Third generation vaccine for world eradication of poliomyelitis

Great efforts have been undertaken by the World Health Organization to achieve eradication of poliomyelitis, a paralytic disease. At present, two different vaccines are available: inactivated polio vaccine (IPV) developed by Salk based on chemical inactivation of the virus and oral polio vaccine (OPV) developed by Sabin based on live attenuated virus strains. The risks associated with IPV concern the safety of the production process as it is based on highly virulent wild type strains, and in contrast, the OPV risks are associated with the reversibility of the attenuated viruses to a transmissible paralytic form. There is therefore a need for a new generation polio vaccines capable to overcome outbreaks and manufacturing risks. With the evolution of molecular virology of Sabin vaccine strains, it is now possible to design extremely genetically stable and hyperattenuated strains without the associated reversion risks. Sabin poliovirus strains were therefore genetically modified giving rise to the third generation of polio vaccine strains [1, 2]. In the present work we have explored the possibility of using the already well-established IPV production process, developed at our site [3] and integrated worldwide [4] for the production and manufacturing of third generation of IPV strains. Specifically, we have produced third generation vaccines in animal component free medium and at 50-L pilot scale. The product obtained did show acceptable yields and was immunogenic in rats. Together, our results indicate that the third generation vaccine strains produced under the flexible platform process are potential candidates which provide increased biosafety during manufacturing which is necessary after polio eradication. In addition, the flexibility and scalability of the process constitute a platform for the production of a large range of vaccines worldwide. 1. Knowlson, S., et al., New Strains Intended for the Production of Inactivated Polio Vaccine at Low-Containment After Eradication. PLoS Pathog, 2015. 11(12): p. e1005316. 2. Macadam, A.J., et al., Rational design of genetically stable, live-attenuated poliovirus vaccines of all three serotypes: relevance to poliomyelitis eradication. J Virol, 2006. 80(17): p. 8653-63. 3. Thomassen, Y.E., et al., Scale-down of the inactivated polio vaccine production process. Biotechnol Bioeng, 2013. 110(5): p. 1354-65. 4. Wezel, v., Monolayer growth systems: Homogeneous unit processes. Spier, R. E. and Griffiths, J. B., eds., 1985: p. 266-281

Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus

Metabolic control of mitochondrial properties by adenine nucleotide translocator determines palmitoyl-CoA effects.

Inhibition of the mitochondrial adenine nucleotide translocator (ANT) by long-chain acyl-CoA esters has been proposed to contribute to cellular dysfunction in obesity and type 2 diabetes by increasing formation of reactive oxygen species and adenosine via effects on the coenzyme Q redox state, mitochondrial membrane potential (Δψ) and cytosolic ATP concentrations. We here show that 5 μm palmitoyl-CoA increases the ratio of reduced to oxidized coenzyme Q (Q

Functioning phosphorylation in liver mitochondria of high-fat diet fed rats.

AbstractWe proposed that inhibition of mitochondrial adenine nucleotide translocator (ANT) by long chain acyl-CoA (LCAC) underlies the mechanism associating obesity and type 2 diabetes. Here we test that after long-term exposure to a high-fat diet (HFD): (i) there is no adaptation of the mitochondrial compartment that would hinder such ANT inhibition, and (ii) ANT has significant control of the relevant aspects of oxidative phosphorylation. After 7 weeks, HFD induced a 24±6% increase in hepatic LCAC concentration and accumulation of the oxidative stress marker Nε-(carboxymethyl)lysine. HFD did not significantly affect mitochondrial copy number, oxygen uptake, membrane potential (Δψ), ADP/O ratio, and the content of coenzyme Q9, cytochromes b and a+a3. Modular kinetic analysis showed that the kinetics of substrate oxidation, phosphorylation, proton leak, ATP-production and ATP-consumption were not influenced significantly. After HFD-feeding ANT exerted considerable control over oxygen uptake (control coefficient C=0.14) and phosphorylation fluxes (C=0.15), extra- (C=0.23) and intramitochondrial (C=−0.56) ATP/ADP ratios, and Δψ (C=−0.11). We conclude that although HFD induces accumulation of LCAC and Nε-(carboxymethyl)lysine, oxidative phosphorylation does not adapt to these metabolic challenges. Furthermore, ANT retains control of fluxes and intermediates, making inhibition of this enzyme a more probable link between obesity and type 2 diabetes

Genome-wide analysis of yeast stress survival and tolerance acquisition to analyze the central trade-off between growth rate and cellular robustness

A genome-wide analysis of the acquisition of stress cross-tolerance shows that reduction of growth rate is an important determinant of severe stress survival. Cellular functions important for the coupling of growth rate to stress resistance are identified, as are those required for cross-tolerance acquisition independent of growth rate reduction