Construction, phenotypic analysis, and immunogenicity of a UL5/UL29 double deletion mutant of herpes simplex virus 2

Abstract

A number of studies have shown that replication-defective mutant strains of herpes simplex virus (HSV) can induce protective immunity in animal systems against wild-type HSV challenge. However, all of those studies used viruses with single mutations. Because multiple, stable mutations provide optimal levels of safety for live vaccines, we felt that additional mutations needed to be engineered into a candidate vaccine strain for HSV-2 and genital herpes. We therefore isolated an HSV-2 strain with deletion mutations in two viral DNA replication protein genes, UL5 and UL29. The resulting double deletion mutant virus strain, dl5-29, fails to form plaques or to give any detectable single cycle yields in normal monkey or human cells. Nevertheless, dl5-29 expresses nearly the same pattern of gene products as the wild-type virus or the single mutant viruses and induces antibody titers in mice that are equivalent to those induced by single deletion mutant viruses. Therefore, it is feasible to isolate a mutant HSV strain with two mutations in essential genes and with an increased level of safety but which is still highly immunogenic.

FIG. 1

Plasmids used in this study. (A) UL29 gene plasmids. The top line shows the HSV-2 genome from bp 52000 to 66000. The arrow shows the location and orientation of the UL29 ORF encoding ICP8. Letters above the line indicate restriction endonuclease cleavage sites, and numbers below the line show base pair numbers for the cleavage sites. The next six lines diagram the viral DNA inserts from this region in various plasmids, and the bottom two lines show the predicted DNA fragments arising from this region in wt and deletion mutant viral DNAs upon cleavage with SalI. (B) UL5 gene plasmids. The top line shows the HSV-2 genome from bp 6400 to 18842. The arrows show the locations and orientations of the UL4, UL5, and UL6 ORFs. Letters above the line indicate the locations of restriction endonuclease cleavage sites, and numbers denote the base pair number for the cleavage site. The next six lines diagram the viral DNA inserts from this region in various plasmids, and the bottom two lines show the predicted DNA fragments from this region upon cleavage of wt or deletion mutant viral DNAs with MluI. Restriction sites: B, BamHI; S, SalI; K, KpnI; Ev, EcoRV; Sp, SpeI; Sm, SmaI; H, HindIII; M, MluI; Bg, BglII; D, DraI; E, EcoRI.

FIG. 2

Genome structure of wt and mutant HSV-2 strains. (A) Genome structure of the wt virus. Boxes represent repeated sequences in the viral genome, and lines represent the unique sequences. (B) Expanded regions showing sequence features in the vicinity of the UL5 (left) and UL29 (right) viral genes. Boxes indicate the locations and orientations of ORFs. Arrows indicate the start sites and direction of transcription. (C) Genomic locations of the dl5 (left) and dl29 (right) deletion mutations. Numbers correspond to base pairs in the HSV-2 strain HG-52 sequence (10). (D) Sequence coordinates of the HSV-2 UL5 gene (left) and HSV-1 UL29 gene (right) transformed into Vero cells to make V529 cells.

FIG. 3

Southern blot analysis of deletion mutations in dl5-29. (A) UL5; viral DNAs digested with MluI and probed with plasmid pEH49. Lane 1, dl5; lane 2, dl29; lanes 3 and 4, dl5-29 (two isolates); lane 5, 5BlacZ; lane 6, wt HSV-2. (B) UL29; viral DNA digested with SalI and probed with plasmid pGEM5B. Lane 1, dl5; lane 2, dl29; lanes 3 and 4, dl5-29 (two isolates); lane 5, 5BlacZ; lane 6, wt HSV-2.

FIG. 4

Southern blot analysis of the oriL region in dl5-29. Viral or plasmid DNA was digested with specific restriction endonucleases, separated electrophoretically, transferred, and probed with a purified 1.1-kb SalI/SphI fragment of plasmid pEH51 containing the HSV-2 oriL region. Lane 1, wt HSV-2 DNA digested with SalI and SphI; lane 2, dl5-29 DNA digested with SalI, SpeI, and SphI; lane 3, pEH51 digested with SalI and SphI.

FIG. 5

(A) Sequence of the dl5 deletion site in dl5-29 viral DNA; (B) sequence of the dl29 deletion site in dl5-29 viral DNA.

FIG. 6

Protein expression by wt and mutant viruses in infected Vero cells. Vero cells were infected with the indicated viruses and labeled from 6 to 6.5 or 9.5 to 10 hpi. Lysates were prepared, and the proteins were resolved by SDS-PAGE. An autoradiogram is shown. Lanes: 1, dl5, 6 to 6.5 hpi; 2, dl29, 6 to 6.5 hpi; 3, 5BlacZ, 6 to 6.5 hpi; 4, dl5-29, 6 to 6.5 hpi; 5, wt, 6 to 6.5 hpi; 6, dl5, 9.5 to 10 hpi; 7, dl29, 9.5 to 10 hpi; 8, 5BlacZ, 9.5 to 10 hpi; 9, dl5-29, 9.5 to 10 hpi; 10, wt, 9.5 to 10 hpi; 11, mock-infected cells, 9.5 to 10 hpi. The positions of certain viral proteins are indicated at the left.

FIG. 7

Western blot analysis of gB and gD expression. Proteins from infected cell lysates were electroblotted from SDS-polyacrylamide gels onto nitrocellulose membranes and probed with either anti-gB (A) or anti-gD (B) antibody. Arrowheads indicate the specific glycoprotein bands. (A) Lanes: 1, dl5; 2, dl29; 3, 5BlacZ; 4, dl5-29; lane 5, wt HSV-2; lane 6, mock-infected cells. (B) Lanes: 1, mock-infected cells; 2, wt HSV-2; 3, dl5-29; 4, 5BlacZ; lane 5, dl29; lane 6, dl5. Positions of molecular weight markers are indicated on the right.

FIG. 8

Protein expression by wt and dl5-29 viruses in human cell lines. The indicated cells were infected with wt or dl5-29 virus at an MOI of 10 for 9.5 h and pulse-labeled with from 9.5 to 10 hpi. Lysates were prepared, and the proteins were resolved by SDS-PAGE. An autoradiogram is shown. Lane 1, HEL299 cells mock infected; lane 2, HEL299 cells infected with wt virus; lane 3, HEL299 cells infected with dl5-29; lane 4, MRC-5 cells mock infected; lane 5, MRC-5 cells infected with wt virus; lane 6, MRC-5 cells infected with dl5-29.

FIG. 9

Antibody responses to deletion mutant viruses. BALB/c mice were inoculated with 2 × 10⁶ PFU of the indicated virus or control cell extract at days 0 and 28. Sera were obtained at day 21 (Primary) and, after a boost, at day 37 (Secondary). Individual samples were analyzed for anti HSV-2 antibody titer by ELISA as described in Materials and Methods. Values shown indicate the geometric mean (log₂) ± standard deviation (n = 6).