Virus-specific memory CD8 T cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) caused an acute human respiratory illness with high morbidity and mortality in 2002-2003. Several studies have demonstrated the role of neutralizing antibodies induced by the spike (S) glycoprotein in protecting susceptible hosts from lethal infection. However, the anti-SARS-CoV antibody response is short-lived in patients who have recovered from SARS, making it critical to develop additional vaccine strategies. SARS-CoV-specific memory CD8 T cells persisted for up to 6 years after SARS-CoV infection, a time at which memory B cells and antivirus antibodies were undetectable in individuals who had recovered from SARS. In this study, we assessed the ability of virus-specific memory CD8 T cells to mediate protection against infection in the absence of SARS-CoV-specific memory CD4 T or B cells. We demonstrate that memory CD8 T cells specific for a single immunodominant epitope (S436 or S525) substantially protected 8- to 10-month-old mice from lethal SARS-CoV infection. Intravenous immunization with peptide-loaded dendritic cells (DCs) followed by intranasal boosting with recombinant vaccinia virus (rVV) encoding S436 or S525 resulted in accumulation of virus-specific memory CD8 T cells in bronchoalveolar lavage fluid (BAL), lungs, and spleen. Upon challenge with a lethal dose of SARS-CoV, virus-specific memory CD8 T cells efficiently produced multiple effector cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin 2 [IL-2]) and cytolytic molecules (granzyme B) and reduced lung viral loads. Overall, our results show that SARS-CoV-specific memory CD8 T cells protect susceptible hosts from lethal SARS-CoV infection, but they also suggest that SARS-CoV-specific CD4 T cell and antibody responses are necessary for complete protection.

Importance: Virus-specific CD8 T cells are required for pathogen clearance following primary SARS-CoV infection. However, the role of SARS-CoV-specific memory CD8 T cells in mediating protection after SARS-CoV challenge has not been previously investigated. In this study, using a prime-boost immunization approach, we showed that virus-specific CD8 T cells protect susceptible 8- to 10-month-old mice from lethal SARS-CoV challenge. Thus, future vaccines against emerging coronaviruses should emphasize the generation of a memory CD8 T cell response for optimal protection.

FIG 1

Prime-boost immunization induces a strong CD8 T cell response in 8-month-old mice. (A) Eight-month-old B6 mice were immunized intravenously (i.v.) with DC-loaded peptides and boosted 6 days later with rVV-S436 or rVV-S525 intranasally. Mice were rested for 42 to 45 days and then challenged with a lethal dose (5 × 10⁴ PFU) of MA15 (i.n.). Lungs and BAL were harvested 5 days postchallenge for further analysis. dpi, days postinfection. (B) FACS plots show percentages of S436- and S525-specific IFN-γ⁺ CD8 T cells in the blood (after direct ex vivo stimulation with respective peptides) on 0, 4, and 6 days after DC-peptide immunization. (C) Mean percentages (top) and numbers (bottom) of S436- and S525-specific CD8 T cells in the blood are shown. (D) FACS plots represent percentages of S436- and S525-specific IFN-γ⁺ CD8 T cells (after direct ex vivo stimulation with respective peptides) in BAL, lungs, and spleen 8 days after rVV-minigene boosting. (E and F) Bar graphs show mean percentages (E) and numbers (F) of S436- and S525-specific IFN-γ⁺ CD8 T cells (after in vitro stimulation with respective peptides) in BAL, lungs, and spleen 8 days after rVV-minigene boosting. Data are representative of 2 independent experiments with 3 or 4 mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by unpaired two-tailed Student's t test).

FIG 2

Identification of SARS-CoV-specific memory CD8 T cells. Prime-boost-immunized mice were rested for 42 to 45 days, and the percentages and numbers of S436 and S525-specific memory CD8 T cells were determined in BAL, lungs, and spleen. (A and B) The bar graphs show mean percentages (A) and numbers (B) of S436- and S525-specific IFN-γ⁺ CD8 T cells in the BAL, lungs, and spleen 42 to 45 days after rVV-minigene boosting. (C) The bar graphs show mean percentage of polyfunctional S436- and S525-specific IFN-γ⁺ CD8 T cells in the BAL, lungs, and spleen. Data are means of cytokine-positive CD8 T cells obtained by Boolean gating. (D) Scatter plots represent the percentages of S436 and S525 CD8 T cells that express the indicated marker. Data are representative of 2 or 3 independent experiments with 3 or 4 mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by unpaired two-tailed Student's t test).

FIG 3

Immunization induces polyfunctional secondary effector CD8 T cells. Prime-boost-immunized mice were rested for 42 to 45 days and then challenged with a lethal dose (5 × 10⁴ PFU) of MA15 (i.n.). Lungs and BAL were harvested 5 days postchallenge, and the percentage and number of epitope-specific CD8 T cells were determined. (A and B) The bar graphs show mean percentages (A) and numbers (B) of S436- and S525-specific IFN-γ⁺ CD8 T cells (after direct ex vivo stimulation with respective peptides) in the BAL and lungs 5 days after MA15 challenge. (C) IFN-γ⁺ CD8 T cells were further gated for TNF-α and IL-2 expression to determine polyfunctionality in the BAL and lungs. Data are representative of 3 independent experiments with 3 or 4 mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by unpaired two-tailed Student's t test).

FIG 4

Increased granzyme B production and cytotoxicity after challenge. (A and B) Histograms represent percent granzyme B⁺ CD8 T cells in the BAL and lungs at day 5 after MA15 challenge (A). (B) Bar graphs represent mean percentage of granzyme B⁺ CD8 T cells (after direct ex vivo stimulation with respective peptide). (C and D) In vivo cytotoxicity assays were performed 5 days after MA15 challenge, and the percent killing was calculated as described in Materials and Methods (numbers represent the percentage of cells labeled with different concentrations of CFSE). n = 4 or 5 mice/group/experiment. Data are representative of 2 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by unpaired two-tailed Student's t test).

FIG 5

Virus-specific memory CD8 T cells protect mid-aged mice from lethal MA15 infection. Prime-boost-immunized mice were rested for 42 to 45 days and then challenged with a lethal dose (5 × 10⁴ PFU) of MA15 intranasally. Percent initial body weight (A) and survival curves (B) are shown. The experiment shows data combined from three independent experiments, with 8 mice in the naive group and 15 or 16 mice in all other groups. Data are expressed as the percent initial weight ± the SEM. (C) Virus titers in lungs. Data correspond to means for 4 mice per group ± SEM and are representative of 2 or 3 independent experiments. (D) Lung sections from control and immunized mice are shown at days 0, 4, and 7 postchallenge. Stars represent alveolar and bronchiolar edema and arrowheads show peribronchial lymphocyte infiltration. DPI, days postinfection. *, P < 0.05; ** P < 0.01; ***, P < 0.001 (by unpaired Student's t test).