Development of a Multivalent Kunjin Virus Reporter Virus-Like Particle System Inducing Seroconversion for Ebola and West Nile Virus Proteins in Mice

Abstract

Kunjin virus (KUNV) is an attenuated strain of the severe neurotropic West Nile virus (WNV). The virus has a single-strand positive-sense RNA genome that encodes a polyprotein. Following gene expression, the polyprotein is cleaved into structural proteins for viral packaging and nonstructural proteins for viral replication and expression. Removal of the structural genes generate subgenomic replicons that maintain replication capacity. Co-expression of these replicons with the viral structural genes produces reporter virus-like particles (RVPs) which infect cells in a single round. In this study, we aimed to develop a system to generate multivalent RVPs based on KUNV to elicit an immune response against different viruses. We selected the Ebola virus (EBOV) glycoprotein (GP) and the matrix protein (VP40) genes, as candidates to be delivered by KUNV RVPs. Initially, we enhanced the production of KUNV RVPs by generating a stable cell line expressing the KUNV packaging system comprising capsid, precursor membrane, and envelope. Transfection of the DNA-based KUNV replicon into this cell line resulted in an enhanced RVP production. The replicon was expressed in the stable cell line to produce the RVPs that allowed the delivery of EBOV GP and VP40 genes into other cells. Finally, we immunized BALB/cN mice with RVPs, resulting in seroconversion for EBOV GP, EBOV VP40, WNV nonstructural protein 1, and WNV E protein. Thus, our study shows that KUNV RVPs may function as a WNV vaccine candidate and RVPs can be used as a gene delivery system in the development of future EBOV vaccines.

Keywords: Ebola virus; Kunjin virus; glycoprotein (GP); matrix protein (VP40); packaging system; replicons; reporter virus-like particles (RVPs); seroconversion; stable cell line; vaccines.

Figure 1

Establishment of a stable cell line expressing the Kunjin virus (KUNV) packaging system of capsid (C), precursor membrane (prM), and envelope (E), C-prM-E. (A) Schematic illustration of the gene construct expressing C-prM-E having G418 antibiotic resistance. Gene expression is driven by the CAG promoter. The sequence coding C-prM-E was fused with an internal ribosome entry site (IRES) sequence and the neomycin/kanamycin resistance (NeoR/KanR) gene, followed by the polyadenylation signal (pA). To generate baby hamster kidney cells (BHK)-21 stably expressing KUNV C-prM-E, the gene construct was transfected into BHK-21 cells, followed by selection for transfected cells with G418. Cells were then separated into single cells and grown as individual clones. The schema was generated by using the Biorender web tool. (B) Immunoblotting of the cell lysates of each clone (1–11) to examine the expression level of E protein. The expression levels were normalized using the endogenous Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein as control. (C) Immunofluorescence staining of cells from clone 9 with the E antibody. The nucleus was counterstained by DAPI (blue). The bar scales represent 20 µm.

Figure 2

Construction of an enhanced KUNV reporter virus-like particle (RVP) production system. (A) The scheme illustrates the infectious cycles to form RVPs. (1) The DNA KUNV replicon is transfected into the stable cell line expressing C-prM-E. The RNA replicon is then expressed and replicated inside replication compartments. The RNA replicon is packaged by C, prM, and E, forming RVPs (2). The RVP progeny may infect and replicate in neighboring C-prM-E expressing cells, forming additional RVPs. (B) Schematic illustration of the DNA-based KUNV replicon construct. The replicon is driven by the Cytomegalovirus (CMV) promoter expressing an open reading frame flanked by the 5′- untranslated region (UTR) and the 3′-UTR comprising: first, 81 nucleotides of the C gene fused in frame with the firefly luciferase gene (Luc) as a reporter gene, the foot-and-mouth disease virus autoprotease 2a (FMDV 2A) and, last, 84 nucleotides of the E gene and all the nonstructural proteins. The antigenomic hepatitis delta virus ribozyme (HDVr) sequence was inserted immediately downstream of the KUNV 3′-UTR, followed by the Simian virus 40 (SV40) polyadenylation signal (pA). (C) Immunofluorescence labeling of the C-prM-E stable cells transfected with the replicon construct 3 days and 5 days post-transfection with an antibody against dsRNA (red). The nucleus was counterstained by DAPI (blue). Bar scales represent 20 µm. (D) Relative luciferase units (RLU) from cell lysates 3–7 days post-transfection. (E) Gene copy numbers of replicon from cell lysates, and cell culture supernatants 3–7 days post-transfection as measured by qPCR. The experiments were conducted independently three times with two technical repeats. The p values are indicated using ** p < 0.01 and *** p < 0.001.

Figure 3

Transmission electron microscopy images of the RVP production system. (A–D) Images representing the C-prM-E stable BHK-21 cell line transfected with the KUNV replicon construct. (A,C) represent different subcellular areas, whereas (B,D) show higher magnification images of the yellow boxes indicated in the (A) and the (C), respectively. (E) The image represents BHK-21 cells used as control. (F) The image of cell culture supernatants from the C-prM-E stable cells transfected with the KUNV replicon. ER: the endoplasmic reticulum; Nu: nucleus; M: mitochondria; CM/PC: convoluted membranes, paracrystalline structure; Vp: vesicle packets; Ve: virus-induced vesicles in the ER; VLP/RVP: virus-like particle, reporter virus-like particles; Ri: ribosome. Scale bars represent 500 nm in (A,C,E) and 100 nm in (B,D,F).

Figure 4

KUNV RVPs infect cells in a single round. (A) Schematic illustration of the process of RVP infection of BHK-21 cells. Hereby, the absence of a packaging system in the infected cells limits additional RVP production. (B) Gene copy numbers of the KUNV replicon in cell lysates after first- and second-round infection and uninfected control lysates as measured by qPCR. The experiments were conducted independently three times with two technical repeats. The p values are indicated using *** p < 0.001.

Figure 5

Expression of EBOV vaccine candidates using the KUNV replicon. (A) The luciferase reporter gene (Luc) was substituted with genes coding EBOV GP or VP40 in the DNA replicon. (B) Immunoblotting of cell lysates 2 days after transfection with EBOV GP or EBOV VP40 replicons versus the untransfected cell control.

Figure 6

Immunofluorescence labeling of BHK-21 C-prM-E cells after transfection with the KUNV replicons expressing EBOV GP or VP40 proteins (A,B), respectively, followed by two cycles of RVP infections of BHK-21 cells. The cells were visualized with the antibodies anti-EBOV GP (green), EBOV VP40 (green) (A,B), respectively, and the antibodies anti-dsRNA (red) and KUNV NS1 (red). The nucleus was counterstained with DAPI (blue). Bar scales represent 20 µm.

Figure 7

RVPs induced seroconversion. (A) Schematic illustration of the mice immunization schedule. Mice sera from the study groups were assayed with enzyme-linked immunosorbent assays (ELISA) to measure antibodies against EBOV GP (B), EBOV VP40 (C), WNV NS1 (D), and WNV E (E). The dashed line indicates the detection limit as suggested by the kit manufacturers. The assays were conducted with two technical repeats. The p values are indicated using * p < 0.05 and ** p < 0.01.