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Defining the Multiplicity and Time of Infection for the Production of Zaire Vaccine 37 (2019) 6962–6969 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Defining the multiplicity and time of infection for the production of Zaire Ebola virus-like particles in the insect cell-baculovirus expression system Ana Ruth Pastor a,1, Gonzalo González-Domínguez a,b,1, Marco A. Díaz-Salinas c, Octavio T. Ramírez a, ⇑ Laura A. Palomares a, a Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ave. Universidad 2001, Cuernavaca, Morelos 62210, Mexico b Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico c Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ave. Universidad 2001, Cuernavaca, Morelos 62210, Mexico article info abstract Article history: The Ebola virus disease is a public health challenge. To date, the only available treatments are medical Available online 28 June 2019 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) Keywords: are a promising alternative. The Zaire Ebola virus serotype (ZEBOV) is the most aggressive with the high- Ebola virus disease est mortality rates. Production of ZEBOV-VLP has been accomplished in mammalian and insect cells by Virus-like particles the recombinant coexpression of three structural proteins, the glycoprotein (GP), the matrix structural ZEBOV-VLP protein VP40, and the nucleocapsid protein (NP). However, specific conditions to manipulate protein con- Design of experiments centrations 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 identi- fied 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. Ó 2019 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/). 1. Introduction aggressive serotype and has the highest mortality rates. Clinical trials have shown the efficacy of several vaccines, among them a The Ebola virus disease continues to be a challenge to human vectored vaccine based on chimpanzee adenovirus [1] and a vesic- health. In particular, the Zaire Ebola virus (ZEBOV) is the most ular stomatitis virus (VSV) chimeric vaccine [2]. These vaccines are very promising. However, both vaccines are live virus vaccines that may be safe when administered to healthy individuals, but that Abbreviations: DoE, design of experiments; GP, ZEBOV glycoprotein; hpi, hours may pose risks to immunodeficient patients, malnourished chil- post first infection; MOI, multiplicity of infection; NP, ZEBOV nucleoprotein; pfu, dren and other fragile populations. Therefore, the development of plaque forming units; TEM, transmission electron microscopy; TOI, time of infection; VLP, virus-like particles; ZEBOV, Zaire serotype Ebola virus. a recombinant, protein-based vaccine with very high purity and ⇑ Corresponding author. low adverse effects is desirable. A vaccine with such characteristics E-mail addresses: [email protected] (A.R. Pastor), gonzalo030793@gmail. would be used not for emergency situations, but to provide long- com (G. González-Domínguez), [email protected] (M.A. Díaz-Salinas), lasting protection before an outbreak starts. An alternative that [email protected] (O.T. Ramírez), [email protected] (L.A. Palomares). has shown to be safe and efficacious is the use of virus-like 1 Both authors contributed equally. https://doi.org/10.1016/j.vaccine.2019.06.029 0264-410X/Ó 2019 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). A.R. Pastor et al. / Vaccine 37 (2019) 6962–6969 6963 particles (VLPs), which are obtained when the structural proteins The results presented here are useful for the design of production of a virus are recombinantly expressed and assembled into struc- strategies of Ebola VLP. tures similar to the native virus [3,4]. Ebola virus is an enveloped filamentous negative-stranded RNA 2. Materials and methods virus formed by seven structural proteins [5]. The three most abun- dant structural proteins are VP40, the Ebola virus glycoprotein GP, 2.1. Cell line and culture medium and the nucleoprotein (NP); with a content of each protein in the capsid of approximately 37.7%, 4.7% and 17%, for VP40, GP and Sf9 (Spodoptera frugiperda) insect cells were routinely subcul- NP, respectively. VP40, the most abundant viral protein, has a tured to 0.6 Â 106 cells/mL every 3–4 days in 250 mL shake flasks molecular weight of 36 kDa. It promotes assembly of the viral cap- with 50 mL of working volume, maintained at 27 °C and 110 rpm sid by interacting with host proteins, and its recombinant expres- using serum-free Protein Sciences Formulary Medium (PSFM) (SAFC sion results in the formation of filamentous particles [6]. VP40 BiosciencesTM, Cat 67916 – 200L3216) for protein production, and interacts with the cell membrane to promote virus release from SF900II (Gibco, Cat 10902-104) for baculovirus amplification and the host cell. Coexpression of VP40 with GP results in the formation titration. of filamentous enveloped structures with GP incorporated into them, and a morphology similar to that of the native virus [7]. These 2.2. Baculovirus amplification and titration particles induce higher humoral and cellular protective responses than particles formed only by VP40, highlighting the importance Recombinant baculovirus containing: (i) gp gene (named bac- of GP for a potential vaccine [8]. In the native virus, NP forms the GP), (ii) vp40 gene (bac-VP40) and (iii) np gene (bac-NP) of ZEBOV viral nucleocapsid that protects genomic RNA. NP assembles into were designed by Dr. Carlos F. Arias and Dr. Susana López helical tubular structures covered by the envelope that contains (IBt-UNAM), and constructed using the Bac to Bac system (Invitro- VP40 and GP [9]. It has been shown that coexpression of GP or NP gen). Baculovirus stocks were amplified in Sf9 cells infected at with VP40 increases budding of VLP by 40% [10]. Interestingly, 1.5 Â 106 cells/mL at a MOI of 0.1 plaque forming units (pfu)/cell. the composition of the ZEBOV VLP not only determines their When the cell viability reached 30–40%, cultures were harvested immunogenicity but also their productivity by promoting budding. and centrifuged at 2000g for 10 min at 4 °C. The pellet was discarded Based on information from previous reports, we decided that an and the supernatant was stored at 4 °C until further use. Baculovirus Ebola VLP vaccine candidate should include NP, GP, and VP40. NP titers were determined using a cell viability MTT assay [18]. will increase VLP budding and provide stability, GP will induce neutralizing humoral and cellular responses, and VP40 will form the VLP as the main structural protein. The insect cell- 2.3. Design of experiments (DoE) and coinfected cultures baculovirus expression system was selected to produce ZEBOV VLP because of its high productivity, versatility and the efficient To identify the effects of MOI and TOI on recombinant ZEBOV coexpression of several proteins, which has been previously protein production and VLP assembly, a multi factorial design reported. These characteristics make the baculovirus expression was generated using the software Statgraphics Centurion XVI.II. system particularly suitable for the production of complex VLPs Two levels of MOI, 0.1 and 5 pfu/cell, and of time of infection [3,4,11]. ZEBOV VLPs produced by insect cells have been shown (TOI), simultaneous infection or consecutive infection (addition to be as immunogenic as VLPs produced by mammalian cells of one baculovirus at 6 h post the first infection (hpi)), were set [12,13,14]. However, a potential disadvantage of the baculovirus as factors, resulting in 32 unique experiments listed in Table 1. expression system is the need to generate large volumes of bac- Responses were cell growth and presence of each recombinant ulovirus stocks for large scale manufacturing, which can increase protein. Results were analyzed by analyses of variance (ANOVA), the appearance of defective baculovirus particles upon repeated with a significance of p < 0.05. 6 passaging [11]. Low multiplicities of infection (MOI) would then For infection, Sf9 cells were seeded at 1 Â 10 cells/mL in 50 mL be preferred for recombinant protein production, but the use of of PSFM using 250 mL shake flasks and infected with the three bac- 6 low MOI is challenging for the production of VLPs formed by sev- uloviruses when cultures reached 2 Â 10 cells/mL, as described in eral proteins, as only a fraction of the population is infected upon Table 1. Samples from each experiment were collected every 12 h virus addition, and secondary infection is needed for the infection and centrifuged at 2000g for 10 min at 4 °C. Pellets were stored at of all cells in the culture [11,15–17].
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