Highly conserved protective epitopes on influenza B viruses

Abstract

Identification of broadly neutralizing antibodies against influenza A viruses has raised hopes for the development of monoclonal antibody-based immunotherapy and "universal" vaccines for influenza. However, a substantial part of the annual flu burden is caused by two cocirculating, antigenically distinct lineages of influenza B viruses. Here, we report human monoclonal antibodies, CR8033, CR8071, and CR9114, that protect mice against lethal challenge from both lineages. Antibodies CR8033 and CR8071 recognize distinct conserved epitopes in the head region of the influenza B hemagglutinin (HA), whereas CR9114 binds a conserved epitope in the HA stem and protects against lethal challenge with influenza A and B viruses. These antibodies may inform on development of monoclonal antibody-based treatments and a universal flu vaccine for all influenza A and B viruses.

Fig. 1. In vitro binding and neutralizing activity of CR8033, CR8071 and CR9114

(A) Relative binding potency (Kd) for binding of CR8033, CR8071 and CR9114 to a wide range of influenza A and B hemagglutinins (HAs) as determined using a fluorescence-based plate assay. The HAs were selected to cover the two lineages and main phylogenetic branches of influenza B, as well as the major subtypes of influenza A that have infected humans. Values are from one representative of three independent experiments and reported in nM. (B) Dendrogram of all non-redundant, full-length influenza B HA sequences in the National Center for Biotechnology Information Flu database (85). The positions in the phylogenetic tree of the influenza B HAs used in these studies are indicated. (C) Fifty percent inhibitory concentrations (IC₅₀, μg/ml) of CR8033, CR8071 and CR9114 against representative strains from the Yamagata and Victoria lineages of influenza B and the H1N1 and H3N2 subtypes of influenza A as determined by means of microneutralization (MN) and hemagglutination inhibition (HI) assays. Values are representative of three independent experiments and reported in micrograms per milliliter.n.a. = not applicable.

Fig. 2. In vivo efficacy of CR8033, CR8071 and CR9114

Efficacy of CR8033 (A), CR8071 (B), and CR9114 (C), against lethal challenge with mouse-adapted B/Florida/4/2006 (left panels), or B/Malaysia/2506/2004 (right panels) virus, and (D) of CR9114 against mouse-adapted A/Puerto Rico/8/1934 (left panel), or A/Hong Kong/1/1968 virus (right panel). Survival curves of mice (10 animals per group in A, B and C, 8 per group in D) treated with the indicated doses of CR8033, CR8071, or CR9114 are shown, or vehicle control 24 hours before challenge by intranasal inoculation (at day 0). Asterisks indicate significant improvements in survival proportions at day 21, compared to vehicle control (p<0.05).

Fig. 3. CR8033, CR8071 and CR9114 bind to distinct epitopes on influenza B HA and conservation of the CR9114 neutralizing epitope on influenza A and B

(A) Surface representation illustrating the neutralizing epitopes on HA B/Brisbane/60/2008 of CR8033, CR8071 and CR9114 as determined from the EM structures in (B) and the crystal structures of CR9114 in Fig 3C and CR8071 in fig. S8. The EM density maps allow unambiguous fits of known structures with good correspondence with the CR9114 epitope defined by both x-ray crystallography and EM, despite differences in resolution. The structure is colored by conservation of contact residues across all available influenza B virus sequences. Red = over 98% conserved; orange = 75-98% conserved; yellow = 50-75% conserved (B) Negative stain EM reconstructions (gray mesh) of CR8033 (left), CR8071 (middle) and CR9114 (right) in complex with B/Florida/4/2006. Side (top) and overhead (bottom) views show the fits of the individual crystal structures of the Fabs and flu B HA to the EM density. (C) Crystal structure of CR9114 in complex with H5 HA (group 1). One HA/Fab protomer of the trimeric complex is colored with HA1 in magenta, HA2 in cyan, Fab heavy chain in green, Fab light chain in yellow, and N-linked glycans in colored balls representing their atom type. The other two protomers are colored in gray. (D) Conservation of CR9114 contact residues across all 16 influenza A subtypes (left) and between influenza A and B viruses (right). Red, orange, yellow correspond to coloring used in Fig. 3A with green = 25-50% conserved. Carbohydrates positions are represented by a cross colored in black (group 1), cyan (group 2), green (group 1+2), orange (influenza B), and blue (influenza A and B). Residue numbers are shown with HA1 in magenta and HA2 in black. (E) Illustration of cross-reactivity of CR9114 for influenza A H1, H3, H7 and H9 subtypes using negative stain EM (from left to right). Single particle reconstructions of negatively stained CR9114 Fabs bound to HA trimers from four major subtypes of influenza A that have infected humans (SC1918/H1, group 1; HK68/H3, group 2; Neth03/H7, group 2; and Wisc66/H9, group 1). The HA trimers are colored in different shades of blue. The Fabs of CR9114 (three per trimer) are colored in purple (the third Fab, at the back, has been omitted for clarity). Broadly neutralizing CR9114 binds in a structurally similar manner to the stem region across all subtypes, groups and classes of HA. Additional density in the HA not accounted for by the protein likely corresponds to glycosylation.

Fig. 4. Neutralization mechanisms of CR8033, CR8071 and CR9114

(A) CR9114 protects HAs of group 1 (A/South Carolina/1/1918 (H1N1) and A/Vietnam/1203/2004 (H5N1)) and group 2 (A/Netherlands/219/2003 (H7N7) and A/Hong Kong/1/1968 (H3N2)) influenza A viruses from the pH-induced protease sensitivity associated with membrane fusion. Exposure to low pH converts the HAs to the post-fusion state, rendering them sensitive to trypsin digestion (lane 1 vs. 3), but CR9114 prevents this conversion, retaining the HA in the protease-resistant, pre-fusion form (lane 2). CR8033 and CR8070 do not prevent this conversion at low pH (right panel) (N=4). (B) Expression of influenza NP in monolayers of MDCK cells 16-18 hrs after inoculation with B/Florida/4/2006 or B/Malaysia/2506/2004 viruses that were pre-incubated with CR8033, CR8071, CR9114, or polyclonal sheep sera directed against B/Florida/4/2006 or B/Malaysia/2506/2004, as indicated. NP expression is determined by immunofluorescence. Representative images of three independent experiments are shown. (C) Immunoblots of uncleaved HA (HA0) detected in the lysate and supernatant of MDCK cells infected with B/Florida/4/2006 virus and subsequently (from 3 to 20 hours post infection) incubated with different concentrations of antibodies or zanamivir as indicated. HA0 was detected using rabbit serum against B/Jiangsu/10/03 (Yamagata lineage). Concentrations are in μg/ml and μM for antibodies and zanamivir, respectively. Results from one representative of two independent experiments are shown. (D) SEM images of the surface of MDCK cells infected with B/Florida/4/2006 virus and subsequently (from 3 to 20 hours post infection) incubated with CR8071 (10 μg/ml), CR8033 (2.5 μg/ml), or zanamivir (60 μM). Representative images of three independent experiments are shown. Scale bar 1 μm.