Highly efficient induction of protective immunity by a vaccinia virus vector defective in late gene expression

Abstract

Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10(4) PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10(3) PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy.

FIG. 1

Map of the D4R gene regions in wt vaccinia virus (WR-wt) and in the defective viruses vD4-vA and vD4-prME. In vD4-vA, the D4R ORF is partially substituted by a 400-bp DNA fragment of phage φX. In vD4-prME, the substitution comprises the TBE virus prM/E, lacZ, and gpt ORFs. The direction of transcription is indicated by arrows.

FIG. 2

Growth of defective and wt viruses. Infected cells were harvested at the indicated time points and titered on the complementing cell line RK-D4R-44.20 (RK44) for the defective viruses vD4-vA and vD4-prME and on wt cells (RK13) for the WR wt virus.

FIG. 3

β-Gal production by defective and wt viruses in noncomplementing RK13 cells. The cultures were infected with 10 PFU of the respective virus per cell, and β-Gal was measured at the indicated time points. The viruses contain the same promotor-lacZ gene construct. Average values of three independent experiments are shown. OD₄₁₅, optical density at 415 nm.

FIG. 4

Expression of TBE virus prM/E antigens. Cell cultures were infected with 0.1 (A) or 1.0 (B) PFU/cell, and total proteins were harvested 72 h postinfection. Lane 1, size marker (positions are indicated in kilodaltons); lanes 2 to 5, complementing cells (A; RK-D4R-44) or cells nonpermissive for defective virus (B; RK-13) infected with the defective virus clones vD4-prME#6 (lane 2) and vD4-prME#9 (lane 3), negative control defective virus vD4-vA (lane 4), and positive control wt virus varec280 (lane 5); lane 6, CV-1 cells infected with varec280; lane 7, purified TBE virus glycoprotein E. The upper arrow points to the prM/E fusion protein and the lower arrow points to glycoprotein E.

FIG. 5

Vaccination of mice with defective vaccinia viruses expressing the TBE virus prM/E antigen and protection against a lethal TBE virus challenge. Groups of 10 mice were vaccinated once with the viruses and doses indicated and challenged 21 days later with 100 LD₅₀ of TBE virus. The y axis represents the number of survivors 3 weeks after challenge. Mean values of two consecutive experiments are shown. (A) Intramuscular vaccination; (B) Subcutaneous vaccination.

FIG. 6

Rapid clearance of the defective virus in vaccinated animals. (A) Replication of the vaccine preparations in cyclophosphamide-treated mice was assayed by injecting two different doses of the defective virus vD4-prME (10⁶ or 10⁸ PFU/animal [d6 or d8]) and one dose of replicating virus varec280 (10⁶ PFU/animal [r6]). Virus was isolated from muscle preparations (site of inoculation) at the times indicated. Titration of the defective virus was done in the complementing cell line RK-D4R-44; the varec280 virus was titered in RK13 cells. Average titers obtained from three animals are shown. (B) Titer and genomic DNA load in the spleens of vaccinated animals. After injection of defective virus (10⁸ PFU/animal), spleens were isolated at the indicated times and the virus titer was determined (d8). From the same preparations, the amount of vaccinia DNA, given in genomic equivalents (ge), was determined by quantitative PCR. Titers below the detection limit (dashed line) are indicated by open symbols.