Biology subjects

Guthmiller, J. J.

Publications and source records attributed to Guthmiller, J. J..

8 recordsLinked to original sources

bioRxiv2026

Structural insights into antibody responses against influenza A virus in its natural reservoir

While influenza A virus undergoes rapid antigenic drift in humans, at least some subtypes, such as H3, have relatively stable antigenicity in natural waterfowl reservoirs, despite the presence of immune pressure. However, the underlying mechanisms remain poorly understood. This study identified and characterized 187 antibodies to H3 hemagglutinin from experimentally infected mallard ducks, 18 of which were further analyzed by cryo-EM. Compared with human H3 antibodies, duck H3 antibodies exhibited higher glycan-binding propensity, more balanced immunodominance hierarchy, and targeted distinct epitopes. Other unique features of duck H3 antibodies included a convergent CDR H3-independent heavy chain-only binding mode and an N-glycosylated CDR H3 as decoy receptor. By annotating duck immunoglobulin germline genes, we also demonstrated the importance of gene conversion in duck H3 antibodies. Overall, our findings provide insights into how millennia of coevolution have shaped the interplay between influenza A virus antigenic drift and antibody responses in the natural reservoir.

immunology↗

bioRxiv2024

Virus-free continuous directed evolution in human cells using somatic hypermutation

The B cells of the human immune system have evolved somatic hypermutation (SHM) mechanisms that introduce mutations at the immunoglobulin genomic loci at a significantly higher frequency than the rest of the genome thereby allowing them to evolve antibody sequences without compromising fitness due to genome-wide mutations. Inspired by these observations, here we developed a continuous directed evolution platform in human B cell lines (CODE-HB) that repurposes the SHM mechanisms to a stable, non-immunoglobulin genomic locus of the human B cell lines to continuously evolve cytosolic and surface displayed proteins with a broad mutational spectrum comprising of substitutions, deletions and insertions. We developed a human B cell surface display platform and used CODE-HB to evolve neutralizing antibodies targeting avian influenza and escape variants of influenza. Given the modularity and simplicity of CODE-HB, we anticipate that this platform can be used for rapidly evolving biotechnologically relevant biomolecules directly in human cells.

synthetic biology↗

bioRxiv2024

A single mutation in dairy cow-associated H5N1 viruses increases receptor binding breadth

Clade 2.3.4.4b H5N1 is causing an unprecedented outbreak in dairy cows in the United States. To understand if recent H5N1 viruses are changing their receptor use, we screened recombinant hemagglutinin (HA) from historical and recent 2.3.4.4b H5N1 viruses for binding to distinct glycans bearing terminal sialic acids. We found that H5 from A/Texas/37/2024, an isolate from the dairy cow outbreak, has increased binding breadth to glycans bearing terminal 2,3 sialic acids, the avian receptor, compared to historical and recent 2.3.4.4b H5N1 viruses. We did not observe any binding to 2,6 sialic acids, the receptor used by human seasonal influenza viruses. We identified a single mutation outside of the receptor binding site, T199I, was responsible for increased binding breadth, as it increased receptor binding site flexibility. Together, these data show recent H5N1 viruses are evolving increased receptor binding breadth which could impact the host range and cell types infected with H5N1.

microbiology↗

bioRxiv2021

Librator, a platform for optimized sequence editing, design, and expression of influenza virus proteins

Artificial mutagenesis and chimeric/mosaic protein engineering have laid the foundation for antigenic characterization1 and universal vaccine design2-4 for influenza viruses. However, many methods used for influenza research and vaccine development require sequence editing and protein expression, limiting their applicability and the progress of related research to specialists. Rapid tools allowing even novice influenza researchers to properly analyze and visualize influenza protein sequences with accurate nomenclature are needed to expand the research field. To address this need, we developed Librator, a system for analyzing and designing protein sequences of influenza virus Hemagglutinin (HA) and Neuraminidase (NA). With Librators graphical user interface (GUI) and built-in sequence editing functions, biologists can easily analyze influenza sequences and phylogenies, automatically port sequences to visualize structures, then readily mutate target residues and design sequences for antigen probes and chimeric/mosaic proteins efficiently and accurately. This system provides optimized fragment design for Gibson Assembly5 of HA and NA expression constructs based on peptide conservation of all historical HA and NA sequences, ensuring fragments are reusable and compatible, allowing for significant reagent savings. Use of Librator will significantly facilitate influenza research and vaccine antigen design.

microbiology↗

bioRxiv2021

An egg-derived sulfated N-Acetyllactosamine glycan is an antigenic decoy of influenza virus vaccines

Influenza viruses grown in eggs for the purposes of vaccine generation often acquire mutations during egg adaptation or possess differential glycosylation patterns than viruses circulating amongst humans. Here, we report that seasonal influenza virus vaccines possess an egg-derived sulfated N-acetyllactosamine (LacNAc) that is an antigenic decoy. Half of subjects that received an egg-grown vaccine mounted an antibody response against this egg-derived antigen. Egg-binding monoclonal antibodies specifically bind viruses grown in eggs, but not viruses grown in other chicken derived cells, suggesting only egg-grown vaccines can induce anti-LacNAc antibodies. Notably, antibodies against the sulfated LacNAc utilized a restricted antibody repertoire and possessed features of natural antibodies, as most antibodies were IgM and have simple heavy chain complementarity determining region 3. By analyzing a public dataset of influenza virus vaccine induced plasmablasts, we discovered egg-binding public clonotypes that were shared across studies. Together, this study shows that egg-grown vaccines can induce antibodies against an egg-associated glycan, which may divert the host immune response away from protective epitopes.

immunology↗

bioRxiv2021

A public broadly neutralizing antibody class targets a membrane-proximal anchor epitope of influenza virus hemagglutinin

Broadly neutralizing antibodies against influenza virus hemagglutinin (HA) have the potential to provide universal protection against influenza virus infections. Here, we report a distinct class of broadly neutralizing antibodies targeting an epitope toward the bottom of the HA stalk domain where HA is "anchored" to the viral membrane. Antibodies targeting this membrane-proximal anchor epitope utilized a highly restricted repertoire, which encode for two conserved motifs responsible for HA binding. Anchor targeting B cells were common in the human memory B cell repertoire across subjects, indicating pre-existing immunity against this epitope. Antibodies against the anchor epitope at both the serological and monoclonal antibody levels were potently induced in humans by a chimeric HA vaccine, a potential universal influenza virus vaccine. Altogether, this study reveals an underappreciated class of broadly neutralizing antibodies against H1-expressing viruses that can be robustly recalled by a candidate universal influenza virus vaccine.

immunology↗

bioRxiv2020

SARS-CoV-2 infection severity is linked to superior humoral immunity against the spike

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity and kinetics of the antibody response mounted against this novel virus are not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and non-structural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.

immunology↗

bioRxiv2020

Biochemical Patterns of Antibody Polyreactivity Revealed Through a Bioinformatics-Based Analysis of CDR Loops

Antibodies are critical components of adaptive immunity, binding with high affinity to pathogenic epitopes. Antibodies undergo rigorous selection to achieve this high affinity, yet some maintain an additional basal level of low affinity, broad reactivity to diverse epitopes, a phenomenon termed "polyreactivity". While polyreactivity has been observed in antibodies isolated from various immunological niches, the biophysical properties that allow for promiscuity in a protein selected for high affinity binding to a single target remain unclear. Using a database of nearly 1,500 polyreactive and non-polyreactive antibody sequences, we created a bioinformatic pipeline to isolate key determinants of polyreactivity. These determinants, which include an increase in inter-loop crosstalk and a propensity for an "inoffensive" binding surface, are sufficient to generate a classifier able to identify polyreactive antibodies with over 75% accuracy. The framework from which this classifier was built is generalizable, and represents a powerful, automated pipeline for future immune repertoire analysis.

immunology↗