HLA-A2-restricted protection against lethal lymphocytic choriomeningitis

Abstract

The consequences of human lymphocytic choriomeningitis virus (LCMV) infection can be severe, including aseptic meningitis in immunocompetent individuals, hydrocephalus or chorioretinitis in fetal infection, or a highly lethal outcome in immunosuppressed individuals. In murine models of LCMV infection, CD8(+) T cells play a primary role in providing protective immunity, and there is evidence that cellular immunity may also be important in related arenavirus infections in humans. For this reason, we sought to identify HLA-A2 supertype-restricted epitopes from the LCMV proteome and evaluate them as vaccine determinants in HLA transgenic mice. We identified four HLA-A*0201-restricted peptides-nucleoprotein NP(69-77), glycoprotein precursor GPC(10-18), GPC(447-455), and zinc-binding protein Z(49-58)-that displayed high-affinity binding (< or =275 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LCMV antigens in HLA-restricted human antigen presenting cells. One of the epitopes (GPC(447-455)), after peptide immunization of HLA-A*0201 mice, induced CD8(+) T cells capable of killing peptide-pulsed HLA-A*0201-restricted target cells in vivo and protected mice against lethal intracranial challenge with LCMV.

FIG. 1.

Identification of immunogenic LCMV peptides after infection with LCMV. HLA-A*0201 mice (A), C57BL/6 mice (B), or BALB/c mice (C) were inoculated with 2 × 10⁵ PFU of LCMV strain Armstrong 53b via i.p. inoculation. Splenic CD8⁺ T cells were isolated 10 days later and exposed to JA2.1 target cells that had been pulsed with 10⁻⁵ M of each of the listed peptides in an ex vivo IFN-γ ELISPOT assay. Peptides that induced significant IFN-γ spot formation compared to an irrelevant HLA-A*0201-restricted peptide are denoted by an asterisk; the dotted line indicates the maximum number of spots produced by CD8⁺ T cells exposed to the irrelevant peptide.

FIG. 2.

Avidity of CD8⁺ T-cell responses against immunogenic LCMV peptides in HLA-A*0201 mice after peptide immunization. HLA-A*0201 mice were immunized with the peptides listed above each graph (NP_69-77, NP_101-109, NP_240-248, NP_315-323, GPC_10-18, GPC_447-455, Z_49-58, L_1189-1197, or L_1734-1742). Splenic CD8⁺ T cells were isolated 11 to 14 days postimmunization and exposed to either JA2.1 cells or LPS blasts from CB6F1 mice that had been pulsed with gradient doses of the immunizing LCMV peptide in an ex vivo IFN-γ ELISPOT assay. An asterisk denotes the lowest concentration of peptide that yielded a significant response.

FIG. 3.

Natural processing and recognition of immunogenic LCMV peptides in HLA-A*0201-restricted human target cells that endogenously express native LCMV antigens. HLA-A*0201 mice were immunized with the peptides listed above each graph (NP_69-77, NP_235-244, NP_240-248, GPC_10-18, GPC_447-455, Z_49-58, L_1189-1197, or L_1734-1742). Splenic CD8⁺ T cells were isolated 11 to 14 days later and exposed to JA2.1 cells that had been pulsed with peptide (LCMV peptide or irrelevant peptide) or infected with rVV (irrelevant or relevant rVV) in an ex vivo IFN-γ ELISPOT assay. Peptides or rVV were considered immunogenic if they induced IFN-γ spot formation that was significant compared to JA2.1 target cells that had been pulsed with an irrelevant HLA-A*0201-restricted peptide or infected with an irrelevant VV construct. Immunogenic responses are denoted by an asterisk.

FIG. 4.

Peptide immunization of HLA-A*0201 mice results in significant reduction in virus titer after LCMV challenge. (A) The logistics of the challenge experiment are outlined. HLA-A*0201 mice (n = 5 per group) were immunized with adjuvant alone (control), GPC_33-41, GPC_447-455, NP_69-77, Z_49-58, or GPC_10-18 as described in Materials and Methods. On day 14 postimmunization, mice were inoculated i.p. with 2 × 10⁵ PFU of LCMV strain Armstrong 53b. (B) Spleens were harvested on day 4 postchallenge and virus titers were determined via plaque assay. Mean titers from each peptide immunized group were compared to the control group by using the Student t test to determine whether differences were significant. Significant reductions in virus titer are indicated (*, P = 0.02; **, P = 0.002; ***, P = 0.000002).

FIG. 5.

Challenge with LCMV increases the frequency of epitope-specific CD8⁺ T cells in peptide immunized HLA-A*0201 mice. HLA-A*0201 mice were immunized with GPC_33-41, GPC_447-455, NP_69-77, Z_49-58, or GPC_10-18. On day 14 postimmunization, a subset of mice in each immunization group were inoculated i.p. with 2 × 10⁵ PFU of LCMV Armstrong 53b. On day 18 postimmunization, splenic CD8⁺ T cells were isolated from mice that were immunized without an accompanying viral challenge (□) or immunized and challenged with LCMV (▪). CD8⁺ T cells were exposed to JA2.1 target cells that had been pulsed with 10⁻⁵ M of the immunizing peptide in an ex vivo IFN-γ ELISPOT assay. The fold increases in epitope-specific CD8⁺ T-cell frequencies in challenged mice compared to unchallenged mice are indicated.

FIG. 6.

Protection is HLA-A*0201-restricted. (A) The logistics of the challenge experiment are outlined. CB6F1 mice (n = 5 per group) were immunized with adjuvant alone (control), GPC_33-41, GPC_447-455, or NP_69-77 as described in Materials and Methods. On day 14 postimmunization, mice were inoculated i.p. with 2 × 10⁵ PFU LCMV strain Armstrong 53b. (B) Spleens were harvested on day 4 postchallenge, and virus titers were determined via plaque assay. Mean titers from each peptide-immunized group were compared to the control group by using the Student t test to determine whether differences were significant. Significant reductions in virus titer are indicated (*, P = 0.0004). (C) In each group of mice, splenic CD8⁺ T cells were isolated 4 days after challenge and exposed to 10⁻⁵ M of either the immunizing peptide (▪) or an irrelevant peptide (□) in an ex vivo IFN-γ ELISPOT assay. We compared CD8⁺ T cells for their ability to produce IFN-γ in response to the immunizing peptide versus the irrelevant peptide by using the Student t test. Significant increases in epitope-specific CD8⁺ T-cell frequencies are indicated by an asterisk.

FIG. 7.

In vivo killing of peptide-pulsed target cells after peptide immunization of HLA-A*0201 mice. HLA-A*0201 mice were immunized with GPC_10-18, GPC_33-41, GPC_447-455, NP_69-77, Z_49-58, or adjuvant alone (control). Ten days later, CFSE-labeled target cells (CFSE^hi, pulsed with the immunizing peptide; CFSE^lo, pulsed with an irrelevant peptide) were delivered to recipient mice by i.v. injection. After 18 h, CFSE-labeled cells were recovered from the spleens of recipient mice and enumerated. The numbers represent the percentage of peptide-pulsed target cells killed.