Herpes simplex virus vectors elicit durable immune responses in the presence of preexisting host immunity

Abstract

Herpes simplex virus (HSV) recombinants are being developed as vaccine vectors for the expression of heterologous antigens. There is concern, however, that preexisting HSV immunity may decrease their effectiveness. We have addressed this issue in an animal model. Immunized mice were inoculated with a replication-defective HSV-1 vector that expressed the Escherichia coli beta-galactosidase protein as a model antigen. We assessed vector efficacy by analyzing the immunoglobulin G (IgG) antibody response and cellular proliferative response directed against beta-galactosidase. We report that the ability of the vector to induce antibody or proliferative responses was not diminished by preexisting immunity to HSV. Of further note, the anti-HSV and anti-beta-galactosidase IgG responses following vector administration were extremely durable in both immunized and naive mice. These results indicate that the ability of a replication-defective HSV-derived vaccine vector to elicit long-lived immune responses in mice is not impaired by prior HSV exposure.

FIG. 1.

HSV-1 recombinants used in this study. HSV-1 viruses included the wild-type (wt) laboratory strain KOS1.1 and replication-defective KOS1.1-derived recombinants d301, d102, and HD-2. d301 and d102 each contain large deletions within the U_L29 gene encoding the ICP8 protein. HD-2 encodes a truncated ICP8 protein fused to E. coli β-galactosidase (β-gal).

FIG. 2.

Antibody responses as a function of viral dose. Groups of six mice were inoculated with various doses of HD-2 (0 to 10⁸ PFU) or UV-treated HD-2 (10⁸ PFU prior to treatment, 5 × 10⁴ PFU following treatment), at weeks 0 and 3. Serum samples were collected at weeks 3 (primary) and 5 (secondary). HSV-1- (A) and β-galactosidase- (B) specific IgG titers were determined by ELISA, and results are shown as mean titer (log₂) ± standard deviation.

FIG. 3.

Induction and durability of IgG responses in mice. The generation of IgG antibody specific for HSV-1 (A) and β-galactosidase (B) and the durability of the IgG responses directed towards HSV-1 (C) and β-galactosidase (D) antigens are shown. Groups of six mice were immunized once with 2 × 10⁶ PFU virus (solid circles) using either d301 (solid lines) or d102 (dashed lines) or mock infected (open squares) at week 0 (open arrow). At weeks 4 and 8, all mice were inoculated with 2 × 10⁶ PFU of HD-2, expressing β-galactosidase (solid arrows). Serum samples were collected at the indicated times, and HSV-1- and β-galactosidase-specific IgG antibody titers were determined by ELISA. Results are shown as the mean reciprocal dilution (log₂) ± standard deviation.

FIG. 4.

Induction of β-galactosidase IgG after two HSV-1 immunizations. Titers of IgG specific for HSV-1 (A) and β-galactosidase (B) are shown. Groups of six mice were immunized twice, at weeks 0 and 3 (open arrows), with 2 × 10⁶ PFU of d301 virus (solid circles) or mock infected (open squares). At weeks 7, 10, and 13, all mice were inoculated with 2 × 10⁶ PFU of HD-2 virus, expressing β-galactosidase (solid arrows). Serum samples were collected at the indicated times, and IgG was detected by ELISA. Results are shown as the mean reciprocal dilution (log₂) ± standard deviation.

FIG. 5.

Induction of β-galactosidase IgG by cell-free HD-2 virus after one or two immunizations. (A and B) Groups of six mice were immunized once with 2 × 10⁶ PFU of d301 (solid bars) or mock infected (open bars) at week 0. At weeks 4 and 7, all mice were inoculated with 2 × 10⁶ PFU of cell-free HD-2 virus, expressing β-galactosidase. IgG antibodies specific for HSV-1 (A) and β-galactosidase (B) were determined by ELISA using serum collected prior to week 0 (naïve), at week 3 (immune), at week 7 (primary), and at week 9 (secondary). (C and D) Groups of six mice were immunized twice with 2 × 10⁶ PFU of d301 virus (solid bars) or mock infected (open bars) at weeks 0 and 3. All mice received 2 × 10⁶ PFU of cell-free HD-2 at weeks 7, 10, and 13. Serum samples were collected prior to week 0 (naive), at week 6 (immune), at week 10 (primary), at week 12 (secondary), and at week 15 (tertiary). Titers of HSV-1- (C) and β-galactosidase- (D) specific IgG antibody were determined by ELISA. Results are shown as the mean reciprocal dilution (log₂) ± standard deviation.

FIG. 6.

Induction of β-galactosidase IgG by cell-free HD-2 virus after immunization with replication-competent HSV-1. HSV-1- (A) and β-galactosidase- (B) specific IgG antibody responses are shown. Groups of six mice were immunized once with 2 × 10⁶ PFU of either a replication-competent virus, KOS1.1 (hatched bars), or a replication-defective virus, d301 (solid bars), or mock infected (open bars) at week 0. All mice were inoculated with 2 × 10⁶ PFU of cell-free HD-2 virus at weeks 4 and 7. Serum samples were collected prior to week 0 (naïve) and at week 3 (immune), week 7 (primary), and week 10 (secondary). IgG titers were determined by ELISA, and results are shown as the mean reciprocal dilution (log₂) ± standard deviation.

FIG. 7.

Cellular proliferative responses to β-galactosidase in immune mice. Proliferation was measured in the presence of HSV and β-galactosidase antigens. Mice were immunized once with 2 × 10⁶ PFU of d301 or mock infected at week 0. At weeks 4 and 7, all mice were inoculated with 2 × 10⁶ PFU of cell-free HD-2 virus. Spleens were harvested from two mock-immune (open bars) and two d301- immune (solid bars) mice and pooled for analysis. Single-cell suspensions were generated, and 2 × 10⁵ cells were cultured in the presence of HSV or β-galactosidase antigen (5 μg per well) or medium alone for 4 days at 37°C. BrdU was added to each culture for the final 6 h, cells were fixed and lysed, and BrdU incorporation was measured in an ELISA-based assay. Results are shown as the mean fold proliferation (BrdU incorporation in the presence of antigen divided by incorporation in medium alone) of quadruplicate samples ± standard deviation.