Arenavirus reverse genetics for vaccine development

Abstract

Arenaviruses are important human pathogens with no Food and Drug Administration (FDA)-licensed vaccines available and current antiviral therapy being limited to an off-label use of the nucleoside analogue ribavirin of limited prophylactic efficacy. The development of reverse genetics systems represented a major breakthrough in arenavirus research. However, rescue of recombinant arenaviruses using current reverse genetics systems has been restricted to rodent cells. In this study, we describe the rescue of recombinant arenaviruses from human 293T cells and Vero cells, an FDA-approved line for vaccine development. We also describe the generation of novel vectors that mediate synthesis of both negative-sense genome RNA and positive-sense mRNA species of lymphocytic choriomeningitis virus (LCMV) directed by the human RNA polymerases I and II, respectively, within the same plasmid. This approach reduces by half the number of vectors required for arenavirus rescue, which could facilitate virus rescue in cell lines approved for human vaccine production but that cannot be transfected at high efficiencies. We have shown the feasibility of this approach by rescuing both the Old World prototypic arenavirus LCMV and the live-attenuated vaccine Candid#1 strain of the New World arenavirus Junín. Moreover, we show the feasibility of using these novel strategies for efficient rescue of recombinant tri-segmented both LCMV and Candid#1.

Fig. 1.

The polymerase I terminator (Pol-I T) is more efficient than the hepatitis delta virus ribozyme (HDVR) in promoting RNA replication and transcription of a LCMV S genome analogue. (a) Schematic representation of the reporter gene expression plasmids. Dual-reporter LCMV S RNA analogue plasmids encoding Pur-GFP and Gluc instead of the viral NP and GP, respectively, were inserted between the human RNA polymerase I promoter (hPol-I) and terminator (T, top) or the HDVR (bottom) sequences. Viral untranslated regions (UTR) and intergenic region (IGR) are indicated. (b, c) Comparison of the Pol-I T and HDVR sequences. Human 293T cells were co-transfected with the hpPol-I Gluc/Pur-GFP HDVR or hpPol-I Gluc/Pur-GFP T dual-reporter plasmids together with the pC expression plasmids for LCMV NP and L, together with pSV40-Cluc to normalize transfection efficiencies. As negative control cells were transfected without pC-NP, using empty pC to keep the total amount of transfected DNA constant. At 48 h p.t., GFP-expressing cells were detected by fluorescence microscopy (b). TCSs from the same transfections were analysed for levels of Gluc and Cluc activities (c); representative GFP expression images and Gluc fold induction over the negative controls for three independent experiments are shown. Scale bar, 100 µm.

Fig. 2.

Specificity of the Pol-I promoter. (a) Schematic representation of plasmids expressing dual-reporter LCMV S RNA analogues (MG) under the hPol-I (top) or mPol-I (bottom) promoters: dual-reporter LCMV S RNA analogue plasmids encoding Pur-GFP and Gluc instead of the viral NP and GP, were inserted between the human RNA polymerase I promoter (hPol-I, top) or the murine RNA polymerase I promoter (mPol-I, bottom) and the polymerase terminator (Pol-I T) sequences. (b, c) Species specificity of the Pol-I promoter. Human 293T cells and rodent BHK-21 cells were co-transfected with the pC LCMV NP and L expression plasmids together with hPol-I or mPol-I Gluc/Pur-GFP reporter plasmids, together with pSV40-Cluc vector to normalize transfection efficiencies. As negative control, cells were co-transfected only with LCMV pC-L, using empty pC to keep constant the total amount of transfected DNA. At 48 h p.t., GFP expression was detected by fluorescence microscopy (b) and TCSs were analysed for the presence of secreted Gluc and Cluc (c); representative GFP expression images and Gluc fold induction over the negative controls for three independent experiments are shown. Scale bar, 100 µm.

Fig. 3.

Rescue of recombinant LCMV and Candid#1 in 293T and Vero cell lines. (a) Schematic representation of the plasmids used for the generation of rLCMV and rCandid#1. Protein expression plasmids (pC) encoding NP and L were co-transfected together with the respective LCMV or Candid#1 hpPol-I S and hpPol-I L plasmids into 293T or Vero cells. At 72 h p.t., cells were passed to 10 cm dishes for an additional 72 h before TCSs were collected and assessed for viral rescue. (b, c) Virus rescue. TCSs from transfected 293T (b) and Vero (c) cells were evaluated for the presence of infectious virus progeny by immunofluorescecence after infection of fresh Vero cells. Mock-infected cells were included as controls. Representative images of at least three independent virus rescues are shown. Scale bars, 100 µm.

Fig. 4.

Rescue of recombinant tri-segmented (r3) LCMV and Candid#1 in Vero cells. (a) Schematic representation of the plasmid used for the generation of r3LCMV and r3Candid#1. (b, c) Virus rescue. Protein expression plasmids (pC) encoding NP and L were co-transfected together with the respective LCMV (b) or Candid#1 (c) hpPol-I GP/GFP, hpPol-I Gluc/NP and hpPol-I L plasmids into Vero cells. At 72 h p.t., cells were transferred into 10 cm dishes for an additional 72 h before TCSs were collected and assessed for virus rescue based on their infectivity in fresh Vero cells by GFP expression [b(i) and c(i)]. Representative images of at least three independent virus rescues are shown. Scale bar, 100 µm. TCSs from infected Vero cells were collected to assess levels of Gluc activity [b(ii) and c(ii)]. Mock-infected cells were included as negative controls. Cell lysates were prepared and analysed for protein expression by WB [b(iii) and c(iii)] using a monoclonal antibody for GFP or a polyclonal antibody for Gluc. LCMV and Candid#1 NP expressions were assessed with monoclonal antibodies. GAPDH protein expression was included as a loading control.

Fig. 5.

A two-plasmid arenavirus rescue system. (a) Schematic representation of the two-plasmid system for the generation of rLCMV and rCandid#1: Plasmids encoding either NP and the vRNA L segment (pC-NP/hpPol-I L), or the L protein and the vRNA S segment (pC-L/hpPol-I S) for the generation of recombinant arenavirus using the two-plasmid approach are indicated. (b, c) Comparative activity of pC and pC/hpPol-I-based expression plasmids in a MG assay. Human 293T cells were co-transfected with the LCMV dual-reporter MG plasmid and the pC expression plasmids encoding the viral NP and L (four-plasmid viral rescue) or the pC/hpPol-I (two-plasmid viral rescue) expression plasmids for LCMV, together with pSV40-Cluc vector to normalize transfection efficiencies. As a negative control, cells were co-transfected only with pC-L or pC-L/hpPol-I S (-NP), using empty pC to keep constant the total amount of transfected DNA. At 48 h p.t., MG expression was analysed by GFP using fluorescence microscopy (b) and TCSs were collected and analysed for Gluc and Cluc expression (c). Representative images of three independent experiments and fold induction over the negative controls are represented. Scale bar, 100 µm.

Fig. 6.

Rescue of rLCMV and rCandid#1 in Vero cell using a two-plasmid-based approach. (a) Virus rescue. Vero cells were co-transfected with plasmids illustrated in Fig. 5(a). Six days after transfection, TCSs were assessed for rLCMV and rCandid#1 viral rescues by infecting fresh monolayers of Vero cells and detection of viral antigen by immunofluorescence. Mock-infected cells were included as controls. Representative images of at least three independent virus rescues are shown. Scale bar, 100 µm. Comparison between pC+pPol-I (four-plasmid) and pC/hpPol-I (two-plasmid) based systems for the generation of rLCMV (b) and rCandid#1 (c). Viral titres in TCSs from transfected 293T and Vero cells were determined by immunofocus centre assay (FFU ml⁻¹) in Vero cells.