- Top of page
- Materials and methods
The A/Victoria/3/75 (H3N2-subtype ) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiaeα-mating factor secretion signal and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol-containing medium resulted in the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA-conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10–12 glycosyl residues.
Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse-adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.
Influenza is a well-known viral pathogen, which causes epidemics and pandemics in humans and animals. In humans, an influenza infection is normally restricted to the upper respiratory tract, and often results in severe morbidity, many hospitalizations, and even mortality. Two types of antigenic variation of the influenza virion have been recognized, namely ‘drift’ and ‘shift’, which are the underlying causes of recurrent epidemics and pandemics, respectively [1–4]. Because of this frequent and unpredictable antigenic variability, the design of a universal influenza vaccine has so far not been realized. Current prophylaxis against influenza is via parenteral vaccination with inactivated or subunit virus grown in embryonated chicken eggs, despite their potentially serious limitations as a host system [5–9]. A number of alternative approaches have been pursued to achieve protective immunity including peptide vaccination, DNA vaccination and recombinant vaccinia virus technology [10–13]. However, concerns about safety, pre-existing immunity in people, immune responses against the vector itself and, in some instances, an insufficient protective immune response, have limited the usefulness of these approaches. The use of purified, recombinant influenza membrane proteins appear to be a promising alternative [14–18].
Three membrane proteins are present on the influenza virion: hemagglutinin (HA), neuraminidase and the M2 protein. Although neuraminidase, a type-II membrane glycoprotein, displays similar amounts of antigenic variation to HA, antibodies specific to this protein, do not neutralize influenza infection, even though they do minimize viral spread. The conserved influenza M2 protein is only present in minute amounts on the viral surface and is therefore incapable of eliciting neutralizing immunity .
HA, a homotrimeric class I membrane glycoprotein, is quantitatively the major surface protein of influenza virus and the major antigen against which neutralizing antibodies are elicited . Therefore, recombinant HA is a very favorable antigen as a candidate influenza vaccine. HA mediates the attachment of the virus to the target cell through specific binding with sialic acid-containing determinants and, following internalization, the release of the viral content into the attacked cell [19, 20]. HA-specific antibodies are protective as a result of their ability to prevent virus attachment and penetration of the host cell, or presumably by interfering with the low-pH-induced conformational change of the HA molecule needed for fusion [21–24]. Because of the immune selection pressure, HA is the viral component which is most important in antigenic drift.
The HA monomer is synthesized as a single polypeptide chain which undergoes post-translational cleavage at two sites: the N-terminal signal sequence is removed and, depending on the host cell and virus strain, the molecule is cleaved, with the removal of one or more intervening residues, resulting in two polypeptide chains called HA1 (36 kDa) and HA2 (27 kDa), linked via a disulfide bridge [25–27]. A C-terminal stretch of hydrophobic amino acids anchors HA to the viral membrane and, though not essential for secretion, this sequence plays a major role in the trimerization process .
The use of transformed mammalian, avian or insect cells for expression is a serious constraint, both practically and economically, for production of an influenza vaccine. Therefore, the use of a recombinant organism, such as yeast that is easy and economical to grow on a large scale in a fermentor, could be a major advantage for production of an influenza HA vaccine. Recently, we described partial protective immunity in mice with soluble influenza neuraminidase expressed by Pichia pastoris. Here we report the expression, characterization and immuno-protective potential of glycosylated, secreted influenza H3-subtype HA in P. pastoris. The purified protein, administered in mice in combination with proper adjuvants, is capable of eliciting a fully protective antibody response in mice against a lethal viral challenge.
- Top of page
- Materials and methods
The methylotrophic yeast P. pastoris has been developed as a host for the efficient production of heterologous proteins . Interest in this eukaryotic methylotropic organism as a heterologous protein production system has grown, since it has the potential of high-level expression and rapid growth to very high cell densities in inexpensive media . Jabbar and Nayak (1987) reported the (low) expression of truncated, hyperglycosylated, and mainly cell-associated, A/WSN/33 immuno-reactive influenza HA in S. cerevisiae without, however, demonstrating its applicability in vivo. Here we demonstrate that P. pastoris is capable of expressing a soluble form of the influenza A virus HA with near native antigenic structure. Furthermore, purified HA0s, although monomeric, was capable of eliciting a protective antibody response against a lethal influenza challenge in mice.
The expression level of anchor-free HA from P. pastoris was markedly affected by the pH of the induction medium, with pH 8.0 being optimal. The influence of the extracellular pH on trimerization and transport of HA in a mammalian cell expression system has been reported previously . The monomeric structure of the purified HA0s is not unexpected, in view of the observations by Singh et al. who demonstrated that recombinant, anchor-free X31 [A/Aichi/68 HA (H3-subtype)] was secreted as a monomer from CV-1 cells . Anchor-free A/Japan/305/57 HA (H2), on the other hand, formed monomeric, trimeric and higher order complexes in the Golgi complex or in secretory vesicles, when expressed in the same cells. Vanlandschoot et al. reported on the expression, in insect cells, of aggregated and monomeric soluble A/Victoria/3/75 HA0s at pH 6.0, and monomeric HA0s at pH 8.0, with a minor trimeric fraction being observed under both conditions [34, 43]. Although we did not analyze the oligomeric state of the low-pH-expressed HA0s in P. pastoris, it seems unlikely that aggregated HA0s molecules of a similar size (> 1.5 × 106 Da) could traverse the yeast cell wall.
Analysis of the N-linked carbohydrates showed the presence, predominantly, of (N-acetylglucosamine)2Man8–10 residues ( Fig. 4). This result is in agreement with the reported average 8–14 mannose residues added post-translationally by P. pastoris, and is in striking contrast with the observation of the rather exceptional hyperglycosylated nature of soluble recombinant neuraminidase containing N-glycans with 30–40 mannose residues, from the same organism . Although the molecular mechanisms determining the outcome of the glycosylation pattern of a glycoprotein in a particular eukaryotic host organism remain enigmatic, one might speculate that the folding kinetics play a role. Glycoproteins that spend longer in the early exocytic vesicles might be more susceptible as as substrate for glycosyltransferase activity. Recognition of P. pastoris-secreted HA0s by a panel of mAbs implies that at least part of the molecule is correctly folded.
The amino terminus of HA0s was incompletely processed by the STE13-like dipeptidyl aminopeptidase A (DPAPA) activity from P. pastoris, resulting in an approximately equimolar mixture of fully, partially and unprocessed HA0s. Incomplete maturation by DPAPA of recombinantly expressed proteins has often been observed in yeast [29, 50]. The presence of a spacer peptide has been reported to improve the cleavage by Kex2p at the dibasic site of the α-factor leader sequence, but this advantage may be outweighed by incomplete removal of the GluAla dipeptides . Immunization of mice with purified HA0s in combination with adjuvant resulted in a strong virus-specific antibody response. A drawback of most soluble antigens is the requirement of an adjuvant to elicit immunity. Monophosphoryl lipid A was chosen in our experiments because it has already been successfully evaluated in clinical trials [52, 53].
The serum response obtained was fully protective against a homologous X47 challenge and partially against a heterologous X31 challenge. Furthermore, immunized mice showed less severe symptoms of infection than control mice. This partial cross-protection in mice is not completely unexpected, since we previously described the isolation of a cross-neutralizing mAb after immunization with the homologous HA0s expressed in COS cells and challenge with the X31 virus .
Until now, only inactivated vaccines, isolated from embryonated chicken eggs, have gained widespread use against influenza. However, their effectiveness is compromised by the frequent antigenic drift of the influenza virus, the limited supplies of high-quality eggs and the susceptibility of the eggs to avian influenza infection, and the danger of variant selection in the avian host. We here describe a alternative method to produce an influenza vaccine, based on the synthesis of recombinant HA0s in P. pastoris. This fermentable, methylotrophic yeast has proven to be an excellent and cost-effective host for the production of heterologous proteins . Although membrane-bound HA has already been successfully expressed in Sf9 insect cells  and has been shown to elicit protective antibodies , the expression of protective secreted HAs from a monocellular organism has so far not been reported. We have shown that the immunogenic potential of yeast-derived HA0s as described here, may be appropriate for the development of an easily adaptable, safe and economic alternative to the currently used influenza vaccine. Furthermore, being a recombinant expression system, it may be possible to improve its protective properties by genetic engineering. For example, one could produce an HA0s molecule with altered or omitted variable and immuno-dominant epitopes, which might result in an antigen with broadened protection potential. Although the mouse model used here is commonly accepted to evaluate experimental influenza vaccines, the results described should only be regarded as an initial proof of principle.
Finally, the flexibility and the potential speed associated with a yeast expression system, may prove to be indispensable at the time of an emerging pandemic. Indeed, it has been calculated that the outbreak of a new influenza pandemic will not allow sufficient time and number of embryonated eggs to produce vaccines for a large population at risk. Alternative antiviral medication will probably not be available in sufficient amounts. The H5N1 influenza subtype recently isolated from patients in Hong Kong, although apparently under control by now, is a reminder of the always looming, potential global threat . In this case, even the production of vaccine in embryonated eggs was compromised because the seed virus killed the embryos. In the USA, an influenza pandemic vaccination policy is being worked out for distribution of vaccines according to a predetermined priority system [56, 57]. The availability of a flexible influenza vaccine-producing system and that could be scaled up quickly may be the key step that would allow implementation of such a selective vaccination plan. A comparative study with conventional influenza vaccines and different viral strains will be necessary to be able to further evaluate the potential of the described influenza virus HA expression system.