Disruption of virion host shutoff activity improves the immunogenicity and protective capacity of a replication-incompetent herpes simplex virus type 1 vaccine strain

Abstract

The virion host shutoff (vhs) protein encoded by herpes simplex virus type 1 (HSV-1) destabilizes both viral and host mRNAs. An HSV-1 strain with a mutation in vhs is attenuated in virulence and induces immune responses in mice that are protective against corneal infection with virulent HSV-1, but it has the capacity to establish latency. Similarly, a replication-incompetent HSV-1 strain with a mutation in ICP8 elicits an immune response protective against corneal challenge, but it may be limited in viral antigen production. We hypothesized therefore that inactivation of vhs in an ICP8(-) virus would yield a replication-incompetent mutant with enhanced immunogenicity and protective capacity. In this study, a vhs(-)/ICP8(-) HSV-1 mutant was engineered. BALB/c mice were immunized with incremental doses of the vhs(-)/ICP8(-) double mutant or vhs(-) or ICP8(-) single mutants, or the mice were mock immunized, and protective immunity against corneal challenge with virulent HSV-1 was assessed. Mice immunized with the vhs(-)/ICP8(-) mutant showed prechallenge serum immunoglobulin G titers comparable to those immunized with replication-competent vhs(-) virus and exceed those of mice immunized with the ICP8(-) single mutant. Following corneal challenge, the degrees of protection against ocular disease, weight loss, encephalitis, and establishment of latency were similar for vhs(-)/ICP8(-) and vhs(-) virus-vaccinated mice. Moreover, the double deleted vhs(-)/ICP8(-) virus protected mice better in all respects than the single deleted ICP8(-) mutant virus. The data indicate that inactivation of vhs in a replication-incompetent virus significantly enhances its protective efficacy while retaining its safety for potential human vaccination. Possible mechanisms of enhanced immunogenicity are discussed.

FIG. 1

Southern blot analysis of recombinant virus genomes. Viral DNAs were digested with EcoRV, electrophoresed, and transferred to nitrocellulose membranes. The blot was probed with a 2-kb fragment of UL29 that was expected to hybridize to 4.6- and 13.3-kb fragments in a mutant virus and a single 14.8-kb fragment in wild-type virus. Lanes are as indicated.

FIG. 2

Multistep growth curve in cultured cells. Replicate monolayers of S2 cells were infected with approximately 100 PFU of HSV-1 strain KOS, KOS1.1, HD-2, BGS41, or Δ41Δ29 and incubated for the indicated periods of time. Monolayers were then collected, and viral titers were determined by standard plaque assay. Two independent experiments gave similar results.

FIG. 3

RNA degradation assay by Northern blot analysis. Graph shows the percent of GAPDH RNA remaining from 28S-normalized KOS-, KOS1.1-, HD-2-, BGS41-, and Δ41Δ29-infected Vero cells at 8 h postinfection, relative to mock-infected cells in the presence of actinomycin D. Two independent experiments gave similar results.

FIG. 4

Prechallenge HSV-1-specific serum IgG titers. Serum was collected from each of the mice immunized with 4 × 10⁵ PFU (filled bars), 1 × 10⁵ PFU (striped bars), or 2.5 × 10⁴ PFU (open bars) of the indicated viruses and analyzed for HSV-specific IgG by ELISA. C.S., control supernatant. Geometric mean titers ± SEM are from groups of six mice and are shown for one representative experiment of three performed. The difference in means was tested for significance by the Student t test (BGS41 or Δ41Δ29 versus HD-2).

FIG. 5

Acute replication of challenge virus in the corneal epithelium. Mouse eyes were swabbed at the indicated times after corneal challenge with 8 × 10⁵ PFU of HSV-1 strain mP. Immunizing doses of (A) 4 × 10⁵ PFU, (B) 1 × 10⁵ PFU, and (C) 2.5 × 10⁴ PFU are shown. Virus content in the tear film was assessed by standard plaque assay. Values represent the geometric mean titer ± SEM from groups of six mice and are from the same experiment as that shown in Fig. 4.

FIG. 6

Change in body weight postchallenge. Baseline weights of mice (∼20 g) were obtained prior to corneal challenge with 8 × 10⁵ PFU of HSV-1, and mice were weighed each day following challenge through day 8. Immunizing doses of (A) 4 × 10⁵ PFU, (B) 1 × 10⁵ PFU, and (C) 2.5 × 10⁴ PFU are shown. Values represent the mean weight change per group of six mice and are from the same experiment as that shown in Fig. 4.

FIG. 7

Severity of blepharitis postchallenge. Blepharitis was scored daily postchallenge in masked fashion. Immunizing doses of (A) 4 × 10⁵ PFU, (B) 1 × 10⁵ PFU, and (C) 2.5 × 10⁴ PFU are shown. Values represent the mean score ± SEM for six mice per group and are from the same experiment as that shown in Fig. 4 and 5.