Expression of recombinant HA proteins in bacteria could provide a rapid and economical approach for early response to impending influenza virus pandemic. Early studies demonstrated that protective influenza virus antigenic sites are conformation dependent and map primarily to the HA1 globular domain. Therefore, producing HA1 proteins in a properly folded state is imperative to eliciting protective antibody responses.
In the present study we dissected the structure-function requirements of bacterially expressed HA1 proteins and evaluated their potential use as prophylactic vaccines against HP H5N1 AIV. The main findings are as follows. (i) A panel of H5N1-A/Vietnam/1203/2004 HA1 proteins with N- and C-terminal deletions purified from E. coli under careful redox conditions were shown to be properly folded by binding to conformation-dependent huMAb. (ii) HA1 with an intact N terminus contained oligomers in addition to monomers, whereas HA1 with N-terminal deletions contained only monomers. (iii) A fetuin receptor-binding assay demonstrated that only HA1 proteins with intact N termini, containing oligomers, bound receptors. (iv) Hemagglutination required oligomeric HA1. (v) Site-directed mutagenesis of Ile-Cys-Ile residues at positions 3 to 5 disrupted oligomer formation, fetuin binding, and RBC agglutination, with no effect on HA1 folding. (vi) In rabbits, properly folded HA1 containing oligomers generated more rapid potent neutralizing antibodies than monomeric HA1 and cross-neutralized several H5N1 clades, including A/Indoensia/5/2005. (vii) Vaccination of ferrets with HA1(1-320) at either 3 or 15 μg of protein per dose protected animals from lethality and morbidity after challenge with homologous (A/Vietnam/1203/2004) or heterologous (A/Whooperswan/Mongolia/244/2005) HP AIV challenge. In contrast, monomeric HA1(28-320) was not immunogenic in ferrets at the same doses and did not protect animals from H5N1 challenge.
The structure of HA from highly pathogenic H5N1 A/Vietnam/1203/2004 resembles the 1918 and other human H1 HAs (21
). Most of the intersubunit salt bridges and hydrophobic interactions are between the HA2 chains due to a coiled-coil structure which forms the stem of the HA trimer (4
). These earlier HA-structural studies did not describe the oligomerization signal in the HA1 globular domain identified in the present study, suggesting that in the presence of HA2 the N-terminus β-sheet structure is engaged in an HA1-HA2 bridge and not in HA1 oligomerization. This may explain why most recombinant HA ectodomain proteins exist as monomers and require the addition of multimerization sequences such as “foldon” at the C terminus in order to produce stable oligomeric structures (3
). This was further confirmed in a recent study in our laboratory with bacterially expressed HA proteins from the novel H1N1 A/California/04/2009 comparing the composition and immunogenicity of globular HA1(1-330) to that of the HA ectodomain (1-480). Both proteins were properly folded. However, only the HA1 globular head (1-330) formed oligomers and agglutinated human RBCs, whereas the HA ectodomain (1-480) contained only monomers and did not agglutinate RBC (13
). It is likely that in the native spikes the N-terminal β-sheets of the three HA1 globular domains are not in sufficient proximity to form oligomers, but in the absence of HA2 they are free and close enough to provide the needed oligomerization signal. This was confirmed by our finding that an N-terminal fragment HA1(1-104) without the RBD appeared primarily as oligomers in gel filtration chromatography (Fig. ).
The oligomeric forms were not simply aggregates of monomers but contained multiples of functional trimers, as determined by equilibrium ultracentrifugation. In the native structure, the Cys4 in of HA1 is engaged in a disulfide bond with Cys462 in the HA2. Therefore, it was possible that during refolding this unpaired Cys4 form bonds with other Cys4 residues in adjacent HA1 molecules. To that end, we mutated Cys4 and found that the HA1 (Cys4Ala) mutant protein still contained a prominent oligomeric structure (60 to 75% of the total protein) that ran similar to the unmutated HA1 oligomers on gel filtration chromatography. More importantly, the Cys4Ala mutant bound well to receptor in the fetuin SPR and agglutinated RBCs. In contrast, triple mutants with Ile3-Cys4-Ile5 converted to either Ala-Ala-Ala or Gly-Gly-Gly contained only monomers and lost receptor binding and hemagglutination.
In mammalian and eukaryotic cells, posttranslational glycosylation of HA was shown to play an important role in proper folding, trimer stabilization, and transport to the cell outer membrane (5
). On the other hand, we have demonstrated here and in previous studies that bacterially expressed unglycosylated HA1 (and HA0) can be purified as properly folded proteins, as determined by CD spectra analysis and binding to conformation-dependent neutralizing antibodies (12
), in agreement with the findings in another recent study (1
Importantly, our present study demonstrated that, in addition to proper folding, HA1 oligomers were required for high-avidity receptor (fetuin) binding and for cross-linking of RBCs, resulting in hemagglutination. Importantly, the traditional inactivated subunit vaccine generated from egg-grown virus contains primarily oligomeric forms (Fig. ). Previous reports on the production of recombinant HA in mammalian cells, insect cells, or bacterial systems did not provide information on the presence and function of oligomers versus monomeric forms of HA (1
). More recent publications emphasized the importance of high-MW oligomers for optimal immunogenicity of influenza virus recombinant HA proteins. Trimerization domains such as GCN4-pII and foldon were added to the C termini of HA ectodomains produced in mammalian or insect cells in order to produce stable oligomeric structures and to elicit optimal neutralizing antibody titers (3
). On the other hand, our study demonstrates oligomer formation with bacterially expressed HA1 with no requirement for the addition of any foreign trimerization sequence. Therefore, our bacterially expressed and properly folded HA1 proteins with intact N termini behave similarly to inactivated H5N1 subunit vaccine in terms of in vitro
functions, including receptor binding and RBC agglutination.
The ferret protection data with highly pathogenic avian H5N1 studies provide strong evidence that bacterially expressed HA1 proteins, which are properly folded and contain functional oligomers, are potent inducers of protective immunity against pathogenic influenza viruses. Although all H5N1 viruses are between 95 and 98% identical regardless of clade, there is poor cross-reactivity between antibodies elicited to clade 1 HP H5N1 viruses, such as A/Vietnam/04, and clade 2 H5N1 viruses that predominate among recently transmitted strains, resulting in high human lethality. The cross-protection against heterologous strains is important since it is not certain which of the avian H5N1 influenza virus strains will adapt to human-to-human transmission. The level of protection achieved with the oligomeric forms of H5N1 HA1 bacterial proteins was equivalent to that observed after vaccination with inactivated H5N1 vaccine.
The combination of recombinant technology and improved purification approaches, combined with analytical assays to confirm proper folding and higher-order quaternary structures, will facilitate the large-scale production of HA in bacterial systems. Within 2 weeks of pandemic strain isolation, large quantities of HA1 proteins can be produced (currently 40 to 50 mg/liter in a batch culture; with 8- to 10-fold-higher yields in small-scale continuous fermentation cultures). Thus far, we have generated bacterially expressed properly folded HA1 from two H5N1 strains (A/Vietnam/1203/2004 [clade 1] and A/Indonesia/5/2005 [clade 2.1]), novel H1N1 (A/California/04/2009), H3N2 (A/Wisconsin/15/2009 and A/Victoria/210/2009), and H7N7 (A/Netherlands/219/03), and all were shown to form functional oligomers (≥70%), with lot-to-lot consistency.
Therefore, the production of HA1(1-320) proteins in bacterial systems is a viable and scalable approach for rapid vaccine production in response to emerging influenza virus strains with little or no preexisting immunity (such as H5N1 influenza virus), especially for individuals with known egg allergies.