Protection from bacterial infection by a single vaccination with replication-deficient mutant herpes simplex virus type 1

Abstract

Adaptive immune responses in which CD8(+) T cells recognize pathogen-derived peptides in the context of major histocompatibility complex class I molecules play a major role in the host defense against infection with intracellular pathogens. Cells infected with intracellular bacteria such as Listeria monocytogenes, Salmonella enterica serovar Typhimurium, or Mycobacterium tuberculosis are directly lysed by cytotoxic CD8(+) T cells. For this reason, current vaccines for intracellular pathogens, such as subunit vaccines or viable bacterial vaccines, aim to generate robust cytotoxic T-cell responses. In order to investigate the capacity of a herpes simplex virus type 1 (HSV-1) vector to induce strong cytotoxic effector cell responses and protection from infection with intracellular pathogens, we developed a replication-deficient, recombinant HSV-1 (rHSV-1) vaccine. We demonstrate in side-by-side comparison with DNA vaccination that rHSV-1 vaccination induces very strong CD8(+) effector T-cell responses. While both vaccines provided protection from infection with L. monocytogenes at low, but lethal doses, only rHSV-1 vaccines could protect from higher infectious doses; HSV-1 induced potent memory cytotoxic T lymphocytes that, upon challenge by pathogens, efficiently protected the animals. Despite the stimulation of relatively low humoral and CD4-T-cell responses, rHSV-1 vectors are strong candidates for future vaccine strategies that confer efficient protection from subsequent infection with intracellular bacteria.

FIG. 1.

Visualization of antigen-specific CTL expansion after vaccination. PBL were stained with anti-CD8-APC and H-2K^b/OVA_257-264 tetramers-phycoerythrin and analyzed by flow cytometry before (a) and 7 days after vaccination with pcDNA3-OVA (b), T0H-OVA (c), or T0-GFP (4 × 10⁶ virus particles i.v.) (d). The percentage of CD8⁺ Tet⁺ CTL among PBL is indicated on each plot. (e) The frequency of antigen-specific cells among peripheral blood CD8⁺ T cells was determined by H-2K^b/OVA_257-264 tetramer staining for pcDNA3-OVA-, T0H-OVA-, and T0-GFP-immunized mice as shown (a to d). Data represent average values ± standard deviations (error bars) obtained from five mice per group at each time point. p.i., postimmunization.

FIG. 2.

Protection against L.m.-OVA infection. (a) C57BL/6 mice were left unimmunized or vaccinated with pcDNA3-OVA, T0H-OVA, or T0-GFP (4 × 10⁶ virus particles i.v.) (day 0) and infected i.v. with 5 × 10⁴ (two times the LD₅₀) L.m.-OVA cells 8 days later. H-2K^b/OVA_257-264 tetramer staining for OVA-specific CD8⁺ T cells in blood was performed at day 7 postvaccination (b), and survival of the mice was monitored for 10 days after infection (c). At day 10 postinfection (day 18 postimmunization), spleens of surviving mice were analyzed for presence of Tet⁺ CTL (d). The bar graphs (b and d) depict the percentage (mean ± standard deviation [error bars]) of Tet-positive cells among total CD8⁺ T cells (five mice/group). (e) As a control for the antigen-specificity of protection, vaccinated mice were infected with 5 × 10⁴ wild-type L. monocytogenes cells (not expressing OVA), and survival was monitored. Abbreviations: no imm, no immunization; p.i., postimmunization.

FIG. 3.

Protection against infection with high dose of L.m.-OVA. C57BL/6 mice were vaccinated with T0H-OVA, T0-GFP (4 × 10⁶ virus particles i.v.), or pcDNA3-OVA and infected with 10⁵ (four times the LD₅₀) L.m.-OVA cells 7 days postvaccination. Mice were sacrificed 3 days after infection, and bacterial counts in spleen (a) and livers (b) were determined from organ homogenates (n = 3 to 4 mice per group). (c) Differentially vaccinated mice (three or four per group) were infected with 5 × 10⁵ (20 times the LD₅₀) L.m.-OVA cells, and survival was monitored for 13 days.

FIG. 4.

Long-term protection against L.m.-OVA infection. (a) Experimental protocol: C57BL/6 mice were vaccinated with pcDNA3-OVA, T0H-OVA, or T0-GFP (4 × 10⁶ virus particles i.v.) and infected i.v. with 5 × 10⁴ (two times the LD₅₀) L.m.-OVA cells 6 weeks later. (b) The percentage of H-2K^b/OVA_257-264 tetramer-specific cells among CD8⁺ T lymphocytes in the blood was determined by flow cytometry at the days indicated. (c) Survival of the mice was monitored daily; no death occurred after day 7 postinfection. (d) On day 7 after infection (day 49 postvaccination), spleen, mesenteric (mes), inguinal, and brachial lymph nodes were isolated and analyzed by flow cytometry; inguinal and brachial lymph nodes (ing. and bra. LN) were pooled for each mouse. The bar graphs depict the percentage (mean ± standard deviation [error bars]) of H-2K^b/OVA_257-264 tetramer-positive cells among CD8⁺ T cells (n = 4 to 5 per group).

FIG. 5.

Monitoring of OVA-specific CD4⁺-T-cell responses in vivo following rHSV-1 vaccination. BALB/c mice received 2.5 × 10⁶ naive DO11.10 cells at day −1 and were immunized with gene gun (pcDNA3-OVA), T0H-OVA, or T0-GFP (4 × 10⁶ virus particles) i.v. at day 0. The frequency of DO11.10 T cells present in the inguinal lymph nodes and spleen was measured by flow cytometry. (a to c) Cell suspensions were stained with anti-CD4, and KJ1-26-fluorescein isothiocyanate (DO11.10 TCR-specific). The percentages of CD4⁺/KJ1-26⁺ cells in lymph nodes of control (T0-GFP)-, pcDNA3-OVA-, and T0H-OVA-immunized mice at day 5 postimmunization are indicated in the dot plots. Kinetics of DO11.10 T-cell expansion is shown for lymph nodes (d) and spleen (e). In panels d and e, the total cell numbers ± standard deviations (error bars) of CD4⁺ KJ1-26⁺ cells are indicated (three to four animals per group).

FIG. 6.

OVA-specific antibody responses in immunized BALB/c mice. Sera were obtained from mice immunized with pcDNA3-OVA, T0H-OVA, or T0-GFP (4 × 10⁶ virus particles i.v.) at the indicated time points postvaccination, and OVA-specific antibody serum levels were determined by enzyme-linked immunosorbent assay. Preimmune serum (day 0) from each group was determined with a pool of sera. Results are expressed as the mean of optical density at 450 nm (O.D. 450 nm) ± standard deviation (error bars) from at least three individual mice per group.