Abstract
Virus-like particles (VLPs) play a prominent role in vaccination as safe and highly versatile alternatives to attenuated or inactivated viruses or subunit vaccines. We present here two innovations, VLP-factory™ and ADDomer© , for creating VLPs displaying entire proteins or peptide epitopes as antigens, respectively, to enable efficient vaccination. For producing these VLPs, we use MultiBac, a baculovirus expression vector system (BEVS) that we developed for producing complex protein biologics in insect cells transfected with an engineered baculovirus. VLPs are protein assemblies that share features with viruses but are devoid of genetic material, and thus considered safe. VLP-factory™ represents a customized MultiBac baculovirus tailored to produce enveloped VLPs based on the M1 capsid protein of influenza virus. We apply VLP-factory™ to create an array of influenza-derived VLPs presenting functional mutant influenza hemagglutinin (HA) glycoprotein variants. Moreover, we describe MultiBac-based production of ADDomer© , a synthetic self-assembling adenovirus-derived protein-based VLP platform designed to display multiple copies of pathogenic epitopes at the same time on one particle for highly efficient vaccination. © 2021 The Authors. Basic Protocol 1: VLP-factory™ baculoviral genome generation Basic Protocol 2: Influenza VLP array generation using VLP-factory™ Basic Protocol 3: Influenza VLP purification Basic Protocol 4: ADDomer© BioBrick design, expression, and purification Basic Protocol 5: ADDomer© candidate vaccines against infectious diseases.
Highlights
Recombinant protein production is an essential prerequisite for cost-efficient and versatile production of complex biologics which can be used as scaffolds to display proteins and peptides that can represent antigenic epitopes from pathogens including viruses that cause infectious diseases
Virus-like particles (VLPs) are used for vaccination against a range of infectious diseases including influenza and viral infection−based malignancies such as cervical cancer caused by papilloma virus (Charlton & Lua, 2017; Cox & Hollister, 2000; Jeong & Seong, 2017; Pouyanfard & Muller, 2017; Schiller & Lowy, 2006; Temchura & Uberla, 2017)
We developed MultiBac, a modular baculovirus-based expression system suited for efficiently producing challenging protein assemblies for a wide range of applications, as we and others have described in considerable detail (e.g., Barford, Takagi, Schultz, & Berger, 2013; Berger et al, 2013; Berger, Fitzgerald, & Richmond, 2004; Bieniossek, Imasaki, Takagi, & Berger, 2012; Bieniossek, Richmond, & Berger, 2008; Fitzgerald et al, 2006; Fitzgerald et al, 2007; Nie et al, 2009; Sari et al, 2016; Trowitzsch, Bieniossek, Nie, Garzoni, & Berger, 2010; Trowitzsch, Palmberger, Fitzgerald, Takagi, & Berger, 2012; Vijayachandran et al, 2011)
Summary
Recombinant protein production is an essential prerequisite for cost-efficient and versatile production of complex biologics which can be used as scaffolds to display proteins and peptides that can represent antigenic epitopes from pathogens including viruses that cause infectious diseases. Any design in this part that would abolish immune suppression and enhance antigenicity would need to be carefully crafted to maintain HA functionality in membrane fusion To identify such mutants, an array of influenza VLPs containing randomized amino acids at certain positions in the ISD were required (Sari-Ak et al, 2019). Capsids formed by H1N1 M1 are readily released from the cells in the expression culture, acquiring the lipid bilayer envelope comprising heterologously expressed membrane proteins during the budding process Because of these characteristics of H1N1 M1, our VLP-factoryTM is not limited to flu VLPs with HA in the envelope, but can be used to produce VLPs displaying diverse viral envelope proteins, one or several simultaneously, from diverse influenza subtypes or even from other pathogens, that are expressed from genes inserted in addition to M1 and mCherry into the VLP-factoryTM baculovirus. In Basic Protocol 5, the approach to engineer the ADDomer© platform for vaccine development against an infectious disease is explained
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