Biology subjects

Czudnochowski, N.

Publications and source records attributed to Czudnochowski, N..

12 recordsLinked to original sources

bioRxiv2026

Pan-neutralization of parainfluenza viruses by a hemagglutinin-neuraminidase antibody

Human parainfluenza viruses (HPIVs) can cause severe respiratory illnesses, such as croup, bronchiolitis, and pneumonia, particularly in children, the elderly and immunocompromised individuals. No vaccines or specific therapeutics are available for use in humans. Here, we report the discovery of a human monoclonal antibody designated PVA269 that broadly and potently neutralizes all four HPIV subtypes, PIV5, and Sendai virus by targeting the hemagglutinin-neuraminidase (HN) glycoprotein. We show that PVA269 inhibits neuraminidase activity and hemagglutination of erythrocytes through insertion of a long heavy chain complementary-determining region 3 in the enzyme active site. We reveal that the antibody markedly remodels its interactions to accommodate distinct viral features across HPIV subtypes, such as HPIV2 N-linked glycans. These results define the molecular basis for the unique PVA269 pan-neutralizing activity of human and animal viruses spanning two genera. PVA269 provides potent prophylactic activity against HPIV3 replication in the upper and lower airways of the clinically predictive cotton rat model, thus supporting translation of its protective efficacy to humans. These data establish PVA269 as a best-in-class monoclonal antibody and a promising clinical candidate to prevent HPIV infection, transmission, and disease in vulnerable populations.

immunology↗

bioRxiv2023

Therapeutic and vaccine-induced cross-reactive antibodies with effector function against emerging Omicron variants

Currently circulating SARS-CoV-2 variants acquired convergent mutations at receptor-binding domain (RBD) hot spots1. Their impact on viral infection, transmission, and efficacy of vaccines and therapeutics remains poorly understood. Here, we demonstrate that recently emerged BQ.1.1. and XBB.1 variants bind ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1 and XBB.1 RBDs bound to human ACE2 and S309 Fab (sotrovimab parent) explain the altered ACE2 recognition and preserved antibody binding through conformational selection. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1, the variant displaying the greatest loss of neutralization. Moreover, in several donors vaccine-elicited plasma antibodies cross-react with and trigger effector functions against Omicron variants despite reduced neutralizing activity. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring persistent immune imprinting. Our findings suggest that this previously overlooked class of cross-reactive antibodies, exemplified by S309, may contribute to protection against disease caused by emerging variants through elicitation of effector functions.

immunology↗

bioRxiv2022

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month timeframe. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both pre- and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sub-lineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly-reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

immunology↗

bioRxiv2021

Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement

The SARS-CoV-2 Omicron variant of concern evades antibody mediated immunity with an unprecedented magnitude due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and X-ray crystal structures of the spike and RBD bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a structural framework for understanding the marked reduction of binding of all other therapeutic mAbs leading to dampened neutralizing activity. We reveal electrostatic remodeling of the interactions within the spike and those formed between the Omicron RBD and human ACE2, likely explaining enhanced affinity for the host receptor relative to the prototypic virus.

biochemistry↗

bioRxiv2021

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift

The recently emerged SARS-CoV-2 Omicron variant harbors 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Here, we show that the Omicron RBD binds to human ACE2 with enhanced affinity relative to the Wuhan-Hu-1 RBD and acquires binding to mouse ACE2. Severe reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for vaccinated and convalescent individuals. Most (26 out of 29) receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only three mAbs, including the ACE2-mimicking S2K146 mAb1, retaining unaltered potency. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs recognizing antigenic sites outside the RBM, including sotrovimab2, S2X2593 and S2H974, neutralized Omicron. The magnitude of Omicron-mediated immune evasion and the acquisition of binding to mouse ACE2 mark a major SARS-CoV-2 mutational shift. Broadly neutralizing sarbecovirus mAbs recognizing epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

immunology↗

bioRxiv2021

A human antibody that broadly neutralizes betacoronaviruses protects against SARS-CoV-2 by blocking the fusion machinery

The repeated spillovers of {beta}-coronaviruses in humans along with the rapid emergence of SARS-CoV-2 escape variants highlight the need to develop broad coronavirus therapeutics and vaccines. Five monoclonal antibodies (mAbs) were isolated from COVID-19 convalescent individuals and found to cross-react with multiple {beta}-coronavirus spike (S) glycoproteins by targeting the stem helix. One of these mAbs, S2P6, cross-reacts with more than twenty human and animal {beta}-coronavirus S glycoproteins and broadly neutralizes SARS-CoV-2 and pseudotyped viruses from the sarbecovirus, merbecovirus and embecovirus subgenera. Structural and functional studies delineate the molecular basis of S2P6 cross-reactivity and broad neutralization and indicate that this mAb blocks viral entry by inhibiting membrane fusion. S2P6 protects hamsters challenged with SARS-CoV-2 (including the B.1.351 variant of concern) through direct viral neutralization and Fc-mediated effector functions. Serological and B cell repertoire analyses indicate that antibodies targeting the stem helix are found in some convalescent donors and vaccinees but are predominantly of narrow specificity. Germline reversion of the identified cross-reactive mAbs revealed that their unmutated ancestors are specific for the endemic OC43 or HKU1 viruses and acquired enhanced affinity and breadth through somatic mutations. These data demonstrate that conserved epitopes in the coronavirus fusion machinery can be targeted by protective antibodies and provide a framework for structure-guided design of pan-{beta}-coronavirus vaccines eliciting broad protection.

immunology↗

bioRxiv2021

Antibodies to the SARS-CoV-2 receptor-binding domain that maximize breadth and resistance to viral escape

An ideal anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse SARS-related coronaviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics and guide vaccine design. Here, we comprehensively characterize escape, breadth, and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD), including S3094, the parental antibody of the late-stage clinical antibody VIR-7831. We observe a tradeoff between SARS-CoV-2 in vitro neutralization potency and breadth of binding across SARS-related coronaviruses. Nevertheless, we identify several neutralizing antibodies with exceptional breadth and resistance to escape, including a new antibody (S2H97) that binds with high affinity to all SARS-related coronavirus clades via a unique RBD epitope centered on residue E516. S2H97 and other escape-resistant antibodies have high binding affinity and target functionally constrained RBD residues. We find that antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency, but we identify one potent RBM antibody (S2E12) with breadth across sarbecoviruses closely related to SARS-CoV-2 and with a high barrier to viral escape. These data highlight functional diversity among antibodies targeting the RBD and identify epitopes and features to prioritize for antibody and vaccine development against the current and potential future pandemics.

microbiology↗

bioRxiv2021

Structural basis for broad sarbecovirus neutralization by a human monoclonal antibody

The recent emergence of SARS-CoV-2 variants of concern (VOC) and the recurrent spillovers of coronaviruses in the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here, we describe a human monoclonal antibody (mAb), designated S2x259, recognizing a highly conserved cryptic receptor-binding domain (RBD) epitope and cross-reacting with spikes from all sarbecovirus clades. S2x259 broadly neutralizes spike-mediated entry of SARS-CoV-2 including the B.1.1.7, B.1.351, P.1 and B.1.427/B.1.429 VOC, as well as a wide spectrum of human and zoonotic sarbecoviruses through inhibition of ACE2 binding to the RBD. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2x259 possesses a remarkably high barrier to the emergence of resistance mutants. We show that prophylactic administration of S2x259 protects Syrian hamsters against challenges with the prototypic SARS-CoV-2 and the B.1.351 variant, suggesting this mAb is a promising candidate for the prevention and treatment of emergent VOC and zoonotic infections. Our data unveil a key antigenic site targeted by broadly-neutralizing antibodies and will guide the design of pan-sarbecovirus vaccines.

immunology↗

bioRxiv2021

The dual function monoclonal antibodies VIR-7831 and VIR-7832 demonstrate potent in vitro and in vivo activity against SARS-CoV-2

Sotrovimab (VIR-7831) and VIR-7832 are dual action monoclonal antibodies (mAbs) targeting the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sotrovimab and VIR-7832 were derived from a parent antibody (S309) isolated from memory B cells of a 2003 severe acute respiratory syndrome coronavirus (SARS-CoV) survivor. Both mAbs contain an "LS" mutation in the Fc region to prolong serum half-life. In addition, VIR-7832 encodes an Fc GAALIE mutation that has been shown previously to evoke CD8+ T-cells in the context of an in vivo viral respiratory infection. Sotrovimab and VIR-7832 neutralize wild-type and variant pseudotyped viruses and authentic virus in vitro. In addition, they retain activity against monoclonal antibody resistance mutations conferring reduced susceptibility to previously authorized mAbs. The sotrovimab/VIR-7832 epitope continues to be highly conserved among circulating sequences consistent with the high barrier to resistance observed in vitro. Furthermore, both mAbs can recruit effector mechanisms in vitro that may contribute to clinical efficacy via elimination of infected host cells. In vitro studies with these mAbs demonstrated no enhancement of infection. In a Syrian Golden hamster proof-of concept wildtype SARS-CoV-2 infection model, animals treated with sotrovimab had less weight loss, and significantly decreased total viral load and infectious virus levels in the lung compared to a control mAb. Taken together, these data indicate that sotrovimab and VIR-7832 are key agents in the fight against COVID-19.

immunology↗

bioRxiv2020

The circulating SARS-CoV-2 spike variant N439K maintains fitness while evading antibody-mediated immunity

SARS-CoV-2 can mutate to evade immunity, with consequences for the efficacy of emerging vaccines and antibody therapeutics. Herein we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is the most divergent region of S, and provide epidemiological, clinical, and molecular characterization of a prevalent RBM variant, N439K. We demonstrate that N439K S protein has enhanced binding affinity to the hACE2 receptor, and that N439K virus has similar clinical outcomes and in vitro replication fitness as compared to wild- type. We observed that the N439K mutation resulted in immune escape from a panel of neutralizing monoclonal antibodies, including one in clinical trials, as well as from polyclonal sera from a sizeable fraction of persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.

microbiology↗

bioRxiv2019

A large inner membrane pore defines the ESX translocon

The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane; however, the architecture and mechanism of translocation has remained elusive. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the model organism Mycobacterium smegmatis. The structure reveals two large -helical membrane pores of sufficient diameter to secrete folded substrates. A complex, asymmetric, multimeric cytoplasmic domain is poised to gate and regulate the pores function. Our study provides mechanistic insights into the ESX systems and will guide structure-based design of drugs targeting this unique bacterial translocon.\n\nOne Sentence SummaryThe structure of the ESX-3 secretion system reveals a pore of sufficient size for the transit of folded substrates and a complex, cytoplasmic regulatory apparatus.

microbiology↗

bioRxiv2019

Two accessory proteins govern MmpL3 mycolic acid transport in mycobacteria

Mycolic acids are the signature lipid of mycobacteria and constitute an important physical component of the cell wall, a target of mycobacterial specific antibiotics, and a mediator of M. tuberculosis pathogenesis. Mycolic acids are synthesized in the cytoplasm and are thought to be transported to the cell wall as a trehalose ester by the MmpL3 transporter, an antibiotic target for M. tuberculosis. However, the mechanism by which mycolate synthesis is coupled to transport, and the full MmpL3 transport machinery, is unknown. Here we identify two new components of the MmpL3 transport machinery in mycobacteria. The protein encoded by MSMEG_0736/Rv0383c is essential for growth of M. smegmatis and M. tuberculosis, is anchored to the cytoplasmic membrane, physically interacts with and colocalizes with MmpL3 in growing cells, and is required for trehalose monomycolate transport to the cell wall. In light of these findings we propose Msmeg_0736/Rv0383c be named \"TMM transport factor A\", TtfA. The protein encoded by MSMEG_5308 also interacts with the MmpL3 complex, but is nonessential for growth or TMM transport. However, MSMEG_5308 accumulates with inhibition of MmpL3 mediated TMM transport and stabilizes the MmpL3/TtfA complex, indicating that it stabilizes the transport system during stress. These studies identify two new components of the mycobacterial mycolate transport machinery, an emerging antibiotic target in M. tuberculosis.

microbiology↗