Deep mutational scanning of rabies glycoprotein defines mutational constraint and antibody-escape mutations.

Rabies virus causes nearly 60,000 human deaths annually. Antibodies that target the rabies glycoprotein (G) are being developed as post-exposure prophylactics, but mutations in G can render such antibodies ineffective. Here, we use pseudovirus deep mutational scanning to measure how all single amino-acid mutations to G affect cell entry and neutralization by a panel of antibodies.

Defining a highly conserved B cell epitope in the receptor binding motif of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 mRNA vaccines induce robust and persistent germinal centre (GC) B cell responses in humans. It remains unclear how the continuous evolution of the virus impacts the breadth of the induced GC B cell response. Using ultrasound-guided fine needle aspiration, we examined draining lymph nodes of nine healthy adults following bivalent booster immunization.

High-throughput sequencing-based neutralization assay reveals how repeated vaccinations impact titers to recent human H1N1 influenza strains.

Unlabelled: The high genetic diversity of influenza viruses means that traditional serological assays have too low throughput to measure serum antibody neutralization titers against all relevant strains. To overcome this challenge, we developed a sequencing-based neutralization assay that simultaneously measures titers against many viral strains using small serum volumes using a workflow similar to traditional neutralization assays. The key innovation is to incorporate unique nucleotide barcodes into the hemagglutinin (HA) genomic segment, and then pool viruses with numerous different barcoded HA variants and quantify the infectivity of all of them simultaneously using next-generation sequencing.

Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance.

H5 influenza is a potential pandemic threat. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic risk, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. Here we use pseudovirus deep mutational scanning to measure how all mutations to a clade 2.

Bispecific antibodies with broad neutralization potency against SARS-CoV-2 variants of concern.

The ongoing emergence of SARS-CoV-2 variants of concern (VOCs) that reduce the effectiveness of antibody therapeutics necessitates development of next-generation antibody modalities that are resilient to viral evolution. Here, we characterized N-terminal domain (NTD) and receptor binding domain (RBD)-specific monoclonal antibodies previously isolated from COVID-19 convalescent donors for their activity against emergent SARS-CoV-2 VOCs. Among these, the NTD-specific antibody C1596 displayed the greatest breadth of binding to VOCs, with cryo-EM structural analysis revealing recognition of a distinct NTD epitope outside of the site i antigenic supersite.

Structural basis of broad SARS-CoV-2 cross-neutralization by affinity-matured public antibodies.

Descendants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant now account for almost all SARS-CoV-2 infections. The Omicron variant and its sublineages have spike glycoproteins that are highly diverged from the pandemic founder and first-generation vaccine strain, resulting in significant evasion from monoclonal antibody therapeutics and vaccines. Understanding how commonly elicited antibodies can broaden to cross-neutralize escape variants is crucial.

Computationally restoring the potency of a clinical antibody against Omicron.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs and revealed how quickly viral escape can curtail effective options. When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination and is challenging to replace with existing approaches.

Structural and genetic diversity in the secreted mucins, and .

The secreted mucins MUC5AC and MUC5B play critical defensive roles in airway pathogen entrapment and mucociliary clearance by encoding large glycoproteins with variable number tandem repeats (VNTRs). These polymorphic and degenerate protein coding VNTRs make the loci difficult to investigate with short reads. We characterize the structural diversity of and by long-read sequencing and assembly of 206 human and 20 nonhuman primate (NHP) haplotypes.

Hybrid immunity to SARS-CoV-2 arises from serological recall of IgG antibodies distinctly imprinted by infection or vaccination.

Unlabelled: We used plasma IgG proteomics to study the molecular composition and temporal durability of polyclonal IgG antibodies triggered by ancestral SARS-CoV-2 infection, vaccination, or their combination ("hybrid immunity"). Infection, whether primary or post-vaccination, mainly triggered an anti-spike antibody response to the S2 domain, while vaccination predominantly induced anti-RBD antibodies. Immunological imprinting persisted after a secondary (hybrid) exposure, with >60% of the ensuing serological response originating from the initial antibodies generated during the first exposure.

High-throughput sequencing-based neutralization assay reveals how repeated vaccinations impact titers to recent human H1N1 influenza strains.

The high genetic diversity of influenza viruses means that traditional serological assays have too low throughput to measure serum antibody neutralization titers against all relevant strains. To overcome this challenge, we have developed a sequencing-based neutralization assay that simultaneously measures titers against many viral strains using small serum volumes via a workflow similar to traditional neutralization assays. The key innovation is to incorporate unique nucleotide barcodes into the hemagglutinin (HA) genomic segment, and then pool viruses with numerous different barcoded HA variants and quantify infectivity of all of them simultaneously using next-generation sequencing.

Deep mutational scanning reveals functional constraints and antigenic variability of Lassa virus glycoprotein complex.

Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we use pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affect cell entry and antibody neutralization.

Importance of quantifying the number of viral reads in metagenomic sequencing of environmental samples from the Huanan Seafood Market.

In March 2023, the Chinese CDC publicly released raw metagenomic sequencing data for environmental samples collected in early 2020 from the Huanan Seafood Market. Prior to that data release, some scientists had suggested that these samples could be informative for establishing if animals such as raccoon dogs had been infected with severe acute respiratory syndrome virus 2 (SARS-CoV-2). However, no one had analyzed how much SARS-CoV-2 was actually present in the metagenomic sequencing data.

: Structure-informed visualizations for deep mutational scanning and other mutation-based datasets.

Understanding how mutations impact a protein's functions is valuable for many types of biological questions. High-throughput techniques such as deep-mutational scanning (DMS) have greatly expanded the number of mutation-function datasets. For instance, DMS has been used to determine how mutations to viral proteins affect antibody escape (Dadonaite et al.

Dynamics and durability of HIV-1 neutralization are determined by viral replication.

Human immunodeficiency virus type 1 (HIV-1)-neutralizing antibodies (nAbs) that prevent infection are the main goal of HIV vaccine discovery. But as no nAb-eliciting vaccines are yet available, only data from HIV-1 neutralizers-persons with HIV-1 who naturally develop broad and potent nAbs-can inform about the dynamics and durability of nAb responses in humans, knowledge which is crucial for the design of future HIV-1 vaccine regimens. To address this, we assessed HIV-1-neutralizing immunoglobulin G (IgG) from 2,354 persons with HIV-1 on or off antiretroviral therapy (ART).

The effect of single mutations in Zika virus envelope on escape from broadly neutralizing antibodies.

The wide endemic range of mosquito-vectored flaviviruses-such as Zika virus and dengue virus serotypes 1-4-places hundreds of millions of people at risk of infection every year. Despite this, there are no widely available vaccines, and treatment of severe cases is limited to supportive care. An avenue toward development of more widely applicable vaccines and targeted therapies is the characterization of monoclonal antibodies that broadly neutralize all these viruses.