Production and characterization of vaccines based on flaviviruses defective in replication

Abstract

To develop new vaccine candidates for flavivirus infections, we have engineered two flaviviruses, yellow fever virus (YFV) and West Nile virus (WNV), that are deficient in replication. These defective pseudoinfectious viruses (PIVs) lack a functional copy of the capsid (C) gene in their genomes and are incapable of causing spreading infection upon infection of cells both in vivo and in vitro. However, they produce extracellular E protein in form of secreted subviral particles (SVPs) that are known to be an effective immunogen. PIVs can be efficiently propagated in trans-complementing cell lines making high levels of C or all three viral structural proteins. PIVs derived from YFV and WNV, demonstrated very high safety and immunization produced high levels of neutralizing antibodies and protective immune response. Such defective flaviviruses can be produced in large scale under low biocontainment conditions and should be useful for diagnostic or vaccine applications.

FIG. 1

Schematic representation of flavivirus PIV replication in the cells producing C or all of the viral structural proteins for trans-complementation of the defect. Replication of PIVs in normal cells in vivo or in vitro leads to release SVPs having no nucleocapsid.

FIG. 2

YFV C and YFV C, prM and E-expressing cell lines can complement replication of YF PIV. (A) Schematic representation of YFV and GFP-expressing YF PIV genome (see Materials and Methods for details). (B) Schematic representation of VEEV replicons expressing Pac gene and YFV C with the signal peptide of prM (anchored C; VEErep/C1/Pac), or anchored C with 20 a. a. of prM (VEErep/C2/Pac), or all of the YFV structural proteins (VEErep/C-prM-E/Pac). (C) The release of YF PIV by the cell lines transfected with in vitro-synthesized PIV genome. Media were replaced at the indicated time points, and titers of PIVs were determined as described in Materials and Methods. Arrows indicate time points when cells were subpassaged at a 1:5 ratio.

FIG. 3

Growth curves of YF PIV on the packaging cell lines. BHK-21 cells containing VEErep/C2/Pac and VEErep/C-prM-E/Pac replicons were infected with YF PIV at indicated MOIs. At the indicated times, media were replaced and titers of released PIV were determined as described in Materials and Methods. One of several similar experiments is displayed here. Arrows indicate time points when cells were subpassaged at 1:5 ratio.

FIG. 4

The use of cells expressing codon-optimized C gene for production of YF PIV. (A) Nucleotide sequence of synthetic gene. The introduced mutations are indicated by lowercase letters. (B) Growth curves of YF PIV on the packaging cell lines. BHK-21 cells containing VEErep/C2/Pac, VEErep/C-prM-E/Pac, VEErep/C2opt/Pac and VEErep/Copt-prM-E/Pac replicons were transfected with in vitro-synthesized YF PIV RNA as described in Materials and Methods. At the indicated times, media were replaced and titers of released PIV were determined as described in Materials and Methods. (C) Plaques developed in VEErep/C2opt/Pac-containing cell line by YFV and YF PIV after 4 days of incubation at 37°C.

FIG. 5

WN PIV develops spreading infection in packaging cells. (A) Schematic representation of WN PIV genome and VEEV replicon expressing WNV structural proteins (see Materials and Methods for details). (B) WN PIV produced foci of WNV antigen-positive cells (revealed with an antibody to NS1; see Materials and Methods for details) upon infection of BHK(VEErep/C*-E*-Pac) cells after 70 hours of incubation. (C) The same WN PIV preparations produced only single infected cells upon infection of Vero cell monolayers (revealed at 70 hours post infection).

FIG. 6

Detection of E protein released from cells infected with YF and WN PIVs. (A) BHK-21 cells were infected with YF PIV at an MOI of 5 inf.u/cell. The released SVPs were harvested and purified by ultracentrifugation as described in Materials and Methods. Samples were resolved by SDS PAGE, transferred to filters, and E protein was detected by D1-4G2 MAB. Media harvested from uninfected cells, lane 1; media harvested from the cells infected with YF PIV at 48 h post infection, lane 2; media harvested from the cells infected with YF PIVs at 72 h post infection, lane 3; YFV (2 × 10⁷ PFU), lane 4. (B) Vero cells were infected with WN PIV for 24 h, and then portions of the clarified culture fluid (collected before any cell lysis was detected), were resolved by SDS PAGE, transferred to filters, and reacted with an E-specific MAB (7H2; Bioreliance). Reaction of the same samples with polyclonal sera failed to reveal any cell-associated non-structural proteins in this preparation (results not shown) confirming that the E protein was actively secreted. Sample of WNV, lane 1; media harvested from uninfected cells, lane 2; media harvested from the cells infected with WN PIV at 48 h post infection, lane 3. (C) Western blot showing E protein content of fractions prepared from a sucrose density gradient obtained from SVPs harvested from normal (non-packaging) BHK cells electroporated with YFV PIV RNA (see Methods). The peak of E protein reactivity (at 32% sucrose) corresponded to the density of SVPs, and in agreement with this fact, migrated more slowly than YFV run in aside-by-side analyses (42%; see text).