An Alternative Platform for Rapid Production of Effective Subunit Vaccines - Tetrahymena thermophila offers numerous advantages as an expression system, including rapid cell growth and high cell densi

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An Alternative Platform for Rapid Production of Effective Subunit Vaccines
Tetrahymena thermophila offers numerous advantages as an expression system, including rapid cell growth and high cell densities, eukaryotic protein folding, and active synthesis of membrane and secreted proteins.


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Results and Discussion


Figure 2. Recognition of recombinant H5 by neutralizing monoclonal antibodies. Whole cell lysates from transformed T. thermophila, expressing the full-length recombinant H5, were tested in Western blots with a panel of MAbs that are neutralizing for H5N1 avian influenza virus (see Table 1). Cells were lysed in SDS buffer containing either 0% (lane 1), 0.25% (lane 2), or 4% (lane 3) β-mercaptoethanol and proteins run on 12% polyacrylamide gels. Primary antibodies were diluted 1:400 in Tris-buffered saline (TBS) containing 20% nonfat dry milk, and secondary antibody (goat antimouse HRPO conjugate) diluted 1:10,000 in the same buffer. Bands corresponding to the expected sizes of HA monomers (M), dimers (D), and trimers (T) were seen. Recognition of the recombinant antigen was sensitive to reducing agent in all cases.
To assess the utility of T. thermophila as an expression system for subunit vaccines, genes for candidate antigens from viral and protozoan pathogens were introduced into the macronuclear genome, either by homologous recombination or using ribosomal DNA (rDNA) vectors that replicate to high copy number (~9,000) in these cells. The antigens in each case were a 48 kDa GPI-anchored surface protein (i-antigen) from the parasitic protozoan, Ichthyophthirius multifiliis,25 and the H5 hemagglutinin from H5N1 (Vietnam) subtype avian influenza virus. In both instances, high-level expression of the respective proteins was detected. Based on quantitative Western blotting, recombinant proteins reached ~3–5% of total cell protein (equivalent to ~60–100 mg/L without optimization; data not shown) and they correctly localized to ciliary or plasma membranes (Figure 1). More importantly, both proteins were properly folded as determined by their ability to bind neutralizing and protective MAbs against conformational epitopes dependent on disulfide bond formation. As shown in Figure 2, among seven different neutralizing MAbs against the H5N1 virus (Table 1), all seven recognized the recombinant protein expressed in Tetrahymena. Bands corresponding to the H5 monomer, dimer, and trimer were present suggesting that the protein assumed correct quaternary structure as well (Figure 2). Although H5 was present primarily on the plasma membrane, the recombinant i-antigen was localized on both ciliary and plasma membranes, resulting in complete arrest of cell movement on adding protective MAbs.26


Figure 3. Targeting to the constitutive secretory pathway. Truncated genes lacking the coding sequences for the hydrophobic GPI-anchor signal sequence of the 48 kDa i-antigen (i-ag) of I. multifiliis, and the transmembrane spanning domain of H5 hemagglutinin of avian influenza virus were cloned and expressed in Tetrahymena (Panels A and B, respectively). Panel A is a Coomassie blue stained gel of concentrated culture supernatant fractions from cells before (lane 1) and after (lane 2) induction of gene expression. The secreted i-ag is clearly present in this fraction following induction of gene expression. Panel B is an immunofluorescence image of Tetrahymena expressing the truncated H5 hemagglutinin. The viral gene product was present at low levels in culture supernatant fractions (not shown) and appeared as scattered punctate staining within transport vesicles or the ER. Magnification bar = 10μm.
Purification of the full-length membrane-associated antigens required detergent lysis and chromatographic separation on multiple resins. Nevertheless, because both proteins are linked to the lipid bilayer by hydrophobic domains at their C-termini (a single transmembrane-spanning domain in the case of H5, and a GPI-anchor in the case of the i-antigen), we reasoned that purification could be simplified by directing truncated versions lacking C-termini to the constitutive secretory pathway, where they could be readily harvested from culture supernatants. Following transformation and expression, the truncated i-antigen could be detected as a Coomassie blue stainable band in culture supernatant fractions (Figure 3A) allowing straightforward downstream purification. Truncated H5, on the other hand, was detectable only at low levels in culture supernatant fractions, and showed punctate staining throughout the cytoplasm, possibly because of aggregation (Figure 3B). This is consistent with previous reports using insect and mammalian cells in which C-terminal deletions of certain HA subtypes failed to secrete as a result of inappropriate folding or assembly.27,28


Figure 4. Stimulus-dependent secretion in T. thermophila. As depicted in panel A), adding secretagogues to the culture medium results in an explosive discharge of cortical secretory granules (mucocysts) from Tetrahymena that is complete within seconds of the stimulus. The material released from granules takes the form of a proteinaceous gel that can be readily harvested by low speed centrifugation. The tube on the right contains cells from a 50 mL culture that had been induced to secrete and spun at 6,000g for 5 min. The mucocyst gel, termed PRISM (P) is shown in brackets just above the white cell pellet. In panel B, cells were induced to secrete on an EM grid and gently washed off. The grid was then stained with uranyl acetate and examined under the electron microscope. Note the underlying crystalline structure of the hydrated PRISM matrix. Magnification bar = 0.5 μm.
Although most eukaryotic cells allow secretion by the constitutive pathway, specialized cells have an additional pathway in which proteins are stored in cortical secretory granules and then rapidly released in response to specific stimuli. Regulated, stimulus-dependent secretion is evolutionarily conserved (from protozoa to mammals)29 and offers an attractive alternative to constitutive secretion as a means of protein production and purification.30 Tetrahymena has an elaborate apparatus for regulated secretion consisting of large numbers of crystalline, dense core granules known as mucocysts that store upwards of 20% of total cell protein.14,15 Mucocyst granules remain docked at the plasma membrane but discharge their contents en masse within seconds of adding appropriate stimuli. The material released from the granules takes the form of a proteinaceous gel (termed PRISM) that can be harvested by low-speed centrifugation (Figure 4). With the idea that this pathway could be harnessed for the production and purification of H5, we linked the viral hemagglutinin to a member of a small family of granule-associated proteins known as Grls, with the hope of targeting it to mucocysts. As shown in Figure 5, the viral gene product was in fact localized to dense core granules following transformation and induction of the chimeric gene product. Furthermore, when cells were induced to secrete in response to dibucaine, H5 was found exclusively in the PRISM phase (Figure 5).


Figure 5. Targeting to the regulated secretory pathway. Panels A and B are immunofluorescence images showing localization of recombinant H5 in cortical mucocysts of transformed Tetrahymena labeled with MAb 5C5. Panel A is a stacked Z-series, while panel B is single Z-section through the same cells. Antibody concentrations were the same as in Figure 1. Note the obvious punctate staining of mucocysts at the cell periphery. Panel C is a stacked Z-series showing mucocyst granules in wild-type (CU428) cells stained with MAb 5E9, which recognizes the granule-lattice protein Grl3p (generous gift of Aaron Turkewitz, University of Chicago). Panel D shows a Western blot reacted with MAb 5C5 containing fractions from cells induced to secrete by adding dibucaine to the culture medium. Lanes contain, in order: 1) cells bodies after secretion; 2) PRISM; 3) original cell culture medium before secretion; 4) 6,000g supernatant from washed cells before secretion; 5) 6,000g supernatant from cells after secretion. Lanes were loaded equally by volume.
The ability to target H5 to mucocysts for rapid secretion represents a first-step toward streamlined purification of the antigen itself. Although the recombinant protein bound neutralizing antibodies, it remained to be determined whether it could elicit protective antibodies in animals. Because the chimeric H5 was abundantly expressed in PRISM, the entire matrix containing the recombinant antigen was injected intaperitoneally into rats and the resulting antisera tested for both cross-reactivity against baculovirus-expressed H5 in Western blots, and for its ability to neutralize live virus in cell culture. Rat antisera against the chimeric antigen showed strong binding to the purified protein in Western blots, whereas no signals were obtained with nonimmune sera or secondary antibodies alone (Figure 6). More importantly, sera from two of three animals injected with chimeric H5 showed extremely high neutralizing titers in assays with reassortant A/Vietnam/1203/2004(H5N1) influenza virus on cultured MDCK cells (Table 2).


Figure 6. Recombinant H5 in PRISM induces antibody responses in animals. Adult rats were injected with the PRISM matrix containing recombinant H5 in different orientations relative to the granule targeting protein as described in the text. Resulting antisera (diluted 1:1,000) were then tested in Western blots against purified H5 expressed in insect cells (Protein Sciences). Lanes 1–3 correspond to serum samples 1–3, respectively, in Table 2. Signals corresponding to the H5 monomer and higher order aggregates were visible in each case following addition of secondary goat anti-rat IgG (1:3,000 dilution, HRP conjugate [SouthernBiotech]). No signals were obtained when the same protein was reacted with pooled nonimmune rat serum followed by secondary antibody (lane 4), or with secondary antibody alone (lane 5).
The ability to direct foreign proteins to the regulated secretory pathway of Tetrahymena has clear advantages from the standpoint of protein production. First, cells can be grown to high biomass, harvested in buffer without contaminating media or secreted proteins, and then induced to secrete within a very narrow time frame. The material stored in mucocysts comprises small families of granule-lattice and b/g-crystalline-like proteins, the majority of which are insoluble and form the PRISM matrix.31 Soluble mucocyst proteins can be easily removed by low-speed centrifugation, and recombinant proteins of interest harvested with the matrix itself. Methods have now been devised that allow rapid separation of recombinant proteins from PRISM, making this a highly streamlined purification system that is tantamount to performing affinity chromatography in vivo (Clark, et al, in preparation).


Table 2. Virus neutralization titers of rat antisera against recombinant H5
Although the number of animals used in this study was limited, we were surprised that the neutralizing antibody titers against PRISM-linked H5 reached such high levels (Table 2). Dense core mucocysts of T. thermophila are highly crystalline,29 and the PRISM matrix retains its underlying crystalline structure even after mucocyst discharge (Figure 4). We speculate that the arrangement of recombinant antigens within this crystalline array provides a repetitive framework that can strongly cross-link the Ig receptor on B-cells. Furthermore, the particulate nature of the gel itself could allow target proteins to be avidly taken up by antigen-presenting cells promoting strong T-cell responses as well. If these hypotheses are born out, PRISM may offer a powerful low-cost alternative to VLPs and other particle-based delivery systems for producing highly protective subunit vaccines.


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