Process intensification for the production of yellow fever virus‐like particles as potential recombinant vaccine antigen

Abstract

Yellow fever (YF) is a life‐threatening viral disease endemic in parts of Africa and Latin America. Although there is a very efficacious vaccine since the 1930s, YF still causes 29,000–60,000 annual deaths. During recent YF outbreaks there were issues of vaccine shortage of the current egg‐derived vaccine; rare but fatal vaccine adverse effects occurred; and cases were imported to Asia, where the circulating mosquito vector could potentially start local transmission. Here we investigated the production of YF virus‐like particles (VLPs) using stably transfected HEK293 cells. Process intensification was achieved by combining sequential FACS (fluorescence‐activated cell sorting) rounds to enrich the stable cell pool in terms of high producers and the use of perfusion processes. At shaken‐tube scale, FACS enrichment of cells allowed doubling VLP production, and pseudoperfusion cultivation (with daily medium exchange) further increased VLP production by 9.3‐fold as compared to batch operation mode. At perfusion bioreactor scale, the use of an inclined settler as cell retention device showed operational advantages over an ATF system. A one‐step steric exclusion chromatography purification allowed significant removal of impurities and is a promising technique for future integration of upstream and downstream operations. Characterization by different techniques confirmed the identity and 3D‐structure of the purified VLPs.

From a recombinant key antigen to an accurate, affordable serological test: Lessons learnt from COVID-19 for future pandemics

“…1 A, left panel). However, differently from previous studies that adopted transient protein expression techniques [13] , [4] , we focused on stable and constitutive gene expression because transgene integration in the cell genome enhances scalability and significantly decreases costs of recombinant protein production in mammalian cells [3] . Due to the challenges posed in the early months of a pandemic, such as the urgency and the disruption of international supply chains for reagents and synthetic gene constructs, we decreased time and costs by using an old-fashioned technique of co-transfecting the plasmid containing the S gene with an intellectual property-free plasmid (pCIneo, Promega) containing an eukaryotic selection marker.…”

Nanovaccines for Advancing Long-Lasting Immunity against Infectious Diseases

“…Self-assembling or particulate protein immunogens are validated vaccine modalities that are clinically safe and effective . VLPs are examples of such protein-based NPs, with a size distribution from 20 to 800 nm, and lacking any viral genetic material. , Furthermore, VLPs act as a platform for homologous or heterogeneous antigens display, represent an alternative approach to vaccine engineering. , For example, some VLP influenza vaccines could induce cross-reactive responses against homo- and heterosubtype viruses, and long-lived immunity as well. − Compared to isolated protein subunits, this design strategy maintains a high antigen density and repetitive antigen display, which are two exclusive characteristics of virus particles. Further, VLPs are more likely to be taken up by antigen-presenting cells (APCs), promote receptor clustering and subsequent B cell activation, which may account for the ability of VLP-based vaccines to induce potent and long-lasting immune responses. , Furthermore, other nanovaccines have advantages including cross-protective immunity against pathogen mutation, intranasal administration for mucosal immune responses, and large-scale production .…”

“…97,98 Furthermore, VLPs act as a platform for homologous or heterogeneous antigens display, 99 represent an alternative approach to vaccine engineering. 100,101 For example, some VLP influenza vaccines could induce cross-reactive responses against homo-and heterosubtype viruses, and long-lived immunity as well. 102−104 Compared to isolated protein subunits, this design strategy maintains a high antigen density and repetitive antigen display, which are two exclusive characteristics of virus particles.…”

The Present and Future of Yellow Fever Vaccines

“…However, challenge experiments are required to assess the efficacy of the potential vaccine in the mouse model. Manufacturing VLPs at scale has recently been reported for such a candidate vaccine [114].…”