Defining the multiplicity and time of infection for the production of Zaire Ebola virus-like particles in the insect cell-baculovirus expression system

The Ebola virus disease is a public health challenge. To date, the only available treatments are medical support or the emergency administration of experimental drugs. The absence of licensed vaccines against Ebola virus impedes the prevention of infection. Vaccines based on recombinant virus-like particles (VLP) are a promising alternative. The Zaire Ebola virus serotype (ZEBOV) is the most aggressive with the highest mortality rates. Production of ZEBOV-VLP has been accomplished in mammalian and insect cells by the recombinant coexpression of three structural proteins, the glycoprotein (GP), the matrix structural protein VP40, and the nucleocapsid protein (NP). However, specific conditions to manipulate protein concentrations and improve assembly into VLP have not been determined to date. Here, we used a design of experiments (DoE) approach to determine the best MOI and TOI for three recombinant baculoviruses: bac-GP, bac-VP40 and bac-NP, each coding for one of the main structural proteins of ZEBOV. We identified two conditions where the simultaneous expression of the three recombinant proteins was observed. Interestingly, a temporal and stoichiometric interplay between the three structural proteins was observed. VP40 was required for the correct assembly of ZEBOV-VLP. High NP concentrations reduced the accumulation of GP, which has been reported to be necessary for inducing a protective immune response. Electron microscopy showed that the ZEBOV-VLP produced were morphologically similar to the native virus micrographs previously reported in the literature. A strategy for producing ZEBOV in insect cells, which consists in using a high MOI of bac-VP40 and bac-GP, and reducing expression of NP, either by delaying infection or reducing the MOI of bac-NP, was the most adequate for the production of VLP.