Antigen pressure from two founder viruses induces multiple insertions at a single antibody position to generate broadly neutralizing HIV antibodies.

Vaccination strategies aimed at maturing broadly neutralizing antibodies (bnAbs) from naïve precursors are hindered by unusual features that characterize these Abs, including insertions and deletions (indels). Longitudinal studies of natural HIV infection cases shed light on the complex processes underlying bnAb development and have suggested a role for superinfection as a potential enhancer of neutralization breadth. Here we describe the development of a potent bnAb lineage that was elicited by two founder viruses to inform vaccine design. The V3-glycan targeting bnAb lineage (PC39-1) was isolated from subtype C-infected IAVI Protocol C elite neutralizer, donor PC39, and is defined by the presence of multiple independent insertions in CDRH1 that range from 1-11 amino acids in length. Memory B cell members of this lineage are predominantly atypical in phenotype yet also span the class-switched and antibody-secreting cell compartments. Development of neutralization breadth occurred concomitantly with extensive recombination between founder viruses before each virus separated into two distinct population "arms" that evolved independently to escape the PC39-1 lineage. Ab crystal structures show an extended CDRH1 that can help stabilize the CDRH3. Overall, these findings suggest that early exposure of the humoral system to multiple related Env molecules could promote the induction of bnAbs by focusing Ab responses to conserved epitopes.

Differences in Protein Capture by SP3 and SP4 Demonstrate Mechanistic Insights of Proteomics Cleanup Techniques.

The goal of proteomics experiments is to identify proteins to observe changes in cellular processes and diseases. One challenge in proteomics is the removal of contaminants following protein extraction, which can limit protein identifications. Single-pot, solid-phase-enhanced sample preparation (SP3) is a cleanup technique in which proteins are captured on carboxylate-modified particles through a proposed hydrophilic-interaction-liquid-chromatography (HILIC)-like mechanism. Recent results have suggested that proteins are captured in SP3 due to a protein-aggregation mechanism. Solvent precipitation, single-pot, solid-phase-enhanced sample preparation (SP4) is a newer cleanup technique that employs protein aggregation to capture proteins without modified particles. We hypothesize that differences in capture mechanisms of SP3 and SP4 affect which proteins are identified by each cleanup technique. Herein, we assess the proteins identified and enriched using SP3 versus SP4 for MCF7 subcellular fractions and correlate protein capture in each method to protein hydrophobicity. Our results indicate that SP3 captures more hydrophilic proteins through a combination of HILIC-like and protein-aggregation mechanisms, while SP4 captures more hydrophobic proteins through a protein-aggregation mechanism. Ultimately, we demonstrate that protein-capture mechanisms are distinct, and the selection of a cleanup technique that yields high proteome coverage is dependent on protein-sample hydrophobicity. Data has been deposited into MassIVE (MSV000094130) and ProteomeXchange (PXD049965).

A Sulfonamide Sialoside Analogue for Targeting Siglec-8 and -F on Immune Cells.

The Siglec family of cell surface receptors have emerged as attractive targets for cell-directed therapies due to their restricted expression on immune cells, endocytic properties, and ability to modulate receptor signaling. Human Siglec-8, for instance, has been identified as a therapeutic target for the treatment of eosinophil and mast cell disorders. A promising strategy to target Siglecs involves the use of liposomal nanoparticles with a multivalent display of Siglec ligands. A key challenge for this approach is the identification of a high affinity ligand for the target Siglec. Here, we report the development of a ligand of Siglec-8 and its closest murine functional orthologue Siglec-F that is capable of targeting liposomes to cells expressing Siglec-8 or -F. A glycan microarray library of synthetic 9-N-sulfonyl sialoside analogues was screened to identify potential lead compounds. The best ligand, 9-N-(2-naphthyl-sulfonyl)-Neu5Acα2-3-[6-O-sulfo]-Galß1-4GlcNAc (6'-O-sulfo NSANeu5Ac) combined the lead 2-naphthyl sulfonyl C-9 substituent with the preferred sulfated scaffold. The ligand 6'-O-sulfo NSANeu5Ac was conjugated to lipids for display on liposomes to evaluate targeted delivery to cells. Targeted liposomes showed strong in vitro binding/uptake and selectivity to cells expressing Siglec-8 or -F and, when administered to mice, exhibit in vivo targeting to Siglec-F+ eosinophils.

Differences in Protein Capture by SP3 and SP4 Demonstrate Mechanistic Insights of Proteomics Clean-up Techniques.

The goal of proteomics experiments is to identify proteins to observe changes in cellular processes and diseases. One challenge in proteomics is the removal of contaminants following protein extraction, which can limit protein identification. Single-pot, solid-phase-enhanced sample preparation (SP3) is a clean-up technique in which proteins are captured on carboxylate-modified particles through a proposed hydrophilic-interaction-liquid-chromatography (HILIC)-like mechanism. However, recent results have suggested that proteins are captured in SP3 due to a protein-aggregation mechanism. Thus, solvent precipitation, single-pot, solid-phase-enhanced sample preparation (SP4) is a newer clean-up technique that employs protein-aggregation to capture proteins without modified particles. SP4 has previously enriched low-solubility proteins, though differences in protein capture could affect which proteins are detected and identified. We hypothesize that the mechanisms of capture for SP3 and SP4 are distinct. Herein, we assess the proteins identified and enriched using SP3 versus SP4 for MCF7 subcellular fractions and correlate protein capture in each method to protein hydrophobicity. Our results indicate that SP3 captures more hydrophilic proteins through a combination of HILIC-like and protein-aggregation mechanisms, while SP4 captures more hydrophobic proteins through a protein-aggregation mechanism. From these results, we recommend clean-up techniques based on protein-sample hydrophobicity to yield high proteome coverage in biological samples.

Evolution of human H3N2 influenza virus receptor specificity has substantially expanded the receptor-binding domain site.

Hemagglutinins (HAs) from human influenza viruses descend from avian progenitors that bind α2-3-linked sialosides and must adapt to glycans with α2-6-linked sialic acids on human airway cells to transmit within the human population. Since their introduction during the 1968 pandemic, H3N2 viruses have evolved over the past five decades to preferentially recognize human α2-6-sialoside receptors that are elongated through addition of poly-LacNAc. We show that more recent H3N2 viruses now make increasingly complex interactions with elongated receptors while continuously selecting for strains maintaining this phenotype. This change in receptor engagement is accompanied by an extension of the traditional receptor-binding site to include residues in key antigenic sites on the surface of HA trimers. These results help explain the propensity for selection of antigenic variants, leading to vaccine mismatching, when H3N2 viruses are propagated in chicken eggs or cells that do not contain such receptors.

Human Influenza Virus Hemagglutinins Contain Conserved Oligomannose N-Linked Glycans Allowing Potent Neutralization by Lectins.

Hemagglutinins (HAs) from human influenza viruses adapt to bind α2-6-linked sialosides, overcoming a receptor-defined species barrier distinct from the α2-3 specificity of avian virus progenitors. Additionally, human-adapted HAs gain glycosylation sites over time, although their biological function is poorly defined. Using quantitative glycomic analysis, we show that HAs from human pandemic viruses exhibit significant proportions of high-mannose type N-linked glycans throughout the head domain. By contrast, poorly adapted avian-origin HAs contain predominately complex-type glycans, which have greater structural diversity. Although oligomannose levels vary, they are present in all tested recombinant HAs and whole viruses and can be specifically targeted for universal detection. The positions of high-mannose glycosites on the HA of human H1N1 and H3N2 strains are conserved. Additionally, high-mannose-binding lectins possess a broad capacity to neutralize and prevent infection with contemporary H3N2 strains. These findings reveal the biological significance of HA glycosylation and therapeutic potential of targeting these structures.