Research Article: Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes

Date Published: June 12, 2014

Publisher: Public Library of Science

Author(s): Javier G. Magadán, Meghan O. Altman, William L. Ince, Heather D. Hickman, James Stevens, Aaron Chevalier, David Baker, Patrick C. Wilson, Rafi Ahmed, Jack R. Bennink, Jonathan W. Yewdell, James E. Crowe.


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.

Partial Text

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 infection-induced 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].

Although many questions remain to be answered, there is a tremendous enthusiasm regarding the potential of StRAbs to improve IAV vaccination. At the very least, StRAbs should be useful therapeutically in severe influenza. At the very most, vaccines that effectively induce StRAbs could provide long lasting protection and greatly diminish the annual toll of seasonal IAV and prevent pandemics arising from introduction of novel HA subtypes into the human population from the enormous animal reservoir [39], [40].




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