for a monoclonal anti-FLAG antibody. We then use a CasPlay library tiling the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteome to evaluate vaccine-induced antibody reactivities. Using a peptide library representing the human virome, we demonstrate the ability of CasPlay to identify epitopes across many viruses from microliters of patient serum. Our results suggest that CasPlay is a viable strategy for customized protein interaction studies from highly complex libraries and could provide an alternative to phage display technologies.