Although egg-based vaccines have been used to combat seasonal flu, the lengthy production cycles and limited manufacturing capacity of egg-based vaccines are not conducive to facilitating a rapid response during a potential influenza virus pandemic. Several clinical studies have shown that recombinant HA-based vaccines purified from baculovirus expression systems are safe and effective against H1N1 and H3N2 influenza viruses (7
). The protein-based approach represents an attractive alternative to egg-based technology, since it uses HA proteins as antigens and does not require the production of potentially dangerous live virus. Also, with this approach, vaccine production is not limited by the supply of eggs and can be easily scaled up in a good-manufacturing-practices facility.
Recently, a recombinant HA vaccine against avian H5N1 influenza virus has demonstrated tolerability in humans (16
). However, this vaccine only induced protective neutralizing antibody titers in 50% of the subjects receiving the highest dose (two doses of 90 μg vaccine). Since it has been previously reported that recombinant HA proteins expressed in insect cells tend to form monomers (13
), the suboptimal immunogenicity of this H5 HA vaccine may be due in part to recombinant HA protein not being presented in its native trimeric conformation. In this study, we cloned the ectodomain of HA from an H5N1 virus (KAN-1) and expressed the HA proteins in mammalian or insect cells. HA proteins initially were purified using a nickel affinity column followed by anion-exchange and gel filtration chromatography. The entire purification process can be completed in 2 to 3 days, and protein production can easily be scaled up. In both Hi5 insect cells and 293F mammalian cells, HA proteins were expressed as high-molecular-weight oligomers and stabilized trimers, demonstrating that the trimerizing foldon sequence indeed prevented the HA from dissociating into monomers. Upon the removal of the foldon sequence by thrombin digestion, only trimers and monomers were present in the insect-expressed proteins, whereas in the mammalian cell-expressed proteins only a small portion of monomer was observed after the removal of the foldon sequence. The discrepancy may be due to the different glycosylation states of proteins derived from insect cells versus proteins produced in mammalian cells, although it is certainly not conclusive.
We then evaluated the immunogenicity of these different forms of HA derived from either insect or mammalian cells using an HA/NA-pseudotyped lentiviral system (24
). In this assay, the neutralization activity can be determined easily by measuring the ability of antisera from mice immunized with recombinant HA proteins to inhibit pseudovirus entry. It has been shown that this pseudotype inhibition assay correlates highly with traditional microneutralization and hemagglutination inhibition assays (6
) and can be easily performed in a conventional biosafety level 2 laboratory with biosafety level 3 practices. Among the proteins produced from mammalian cells, high-molecular-weight oligomers elicited the highest titers of neutralizing antibody, followed by the cleaved trimers and uncleaved trimers. Cleaved monomers failed to induce significant neutralizing antibodies against H5N1 virus, even though anti-H5 antibodies were detected by ELISA. This may be due to the preferential induction of antibodies against epitopes present in the monomeric form and not in the trimer, similarly to that observed with human immunodeficiency virus type 1 gp120 monomers and trimers (reviewed in reference 3
). It also is possible that the monomeric form is less immunogenic than the trimer/oligomer forms of the same protein. In a separate study, antisera from animals immunized with mammalian cell-expressed oligomers or cleaved trimers were examined, and their ability to elicit neutralizing antibodies against different H5N1 strain pseudoviruses was similar (unpublished data), though we cannot exclude the possibility of differences in their fine specificity. It should be noted that, although Ribi adjuvant does not contain any denaturants or reducing agents, its effect on the stability and conformation of HA proteins is unknown. We attempted to analyze this effect biochemically but were unable to extract HA proteins from the lipid-rich components of this adjuvant. Although NA plays an essential role in viral replication and infection, the trimming of terminal sialic acid from the HA proteins by NA did not affect the immunogenicity of recombinant HA oligomers. However, the addition of NA did prevent the precipitation of purified protein and facilitated the production of the HA oligomers (data not shown). The removal of terminal sialic acids by NA appeared to be important for the receptor binding of HA. Glycan binding analyses of HA expressed in the insect cell, which lacks sialic acids, have revealed a similar α2-3 specificity (12
) to the NA-coexpression mammalian HA protein, which bound to α2,3-linked sialic acid oligosaccharides. These findings are consistent with the observation that insect-produced, stabilized trimers elicited substantial levels of neutralizing antibodies (Fig. ) despite the lack of the sialylation of HA in this cell type.
Although previous studies have shown that recombinant HA proteins derived from insect cells elicit immune responses (7
), our data provide evidence that oligomeric or trimeric HA produced in mammalian cells are comparable or slightly better in eliciting neutralizing antibodies against avian H5N1 virus. Further testing will be required to determine whether other adjuvants, such as alum, QS-21, or MF-59, can improve the immunogenicity of recombinant HA proteins. Not only could the potency of these adjuvants differ but also their effects on the stability of the trimer may vary. These results eventually will require validation with the most active and manufacturable forms in human clinical trials. Nonetheless, our data demonstrate that recombinant mammalian cell- or insect-expressed trimeric HA proteins represent a promising approach to the development of vaccines relevant to seasonal and pandemic influenza virus.