Exploring synergies between academia and vaccine manufacturers: a pilot study on how to rapidly produce vaccines to combat emerging pathogens.

BACKGROUND: In spring 2009, a new swine-origin influenza A (H1N1) virus emerged in Mexico. During the following weeks the virus spread worldwide, prompting the World Health Organization to declare the first influenza pandemic of the 21st century. Sustained human-to-human transmission and severe disease progression observed in some patients urged public health authorities to respond rapidly to the disease outbreak and vaccine manufacturers to develop pandemic influenza vaccines for mass distribution. With the onset of the pandemic we began to explore the potential of academic/industrial collaboration to accelerate the production of vaccines during an outbreak of an emerging virus by combining the use of an academic BSL-4 laboratory with the expertise of a commercial vaccine manufacturer. METHODS AND RESULTS: To obtain virus seed stocks used for the production of a vaccine to combat the pandemic H1N1 2009 influenza virus (H1N1pdm), we followed various strategies: (i) optimization of cell culture conditions for growth of wild-type H1N1pdm isolates; (ii) classical reassortment of H1N1pdm and standard influenza vaccine donor strain PR8; and (iii) generation of corresponding reassortant viruses using reverse genetics. To ensure a rapid transition to production, the entire potential seed stock development process was carried out in a certified canine kidney suspension cell line (MDCK 33016-PF) under Good Manufacturing Practice (GMP) conditions. CONCLUSIONS: The outcome of this study indicates that a combination of different experimental strategies is the best way to cope with the need to develop vaccines rapidly in the midst of an emerging pandemic.

Canine MDCK cell lines are refractory to infection with human and mouse prions.

Influenza vaccine production in embryonated eggs is associated with many disadvantages, and production in cell culture systems is a viable alternative. Madin Darby canine kidney (MDCK) cells are permissive for a variety of orthomyxoviruses and have proven particularly suitable for vaccine mass production. However, mammalian cells harboring the Prnp gene can theoretically acquire prion infections. Here, we have attempted to infect MDCK cells and substrains thereof with prions. We found that MDCK cells did not produce any protease-resistant PrP(Sc) upon exposure to brain homogenates derived from humans suffering from Creutzfeldt-Jakob disease (CJD) or from mice infected with Rocky Mountain Laboratory (RML) scrapie prions. Further, transmission of MDCK lysates to N2aPK1 cells did not induce formation of PrP(Sc) in the latter. PrP(C) biogenesis and processing in MDCK cells were similar to those of prion-sensitive N2aPK1 cells. However, steady-state levels of PrP(C) were very low, and PrP(C) did not partition with detergent-resistant membranes upon density gradient analysis. These factors may account for their resistance to infection. Alternatively, prion resistance may be related to the specific sequence of canine Prnp, as suggested by the lack of documented prion diseases in dogs.

DeltaNp73 can modulate the expression of various genes in a p53-independent fashion.

DeltaNp73alpha is an isoform of the p53 homologue p73 that lacks an amino-terminal transactivation domain and antagonizes the induction of gene expression by p53. Here, we examined whether DeltaNp73alpha might also modulate cellular transcription in the absence of p53. The expression of DeltaNp73alpha in the p53-/- cell line H1299 reduced the mRNA levels of p21/CDKN1A, but did not affect other p53-responsive genes. Correspondingly, the p21/CDKN1A promoter was downregulated by DeltaNp73alpha in reporter assays, whereas other p53-responsive promoters were not inhibited. To identify additional genes that respond to DeltaNp73alpha in the absence of p53, microarrays carrying 4600 cDNA clones were hybridized. The expression of 30 genes was found to be altered more than threefold by overexpressed DeltaNp73alpha. For instance, DeltaNp73alpha increased the expression of EGR1 and CDC6, whereas it decreased the mRNA levels of c-MYC, cyclin A2/CCNA2, NF-kappaB1, ODC1, and RET finger protein/RFP. Semiquantitative reverse transcription-PCR confirmed these results and further revealed that the influence of DeltaNp73alpha on the regulation of these genes differs from other p73 isoforms and p53. We conclude that the impact of DeltaNp73alpha on gene expression is not limited to p53-responsive genes. Rather, DeltaNp73alpha can regulate the expression of a variety of genes independently of p53.

Reactivation of mutant p53 by a one-hybrid adaptor protein.

The most frequent genetic alteration in cancer is a mutation of p53. In most cases, this leads to a sharp increase of the p53 protein levels but abolishes p53's function as an activator of transcription. To correct this defect, wild-type p53 is being reintroduced into tumor cells through gene therapy vectors, thereby inducing cell death. However, this effect is not necessarily specific for tumor cells. Furthermore, mutant p53 in tumor cells trans-dominantly impairs the function of wild-type p53. As an approach to overcome these obstacles, we have developed an adaptor protein that reactivates mutant p53 rather than stimulating transcription on its own. The DNA binding and tetramerizing portions of the p53-homologue p73 were fused to the oligomerization domain of p53. This chimera binds to the DNA of p53-responsive promoters through the p73-derived portions, and it binds to mutant p53 by the p53-derived oligomerization domain. Through this one-hybrid system, mutant p53 is re-enabled to activate transcription. When the adaptor was expressed in tumor cells that contain mutant p53, expression of p53-responsive genes was activated, and growth was inhibited. No such effects were observed in cells that contain wild-type p53 or no p53 at all. When the adaptor was expressed through an adenovirus vector, tumor cells containing mutant p53 were specifically induced to undergo apoptosis. This strategy can turn mutant p53 into an inhibitor of tumor cell growth and might enable gene therapy to eliminate cancer cells with specificity.