Stalking influenza.

I nfluenza A virus causes seasonal flu epidemics and periodic worldwide pandemics (e.g., the 1918 Spanish flu, which caused ≈50 million deaths). The viral surface protein HA is the primary target of neutralizing Abs in natural infections (1). At any given time there are a limited number of viral strains circulating in humans, restricting widespread immunity to a small subset of potential viruses. Yearly seasonal epidemics arise from antigenic drift in the sequence of HA of currently circulating viruses, whereas pandemics are caused by the emergence of new, antigenically divergent viruses to which there is little to no immunity in the population (i.e., antigenic shift). The worldwide spread of a new H1N1 virus in 2009 caused the first recorded pandemic in more than 40 years. Current influenza vaccines are primarily produced from killed virus and mimic natural infection, inducing strain-specific, mainly HA-based, neutralization. Vaccine is produced from representative circulating strains grown in chicken eggs in a months-long process. Recent efforts aim to produce a broader influenza vaccine that focuses on common neutralization epitopes shared by multiple influenza strains. Such a vaccine should target a variety of influenza strains and better combat pandemics. A study published in PNAS describes an exciting strategy toward developing a broader influenza vaccine (2). HA is trimeric and comprises a receptorbinding surface subunit (HA1) and a transmembrane subunit (HA2) that mediates entry after exposure to low pH in the endosome. Decades of HA structural characterization have defined the steps of membrane fusion and the epitopes of neutralizing Abs (Fig. 1) (1). HA is produced as a single-chain precursor with an HA1 cap covering an HA2 stalk. Cleavage leads to burial of the N-terminal HA2 fusion peptide and primes HA for fusion. Exposure to low pH induces large conformational changes in HA2 that propel the fusion peptide to the host endosomal membrane and lead to membrane fusion. The epitopes for most strain-specific neutralizing Abs are on the variable HA1 cap, and these Abs typically prevent receptor binding (3, 4). However, a handful of more broadly neutralizing monoclonal Abs have recently been identified that bind to the more conserved HA2 stalk (5–8). Designing an HA2-based immunogen to elicit these broadly neutralizing Abs is a high priority in the field. Without HA1, HA2 folds into its most stable postfusion (low pH) conformation (9), making it challenging to produce HA2 antigen in its prefusion (neutral pH) conformation in the absence of HA1. Using structures of preand postfusion HA as a guide, Bommakanti et al. designed and produced an Escherichia coli-expressed single-chain antigen, HA6 (2). HA6 comprises the majority of the HA2 stalk and two smaller segments of HA1 that stabilize the stalk in the prefusion structure (Fig. 1). Importantly, introduced mutations (i) remove hydrophobic residues that become exposed upon deletion of the remainder of HA to improve solubility, and (ii) place charged residues at buried sites in the lowpH conformation of HA2 to destabilize this state. Standard biochemical and Low pH Pre-Fusion Neutral pH Post-Fusion 1 1 328 221 S S A