Bacterial HA1 vaccine against pandemic H5N1 influenza virus: evidence of oligomerization, hemagglutination, and cross-protective immunity in ferrets

Abstract

The impending influenza virus pandemic requires global vaccination to prevent large-scale mortality and morbidity, but traditional influenza virus vaccine production is too slow for rapid responses. We have developed bacterial systems for expression and purification of properly folded functional hemagglutinin as a rapid response to emerging pandemic strains. A recombinant H5N1 (A/Vietnam/1203/2004) hemagglutinin globular domain (HA1) was produced in Escherichia coli under controlled redox refolding conditions. Importantly, the properly folded HA1(1-320), i.e., HA1 lacking amino acids 321 to 330, contained ≥75% functional oligomers without addition of foreign oligomerization sequence. Site-directed mutagenesis mapped the oligomerization signal to the HA1 N-terminal Ile-Cys-Ile residues at positions 3 to 5. The purified HA1 oligomers (but not monomers) bound fetuin and agglutinated red blood cells. Upon immunization of rabbits, the oligomeric HA1(1-320) elicited potent neutralizing antibodies against homologous and heterologous H5N1 viruses more rapidly than HA1(28-320) containing only monomers. Ferrets vaccinated with oligomeric HA1 (but not monomeric HA1 with the N terminus deleted) at 15 and 3 μg/dose were fully protected from lethality and weight loss after challenge with homologous H5N1 (A/Vietnam/1203/2004, clade 1) virus, as well as heterologous clade 2.2 H5N1 (A/WooperSwan/Mongolia/244/2005) virus. Protection was associated with a significant reduction in viral loads in the nasal washes of homologous and heterologous virus challenged ferrets. This is the first study that describes the presence of an N-terminal oligomerization sequence in the globular domain of influenza virus hemagglutinin. Our findings suggest that functional oligomeric rHA1-based vaccines can be produced efficiently in bacterial systems and can be easily upscaled in response to a pandemic influenza virus threat.

FIG. 1.

Biochemical and functional characterization of bacterially expressed and purified H5N1 HA proteins. (A) A panel of A/Vietnam/1203/2004 (H5N1) HA1 domain (amino acids 1 to 330) and N- and C-terminus deletions were expressed in E. coli as fusion proteins with a His₆ tag at the C termini. The purified proteins ran as single bands at the expected molecular weights in reducing SDS-PAGE (see Fig. S2 in the supplemental material). (B) Steady-state binding equilibrium analysis of human H5N1 neutralizing MAb FLA5.10 (10 μg/ml) to purified bacterially expressed H5N1 HA1 proteins immobilized on a sensor chip through the free amine group and onto a blank flow cell, free of peptide. H5N1 vaccine from the reasserted virus rgH5N1xPR8 (2:6) A/Vietnam/1203/2004 (clade 1) from Sanofi Pasteur was also analyzed. Binding was recorded using a ProteOn system SPR biosensor instrument. Similar results were obtained with two additional broadly neutralizing human MAbs FLD21.140 and FLA3.14 (see Fig. S3 in the supplemental material). (C) Agglutination of human RBC by properly folded bacterial H5N1 HA1(1-330) protein and its deletion derivatives, along with H5N1 vaccine. Serial dilutions of purified HA1 proteins were mixed with washed RBCs, and the hemagglutination was deter- mined after 30 min at room temperature. Reassorted virus rgH5N1xPR8 (2:6) A/Vietnam/1203/2004 (clade 1.0) was used as a positive control. H5N1 vaccine was used at a starting concentration of 1 μg/ml. (D) H5N1-neutralizing MAb FLA 5.10 specifically blocks the agglutination of human RBCs by recombinant HA1(1-330) and HA1(1-320) proteins and of rgH5N1xPR8 virus. Twofold serial dilutions of MAb FLA5.10 were preincubated with purified HA1 proteins or virus before mixing with washed RBCs.

FIG. 2.

Characterization of purified H5N1 HA proteins from E. coli and H5N1 vaccine by gel filtration chromatography, reducing and native gel electrophoresis. Superdex S-200 gel filtration chromatography of bacterial H5N1 HA proteins and H5N1 vaccine was performed. Purified H5N1 HA1 proteins with intact N termini (1-320) (A), HA1 with N-terminal deletions (5-320) (B) and (28-320) (C), HA1 N-terminal peptide (1-104) (D), and H5N1 vaccine from the reassorted virus rgH5N1xPR8 (2:6) A/Vietnam/1203/2004 (clade 1) from Sanofi Pasteur (E) were each subjected to gel filtration. The panels present superimposed elution profiles of purified HA proteins (red line) overlaid with calibration standards (gray line). The elution volumes of protein species are shown in parentheses. (F and G) SDS-PAGE analysis of bacterially purified H5N1 HA1 protein forms and H5N1 vaccine in SDS-reducing gel (F) and native gel (G). Different forms of bacterial produced H5N1 HA 1-320 were purified from Superdex S200 XK 26/60 column (GE Healthcare) and subjected to gel analysis, along with the H5N1 vaccine from the reassorted rgH5N1xPR8 (2:6) A/Vietnam/1203/2004 virus.

FIG. 3.

Functional activities of H5N1 HA1 monomers and oligomers in receptor binding and hemagglutination. (A and B) Binding kinetics of purified H5N1 HA1 proteins and its mutants in an SPR-based receptor-binding assay. Steady-state equilibrium analysis of different H5N1-HA1 proteins to fetuin and its asialylated counterpart (asialo-fetuin) was analyzed at 25°C using a ProteOn SPR biosensor. Samples of purified bacterial H5N1-HA1 proteins and H5N1 vaccine (10 μg/ml) were injected simultaneously over a mock surface to which no protein was bound, followed by fetuin (A) or asialo-fetuin (B) immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of protein. Binding kinetics and data analysis were performed by using a ProteOn system SPR biosensor instrument. (C) Monomers, trimers, and oligomers of properly folded bacterially expressed H5N1 HA1(1-320) were purified using size exclusion chromatography and subjected to an SPR-based fetuin-binding assay. (D) Human RBC hemagglutination with HA1(1-320) monomeric, trimeric, and oligomeric forms isolated by size exclusion chromatography. In the hemagglutination assay, anti-His polyclonal sera were added to purified monomer and trimer fractions to increase the valency of the HA1 molecules.

FIG. 4.

Role of HA1 N-terminal residues in receptor binding and hemagglutination. (A) Binding kinetics of purified H5N1 HA1 proteins and its mutants in a SPR-based receptor-binding assay. Steady-state equilibrium analysis of different H5N1-HA1 proteins to fetuin and its asialylated counterpart (asialo-fetuin) was analyzed at 25°C using a ProteOn SPR biosensor. Samples of purified bacterial H5N1 HA1(1-330) with Cys₄Ala mutation (C4A), and HA1(1-330)-C4A with additional isoleucine double mutations at positions 3 and 5 of HA1 (HA-1-330-I3A-C4A-I5A and HA-1-330-I3G-C4A-I5G), were injected simultaneously over a mock surface to which no protein was bound, followed by fetuin immobilized on a sensor chip through the free amine group and onto a blank flow cell, free of protein. Binding kinetics and data analysis was performed by using a ProteOn system SPR biosensor instrument. (B) Hemagglutination of RBCs with N terminus intact HA1(1-330) and HA1 with C4A mutation (HA-1-330-C4A) (both contain oligomers) and HA1 proteins with double isoleucine mutations: I3A-C4A-I5A and I3G-C4A-I5G (both containing only monomers). Serial dilutions of purified HA proteins were mixed with washed RBCs and incubated for 30 min at room temperature. Reassorted rgH5N1xPR8 (2:6) A/Vietnam/1203/2004 virus (clade 1.0) was used as a control.

FIG. 5.

HA1(1-320) elicits higher neutralizing titers than monomeric HA1(28-320) in rabbits. (A) Animals were immunized with 100 μg of proteins mixed with TiterMax adjuvant every 3 weeks. Sera were collected 8 days after each vaccination and analyzed in a microneutralization assay against various H5N1 virus strains. The results are representative of three experiments. (B and C) H5-Viet-HA 1-320 induces oligomer-specific antibodies. Fivefold diluted postvaccination sera from rabbit K1 (H5N1 HA 1-320) or rabbit K3 (HA 28-320) were added to 0.5 mg of purified HA1(1-320)-His₆ or to HA1(28-320)-His₆ proteins (or PBS) and then incubated for 1 h at room temperature. Nickel-nitrilotriacetic acid (Ni-NTA) magnetic beads (200 μl) were added for 20 min at room temperature on an end-to-end shaker to capture the His-tagged proteins and the antibodies bound to them, followed by magnetic separation. The supernatants containing the unbound antibodies were collected. The pre-and postadsorbed sera were subjected to SPR analysis on purified oligomeric H5N1 HA(1-320) (B) or monomeric H5N1 HA(1-320) protein (C), immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. Binding was recorded by using a ProteOn system SPR biosensor instrument.

FIG. 6.

Challenge of vaccinated and unvaccinated ferrets with H5N1 influenza viruses. After two immunizations, ferrets (five animals per group) were infected intranasally with 10⁶ 50% egg infectious doses (EID₅₀) of A/Vietnam/1203/2004 (clade 1) (A and B) or A/Whooperswan/Mongolia/244/2005 (clade 2.2) (C and D). The animals were scored for the percent original body weight (A and C) and the percent survival (B and D). Viral loads in nasal washes after challenge of vaccinated and unvaccinated ferrets with H5N1 influenza viruses, A/Vietnam/1203/2004 (clade 1) (E and F) or A/Whooperswan/Mongolia/244/2005 (clade 2.2) (G and H), on day 3 (E and G) or day 5 (F and H) after virus challenge. The data are presented for individual animals. Horizontal lines represent the average PFU of virus from the nasal washes of each group (five ferrets per group).