Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game

Abstract

Vaccine manufacturing costs prevent a significant portion of the world's population from accessing protection from vaccinepreventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNA interference (RNAi) screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild-type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers >20-fold and >50-fold, respectively. Host gene knockdown events did not affect virus antigenicity, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced replication of enterovirus 71, a clinically relevant virus to which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing possibilities and should facilitate polio eradication using the inactivated poliovirus vaccine. IMPORTANCEUsing a genome-wide RNAi screen, a collection of host virus resistance genes was identified that, upon silencing, increased poliovirus and enterovirus 71 production by from 10-fold to >50-fold in a Vero vaccine manufacturing cell line. This report provides novel insights into enterovirus-host interactions and describes an approach to developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The results show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin strain) and wild-type polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine as well as further reduce costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine-preventable diseases. Vaccines are important defenses in the fight against infectious disease. Currently, a complex set of factors such as population dynamics and bioproduction costs limit the ability to provide adequate immunization coverage to many economically distressed countries. The current efforts to eradicate poliovirus exemplify these challenges. The oral polio vaccine (OPV), consisting of attenuated Sabin strains, has reduced the total number of poliomyelitis cases by Ͼ99% since the late 1980s. However, live attenuated strains carry the risk of phenotypic reversion to a neurovirulent "vaccine-derived poliovirus" (VDPV) capable of inducing vaccine-associated paralytic poliomyelitis (VAPP) (1, 2). To prevent the emergence and circulation of VDPVs during the polio eradication effort and beyond, OPV must be replaced by the inactivated poliovirus vaccine (IPV) (3). The cost of IPV, which is approximately $3.00 per dose compared to approximately $0.20 for OPV, is a major o...

Vero Cells as a Mammalian Cell Substrate for Human Norovirus

“…While others were unsuccessful in propagating HuNoV in Vero cells [38,44,45], we show that low-passed Vero cells can be used as a cell substrate for HuNoV. We previously used low-passaged Vero cells in the development of vaccine substrates for several viruses [52,54]. To begin to evaluate HuNoV infection, we showed that HuNoV RNAs are present within Vero cells following a 1 h or 6 h incubation by qRT-PCR ( Figure 1A).…”

“…To elucidate some of the antiviral Vero cell genes that modify HuNoV replication, we evaluated siRNA knockdown (KD) of host genes previously identified to be important for influenza virus [76], poliovirus [54], and rotavirus [53] replication. KD of several genes in Vero cells, specifically empty spiracles homeobox 2 (EMX2), fibroblast growth factor 2 (FGF2), neuraminidase 2 (NEU2), pyrroline-5carboxylate reductase 1 (PYCR1), RAD51 associated protein 1 (RAD51AP1), and Sec61 translocon gamma subunit (SEC61G) enhanced HuNoV replication in Vero cells ( Figure 5 and Supplementary Table S2).…”

CRISPR/Cas9-The ultimate weapon to battle infectious diseases?

“…CRISPR/Cas9 technologies also offer the potential to overcome some of our present obstacles in vaccine development, and novel strategies have improved yield and efficiency of vaccine manufacturing (Tang et al, 2016;van der Sanden et al, 2016;Yuan et al, 2015).…”

Poliovirus Studies during the Endgame of the Polio Eradication Program

“…In Phase I, a total of 23 proposals were accepted, and 3 projects were selected for Phase II. The projects selected for Phase II included intense continuous polio surveillance in China (Dr. Yong Zhang) (55), establishment of new highproducing cell lines for a PV vaccine (Dr. Ralph Tripp) (56), and increasing production capacity of empty PV capsids (Dr. Ian Jones) for affordable IPV. We might see a down-to-earth way of the Foundation in the selection of PV studies, but innovation, in terms of drastic breakthroughs for the eradication program endgame, has been limited.…”

“…Major basic studies of PV in the endgame of polio eradication can slightly decrease or increase the resulting neutralizing antibody titer, depending on the PV serotypes, but its actual effectiveness in the suppression of wild PV circulation remained to be verified. Development of engineered knock-out cell lines might increase the PV yield of for IPV production (56). Hyper-attenuated strains have been developed by adaptation of PV to cold temperatures (58), or by modification of the RNA stem-loop in the PV-IRES (59).…”