Abstract
Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.
Highlights
influenza A virus (IAV) is responsible for considerable human morbidity and mortality, with enormous attendant economic costs
Recent findings demonstrate that stem-reactive antibodies (StRAbs), specific Abs to the highly conserved stem region of HA, can protect hosts against a broad variety of influenza virus strains
In investigating the binding of StRAbs to HA during its biogenesis in IAVinfected cells, we find that these Abs can bind HA monomers prior to their trimerization in the Golgi complex (GC)
Summary
IAV is responsible for considerable human morbidity and mortality, with enormous attendant economic costs. Current vaccines are at best effective only for a few years due to the evolution of human IAV strains that escape vaccine- or infectioninduced immunity. The most relevant immune escape occurs in the HA, a homotrimeric viral surface type I integral membrane glycoprotein. HA initiates IAV infection by attaching virus to host cell sialic acid receptors and fusing viral and host membranes in acidifying endosomes. Abs to HA neutralize infectivity by blocking virus attachment or acid-triggered conformational changes on HA that mediate host-viral membranes fusion [1]. Most neutralizing anti-HA Abs induced in experimental animals and humans are specific for the HA globular domain, which correlates well with the focus of the immune driven evolution on residues within this domain, in the major antigenic regions (termed Sa, Sb, Ca1, Ca2, and Cb in H1 subtype HAs). Monoclonal Abs (mAbs) of defined fine-specificity have proven to be invaluable reagents for studying HA antigenicity [3,4,5,6] and HA structure [7], function, and biogenesis in biochemical and immunohistochemical studies [8,9,10]
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