Virus-like particles (VLPs) as a platform for the development of yellow fever and Zika virus vaccine candidate
June 17-22, 2018
Flaviviruses are arboviruses that have been posing serious challenges to global health since 2015. Zika virus (ZIKV) emerged in 2015 in Brazil and quickly spread to over 60 countries in Africa, Asia and the Americas, causing Guillain-Barré syndrome in adults and serious congenital malformations in fetuses of infected mothers. Besides mosquito-borne transmission, zika virus can persist for months in sexual fluids and thus poses risk also to non-endemic countries due to sexual transmission of returning travelers. Although the number of cases decreased significantly due to herd immunity in affected countries, development of a vaccine for ZIKV is of great importance to avoid future resurgence of the virus in endemic areas or future spread to currently non-endemic regions.
Yellow fever (YF) is a “historically devastating disease” (Paules and Fauci, 2017), which in past centuries killed approximately 10% of the population of cities like Philadelphia and Barcelona. Although a very effective vaccine exists for YFV, it can cause fatal adverse effects in a small proportion of vaccinees, and recent outbreaks have shown that due to its limited production in embryonated eggs the risk of serious vaccine shortages is high. Fractionating the vaccine dosis (1/5) was the emergency solution introduced by the WHO in 2016 to stop an outbreak in Africa, and is currently being adopted for mass vaccination in Brazil to try to stop the serious outbreak ongoing since 2017. The potential risk of YFV spreading to highly populated areas with no vaccination coverage, where the mosquito vector is present, such as Asia, makes urgent the development of new YFV vaccines.
In this context, virus-like particles (VLPs) can be a promising platform for developing safe and effective vaccines for YFV, ZIKV and other flaviviruses. In this work, we developed stable recombinant cell lines constitutively expressing the structural prM (pre-membrane) and E (envelope) proteins of ZIKV and YFV. Sucrose cushion ultracentrifugation and TEM images have confirmed that VLPs resembling in both size and shape the respective native viruses are formed.
In order to optimize expression, cell transfection protocol was optimized using different transfection reagents, media and host cell lines, including CHO, HEK293, BHK, MDCK and Vero cells. Stable cell lines derived from CHO-K1 and HEK293-3F6 gave the most promising results and were followed for up to 20 weeks post-transfection in the presence and absence of the selection marker, showing that cells grow to high densities with high viabilities and keeping the expression of VLPs.
The use of FACS to sort for high producer cells allowed obtaining enriched cell pools producing significantly higher amounts of VLPs and confirmed the hypothesis that secreted VLPs can be transiently detected on the cell membrane surface. Kinetic studies to evaluate different culture media and cultivation conditions under batch, pseudoperfusion and perfusion mode were carried out with the final aim of increasing productivity and reducing production costs.
Ongoing studies are focusing, on one hand, to purify the ZIKV and YFV VLPs for immunogenicity studies and, on the other hand, to express VLPs of other flaviviruses circulating in the Americas, as preparedness measure for future threats. VLPs of DENV 1, 2, 3 and 4, as well as of Saint Louis encephalitis (SLEV), Ilheus (ILHV) and Cacipacore (CPCV) viruses have already been successfully expressed by transient transfection.
The authors wish to gratefully acknowledge the Vaccine Research Center of NIAID/NIH (USA) for transferring the DENV, SLEV, ILHV and CPCV gene constructs developed during a sabbatical work of L. R. Castilho and used in this work.
Paules CI, Fauci AS (2017), Yellow Fever - Once Again on the Radar Screen in the Americas, N Engl J Med 376:1397-1399, doi: 10.1056/NEJMp1702172.
Renata Alvim and Leda R. Castilho, "Virus-like particles (VLPs) as a platform for the development of yellow fever and Zika virus vaccine candidate" in "Vaccine Technology VII", Amine Kamen, McGill University Tarit Mukhopadhyay, University College London Nathalie Garcon, Bioaster Charles Lutsch, Sanofi Pasteur Eds, ECI Symposium Series, (2018). http://dc.engconfintl.org/vt_vii/120