1. Introduction
Yellow fever virus (YFV) belongs to the Flavivirus genus and forms a virion of approximately 50 nm that is composed by a capsid, a premembrane/membrane (prM/M) protein and an envelope (E) protein, a single-stranded positive-sense RNA and a lipid envelope (Lindenbach et al., 2007). YFV is an arthropod-borne human pathogen transmitted by mosquitoes that causes a “historically devastating disease” (Paules & Fauci, 2017), for which there is no specific treatment available yet (CDC, 2019).
Before the current live-attenuated egg-derived vaccine became available in the 1930s, yellow fever (YF) was a major health threat, since outbreaks used to kill approximately 10% of the population of affected cities, such as occurred in Philadelphia (USA) in 1793 (Frierson, 2010; Monath & Vasconcelos, 2015; Paules & Fauci, 2017). Despite the availability of a highly protective vaccine, nowadays 29,000-60,000 people die annually from YF worldwide (Garske et al., 2014). Moreover, recent outbreaks in Africa (2016) and Brazil (2017-2018) have shown an inadequate preparedness for YF. In Africa in 2016, depletion of the WHO YF vaccine stockpile along with worldwide vaccine shortage led WHO authorities to start dose fractionation (1/5) as an emergency measure for outbreak control. Despite being the largest world producer of YF vaccine, Brazil also suffered from vaccine shortage in the latest outbreak and started using the 1/5 fractional dose in January 2018 for mass vaccination of the population. Since the African outbreak in 2016, when there were several imported cases to China, representing the first ever documented cases of yellow fever in Asia, concerns have arisen about vaccine availability in case the virus starts to circulate in other world regions where the mosquito vector is present, such as Asia (Paules & Fauci, 2017; WHO, 2016; Wasserman et al., 2016).
Although the existing live-attenuated vaccine confers lifetime immunity in up to 99% of vaccinees (Paules & Fauci, 2017), rare but serious adverse effects have been reported (Seligman, 2014; Porudominsky & Gotuzzo, 2018) and usually become more apparent during mass vaccination campaigns in the setting of outbreaks. Thus, considering the risks of YFV spread to world regions where the population is naïve but the mosquito vector circulates, the recently experienced shortages of the vaccine and the occurrence of rare, but fatal adverse effects demonstrate that there is a need for a new yellow fever vaccine (Shearer et al., 2018; Wong et al., 2019).
In this context, virus-like particles (VLPs) represent a promising alternative, since they are 3D particles formed by structural viral proteins produced in recombinant form, thus mimicking the virus, but lacking its genetic material. Antigens are presented in a repetitive mode, enhancing immunogenicity, so VLPs are considered a vaccine platform that combines good safety and high efficacy (Fuenmayor et al., 2017; Krol et al., 2019; Mohsen et al., 2017; Wang & Roden, 2013).
Vaccines must be affordable and their production needs to be scalable. One way to develop cost-effective production processes is to develop continuous perfusion cultivation processes, using cells stably expressing the product. Perfusion technology usually results in high product quality and high volumetric productivity. However, it is a more complex technology in comparison to batch processes, since cells need to be retained inside the bioreactor while medium is continuously renewed and product is continuously harvested (Castilho, 2015). Finding an appropriate cell retention device may be a challenge, considering that the device needs to work continuously for long periods presenting high efficiency, not causing cell damage and not retaining product in order to allow its continuous recovery in the harvest stream (Bielser et al., 2018; Carvalho & Castilho, 2017).
In the present work we generated a stable recombinant HEK293 cell line, which constitutively secretes yellow fever VLPs comprised of the prM and E proteins. In order to develop an intensified VLP production process, we combined two strategies: the use of FACS to sort recombinant cells for high producers and the use of the resulting enriched cell pools to develop continuous perfusion processes based on two different cell retention devices (ATF and cell settler). In order to better characterize the VLP product, VLPs were purified by steric exclusion chromatography and then analyzed by conventional biochemical techniques and by transmission electron microscopy.