Influenza virus hemagglutinin stalk-based antibodies and vaccines

Abstract

Antibodies against the conserved stalk domain of the hemagglutinin are currently being discussed as promising therapeutic tools against influenza virus infections. Because of the conservation of the stalk domain these antibodies are able to broadly neutralize a wide spectrum of influenza virus strains and subtypes. Broadly protective vaccine candidates based on the epitopes of these antibodies, for example, chimeric and headless hemagglutinin structures, are currently under development and show promising results in animals models. These candidates could be developed into universal influenza virus vaccines that protect from infection with drifted seasonal as well as novel pandemic influenza virus strains therefore obviating the need for annual vaccination, and enhancing our pandemic preparedness.

Figure 1. The structure of the influenza virus hemagglutinin (HA)

A The influenza virus HA can be divided into the highly divergent membrane distal globular head domain (red) and the conserved membrane proximal stalk domain (green). Cysteines 52 and 277 (H3 numbering) form a disulfide bond that serves as demarcation line between the two subdomains (PDB 1RU7). B Structure of homotrimeric HA molecule. Globular head domains are shown in red, stalk domains are shown in green. C Structure of an H2 HA in complex with an Fab fragment of the broadly neutralizing mAb C179 (PDB 4HLZ). The HA1 subunit is shown in purple, HA2 is shown in turquoise. Heavy (red) and light (blue) chain of the antibody make contacts with a conformational epitope on the HA stalk domain that is formed by the HA1 and HA2 subunit (21). D Schematic of the head and stalk domains of the HA. The stalk domain (green) covers the C- and N-terminal part of the HA1 and most of the HA2 (excluding the transmembrane domain, TMD and the cytoplasmic tail, CT). The globular head domain (red) is located between cysteines 52 and 277 (H3 numbering) on the HA1 subunit. E Phylogenetic tree of influenza virus HAs. Influenza A virus HAs can be divided into group 1 and group 2 based on their sequence. Influenza A virus subtypes that currently circulate in humans or that have historically demonstrated their potential to do so are marked in red. Subtypes with pandemic potential are marked in green. The tree was built using ClustalW and was visualized in FigTree. The scale bar represents a 7% change in amino acid.

Figure 2. Alternative mechanisms of neutralization

A Classical HI active antibodies (in blue) neutralize by inhibiting attachment of the viral HA to sialylated host cell receptors and block entry at an early stage. B Stalk-reactive antibodies (green) bind to HA on the virus surface and may be taken up with the virus into the endosome. During acidification of the endosome they may prevent conformational change of the HA and inhibit release of the viral genome into the cytosol. C Broadly neutralizing antibodies may also inhibit viral egress. D Stalk-reactive antibodies may inhibit HA maturation by sterical hindrance of the interaction of host proteases with the HA cleavage site. E Stalk-reactive antibodies may also work through ADCC, infected cells as well as viruses are killed/cleared by macrophages and natural killer (NK) cells. F Stalk-reactive antibodies have been shown to trigger complement mediated lysis of infected cells and could potentially also help to clear influenza virus particles.

Figure 3. Boost of stalk-reactive antibodies in nature during the 2009 H1N1 pandemic

Individuals sequentially exposed (through vaccination or infection) to pre-pandemic seasonal H1N1 strains with slightly drifted globular head domains developed mainly an immune response against the globular head of the HA. Infection with pandemic H1N1 virus which features a very divergent H1 head domain (approx. 30% difference as compared to pre-pandemic seasonal strains) induced only a primary response against the immunodominant globular head domain but boosted immune responses directed against the conserved stalk domain. Structures are based on PDB 1RU7.

Figure 4. Design of stalk-based immunogens

A/E Vaccines based on the HA2 subunit only were the earliest candidate vaccines used to induce cross-reactive antibodies. B/F Headless HA constructs span the whole stalk domain including HA1 and HA2 parts of the stalk. The globular head domain located between cysteines 52 and 277 (H3 numbering) was replaced by a glycine linker. Structures shown in B/F and C/G are likely to be mis-folded if expressed on their own. C/G The long alpha helix (LAH) of the HA2 hosts the epitope of broadly neutralizing antibody 12D1 and was successfully used as vaccine construct. It spans amino acid 404 to 458 (H3 numbering) (28). D/H Chimeric HA molecules consist of either an H1 or an H3 stalk domain combined with ‘exotic’ globular head domains. Here, cH6/1 (H6 head on top of an H1 stalk) is shown as an example. The H6 globular head domain (blue) is located between conserved cysteines 52 and 277 (H3 numbering). TMD is transmembrane domain; CT is cytoplasmic tail. Structures are based on PDB 1RU7.

Figure 5. Vaccination strategies based on chimeric HAs

A Example for a cHA vaccination strategy in naive animals. Animals are primed with a cH9/1 construct (H9 head (black) on top of an H1 stalk (green)). The prime induces a strong primary response against the immunodominant globular head domain and an almost undetectable response against the immunosubdominant stalk domain. Animals are then boosted with a cH5/1 construct (H5 head (golden) on top of an H1 stalk domain (green)). Again, a primary response is developed against the divergent globular head but antibodies against the conserved stalk domain are boosted. A second boost with cH6/1 HA (H6 head (blue) on top of an H1 stalk domain (green)) induces a primary response against the divergent H6 head domain but strongly boosts titers of antibodies directed to the conserved stalk domain. B Similar vaccination strategies could be employed for humans. It is likely that a prime is not necessary since most individuals have been exposed to human influenza viruses and have therefore a low but detectable pre-existing immunity directed toward the stalk domain. Individuals can be boosted once (e.g. with cH5/1 HA) or twice (e.g. with cH5/1 and cH6/1 HA) to induce high, broadly neutralizing antibody titers. Structures are based on PDB 1RU7.