Biology subjects

Schmidt, H. R.

Publications and source records attributed to Schmidt, H. R..

6 recordsLinked to original sources

bioRxiv2025

Protective and pathogenic antibody responses from a primate Shigella outbreak inform vaccine design

There is currently no approved vaccine for Shigella spp., a leading cause of diarrhea that are increasingly resistant to antimicrobials. Shigella vaccine development is complicated in part by an incomplete understanding of the structural and molecular determinants of immunity. To address this, we isolated monoclonal antibodies (mAbs) against candidate Shigella vaccine antigens using samples from a Shigella flexneri outbreak in a non-human primate (NHP) research facility. We found that antibodies targeting the Shigella O-antigen (O-Ag) can undergo significant affinity maturation (>10%) to acquire broad cross-reactivity across S. flexneri serotypes. We also found that the virulence-associated type III secretion system (T3SS) proteins IpaD and IpaB elicit moderate T cell and robust antibody responses. T3SS antibodies either inhibit, or - surprisingly - enhance bacterial virulence in vitro and in vivo depending on their epitope specificity. These findings provide key insights into protective and deleterious immune responses against Shigella that directly inform vaccine immunogen design.

immunology↗

bioRxiv2025

Improvement of affinity and potency of a monoclonal antibody against Shigella flexneri 3a O-antigen via phage display and whole-cell in-solution panning

As rates of antimicrobial resistance (AMR) among bacterial pathogens continue to rise, the discovery and development of novel classes of therapeutics that can serve as alternatives or adjuncts to traditional small-molecule antibiotics, such as monoclonal antibodies (mAbs), is a public health priority. Some of the most promising antigen targets for antibacterial mAbs are surface polysaccharides such as O-antigen (O-Ag), a component of the lipopolysaccharide found on the outer membrane of gram-negative bacteria. However, developing mAbs against bacterial surface polysaccharides with sufficient breadth and potency to be clinically viable is difficult in part because antibodies against polysaccharides are generally low affinity, and the challenging biochemistry of polysaccharides often precludes further affinity maturation of mAbs against these targets in vitro. Here, we use a phage display library and a whole-cell in-solution panning strategy to successfully improve the affinity of a mAb against Shigella flexneri 3a O-Ag in vitro without requiring the purification of the target antigen. We demonstrate that a single mutation can improve apparent affinity approximately 10-fold without detectably increasing polyreactivity, and increased affinity correlates with enhanced potency in antibacterial effector function and anti-virulence assays. In addition, the most potent variants also gained increased breadth, successfully coordinating complement deposition and complement-independent opsonophagocytosis against S. flexneri 3b, a serotype weakly recognized by the parent mAb. Altogether, this work represents an important first step towards expanding the antibody engineering toolkit for bacterial surface polysaccharides, which will aid the development of novel mAb therapeutics against AMR bacterial pathogens.

bioengineering↗

bioRxiv2025

Harnessing mRNA for the expression of monoclonal IgG and IgA in non-human primates.

Monoclonal antibodies (mAbs) are an increasingly essential class of medicines across many disease areas (1). In the human body, there are five antibody isotypes, each with potential prophylactic or therapeutic benefits for different disease indications. However, 97% of all clinically approved mAbs are produced as the IgG isotype, partly due to differences in half-life, but largely due to challenges associated with recombinantly producing non-IgG isotypes like IgM or IgA, which have additional N-linked glycan sites and can present as multivalent oligomers. One potential solution to this challenge is to express mAbs in situ using mRNA encapsulated in lipid nanoparticles (LNP), bypassing the need for recombinant protein production (2). Here, we demonstrate the feasibility of expressing a mAb as both IgG and IgA in non-human primates (NHPs) using mRNA-LNPs. We express ePGDM1400v9, a broadly neutralizing mAb targeting human immunodeficiency virus (HIV), in both IgG1 and IgA2 formats by infusing NHPs with LNPs containing the appropriate mRNA. Though IgG1 expression levels were higher than those of IgA2, both formats were detectable in serum within one day of LNP infusion in all NHPs, and both were detectable in mucosal secretions of most animals. Importantly, serum mRNA-produced IgG1 and IgA2 retained HIV-neutralizing function. Furthermore, mass spectrometry analysis confirmed that mAbs of either isotype produced in situ exhibited glycosylation patterns highly similar to that of native antibody, which is likely to confer therapeutic advantages. Altogether, this work demonstrates the feasibility of using mRNA-LNPs to express native-like mAbs of non-IgG isotypes in primates and enables further development of non-IgG mAb constructs. SignificanceMonoclonal antibodies (mAbs) are a rapidly growing class of essential medicines across diverse disease areas and applications. However, the impact potential of mAbs is limited by challenges in production and purification, which favors the use of the IgG antibody isotype despite the disease-specific advantages that other isotypes might offer. One way to overcome this challenge is to express mAbs in situ using messenger ribonucleic acid delivered by lipid nanoparticles (mRNA-LNPs). Here, we show that an anti-human immunodeficiency virus (HIV) mAb, ePGDM1400v9, can be expressed as two different antibody isotypes, IgG and IgA, in nonhuman primates (NHPs) by mRNA-LNP delivery. We demonstrate that both mAb isotypes retain function and exhibit native-like glycosylation patterns that are not achievable with conventional recombinant mAbs.

immunology↗

bioRxiv2021

Molecular reconstruction of recurrent evolutionary switching in olfactory receptor specificity

Olfactory receptor repertoires exhibit remarkable functional diversity, but how these proteins have evolved is poorly understood. Through analysis of extant and ancestrally-reconstructed drosophilid olfactory receptors from the Ionotropic Receptor (IR) family, we investigated evolution of two organic acid-sensing receptors, IR75a and IR75b. Despite their low amino acid identity, we identify a common "hotspot" in their ligand-binding pocket that has a major effect on changing the specificity of both IRs, as well as at least two distinct functional transitions in IR75a during evolution. Ligand-docking into IR models predicts that the hotspot does not contact odor molecules, suggesting that this residue indirectly influences ligand/receptor interactions. Moreover, we show that odor specificity is refined by changes in additional, receptor-specific sites, including those outside the ligand-binding pocket. Our work reveals how a core, common determinant of ligand-tuning acts within epistatic and allosteric networks of substitutions to lead to functional evolution of olfactory receptors.

neuroscience↗

bioRxiv2019

Virtual screening identifies novel high-affinity σ1 receptor ligands

The {sigma}1 receptor is a transmembrane protein implicated in several pathophysiological conditions, including neurodegenerative disease1, drug addiction2, cancer3, and pain4. However, there are no high-throughput functional assays for {sigma}1 receptor drug discovery. Here, we assessed high-throughput structure-based computational docking for discovery of novel ligands of the {sigma}1 receptor. We screened a library of over 6 million compounds using the Schrodinger Glide package, followed by experimental characterization of top-scoring candidates. 77% of tested candidates bound {sigma}1 with high affinity (10-550 nM). These include compounds with high selectivity for the {sigma}1 receptor compared to the genetically unrelated but pharmacologically similar {sigma}2 receptor, as well as compounds with substantial cross-reactivity between the two receptors. These results establish structure-based virtual screening as a highly effective platform for {sigma}1 receptor ligand discovery.

pharmacology and toxicology↗

bioRxiv2018

Structural basis for sigma-1 receptor ligand recognition

The {sigma}1 receptor is a poorly understood integral membrane protein expressed in most cells and tissues in the human body. It has been shown to modulate the activity of other membrane proteins such as ion channels and G protein-coupled receptors1-4, and ligands targeting the {sigma}1 receptor are currently in clinical trials for treatment of Alzheimers disease5, ischemic stroke6, and neuropathic pain7. Despite its importance, relatively little is known regarding {sigma}1 receptor function at the molecular level. Here, we present crystal structures of the human {sigma}1 receptor bound to the classical antagonists haloperidol and NE-100, as well as the agonist (+)-pentazocine, at crystallographic resolutions of 3.1 [A], 2.9 [A], and 3.1 [A] respectively. These structures reveal a unique binding pose for the agonist. The structures and accompanying molecular dynamics (MD) simulations demonstrate that the agonist induces subtle structural rearrangements in the receptor. In addition, we show that ligand binding and dissociation from {sigma}1 is a multistep process, with extraordinarily slow kinetics limited by receptor conformational change. We use MD simulations to reconstruct a ligand binding pathway that requires two major conformational changes. Taken together, these data provide a framework for understanding the molecular basis for agonist action at {sigma}1.

pharmacology and toxicology↗