Modulation of the immune response to the severe acute respiratory syndrome spike glycoprotein by gene-based and inactivated virus immunization

Abstract

Although the initial isolates of the severe acute respiratory syndrome (SARS) coronavirus (CoV) are sensitive to neutralization by antibodies through their spike (S) glycoprotein, variants of S have since been identified that are resistant to such inhibition. Optimal vaccine strategies would therefore make use of additional determinants of immune recognition, either through cellular or expanded, cross-reactive humoral immunity. Here, the cellular and humoral immune responses elicited by different combinations of gene-based and inactivated viral particles with various adjuvants have been assessed. The T-cell response was altered by different prime-boost immunizations, with the optimal CD8 immunity induced by DNA priming and replication-defective adenoviral vector boosting. The humoral immune response was enhanced most effectively through the use of inactivated virus with adjuvants, either MF59 or alum, and was associated with stimulation of the CD4 but not the CD8 response. The use of inactivated SARS virus with MF59 enhanced the CD4 and antibody response even after gene-based vaccination. Because both cellular and humoral immune responses are generated by gene-based vaccination and inactivated viral boosting, this strategy may prove useful in the generation of SARS-CoV vaccines.

FIG. 1.

Analysis of cellular and humoral immunity after DNA, adenoviral, or inactivated SARS virus, with or without adjuvant. Mice in each test group (n = 10) received injections with DNA (25 μg), rAd-S (10⁹), or with inactivated SARS virus (5 μg), with or without adjuvants as indicated in each column. The control group that was sham injected contained five mice. Immunizations were performed twice at 4-week intervals. Intracellular cytokine staining and serum analysis were performed 10 days after the last injection. Each symbol represents the percent positive cells in the CD4⁺ (left panel) or CD8⁺ (middle panel) T-cell population for one animal. The mean ICS response for the responding animals is indicated by horizontal bars. P values represent comparisons of groups by Student's t test (tail = 2, type = 2). Mouse sera were tested by ELISA against SARS-S protein performed 10 days after the last injection (right panel). The mean value of the optical density at 450 nm (O.D.450) from the animals' sera within the test group is represented by the solid bar, with the upper error bar as the standard deviation.

FIG. 2.

Comparison of adenoviral and inactivated SARS virus boosting, with or without adjuvant following DNA priming. Mice in each test group (n = 10) received three injections at 3-week intervals with DNA (25 μg) and were boosted with rAd-S (10⁹) or with inactivated SARS virus (5 μg), with or without adjuvants 3 weeks after the last DNA injection. The control group that was sham injected contained five mice. Intracellular cytokine staining was performed 10 days after the last injection, and immunoglobulin levels were measured as described in Materials and Methods. Each symbol represents the percent positive cells in the CD4⁺ (left panel) or CD8⁺ (middle panel) T-cell population for one animal. The mean ICS responses for the responding animals are indicated by horizontal bars. P values represent comparisons of groups by Student's t test (tail = 2, type = 2). Mouse sera were tested by ELISA against SARS-S protein performed 10 days after the last injection (right panel). The mean value of the optical density at 450 nm (O.D.450) from the animals' sera within the test group is represented by the solid bar, with the upper error bar as the standard deviation.

FIG. 3.

Comparison of inactivated SARS virus boosting with MF59 and CpG after DNA priming. Mice in each test group (n = 10) received three injections at 3-week intervals with DNA (25 μg) and were boosted with inactivated SARS virus (5 μg), with or without MF59 and the specified CpG adjuvants, 3 weeks after the last DNA injection. The control group that was sham injected contained five mice. Intracellular cytokine staining was performed 10 days after the last injection, and immunoglobulin levels were measured as described in Materials and Methods. Each symbol represents the percent positive cells in the CD4⁺ (left panel) or CD8⁺ (middle panel) T-cell population for one animal. The mean ICS responses for the responding animals are indicated by horizontal bars. P values represent comparisons of groups by Student's t test (tail = 2, type = 2). Mouse sera were tested by ELISA against SARS-S protein performed 10 days after the last injection (right panel). The mean value of the optical density at 450 nm (O.D.450) from the animals' sera within the test group is represented by the solid bar, with the upper error bar as the standard deviation.

FIG. 4.

Effects of secondary boosting by inactivated SARS virus and MF59 with and without CpG oligos: lack of cross-priming of CD8 responses. Mice in each test group (n = 10) received three injections at 3-week intervals with DNA (25 μg) and were boosted with rAd-S (10⁹) 3 weeks later. These animals subsequently received a secondary boost with inactivated SARS virus (5 μg), with or without MF59 and the specified CpG adjuvants, 3 weeks after the last rAd injection. The control group that was sham injected contained five mice. Intracellular cytokine staining was performed 10 days after the last injection, and immunoglobulin levels were measured as described in Materials and Methods. Each symbol represents the percent positive cells in the CD4⁺ (left panel) or CD8⁺ (middle panel) T-cell population for one animal. The mean ICS responses for the responding animals are indicated by horizontal bars. P values represent comparisons of groups by Student's t test (tail = 2, type = 2). Mouse sera were tested by ELISA against SARS-S protein performed 10 days after the last injection (right panel). The mean value of the optical density at 450 nm (O.D.450) from the animals' sera within the test group is represented by the solid bar, with the upper error bar as the standard deviation.

FIG. 5.

Effect of inactivated SARS virus boosting after adenoviral priming. Mice in each test group (n = 10) received an injection of rAd-S (10⁹) and were boosted 3 weeks later with inactivated SARS virus (5 μg), with or without MF59 and the specified CpG adjuvants. The control group that was sham injected contained five mice. Intracellular cytokine staining was performed 10 days after the last injection, and immunoglobulin levels were measured as described in Materials and Methods. Each symbol represents the percent positive cells in the CD4⁺ (left panel) or CD8⁺ (middle panel) T-cell population for one animal. The mean ICS responses for the responding animals are indicated by horizontal bars. P values represent comparisons of groups by Student's t test (tail = 2, type = 2). Mouse sera were tested by ELISA against SARS-S protein performed 10 days after the last injection (right panel). The mean value of the optical density at 450 nm (O.D.450) from the animals' sera within the test group is represented by the solid bar, with the upper error bar as the standard deviation.

FIG. 6.

Antigen specificity of the inactivated SARS virus boosting. Animals were immunized with rAd-S (10⁹) as in Fig. 5 and boosted with inactivated SARS virus (5 μg) or ovalbumin (5 μg; negative control) with MF59 or alum adjuvants as indicated, 4 weeks later. The cellular immune responses of these animals were analyzed with ICS 10 days after the final immunization by flow cytometry, and immunoglobulin levels were measured as described in Materials and Methods. Each symbol represents the percent positive cells in the CD4⁺ (left panel) or CD8⁺ (middle panel) T-cell population for one animal. The mean ICS responses for the responding animals are indicated by horizontal bars. P values represent comparisons of groups by Student's t test (tail = 2, type = 2). Mouse sera were tested by ELISA against SARS-S protein performed 10 days after the last injection (right panel). The mean value of the optical density at 450 nm (O.D.450) from the animals' sera within the test group is represented by the solid bar, with the upper error bar as the standard deviation.