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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Table 1. H5N1 MAbs used in Western blots
For localizing recombinant gene products, cells were fixed either before or after adding primary mouse MAbs in 50 mM HEPES buffer containing 4% paraformaldehyde in the cold. Cells were washed, treated with primary and secondary antibody, and mounted in ProLong Gold anti-fade reagent (Invitrogen). Images were acquired with a Leica SP5 confocal microscope using a 63X water objective. For detecting the IAG48[G1] i-antigen, MAb 10H3 was used.23


Figure 1. Surface expression of viral and parasite antigens in Tetrahymena. Panels A and B: Transformed cell lines were induced with CdCl2 overnight and expression of recombinant antigens was visualized by confocal microscopy. For H5 (panel B), cells were stained with a 1:50 dilution of MAb 5C5 (Figure 2) followed by a 1:300 dilution of goat antimouse IgG coupled to Texas Red. For the IAG48[G1] i-antigen (panel A), cells were stained with a 1:100 dilution of MAb 10H3, followed by the secondary antibody as above. Panel A is a stacked Z-series showing fluorescence on ciliary and plasma membranes, while panel B shows a single section with localization of H5 at the plasma membrane. Magnification bars = 10 μm. Panels C and D: Cells transformed with the full-length H5 gene were induced for varying periods of time and then lysed in SDS-sample buffer in the absence of reducing agent, subjected to SDS-PAGE and transferred to nitrocellulose. Blots were screened with a 1:400 dilution of MAb 5C5, followed by a 1:10,000 dilution of goat antimouse IgG coupled to HRP. Panel C shows bands corresponding to monomeric H5 and higher order aggregates. Blots were scanned and expression quantitated by densitometry. As shown in panel D, H5 gene product accumulated over time through at least 48 h in cell lines containing the transgene at the somatic MTT1 locus.
Animal immunization was carried out as follows. Six-week old male Albino Oxford (AO) rats were anesthetized with Isoflurane and injected intraperitoneally with 250 mL of PRISM matrix containing recombinant H5 diluted 1:3 in PBS. An equal volume of Freund's incomplete adjuvant (FIA, Sigma) was then added and the mixture emulsified. Rats were anesthetized and boosted with the same amount of antigen in FIA four weeks after the initial injection, and blood was collected three weeks later using terminal heart puncture. Microneutralization assays were carried out with A/Vietnam/1203/2004xPR8 (VN04) reassortment virus as described previously.24 Cells were fixed and virus infection was detected by ELISA using antibodies against Influenza A virus nucleoprotein.


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