African swine fever virus (ASFV) is the causal agent of African swine fever, a hemorrhagic and often lethal porcine disease causing enormous economical losses in affected countries. Endemic for decades in most of the sub-Saharan countries and Sardinia, the risk of ASFV-endemicity in Europe has increased since its last introduction into Europe in 2007. Live attenuated viruses have been demonstrated to induce very efficient protective immune responses, albeit most of the time protection was circumscribed to homologous ASFV challenges. However, their use in the field is still far from a reality, mainly due to safety concerns. In this study we compared the course of the in vivo infection caused by two homologous ASFV strains: the virulent E75 and the cell cultured adapted strain E75CV1, obtained from adapting E75 to grow in the CV1 cell-line. Interestingly, the kinetics of both viruses not only differed on the clinical signs that they caused and in the virus loads found, but also in the immunological pathways activated throughout the infections. Furthermore, E75CV1 confirmed its protective potential against the homologous E75 virus challenge and allowed the demonstration of poor cross-protection against BA71, thus defining it as heterologous. The in vitro specificity of the CD8+ T-cells present at the time of lethal challenge showed a clear activation against the homologous virus (E75) but not against BA71. These findings will be of utility for a better understanding of ASFV pathogenesis and for the rational designing of safe and efficient vaccines against this virus.

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Recent progress in mammalian cell culture process has resulted in significantly increased product titers, but also a substantial increase in process-and product-related impurities. Due to the diverse physicochemical properties of these impurities, there is constant need for new technologies that offer higher productivity and improved economics without sacrificing the process robustness required to meet final drug substance specifications. Here, we examined the use of new synthetic adsorptive hybrid filters (AHF) modified with the high binding capacity of quaternary amine (Emphaze TM AEX) and salt-tolerant biomimetic (Emphaze TM ST-AEX) ligands for clearance of process-related impurities like host cell protein (HCP), residual DNA, and virus. The potential to remove soluble aggregates was also examined. Our aim was to develop a mechanistic understanding of the interactions governing adsorptive removal of impurities during filtration by evaluating the effect of various filter types, feed streams, and process conditions on impurity removal. The ionic capacity of these filters was measured and correlated with their ability to remove impurities for multiple molecules. The ionic capacity of AHF significantly exceeded that of traditional adsorptive depth filters (ADF) by 40% for the Emphaze TM AEX and by 700% for the Emphaze TM ST-AEX, providing substantially higher reduction of soluble anionic impurities, including DNA, HCPs and model virus. Nevertheless, we determined that ADF with filter aid provided additional hydrophobic functionality that resulted in removal of higher molecular weight species than AHF. Implementing AHF demonstrated improved process-related impurity removal and viral clearance after Protein A chromatography and enabled a two-step purification process. The consequences of enhanced process performance are far reaching because it allows the downstream polishing train to be restructured and simplified, and chromatographic purity standards to be met with a reduced number of chromatographic steps.

In this paper, we present the protein map corresponding to the porcine peripheral blood mononuclear cells (PBMC) to better understand the role of these cells in the pig immune system. To conform the map, the proteins were separated by 2-DE using a 5-8 range pH gradient in IEF and approximately 800 spots were detected. Due to the high level of indeterminate variability associates to the 2-DE, analytical and biological variances were analyzed. The analytical variance was calculated for 50 proteins in three replicate 2-DE gels from the same protein extract whereas the biological variance was determined by comparison of the patterns obtained for the same 50 proteins in different animals. Values of 15.13 and 33.70% were determined for analytical and biological variances, respectively. These average variances will provide a quantified and statistical basis for future proteomic studies directed to evaluate relevant quantitative changes in the biological response. A representative set of the major proteins was subjected to MALDI-TOF analysis and over 75% of the proteins were identified on the basis of their similarity with its human homologue proteins. A large number of cytoskeletal and metabolic proteins were found as well as some proteins related to cell mobility and immunological functions. Finally, other proteins implicated in the cell signaling process, transport or apoptosis were also identified giving a wide overview of the porcine PBMC protein map.