Efficient, trans-complementing packaging systems for chimeric, pseudoinfectious dengue 2/yellow fever viruses

Abstract

In our previous studies, we have stated to build a new strategy for developing defective, pseudoinfectious flaviviruses (PIVs) and applying them as a new type of vaccine candidates. PIVs combined the efficiency of live vaccines with the safety of inactivated or subunit vaccines. The results of the present work demonstrate further development of chimeric PIVs encoding dengue virus 2 (DEN2V) glycoproteins and yellow fever virus (YFV)-derived replicative machinery as potential vaccine candidates. The newly designed PIVs have synergistically functioning mutations in the prM and NS2A proteins, which abolish processing of the latter proteins and make the defective viruses capable of producing either only noninfectious, immature and/or subviral DEN2V particles. The PIV genomes can be packaged to high titers into infectious virions in vitro using the NS1-deficient YFV helper RNAs, and both PIVs and helpers can then be passaged as two-component genome viruses at an escalating scale.

FIG. 1

Replication of chimeric DEN2V/YFV viruses. (A) Schematic representation of the viral genomes. YFV-specific sequences are indicated by open boxes. DEN2V-specific sequences are indicated by filled boxes. FMDV 2A indicates position of FMDV 2A protease. (B) Replication of chimeric viruses in BHK-21 cells electroporated with 5 μg of in vitro-synthesized RNA. Electroporated cells were seeded into 100-mm dishes and incubated at 37°C. At the indicated time points, media were replaced, and titers of the recombinant viruses were determined by plaque assay on BHK-21 cells as described in the Materials and Methods. Dashed line indicates the limit of detection.

FIG. 2

Packaging of different PIV genomes using C_YF/YFV/Cherry helper. The in vitro-synthesized PIV and helper RNAs were transfected into BHK-21 cells by electroporation (see Materials and Methods for details). Cells were seeded into 100-mm dishes in 10 ml of media and incubated at 37°C. At the indicated time points, media were replaced and titers of packaged PIV and helper genomes were measured by infecting naïve BHK-21 cells with different dilutions of the samples and evaluating the numbers of GFP- and Cherry-positive cells. YFVsp indicates YFV-specific signal peptide. C_YFco indicates position of YFV-specific, synthetic capsid-coding sequence, having optimized codon usage. FMDV 2A indicates positions of FMDV-specific 2A protease. YFV-specific sequences are indicated by open boxes. WNV- and DEN2V-specific sequences are indicated by filled, grey boxes. Dashed line indicates the limit of detection. (A) The results of DEN/YFV/GFP PIV and helper genome packaging. (B) The results of WN/YFV/GFP PIV and helper genome packaging. (C) The results of YF/YFV/GFP PIV and helper genome packaging.

FIG. 3

Packaging of PIV genomes using NS1-deficient helper RNAs encoding C/prM/E. The in vitro-synthesized PIV and helper RNAs were transfected into BHK-21 cells by electroporation (see Materials and Methods for details). Cells were seeded into 100-mm dishes in 10 ml of media and incubated at 37°C. At the indicated time points, media were replaced and titers of packaged PIV and helper genomes were measured by infecting BHK-21 cells, containing VEErep/Pac-2A-NS1 replicon, by different dilutions of the samples and evaluating the numbers of GFP- and Cherry-positive cells. YFVsp indicates YFV-specific signal peptide. C_YFco indicates position of YFV-specific, synthetic capsid-coding sequence, having optimized codon usage. FMDV 2A indicates positions of FMDV-specific 2A protease. YFV-specific sequences are indicated by open boxes. DEN2V-specific sequences are indicated by filled, grey boxes. (A) The results of YF/YFV/GFP PIV and YF/YFV/ΔNS1/Cherry helper genome packaging. (B) The results of DEN/YFV/GFP PIV and DEN/YFV/ΔNS1/Cherry helper genome packaging. (C) The results of DEN/YFV/GFP PIV and YF/YFV/ΔNS1/Cherry helper genome packaging. The figures represent the result of one of three highly reproducible experiments.

FIG. 4

Replication of chimeric PIV2 with mutated furin cleavage site in BHK-21 cells. (A) The schematic representation of C_YF/DEN*/YFV/GFP PIV2 genome. The position of mutations in prM cleavage site is indicated by arrow. Identical amino acids in the prM alignment are denoted by dashes. The introduced mutations are indicated by blue color. YFV-specific sequences in PIV2 and helper genomes are indicated by open boxes. DEN2V-specific sequences are indicated by filled, grey boxes. FMDV 2A indicates position of FMDV 2A protease. (B) BHK-21 cells were electroporated by the in vitro-synthesized PIV2 and helper RNAs (see Materials and Methods for details). Cells were seeded into 100-mm dishes in 10 ml of media and incubated at 37°C. (C and D) BHK-21 cells were infected by the samples harvested at the previous passage at 96 h post transfection or infection, at an MOI of 1 inf.u/cell. Cells were seeded into 100-mm dishes in 10 ml of media and incubated at 37°C. At the indicated time points (B–D), media were replaced and titers of packaged PIV2 and helper genomes were measured by infecting BHK-21 cells, containing VEErep/Pac-2A-NS1 replicon, by different dilutions of the samples and evaluating the numbers of GFP- and Cherry-positive cells.

FIG. 5

Replication of two-component genome viruses having a helper genome with a deleted NS1 and the chimeric PIV2 genomes having either a mutated NS2A or mutations in both the NS2A and furin cleavage sites. (A) The schematic representation of chimeric PIV2 genomes having either mutation in YFV NS2A (C_YF/DEN/YFV*/GFP) or in both YFV NS2A and furin cleavage site of DEN2V-specific prM (C_YF/DEN*/YFV*/GFP), and helper genome YF/YFV/ΔNS1/Cherry. YFV-specific sequences are indicated by open boxes. DEN2V-specific sequences are indicated by filled, grey boxes. Arrows indicate the positions of mutations introduced into prM and NS2A. (B) Alignments of amino acid sequences of the protein fragments containing furin cleavage site and the internal cleavage site of NS2A. Identical amino acids are denoted by dashes. The introduced mutations are indicated by blue. Arrows indicate positions of cleavage. (C) BHK-21 cells were electroporated by indicated PIV2 and helper genomes and seeded into 100-mm dishes. Samples harvested at 96 h post electroporation or after starting passage 1 were used to perform the next passage, to infect naïve BHK-21 cells at an MOI of 1 inf.u/cell. At the indicated time points, media were replaced and titers of packaged PIV2 and helper genomes were measured by infecting BHK-21 cells containing VEErep/Pac-2A-NS1 replicon, by different dilutions of the samples and evaluating the numbers of GFP- and Cherry-positive cells. Dashed lines indicate the limits of detection.

Fig. 6

Analysis of viral and subviral particles released from the cells transfected with viral and PIV2 RNAs. (A) Schematic representation of the RNAs used in the experiments. Arrows indicate positions of the mutations introduced into prM and NS2A. (B) Distribution of viral and subviral particles in the discontinuous sucrose gradients. Transfected cells were seeded into 150-mm dishes and, after 24 h incubation at 37°C, media were replaced to serum-free VP-SF medium. After an additional 24 h-long incubation, 20 ml samples of collected media were clarified by low-speed centrifugation and particles were pelleted by ultracentrifugation through the cushion of 10% sucrose in SW-28 rotor (1h, 25, 000 rpm, 4°C). Pellets were suspended in PBS, loaded onto the discontinuous sucrose gradients (see Materials and Methods for details), and centrifugation was performed at 50,000 rpm for 4 h at 4°C in SW-55 rotor of Beckman Optima L-90K ultracentrifuge. Gradients were fractionated, aliquots were diluted with PBS and viruses and VLPs were pelleted in the Beckman TLA-55 rotor. Pellets were dissolved in SDS-containing protein loading buffer having no β-mercaptoethanol. Samples were analyzed by Western blotting using D1-4G2 monoclonal antibody. Secondary goat anti-mouse antibodies were labeled with IRDye 800. Membranes were analyzed on LI-COR imager.

FIG. 7

Proposed replication strategies of the chimeric PIV2 and helper genome-containing viral particles at high and low MOIs. At high MOI, both genomes, the PIV2 genome (encoding C/prM*/E, which are incapable of forming infectious virions) and NS1-deficient helper genome (encoding functional capsid, prM and E), are delivered to the same cell and produce a complete set of YFV nonstructural and structural proteins required for virus replication. Cells produce a two-component genome virus that can be further passaged at an escalating scale. At low MOI, cells receive only one of the genomes, and those infected with PIV2 produce SVPs and/or immature noninfectious viral particles with the DEN2V envelope.