Effective vaccination against long-term gammaherpesvirus latency

Abstract

The fundamental question of whether a primed immune system is capable of preventing latent gammaherpesvirus infection remains unanswered. Recent studies showing that vaccination can reduce acute replication and short-term latency but cannot alter long-term latency further call into question the possibility of achieving sterilizing immunity against gammaherpesviruses. Using the murine gammaherpesvirus 68 (gammaHV68) system, we demonstrate that it is possible to effectively vaccinate against long-term latency. By immunizing mice with a gammaHV68 mutant virus that is deficient in its ability to reactivate from latency, we reduced latent infection of wild-type challenge virus to a level below the limit of detection. Establishment of latency was inhibited by vaccination regardless of whether mice were challenged intraperitoneally or intranasally. Passive transfer of antibody from vaccinated mice could partially reconstitute the effect, demonstrating that antibody is an important component of vaccination. These results demonstrate the potential of a memory immune response against gammaherpesviruses to alter long-term latency and suggest that limiting long-term latent infection in a clinically relevant situation is an attainable goal.

FIG. 1.

Vaccination prevents reactivation from latency. Limiting dilution reactivation analysis was used to quantify latent infection in B6 mice challenged only, vaccinated only, or challenged and vaccinated 16 days after i.p. challenge. CPE, cytopathic effect. The horizontal line indicates the 63.2% Poisson distribution line. (A) Vaccination with γHV68.v-cyclin.LacZ, challenge with 100 PFU of γHV68. Statistical differences between mock-vaccinated and vaccinated mice were P < 0.0001 and P = 0.002 for peritoneal cells and splenocytes, respectively. (B) Vaccination with γHV68.v-cyclin.LacZ and challenge with 10⁶ PFU of γHV68. The statistical difference between mock-vaccinated and vaccinated mice was P = 0.004 for splenocytes. (C) Vaccination with vaccinia virus and challenge with 100 PFU of γHV68.

FIG. 2.

Limiting dilution PCR assays to detect either challenge virus or vaccine virus. (A) Location of sites of amplification from challenge virus (γHV68) or vaccine virus (γHV68.v-cyclin.LacZ) genome by using PCR primers specific for v-cyclin, LacZ, or gene 50. (B) Limiting dilution PCR with primers specific for v-cyclin detects challenge virus only. PCR analysis of serial dilutions of splenocytes harvested from mice infected with challenge virus for 16 days or vaccine virus for 44 days (28-day vaccination plus 16-day challenge period). The horizontal line indicates the 63.2% Poisson distribution line. (C) Limiting dilution PCR with primers specific for LacZ detects vaccine virus only. (D) Limiting dilution PCR with primers specific for gene 50 detects both challenge virus and vaccine virus.

FIG. 3.

Vaccination prevents establishment of latency. Limiting dilution PCR specific for challenge virus (v-cyclin primers) or vaccine virus (LacZ primers) was used to detect viral genome in splenocytes from B6 mice challenged only (A), vaccinated only (B), or vaccinated and challenged (C) 16 days after i.p. challenge with 100 PFU of γHV68. The horizontal line indicates the 63.2% Poisson distribution line. For challenge virus, the difference between mock-vaccinated and vaccinated mice was statistically significant (P = 0.007). The frequency of cells containing the vaccine virus genome decreased by 84% in challenged mice (1 in 1,400 mock-vaccinated versus 1 in 9,000 vaccinated [P = 0.03]).

FIG. 4.

Vaccination prevents latent infection after i.n. challenge. Limiting dilution reactivation analysis and limiting dilution PCR were used to examine latency in B6 mice challenged only, vaccinated only, or challenged and vaccinated 16 days after i.n. challenge with 400 PFU of γHV68. CPE, cytopathic effect. (A) Limiting dilution reactivation analysis. Differences between mock-vaccinated and vaccinated mice were statistically significant (P = 0.0003 and P = 0.002 for peritoneal cells and splenocytes, respectively). (B) Limiting dilution PCR specific for challenge virus (v-cyclin primers). Differences between mock-vaccinated and vaccinated mice were statistically significant (P = 0.01 and P < 0.0001 for peritoneal cells and splenocytes, respectively).

FIG. 5.

Vaccination is effective against long-term latent infection. Limiting dilution reactivation and limiting dilution PCR analysis were used to examine long-term latency in B6 mice after i.p. challenge with 100 PFU of γHV68. CPE, cytopathic effect. (A) Limiting dilution reactivation analysis was used to determine the status of the latent infection in cells harvested 42 days postchallenge. The difference between mock-vaccinated and vaccinated mice was statistically significant (P = 0.01 for peritoneal cells over the five highest cell dilutions). (B) Limiting dilution PCR specific for challenge virus (v-cyclin primers) was used to detect viral genome in cells harvested 42 days postchallenge. Differences between mock-vaccinated and vaccinated mice were statistically significant (P = 0.02 and P = 0.04 for peritoneal cells and splenocytes, respectively). (C) PCR specific for challenge virus for cells harvested 125 days postchallenge. Bars indicate the percentage of reactions positive for viral genome at 10,000 cells per reaction. Differences between mock-vaccinated and vaccinated mice were statistically significant (P = 0.02 and P = 0.10 for peritoneal cells and splenocytes, respectively).

FIG. 6.

Vaccination is effective against acute infection. Plaque assays were used to determine the acute viral titers in splenocytes from B6 mice after i.p. challenge with 100 PFU of γHV68. The mean titer was 784 ± 293 PFU for mock-vaccinated mice (n = 8). Titers in vaccinated mice were below the 50-PFU level of detection (n = 8). The difference between mock-vaccinated and vaccinated mice was statistically significant (P = 0.02).

FIG. 7.

CD8 T cells are not required for vaccination. Limiting dilution reactivation analysis was used to determine the status of the latent infection in CD8^−/− mice challenged only, vaccinated only, or challenged and vaccinated 16 days after i.p. challenge with 100 PFU of γHV68. Differences between mock-vaccinated and vaccinated mice were statistically significant (P < 0.0001 and P = 0.0005 for peritoneal cells and splenocytes, respectively). All 46 wells containing single reactivation events (wells in cell dilutions that yielded a cytopathic effect [CPE] for that dilution of ≤63.2%) in vaccinated samples stained positive for the presence of β-galactosidase, indicating that reactivation was due to the presence of the vaccine virus rather than the challenge virus.

FIG. 8.

Antibody from vaccinated mice can prevent splenic latency. Limiting dilution reactivation analysis was used to determine the status of the latent infection in B6 mice after mock transfer or passive transfer of naive serum or immune serum from vaccinated mice 1 day prior to and 7 days after challenge. Mice were analyzed 16 days after i.p. challenge with 100 PFU of γHV68. CPE, cytopathic effect. The difference between mock-vaccinated and vaccinated mice was statistically significant (P = 0.008 for splenocytes).