Role of dendritic cells in enhancement of herpes simplex virus type 1 latency and reactivation in vaccinated mice

Abstract

Ocular infection with herpes simplex virus type 1 (HSV-1) frequently leads to recurrent infection, which is a major cause of corneal scarring. Thus, the prevention of the establishment of latency should be a primary goal of vaccination against HSV-1. To this end, we have examined the contribution of dendritic cells (DCs) to the efficacy of a vaccine against ocular HSV-1 infection. Transgenic mice (expressing a CD11c-diphtheria toxin receptor-green fluorescent protein construct) with a BALB/c background were immunized with a vaccine consisting of DNA that encodes five HSV-1 glycoproteins or were immunized with vector control DNA. The vaccinated mice were then depleted of their DCs through the injection of diphtheria toxin before and after ocular challenge with HSV-1. Analyses of HSV-1 replication in the eye, blepharitis, corneal scarring, and the survival of the infected mice upon primary infection indicated that DC depletion neither promoted nor compromised the efficacy of the vaccine. In contrast, DC depletion was associated with an approximately fivefold reduction in the level of latent virus in the trigeminal ganglia (TGs) of latently infected mice, as well as a significant reduction in the reactivation rate of latent virus. The possibility that DCs enhance the latency of HSV-1 in the TGs of ocularly infected mice suggests for the first time that DCs, rather than acting as "immune saviors," can exacerbate disease and compromise vaccine efficacy by enhancing viral latency and reactivation.

FIG. 1.

Virus replication in the eyes of DC-depleted mice following ocular HSV-1 infection. Three weeks after the final 5gP DNA vaccination or vector control DNA vaccination, the mice were depleted of DCs or were mock depleted and were infected ocularly with HSV-1 strain McKrae. The presence of HSV-1 in the tear films of 30 mice/group (60 eyes) was monitored on days 1, 2, 3, 4, 5, 6, and 7 as described in Materials and Methods. For each time point, the virus titer (y axis) represents the average of the titers in tear films from 60 eyes. The error bars indicate the SEM.

FIG. 2.

Effect of DC depletion on the levels of gB (A) and LAT (B) transcripts in the corneas and TGs of vaccinated mice during primary infection. Mice were vaccinated with 5gP DNA or vector control DNA and then subjected to DC depletion or mock depletion and ocularly infected with HSV-1 strain McKrae as described in the legend to Fig. 1. The corneas and TGs were harvested on day 5 postinfection, and TaqMan RT-PCR analysis of total RNA from the tissues of individual mice was performed as described in Materials and Methods. The numbers of copies of gB and LAT RNAs were estimated using pAC-gB1 and pGem-LAT5317-8330, respectively. In all experiments, GAPDH expression was used to normalize the relative expression of gB and LAT in the corneas and TGs. Each bar represents the mean ± SEM of results for five mice.

FIG. 3.

Cytofluorometric analysis of T cells and CD11b⁺ cells in the corneas and spleens of DC-depleted mice. The corneas and spleens of three mice per group that were treated as described in the legend to Fig. 1 were harvested 5 days post-ocular infection. Single-cell suspensions were prepared and allowed to react with monoclonal antibodies to CD4+ T cells and CD8⁺ T cells or CD11b⁺ cells, and FACS analysis was performed as described in Materials and Methods. The numbers of CD4⁺ CD8⁻ T cells (CD4⁺), the numbers of CD4⁻ CD8⁺ T cells (CD8⁺), and the numbers of CD11b⁺ cells in the DC-depleted groups of mice are shown relative to the numbers of these cells in their mock-depleted counterparts (horizontal lines at 100%). Each bar represents the mean of results from three individual FACS analyses.

FIG. 4.

Effect of DC depletion on the levels of LAT in the TGs of latently infected mice. TGs from some of the surviving mice from the experiment summarized in Table 1 were isolated on day 30 postinfection, and TaqMan RT-PCR analysis of the total RNA per mouse TG (with two TGs per mouse) was performed as described in Materials and Methods. The levels of LAT in the DC-depleted groups are shown relative to the level of LAT in the mock-depleted mice. GAPDH expression was used to normalize the relative expression of LAT in the TGs. Each bar represents the mean ± SEM of results from 12 mice per group.

FIG. 5.

Effect of DC depletion on the kinetics of induced reactivation in explanted TGs from latently infected mice. Thirty days postinfection, a time at which latency is already established, individual TGs were obtained from latently infected mice that had been vaccinated with 5gP DNA or vector control DNA and then subjected to DC depletion or mock depletion. The TGs from individual mice (two per mouse) were incubated in 1.5 ml of tissue culture medium at 37°C. A 100-μl aliquot was removed from each culture daily for 12 days and used to infect RS cell monolayers. The RS cells were monitored daily for 5 days for the appearance of a CPE to determine the time of the first appearance of reactivated virus from each TG. The results are plotted as the cumulative percentages of TGs that had reactivated. Numbers within the graphs indicate the average number of days (±SEM) before the TGs from each group first showed a CPE. For the vector DNA-vaccinated mice, 10 TGs (from 5 mice)/group were used, whereas for the 5gP DNA-vaccinated mice, 20 TGs (from 10 mice)/group were used.