VLP-factory™ and ADDomer© : Self-assembling Virus-Like Particle (VLP) Technologies for Multiple Protein and Peptide Epitope Display

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.

Keywords: MultiBac; antigenic epitope; baculovirus expression vector system (BEVS); immunization; protein and peptide display; vaccine; virus-like particle (VLP).

Figure 1

MultiBac‐Based VLP‐factory™. On the right, the MultiBac baculoviral genome customized for enveloped virus‐like particle production is shown schematically. The capsid‐forming influenza H1N1 matrix protein M1 and the fluorescent protein mCherry are integrated into the viral backbone. The mCherry protein is a reporter for tracking virus performance. Influenza surface protein (hemagglutinin HA and/or neuraminidase NA) insertion into this VLP‐factory™ by means of transfer plasmids results in baculoviruses producing influenza virus‐like particles (VLPs). Electron micrographs of a selection of the VLPs is shown below. Recombinant influenza VLPs produced by the customized MultiBac genome (VLP‐factory™) and live influenza virus are close to identical in terms of size, shape, and appearance (inset, image courtesy of R. Ruigrok). Scale bars (50 μm) are drawn in white (adapted from Sari‐Ak et al., 2019).

Figure 2

The ADDomer^©. Electron cryo‐microscopy (Cryo‐EM) map of the synthetic self‐assembling ADDomer^© particle, formed by 60 identical protomers. The protomers assemble into 12 pentons, forming a dodecahedron characterized by remarkable thermostability (Vragniau et al., 2019).

Figure 3

Overview of VLP‐factory™ protocol. VLP‐factory™ was created for the expression of a functional influenza VLP variant assay. A transfer plasmid library including genes encoding wild‐type and mutant influenza HA5 and HAB proteins was transformed into VLP‐factory™ (Basic Protocol 1). Illustrated is the initial virus in 6‐well plates, already giving the signal for the expression of mCherry that we perceive from the red color change (Basic Protocol 2). Parallel expression enables extraction and purification of the influenza VLPs by sedimentation and gradient centrifugation for functional studies (Basic Protocol 3).

Figure 4

Influenza VLPs. A selection of VLP‐factory™‐produced samples were examined by negative‐stain electron microscopy (EM). M1 expression alone already produces budded enveloped capsids (top row) in the absence of influenza envelope proteins. Co‐expression of influenza wild‐type or mutant HA and HAB results in VLPs displaying the characteristic protruding spike‐shaped pattern (middle and bottom rows). Scale bars (50 μm) are colored in white.

Figure 5

VLP‐factory™‐produced influenza VLPs are functionally active. By Western blotting, the presence of HA protein in HA5 M1 VLP preparation was examined with a specific antibody (H5N1 MIA‐0052) showing the presence of specific HA bands (left). The right lane in the Western blot is the control containing VLPs comprising M1 only, showing no HA signal. M stands for molecular weight marker (sizes sin kDa). Recombinant influenza VLP was analyzed in a hemolysis experiment (middle). The y axis denotes the percentage of lysed red blood cells (RBC) in the experiment. The ELISA plot (right) shows antibody titers (IgG1) after two injections (adapted from Sari‐Ak et al., 2019). The y axis represents the level of antibody measured in these experiments.

Figure 6

ADDomer^© VLP production. MultiBac‐based production of ADDomer^©. VLP production (Basic Protocol 4) is shown schematically, including a Coomassie‐stained SDS‐PAGE section of highly purified VLP. Plasmid pACEBac‐ADDomer comprises the synthetic gene encoding ADDomer^© in a BioBrick format as indicated (adapted from Vragniau et al., 2019). EMBacY is a version of the MultiBac baculovirus comprising a YFP reporter gene in the viral backbone. VL, insertion site engineered in variable loop; RGD1 and RDG2, insertion sites in RGD loop; Tn7L, Tn7R and mini‐attTn7, recognition and attachment sequences for Tn7 transposase; LoxP, site‐specific recombination site for Cre enzyme; Gent^R, Gentamicin resistance gene; Kan, Kanamycin; Amp, Ampicillin; LacZ, gene for blue/white screening of composite genome; ori^ColE1, replication origin; YFP, yellow fluorescent protein; M, molecular weight marker (sizes given in kDa).

Figure 7

ADDomer^© VLP scaffold sequence. Three parts in the protomer region which are evolutionarily nonconserved engineered with BioBrick format into exchangeable cassettes. One cassette (colored in red) is placed within the VL region, while two additional cassettes, RGD1 (colored in light blue) and RGD2 (colored in dark blue), are located within the loop including a functional RGD tripeptide sequence. Amino acid residues encoded by restriction enzymatic sites in BioBrick design are indicated (colored in yellow and boxed). The RGD tripeptide sequence is highlighted (colored in green).

Figure 8

Native‐like peptide epitope presentation on ADDomer‐tevCHIK. Chikungunya envelope protein E2 consisting of three domains is shown (A, B, and C). E2EP3 (comprises the N‐terminus of E2 with the amino acid sequence NH2‐STDKDNFNVYKATRPYLAH). This E2E3P peptide, the major neutralizing epitope in the Chikungunya virus, is presented in an exposed native‐like conformation in ADDomer‐tevCHIK, with native‐like meaning that the N‐terminal serine residue of the epitope is accessible exactly as is the case in the Chikungunya viral envelope protein containing the major neutralizing epitope E2EP3. The N‐terminus was liberated by TEV protease cleavage at a specific site as indicated.

Figure 9

Genetically encoded multiepitope display by ADDomer^©. Coomassie‐stained SDS‐PAGE of ADDomer^© VLPs is shown displaying a range of epitope arrangements (see Table 2). CHIMP, chimpanzee; OVA, melanoma model epitope; CHICK, Chikungunya major neutralizing epitope; tev, Tobacco Etch Virus NIa proteolytic site; M1, M2 are molecular weight markers (sizes in kDa). The variability of the migration pattern of the ADDomer band reflects the different sizes of the constructs.