Antigenic fingerprinting of H5N1 avian influenza using convalescent sera and monoclonal antibodies reveals potential vaccine and diagnostic targets

Abstract

Background: Transmission of highly pathogenic avian H5N1 viruses from poultry to humans have raised fears of an impending influenza pandemic. Concerted efforts are underway to prepare effective vaccines and therapies including polyclonal or monoclonal antibodies against H5N1. Current efforts are hampered by the paucity of information on protective immune responses against avian influenza. Characterizing the B cell responses in convalescent individuals could help in the design of future vaccines and therapeutics.

Methods and findings: To address this need, we generated whole-genome-fragment phage display libraries (GFPDL) expressing fragments of 15-350 amino acids covering all the proteins of A/Vietnam/1203/2004 (H5N1). These GFPDL were used to analyze neutralizing human monoclonal antibodies and sera of five individuals who had recovered from H5N1 infection. This approach led to the mapping of two broadly neutralizing human monoclonal antibodies with conformation-dependent epitopes. In H5N1 convalescent sera, we have identified several potentially protective H5N1-specific human antibody epitopes in H5 HA[(-10)-223], neuraminidase catalytic site, and M2 ectodomain. In addition, for the first time to our knowledge in humans, we identified strong reactivity against PB1-F2, a putative virulence factor, following H5N1 infection. Importantly, novel epitopes were identified, which were recognized by H5N1-convalescent sera but did not react with sera from control individuals (H5N1 naïve, H1N1 or H3N2 seropositive).

Conclusion: This is the first study, to our knowledge, describing the complete antibody repertoire following H5N1 infection. Collectively, these data will contribute to rational vaccine design and new H5N1-specific serodiagnostic surveillance tools.

Figure 1. Epitope mapping of broadly neutralizing H5N1 human MAbs.

(A) End-point titers (mean of three replicates) using two H5N1 human MAbs (at 1 mg/ml) in a microneutralization assay performed with rgH5N1×PR8 (2∶6) reassorted viruses are shown. *, The information on in vivo protection of mice against wild-type A/Vietnam/1203/2004 and A/Indonesia/5/05 challenge was previously published . (B) HA segment [(-10)-233] was identified by GFPDL panning using MAb FLA5.10 (boxed). Amino acid number +1 corresponds to H3 (A/California/7/2004) amino acid -10. The critical contact residues for FLA5.10, identified using RPL are shown in red circles. (C) Alignment of the critical residues for MAb FLA5.10 binding on the 3-D structure of the HA monomer (Protein Data Base [PDB] identifier 21BX) with amino acid colors corresponding to (B). The predicted glycosylation sites (NXT/NXS) are shown in blue. (D) HA segment (32–320) identified by GFPDL-panning using MAb FLD21.140 (boxed). The putative contact residues identified using RPL, are shown in red circles. (E) Alignment of the critical residues for MAb FLD21.140 binding on the 3-D structure of the HA monomer (PDB Identifier 2IBX) with amino acid colors corresponding to (D). The predicted glycosylation sites (NXT/NXS) are shown in blue.

Figure 2. Steady-state binding equilibrium analysis of human MAbs to purified bacterially expressed H5 HA[(-10-223)] fragment.

Various concentrations of MAbs FLD21.140 (A) and FLA5.10 (B) were injected simultaneously onto recombinant HA [(-10)-223] (identified in Figure 1B) peptide, immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs). As a control, anti-CCR5 MAb 2D7) was injected at the same concentrations on HA [(-10)-223] coupled chip. RU, resonance units.

Figure 3. Elucidation of the epitope profile in HA and NA proteins recognized by antibodies in individuals that survived H5N1 infections in Vietnam.

(A) Alignment of peptides recognized by pooled sera from H5N1-infected individuals identified using H5 (HA+NA) GFPDL. Bars with arrows represent the identified inserts in 5′–3′ orientation. Numbered segments represent inserts that were selected with high frequencies (≥5; Table S1). These peptides were expressed and purified from E .coli or were chemically synthesized and used in ELISA. The sequences of the influenza encoded fragments are numbered according to the intact complete proteome (Figure S1). Peptide ID numbers are the same in (A) and (B). (B) ELISA reactivity of sera from individual H5N1-infected patients (Viet1–5) or pooled sera from 20 healthy Vietnamese adults (75% had neutralizing titers against either H3N2 influenza strains, H1N1 strains, or both) with H5N1 HA1 peptides (1–8), HA2 peptides (9–14), and NA peptides (15–21) (localization indicated in Figure 4A). An initial serum dilution (1∶100) was followed by serial 5-fold dilutions. End-point titers are reported. Days postadmission represent the time of serum collection for each patient. Six antigenic clusters in HA (cluster-I, 2,359–2,453; cluster-II, 2,454–2,621; cluster-III, 2,627–2,670; cluster-IV, 2,682–2,703; cluster-V, 2,706–2,814; cluster-VI, 2,823–2,816) recognized by the convalescent sera are shown.

Figure 4. Main antigenic clusters in the structures of HA and NA recognized by antibodies from H5N1 virus infected individuals.

(A) Antigenic clusters in HA, as identified in Figure 3B, are shown as surface exposed colored patches on one HA monomer within the HA trimer structure (PDB identifier 2IBX). The antigenic clusters (I–III) in HA1 cover the Antigenic Sites a, b, c, d, and e that have been described in the H3 HA1 based on mouse MAbs , . (B) The immunodominant conformational epitope in the NA (NA-3676-3854, peptide 15 in Figure 3B) is shown in green on the tetrameric NA structure (PDB Identifier 2HTY) with the predicted site of bound sialic acid shown in red. Side view and bird-eye views are shown.

Figure 5. Antibody epitopes in H5N1 internal proteins (FLU-6) recognized by pooled sera from H5N1 infected individuals.

(A) Schematic alignment of the peptides identified using GFPDL (H5N1-FLU-6) expressing all internal proteins of influenza A/Vietnam/1203/2004. The predicted influenza encoded proteins are numbered according to the complete proteome (Figure S1). Bars with arrows indicate identified inserts in the 5′–3′ orientation. Numbered segments represent high frequency clones (≥5; Table S1). These peptides were expressed and purified from E. coli or were chemically synthesized and the numbers correspond to the peptide identifiers in the ELISA assay in (B). (B) Reactivities of sera from individual H5N1-infected patients (Viet 1–5) or sera from healthy Vietnamese adults against peptides derived from: PB2 (1); PB1 (2–3); PB1-F2 (4–7); PA (8); NP (9–12); M1 (13–16); M2e (17); M2 (18); NS1 (19–20); and NS2 (21).