The dominant-negative herpes simplex virus type 1 (HSV-1) recombinant CJ83193 can serve as an effective vaccine against wild-type HSV-1 infection in mice

Abstract

By selectively regulating the expression of the trans-dominant-negative mutant polypeptide UL9-C535C, of herpes simplex virus type 1 (HSV-1) origin binding protein UL9 with the tetracycline repressor (tetR)-mediated gene switch, we recently generated a novel replication-defective and anti-HSV-specific HSV-1 recombinant, CJ83193. The UL9-C535C peptides expressed by CJ83193 can function as a potent intracellular therapy against its own replication, as well as the replication of wild-type HSV-1 and HSV-2 in coinfected cells. In this report, we demonstrate that CJ83193 cannot initiate acute productive infection in corneas of infected mice nor can it reactivate from trigeminal ganglia of mice latently infected by CJ83193 in a mouse ocular model. Given that CJ83193 is capable of expressing the viral alpha, beta, and gamma1 genes but little or no gamma2 genes, we tested the vaccine potential of CJ83193 against HSV-1 infection in a mouse ocular model. Our studies showed that immunization with CJ83193 significantly reduced the yields of challenge HSV in the eyes and trigeminal ganglia on days 3, 5, and 7 postchallenge. Like in mice immunized with the wild-type HSV-1 strain KOS, immunization of mice with CJ83193 prevents the development of keratitis and encephalitis induced by corneal challenge with wild-type HSV-1 strain mP. Delayed-type hypersensitivity (DTH) assays demonstrate that CJ83193 can elicit durable cell-mediated immunity at the same level as that of wild-type HSV-1 and is more effective than that induced by d27, an HSV-1 ICP27 deletion mutant. Moreover, mice immunized with CJ83193 developed strong, durable HSV-1-neutralizing antibodies at levels at least twofold higher than those induced by d27. The results presented in this report have shed new light on the development of effective HSV viral vaccines that encode a unique safety mechanism capable of inhibiting the mutant's own replication and that of wild-type virus.

FIG. 1.

(A) A schematic diagram of genomes of the wild-type HSV-1 and the dominant-negative HSV-1 recombinant CJ83193. The top diagram shows the HSV genome, indicating the unique long (UL) region, the unique short (US) region, the inverted repeat regions (open boxes), and the three origins of DNA replication (gray ovals). The lower diagrams show an expanded SacI-PstI DNA fragment containing the ICP0 open reading frame with flanking sequences in wild-type HSV-1, strain KOS, and the DNA sequences encoding UL9-C535C under the control of the tetO-bearing hCMV major immediate-early promoter with the ICP0 flanking sequences in CJ83193. (B) A likely mechanism of UL9-C535C in preventing HSV viral DNA replication. Binding of UL9-C535C to the HSV-1 origin of DNA replication blocks the binding of wild-type HSV-1 UL9 origin binding protein to the origin of DNA replication, leading to inhibition of viral DNA replication.

FIG. 2.

CJ83193 fails to establish reactivatable latent infection in a mouse ocular model. CD-1 mice were randomly assigned to three groups of six male and six female mice each. Following corneal scarification, individual groups of mice were inoculated with KOS at 10⁶ PFU per eye or with CJ83193 and 7134 at 10⁷ PFU per eye, respectively. (A) Acute viral replication in the eye of CD-1 mice. Both eyes of infected mice were swabbed on days 1, 3, and 5 postinfection. Infectious viruses in eye swab material were assessed by standard plaque assay on Vero cell monolayers for KOS and on U2CEP4R11 cell monolayers for CJ83193 and 7134. Virus titers are expressed as means ± standard error of eye swabs (n = 24) for 12 mice per group. (B) Reactivation of latent infection from trigeminal ganglia of mice following eye inoculation was determined by cocultivation at 30 days postinfection. Vero cell monolayers were used for KOS infection. Presence of reactivatable 7134 and CJ83193 viruses were assayed on U2CEP4R11 cell monolayers.

FIG. 3.

Selective detection of viral gene expression in CJ83193-infected cells. Vero cells were seeded at 3 × 10⁶ cells per 100-mm dish. At 48 h postseeding, cells in duplicate dishes were mock infected or infected with KOS, d27, or CJ83193 at an MOI of 10 PFU/cell. Infected cell extracts were prepared at 15 h postinfection. Proteins in infected cell extracts were resolved on SDS-PAGE, followed by immunoblotting with monoclonal antibodies specific for ICP4 (A) and gB (B) and a polyclonal antibody specific for gD (C).

FIG. 4.

Reduction of challenge wild-type HSV-1 strain mP replication in the eye and trigeminal ganglion of mice immunized with CJ83193. Female BALB/c mice were immunized with either mock-infected cell lysate, KOS, d27, or CJ83193 at 2 × 10⁶ PFU per mouse. Individual groups of mice (n = 12) were boosted with the same virus 2 weeks after primary immunization. At 4 weeks after primary immunization, mice in all groups were challenged following corneal scarification with HSV-1 strain mP. Eye swabs were taken on days 1, 3, 5, and 7 postchallenge, while mouse trigeminal ganglia (n = 8) were prepared on days 3, 5, and 7 postchallenge. Infectious viruses in individual eye swab materials (A) and trigeminal ganglia (B) were assessed by standard plaque assay on Vero cell monolayers. Viral titers are expressed as the mean ± standard error in individual eye swabs and trigeminal ganglia of mice per group.

FIG. 5.

Prevention of mortality and herpetic keratitis in mice immunized with CJ83193 after corneal challenge with wild-type HSV-1 strain mP. Female BALB/c mice were randomly divided into four groups and immunized with either mock-infected cell lysate (n = 28), KOS (n = 16), d27 (n = 20), or CJ83193 (n = 20) as described. Four weeks after initial immunization, both eyes of mice were challenged with HSV-1 strain mP following corneal scarification. (A) Mortality of mice following ocular HSV-1 challenge during a 30-day follow-up period. (B) Ophthalmoscopic examination of mice eyes for signs of herpetic keratitis during the same period. Individual eyes were scored for severity of keratitis. The indicated values represent the mean score ± standard error of all eyes from each group of mice.

FIG. 6.

Reduction of latent infection by challenge virus in trigeminal ganglia of mice immunized with KOS, d27, or CJ83193. Mice described in experiments presented in Fig. 5 and from a similar experiment in which mice were immunized with either mock-infected cell lysate (n = 12) or CJ83193 at 2 × 10⁶ PFU/mouse (n = 4) were sacrificed 30 days after challenge. Trigeminal ganglia of mice from different groups were processed and cocultivated individually onto Vero cell monolayers. After 5 days of cocultivation, explants were harvested, processed, and assayed on Vero cell monolayers for the presence of infectious viruses.

FIG. 7.

Induction of short- and long-term DTH responses by CJ83193. Six-week-old female BALB/c mice were randomly divided into several groups. (A) Mice were immunized with (i) either mock-infected cell lysate (n = 19) or KOS (n = 7), d27 (n = 13), and CJ83193 (n = 13) at a dose of 2 × 10⁶ PFU per mouse and (ii) mock-infected cell lysate (n = 12) or d27 (n = 15) and CJ83193 (n = 15) at a dose of 2 × 10⁵ PFU per mouse. At 14 days postvaccination, the left and right rear footpads of mice in all groups were challenged by injection of 10⁶ PFU of HSV-1 strain mP and PBS, respectively. (B) At about 6 months postvaccination of the mice described in Table 2, the left and right rear footpads of mice in all groups were challenged with either HSV-1 strain mP or PBS as described. Footpad thickness was measured with a micrometer at 40 h postchallenge. HSV-specific footpad swelling was expressed as the mean difference ± standard error between the thickness of the left and right footpads.