Expression of BHRF1 improves survival of murine hybridoma cultures in batch and continuous modes.

Cell death by apoptosis limits growth and productivity in most animal cell cultures. It is therefore desirable to define genetic interventions to generate robust cell lines with superior performance in bioreactors, either by increasing specific productivity, life-span of the cultures or both. In this context, forced expression of BHRF1, an Epstein-Barr virus-encoded early protein with structural and functional homology with the anti-apoptotic protein Bcl-2, effectively protected hybridomas in culture and delayed cell death under conditions of glutamine starvation. In the present study, we explored the potential application of BHRF1 expression in hybridomas for long-term apoptosis protection under different biotechnological process designs (batch and continuous) and compared it to strategies based on Bcl-2 overexpression. Our results confirmed that long-term maintenance of the anti-apoptotic effect of BHRF1 can be obtained using bicistronic configurations conferring enhanced protection compared to Bcl-2, even in the absence of selective pressure. Such protective effect of BHRF1 is demonstrated both in batch and continuous culture. Moreover, a further analysis at high cell densities in semi-continuous perfusion cultures indicated that the mechanism of action of BHRF1 involves cell cycle arrest in G0-G1 state and this is translated in lower numbers of dead cells.

Protective effect of viral homologues of bcl-2 on hybridoma cells under apoptosis-inducing conditions.

Targets for metabolic engineering have been identified in a hybridoma cell line to make it more robust in culture toward potential limitations inducing apoptosis. The cells were genetically modified with plasmids harboring endogenous bcl-2 gene and also with viral Bcl-2 homologues, particularly ksbcl-2 and bhrf-1 genes. When cells were exposed to apoptosis-inducing conditions (i.e., glutamine-free medium), the control cells exhibited a decrease in viable cell number within the first 12 h, whereas, for the bcl-2 and ksbcl-2 transfected cell cultures, the viable cell number did not exhibit any clear decrease until after 60 h. Furthermore, hybridoma cells expressing the viral homologue bhrf-1 were even more resistant to cell death, showing a decrease in viability of only 50% at 72 h of culture in glutamine-deprived medium, substantially lower than the 90% viability decrease observed for the control culture. In addition, and most relevant for further bioprocess applications, the cells genetically modified could be brought back to growth conditions even after being exposed to glutamine-deprived conditions during a significant time window, up to 72 h.

Towards licensing of CP7_E2alf as marker vaccine against classical swine fever-Duration of immunity.

Classical swine fever (CSF) marker vaccine candidate CP7_E2alf was tested in a "duration of immunity" trial according to the World Organisation for Animal Heath (OIE) guidelines. To this means, 15 weaner pigs were either orally or intramuscularly vaccinated with a single dose of CP7_E2alf vaccine produced under Good Laboratory Practice (GLP) conditions. Ten additional pigs were included as controls. Six months later, all animals were oronasally challenged with highly virulent CSF virus (CSFV) strain "Koslov". Upon vaccination, all but one orally and all intramuscularly vaccinated pigs developed rising and later on stable CSFV glycoprotein E2-specific antibodies. In contrast, no CSFV E(rns)-specific "marker" antibodies were detectable prior to challenge infection. None of the co-housed control animals seroconverted. Upon challenge infection, all seropositive animals were protected from lethal challenge, whereas all control animals and the non-responder developed severe signs of CSF. One control animal recovered, the others had to be euthanised due to animal welfare reasons between days 4 and 7 post challenge infection. All protected animals showed quickly rising neutralizing antibodies reaching high titres by the end of the trial. At the end of the trial, the marker ELISA was positive for most challenged animals that survived the CSFV infection (27 out of 30). Using reverse transcription polymerase chain reaction, low level genome detection was seen in all vaccinated animals between days 4 and 10 post challenge infection, but no virus could be isolated from any samples of these animals. The OIE guidelines require seroconversion in at least 8 out of 10 vaccinated animals. This requirement was fulfilled. Moreover, only control animals should die. With this requirement, only the intramuscular vaccination fully complied as one orally vaccinated pig did not respond. Concluding, CP7_E2alf induced stable antibodies that led to protection from lethal challenge with highly virulent CSFV strain "Koslov" six months after vaccination, with the exception of one non-responder after oral vaccination.

Vectored vaccines to protect against PRRSV.

PRRSV is the causative agent of the most important infectious disease affecting swine herds worldwide, producing great economic losses. Commercially available vaccines are only partially effective in protection against PRRSV. Moreover, modified live vaccines may allow virus shedding, and could revert generating virulent phenotypes. Therefore, new efficient vaccines are required. Vaccines based on recombinant virus genomes (virus vectored vaccines) against PRRSV could represent a safe alternative for the generation of modified live vaccines. In this paper, current vectored vaccines to protect against PRRSV are revised, including those based on pseudorabies virus, poxvirus, adenovirus, and virus replicons. Special attention has been provided to the use of transmissible gastroenteritis virus (TGEV) as vector for the expression of PRRSV antigens. This vector has the capability of expressing high levels of heterologous genes, is a potent interferon-α inducer, and presents antigens in mucosal surfaces, eliciting both secretory and systemic immunity. A TGEV derived vector (rTGEV) was generated, expressing PRRSV wild type or modified GP5 and M proteins, described as the main inducers of neutralizing antibodies and cellular immune response, respectively. Protection experiments showed that vaccinated animals developed a faster and stronger humoral immune response than the non-vaccinated ones. Partial protection in challenged animals was observed, as vaccinated pigs showed decreased lung damage when compared with the non-vaccinated ones. Nevertheless, the level of neutralizing antibodies was low, what may explain the limited protection observed. Several strategies are proposed to improve current rTGEV vectors expressing PRRSV antigens.

Modified live marker vaccine candidate CP7_E2alf provides early onset of protection against lethal challenge infection with classical swine fever virus after both intramuscular and oral immunization.

Due to the vast economic consequences of classical swine fever (CSF) outbreaks, emergency vaccination plans are under discussion in European Union Member States. However, animals vaccinated with the conventional C-strain vaccine are subject to trade restrictions. To ease these restrictions, potent marker vaccines are required. One promising candidate is the chimeric pestivirus CP7_E2alf. For emergency vaccination in a CSF outbreak scenario, early onset of immunity is required. Here, the studies performed with a CP7_E2alf virus stock produced under good manufacturing conditions (GMP) are reported. In challenge experiments, CP7_E2alf induced full clinical protection 1 week after intramuscular vaccination, and 2 weeks after oral immunization. Furthermore, even after application of diluted vaccine preparations complete protection could be achieved if challenge infection was carried out 4 weeks after vaccination. In conclusion, GMP-produced CP7_E2alf proved to be a suitable marker vaccine candidate - also for emergency vaccination - both after intramuscular and oral application.

Metabolic engineering of apoptosis in cultured animal cells: implications for the biotechnology industry.

Animal cells have been widely used to obtain a wide range of products for human and animal healthcare applications. However, the extreme sensitivity of these cells in respect to changes experienced in their environment is evidenced by the activation of a gene-encoded program known as apoptosis, resulting in their death and destruction. From the bioprocess angle, losses in cell viability bring lower productivities and higher risks of product degradation. Consequently, many research efforts have been devoted to the development of apoptosis protective mechanisms, including the metabolic engineering of apoptosis pathways, that has proven effective in diminishing programmed cell death in a variety of biotechnological relevant cell lines. This review is focused especially in the encouraging initial results obtained with the over-expression of cloned anti-apoptosis genes, from both endogenous and viral origin interfering at mitochondrial and initiator caspases levels.