Antibody recognition of a highly conserved influenza virus epitope - PubMed (original) (raw)

Antibody recognition of a highly conserved influenza virus epitope

Damian C Ekiert et al. Science. 2009.

Abstract

Influenza virus presents an important and persistent threat to public health worldwide, and current vaccines provide immunity to viral isolates similar to the vaccine strain. High-affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Cocrystal structures were determined at 2.2 and 2.7 angstrom resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to other structurally characterized influenza antibodies, CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1 and HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibody-based therapies for the treatment of influenza.

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Figures

Figure 1

Figure 1. Crystal structures of broadly neutralizing antibody CRF6261 in complex with SC1918/H1 and Viet04/H5 HAs

A) The trimeric CRF6261-SC1918/H1 complex. One HA/Fab protomer is colored with HA1 in purple, HA2 in cyan, Fab heavy chain in yellow, and Fab light chain in orange; the other two HA monomers and Fabs in the trimer are colored in grey for clarity. Glycans are depicted as their component atoms (carbon in green, oxygen in red and nitrogen in blue) with their van der Waals’ radii. B) The trimeric CRF6261-Viet04 complex, colored as in A. The HA1 globular heads are located at the top of the figure and the membrane-proximal stem is at the bottom where the Fab is bound.

Figure 2

Figure 2. Broadly neutralizing CR6261 binds in the HA stem distant from other strain-specific antibodies using only its heavy chain

A) Comparison of the binding sites of Fab CR6261 (yellow and orange, as in Fig. 1)) that recognizes the HA stem and strain-specific antibodies that bind to the HA1 globular head domain The strain-specific antibody structures are depicted in green (BH151, PDB code 1EO8), copper (HC63, PDB code 1KEN), dark red (HC45, PDB code 1QFU), and blue (HC19, PDB code 2VIR). The HA trimer is shown as a surface representation, with the HA1 and HA2 from one protomer colored in purple and cyan, respectively. B) A close up view of the interaction of CR6261 with the A-helix. HA1 and HA2 are colored in purple and cyan, respectively. The HCDRs 1, 2, and 3 of the CR6261 VH domain are highlighted in red, blue, and green, respectively. In addition, the 72–73 loop from FR3 of VH, which is structurally analogous to the CDR HV4 of a TCR, is indicated in purple. CDR1 runs along the side of the A-helix, interacting with five consecutive helical turns. In contrast, the light chain makes no contacts with the HA and is separated from the nearest HA side chain by ∼8 Å.

Figure 3

Figure 3. Interaction of CR6261 with SC1918/H1 and Viet04/H5 HA illustrating the neutralizing epitope

Surface representations of the interaction of CR6261 with SC1918/H1 (A–C) and Viet04/H5 (D–F). The coloring in A, B, D, and E corresponds to Fig. 1, with interacting epitope residues highlighted in a darker shade. Atoms making polar contacts are highlighted in red. C and F depict the electrostatic potential surface of the epitope on the HA (red – negative; blue-positive; grey-neutral) with HCDRs 1, 2, 3, and FR3 (CDR “HV4”) represented as sticks and colored blue, red, green, and purple, as in Fig. 2. Key interacting residues on the antibody and the HA are indicated on the top of the surface with labels.

Figure 4

Figure 4. CR6261 recognizes a functionally conserved epitope in the stalk region and inhibits the pH-induced conformational changes in the SC1918/H1 and Viet04/H5 HAs

CR6261 protects SC1918/H1 (A) and Viet04/H5 (B) HAs from the pH-induced protease sensitivity associated with membrane fusion. Exposure to low pH renders the SC1918-H1 and Viet04-H5 HAs sensitive to trypsin digestion (lanes 7–8 vs. 9), but CR6261 prevents conversion to the protease susceptible conformation (lanes 10–12). Note that the CR6261-SC1918-H1 crystals were grown at pH 5.3 that also indicates CR6261 blocks the extensive pH-induced conformational changes. C) Titration of SC1918-H1 trypsin-resistance versus varying pH-treatments (followed by neutralization to pH 8.0). The pH resulting in 50% conversion to protease sensitive conformation is 5.76 (95% confidence interval, 5.70–5.82). D) Superposition of the A-helix from SC1918/H1 (green) and an H3 HA (yellow, PDB code 2VIU) reveals that the CR6261 interacting surface is highly conserved among all subtypes. Helix positions are labeled according to the SC1918/H1 sequence, with the percent similarity across all subtypes (H1-16, from an analysis of 5261 sequences) indicated in parentheses. E–F) HA2 undergoes a dramatic and irreversible conformational change between the pre- and post-fusion states. This results in the translocation of the A-helix (red) from its initial position near the viral envelope (Fig. E) towards the target membrane at the opposite end of the HA trimer (Fig. F). The zipping up of coil H along the outside of the A and B-helices is thought to drive the fusion reaction. The orientation of helix C (yellow) is roughly identical in Figs. E and F. G) Polar residues on the CR6261-interacting surface of the A-helix form a network of interactions with coil H in the post-fusion state. These residues are well positioned to play a critical role in the late stages of membrane fusion, explaining the exceptional conservation the CR6261 epitope on the A-helix.

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