The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging. Here we present a production platform based on BHK-21 suspension (BHK-21SUS) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1-48PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKVPE) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21SUS cells with ZIKVPE in shake flasks resulted in poor virus replication, with a maximum titer of 8.9×103PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2×107cells/mL, and virus titers of 3.9×107PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards process development for manufacturing inactivated or live-attenuated ZIKV vaccines.

We used the recombinant trimeric spike (S) glycoprotein in the prefusion conformation to immunize horses for the production of hyperimmune globulins against SARS-CoV-2. Serum antibody titers measured by ELISA were above 1:10⁶, and the neutralizing antibody titer against authentic virus (WT) was 1:14,604 (average PRNT₉₀). Plasma from immunized animals was pepsin digested to remove the Fc portion and purified, yielding an F(ab’)₂ preparation with PRNT₉₀ titers 150-fold higher than the neutralizing titers in human convalescent plasma. Challenge studies were carried out in hamsters and showed the in vivo ability of equine F(ab’)₂ to reduce viral load in the pulmonary tissues and significant clinical improvement determined by weight gain. The neutralization curve by F(ab’)₂ was similar against the WT and P.2 variants, but displaced to higher concentrations by 0.39 log units against the P.1 (Gamma) variant. These results support the possibility of using equine F(ab’)₂ preparation for the clinical treatment of COVID patients.

Zika virus (ZIKV) was first detected in Brazil in 2015 and then rapidly spread to more than 80 countries in Africa, Asia and the Americas. ZIKV infection was correlated with severe congenital malformations in newborns from infected mothers, as well as with Guillain-Barré syndrome in adults. Although the number of infected people has declined in the affected countries lately, the development of a vaccine for ZIKV is of great importance to avoid the future resurgence of the virus in endemic areas or the future spread to currently non-endemic regions. Among many different platforms currently under study, virus-like particles (VLPs) are a promising alternative for the development of vaccines, since tridimensional particles mimicking the virus - but lacking its genome - can be produced and present the antigen in a repetitive way, potentially eliciting robust immune responses. In this work, we demonstrated the generation of stably transfected HEK293 cells constitutively expressing Zika VLPs. Small-scale shake flask studies using a stable cell pool enriched by Fluorescence-Activated Cell Sorting (FACS) showed that daily medium exchange (intermittent perfusion) significantly enhances viable cell density and VLP production (∼4-fold) over batch cultures. Continuous perfusion in a controlled bioreactor coupled to an ATF-2 cell retention device resulted in maximum VLP titers similar to those obtained under small-scale intermittent perfusion. Our results show that the use of cell lines constitutively expressing Zika VLPs, cultured in stirred-tank perfusion bioreactors, represents a promising system for the production of a VLP-based Zika vaccine candidate.