Immunization with a single major histocompatibility complex class I-restricted cytotoxic T-lymphocyte recognition epitope of herpes simplex virus type 2 confers protective immunity

Abstract

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8(+) cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8(+) T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8(+) CTL in HSV vaccine design.

FIG. 1

rVVs express CTL recognition epitopes. B6/K-1,4,5 (A and B) or T2/K^b (C and D) cells were infected with rVV-ES-gB498-505, rVV-gB498-505, rVV-ES-IV, rVV-IV, or rVV-SC for 1 h and incubated in DMEM with ⁵¹Cr for an additional 3 h. Virus-infected cells were added to either the gB498-505-specific CTL clone 2D5 (A and C) or the SV40 Tag404-411-specific CTL clone Y-4 (B and D) at the indicated effector-to-target ratios in a standard ⁵¹Cr release assay.

FIG. 2

Immunization with rVV-ES-gB498-505 induces primary and memory CTL responses. C57BL/6 mice were immunized i.n. with 10⁷ PFU of rVV-ES gB498-505. Eight days later, the hilar (A) and submaxillary (B) lymph nodes were removed, and single-cell suspensions were formed. The effector lymph node cells were then tested for lytic activity at the indicated effector-to-target ratios against B6/WT-3 cells pulsed with 1 μM gB498-505 synthetic peptide and mock peptide-pulsed B6/WT-3 cells. For analysis of the CTLm response (C), we immunized i.n. five mice with rVV-ES-gB498-505 and three with rVV-ES-IV. Six weeks later, mice were sacrificed and their splenocytes were incubated for 7 days under LDA conditions as described in Materials and Methods. The graded splenocyte cultures were then tested for lytic activity against ⁵¹Cr-labeled B6/K-1,4,5 cells which were either pulsed with gB498-505 synthetic peptide or mock pulsed. Total CTLm per spleen was calculated as (CTLm frequency) × (viable cell yield per spleen).

FIG. 3

Immunization with either rVV-ES-gB498-505 or rVV-gB498-505 protects mice from lethal challenge of HSV-2. Mice were immunized i.n. with either PBS-BSA (A to C), rVV-ES-gB498-505 (A to C), rVV-gB498-505 (C), or rVV-ES-IV (A and B) as described in Materials and Methods. Four to six weeks later, mice were administered i.n. a lethal challenge of 5 × 10⁵ (A) or 10⁵ (B and C) PFU HSV-2 186. Mice were monitored daily for lethality for 28 days. Panels A and B represent data from three and two independent experiments, respectively.

FIG. 4

Immunization with rVV-ES-gB498-505 reduces the extent of HSV infection. Mice were immunized i.n. with either PBS-BSA or 10⁷ PFU or rVV-ES-gB498-505 or rVV-ES-IV. Four to six weeks later, mice received i.n. a lethal dose of 5 × 10⁵ PFU HSV-2 186. Five days later, the brains (A) and trigeminal ganglia (B) were removed and the level of infectious HSV-2 present in the tissues was determined by plaque analysis. The lower limit of detection (10 PFU/tissue) is indicated by the dashed line. The geometric mean of each immunization group is depicted by the solid horizontal line. The mean level of infectious virus recovered from the rVV-ES-gB498-505-immunized and rVV-ES-IV-immunized mice was compared to that of PBS-BSA-immunized mice, using the nonparametric Mann-Whitney test. Data represent the results of two independent experiments.

FIG. 5

WSN/NA/gB expresses the HSV-1 gB498-505 CTL epitope. ⁵¹Cr-labeled B6/WT-3 cells were infected with either HSV-1, WSN/NA/gB, or WSN. After a 5-h incubation, virus-infected B6/WT-3 cells pulsed with 1 μM gB498-505 synthetic peptide or mock peptide pulsed were added to the gB498-505-specific CTL clone 2D5 at the indicated effector-to-target ratios in a standard ⁵¹Cr release assay.

FIG. 6

Immunization with WSN/NA/gB induces a primary and memory HSV-specific CTL response. For induction of primary CTL, 8 C57BL/6 mice were immunized i.n. with WSN/NA/gB, and 5 days later the mediastinal lymph nodes (A) and spleens (B) were removed. Bulk single-cell suspensions were incubated with no added stimulator cells for 5 days and then tested for lytic activity against HSV-infected ⁵¹Cr-labeled B6/WT-3 cells, B6/WT-3 cells pulsed with 1 μM gB498-505 synthetic peptide, and mock peptide-pulsed B6/WT-3 cells. For analysis of a CTLm response (C), five mice were immunized i.n. with WSN/NA/gB. An unimmunized (naive) mouse served as a negative control. Four weeks later, splenocytes from individual mice were cultured with HSV-1-infected, mitomycin C-treated B6/WT-3 cells and tested in a ⁵¹Cr release assay for lytic activity against HSV-infected B6/WT-3 cells, B6/WT-3 cells pulsed with 1 μM gB498-505 synthetic peptide, and mock peptide-pulsed B6/WT-3 cells. The results represent an effector-to-target ratio of 30:1.

FIG. 7

Immunization with WSN/NA/gB confers protection from lethal HSV-2 infection. Mice were immunized i.n. with either PBS–1% FCS (n = 5) or WSN/NA/gB (n = 8) as described in Materials and Methods. Four weeks later, all mice were administered an i.n. challenge of 10⁵ PFU HSV-2 186. Mice were monitored daily for lethality for 28 days.