The global adoption of vaccines to combat disease is hampered by the high cost of vaccine manufacturing. The work described herein follows two previous publications (van der Sanden et al., 2016; Wu et al., 2017) that report a strategy to enhance poliovirus and rotavirus vaccine production through genetic modification of the Vero cell lines used in large-scale vaccine manufacturing. CRISPR/Cas9 gene editing tools were used to knockout Vero target genes previously shown to play a role in polio- and rotavirus production. Subsequently, small-scale models of current industry manufacturing systems were developed and adopted to assess the increases in polio- and rotavirus output by multiple stable knockout cell lines. Unlike previous studies, the Vero knockout cell lines failed to achieve desired target yield increases. These findings suggest that additional research will be required before implementing the genetically engineered Vero cell lines in the manufacturing process for polio- and rotavirus vaccines to be able to supply vaccines at reduced prices.

The efficient establishment of high protein producing recombinant mammalian cell lines is facilitated by the use of a stringent selection system. Here, we describe two methods to create a stringent selection system based on the Zeocin resistance marker. First, we cloned increasingly longer stretches of DNA, encoding a range of 8–131 amino acids immediately upstream of the Zeocin selection marker gene. The DNA stretches were separated from the open reading frame of the selection marker gene by a stopcodon. The idea behind this was that the translation machinery will first translate the small peptide, stop and then restart at the AUG of the Zeocin marker. This process, however, will become less efficient with increasingly longer stretches of DNA upstream of the Zeocin marker that has to be translated first. This would result in lower levels of the Zeocin selection marker protein and thus a higher selection stringency of the system. Secondly, we performed a genetic screen to identify PCR induced mutations in the Zeocin selection protein that functionally impair the selection marker protein. Both the insertion of increasingly longer peptides and several Zeocin selection protein mutants resulted in a decreasing number of stably transfected colonies that concomitantly displayed higher protein expression levels. When the Zeocin mutants were combined with very short small peptides (8–14 amino acids long), this created a flexible, high stringency selection system. The system allows the rapid establishment of few, but high protein producing mammalian cell lines.

In this work, two different in vitro gastric digestion models were used to evaluate the stability of a live attenuated rotavirus vaccine candidate (RV3-BB) under conditions designed to mimic oral delivery in infants. First, a forced-degradation model was established at low pH to assess the buffering capacity of formulation excipients and to screen for RV3-BB stabilizers. Second, a sequential-addition model was implemented to examine RV3-BB stability under conditions more representative of oral administration to infants. RV3-BB rapidly inactivated at < pH 5.0 (37 C, 1 h) as measured by an infectivity RT-qPCR assay. Pre-neutralization with varying volumes of infant formula (Enfamil ®) or antacid (Mylanta ®) conferred partial to full protection of RV3-BB. Excipients with sufficient buffering capacity to minimize acidic pH inactivation of RV3-BB were identified (e.g., succinate, acetate, adipate), however, they concomitantly destabilized RV3-BB in accelerated storage stability studies. Both effects were concentration dependent, thus excipient optimization was required to design candidate RV3-BB formulations which minimize acidinduced viral inactivation during oral delivery while not destabilizing the vaccine during long-term 2 e8 C storage. Finally, a statistical Design-of-Experiments (DOE) study examining RV3-BB stability in the in vitro sequential-addition model identified key formulation parameters likely affecting RV3-BB stability during in vivo oral delivery.

Highlights A low-cost manufacturing process for a neonatal rotavirus vaccine was developed. The formulation process developed resulted in a stable liquid vaccine at 2–8 °C. No pretreatment of vaccinees with antacid needed before oral administration. Tech. transfer package includes manufacturing, formulation, assays and COGs model.

The use of high stringency selection systems often results in the induction of very few recombinant mammalian cell lines, which limits the ability to isolate a cell line with favorable characteristics. The employment of for instance STAR elements in DNA constructs elevates the induced number of colonies and also the protein expression levels in these colonies. Here, we describe a method to systematically identify genomic DNA elements that are able to induce many stably transfected mammalian cell lines. We isolated genomic DNA fragments upstream from the human Rb1 and p73 gene loci and cloned them around an expression cassette that contains a very stringent selection marker. Due to the stringency of the selection marker, hardly any colony survives without flanking DNA elements. We tested fourteen ~3500 bp DNA stretches from the Rb1 and p73 loci. Only two ~3500 bp long DNA fragments, called Rb1E and Rb1F, induced many colonies in the context of the stringent selection system and these colonies displayed high protein expression levels. Functional analysis showed that the Rb1 DNA fragments contained no enhancer, promoter, or STAR activity. Our data show the potential of a methodology to identify novel gene expression augmenting DNA elements in an unbiased manner.