Immunogenicity and protective efficacy in mice and hamsters of a β-propiolactone inactivated whole virus SARS-CoV vaccine

Abstract

The immunogenicity and efficacy of β-propiolactone (BPL) inactivated whole virion SARS-CoV (WI-SARS) vaccine was evaluated in BALB/c mice and golden Syrian hamsters. The vaccine preparation was tested with or without adjuvants. Adjuvant Systems AS01(B) and AS03(A) were selected and tested for their capacity to elicit high humoral and cellular immune responses to WI-SARS vaccine. We evaluated the effect of vaccine dose and each adjuvant on immunogenicity and efficacy in mice, and the effect of vaccine dose with or without the AS01(B) adjuvant on the immunogenicity and efficacy in hamsters. Efficacy was evaluated by challenge with wild-type virus at early and late time points (4 and 18 wk post-vaccination). A single dose of vaccine with or without adjuvant was poorly immunogenic in mice; a second dose resulted in a significant boost in antibody levels, even in the absence of adjuvant. The use of adjuvants resulted in higher antibody titers, with the AS01(B)-adjuvanted vaccine being slightly more immunogenic than the AS03(A)-adjuvanted vaccine. Two doses of WI-SARS with and without Adjuvant Systems were highly efficacious in mice. In hamsters, two doses of WI-SARS with and without AS01(B) were immunogenic, and two doses of 2 μg of WI-SARS with and without the adjuvant provided complete protection from early challenge. Although antibody titers had declined in all groups of vaccinated hamsters 18 wk after the second dose, the vaccinated hamsters were still partially protected from wild-type virus challenge. Vaccine with adjuvant provided better protection than non-adjuvanted WI-SARS vaccine at this later time point. Enhanced disease was not observed in the lungs or liver of hamsters following SARS-CoV challenge, regardless of the level of serum neutralizing antibodies.

FIG. 1.

(A) Dosing and schedule of immunization, sample collection, and virus challenge in BALB/c mice. Each dose was administered IM in a total volume of 100 μL, except SARS-CoV, which was administered IN in 50 μL total volume (10⁵ TCID₅₀/mouse) for primary infection and for challenge. The samples collected are indicated by the arrows above the horizontal line. Peripheral blood mononuclear cells (PBMCs) were collected 1 wk following dose 2. Immunizations (dose1 and dose2) and virus challenge (challenge) are indicated by arrows below the horizontal line. (B) Dosing and schedule of immunization, sample collection, and virus challenge in golden Syrian hamsters. Each dose was administered IM in a total volume of 100 μL, except for SARS-CoV, which was administered IN in 100 μL total volume (10³ TCID₅₀/hamster) for primary infection and for challenge. The samples collected are indicated by arrows above the horizontal line (vt, lungs taken for viral titer determination [n = 4/group] 2 d after challenge; path, lungs and liver taken for histopathological evaluation [n = 3/group] 5 d after challenge). Immunizations (dose1 and dose2) and virus challenge (challenge) are indicated by arrows below the horizontal line. Early challenge (n = 7/group) occurred 4 wk after dose 2. Late challenge (n = 7/group) occurred 18 wk after dose 2.

FIG. 2.

Serum neutralizing antibody responses to WI-SARS vaccine in BALB/c mice. Vaccine dose and adjuvant are indicated along the x-axis; the dose listed is the amount of SARS-CoV S-protein (“S”) administered to each mouse. A control group (SARS) was infected IN with SARS-CoV. Neutralizing antibody titers are expressed as reciprocal mean titer (log₂) for groups of 5 mice. Data points show mean titers achieved following one and two doses of vaccine as measured prior to the administration of dose 2 and prior to challenge, respectively. Error bars indicate standard error of the mean. The dotted line indicates the lower limit of detection. Kruskal-Wallis testing with Tukey post-test demonstrated statistically significant differences between the groups indicated, prior to challenge (*p ≤ 0.05; ***p ≤ 0.001).

FIG. 3.

Cellular immune responses in mice. Shown are percentages of WI-SARS-specific CD4⁺ T-cell responses (geometric mean titer ± CI 95%) in PBMCs obtained on day 7 post-immunization, and re-stimulated with 1 μg/mL WI-SARS (based on S-protein content). Vaccine dose and adjuvant are indicated along the x-axis. The dose indicates the amount of SARS-CoV S-protein (“S”) as part of the WI-SARS material administered to each mouse. One-way ANOVA with Tukey post-test demonstrated statistically significant differences between the groups indicated (*p ≤ 0.05).

FIG. 4.

Efficacy of WI-SARS vaccine in BALB/c mice. Shown are the mean virus titers in lungs 2 d after challenge with 10⁵ TCID₅₀/mouse SARS-CoV. Vaccine dose and adjuvant are indicated along the x-axis. The dose listed is the amount of SARS-CoV-S-protein (“S”) administered to each mouse. Control groups included mice immunized with a Vero cell-grown BPL-inactivated influenza virus vaccine (Flu-BPL), adjuvants AS01_B and AS03_A alone, PBS, and primary infection with SARS-CoV. For the schedule of immunization and challenge see Fig. 1. The dashed line indicates the limit of detection (10^1.5 TCID₅₀/g). Error bars indicate standard error of the mean (n = 5 per group).

FIG. 5.

Serum neutralizing antibody responses to WI-SARS vaccine in golden Syrian hamsters. Vaccine dose and adjuvant are indicated along the x-axis; the dose listed is the amount of SARS-CoV S-protein (“S”) administered to each hamster. Control groups include hamsters immunized with a Vero cell-grown BPL-inactivated influenza virus vaccine (Flu-BPL), and primary infection with SARS-CoV. Neutralizing antibody titers are expressed as the reciprocal mean titer (log₂) for groups of 3 hamsters. Data points show mean titers achieved following one and two doses of vaccine as measured prior to the administration of dose 2 and prior to challenge, respectively. Error bars indicate standard error of the mean.

FIG. 6.

Neutralizing antibody responses to WI-SARS vaccine in golden Syrian hamsters in sera collected 3 and 16 wk following two doses of vaccine. Vaccine dose and adjuvant are indicated along the x-axis; the dose listed is the amount of SARS-CoV S-protein (“S”) administered to each hamster. Control groups included hamsters immunized with a Vero cell-grown BPL-inactivated influenza virus vaccine (Flu-BPL), and primary infection with SARS-CoV. Neutralizing antibody titers are expressed as reciprocal mean titer (log₂) for groups of 3 hamsters. Data points show mean titers achieved. Sera were tested in the same assay. Error bars indicate standard error of the mean.

FIG. 7.

Vaccine efficacy in golden Syrian hamsters at early and late challenge following two doses of WI-SARS vaccine. Vaccine dose and adjuvant are indicated along the x-axis; the dose listed is the amount of SARS-CoV S-protein (“S”) administered to each mouse. Control groups included hamsters immunized with a Vero cell-grown BPL-inactivated influenza virus vaccine (Flu-BPL), and primary infection with SARS-CoV. Shown are mean virus titers in lungs 2 d after challenge with 10³ TCID₅₀/mouse SARS-CoV (n = 4 per group). For the schedule of immunization and challenge see Fig. 1B. E indicates early challenge, 4 wk following the second immunization; L indicates late challenge, 18 wk after the second immunization. The dashed line indicates the limit of detection (10^1.5 TCID₅₀/g). Error bars indicate standard error of the mean.

FIG. 8.

Histopathological findings in the lungs of hamsters sacrificed on day 5 following late challenge. (A) Diffuse alveolar lesions in the lung of a hamster vaccinated with Flu-BPL with adjuvant AS01_B (hematoxylin & eosin, 40×). (B) Diffuse alveolar and perivascular lesions with abundant viral antigen (brown color) in the lung of a hamster vaccinated with Flu-BPL with adjuvant AS01_B (40×). (C) Few lesions are seen in the lung of a hamster vaccinated with 0.5 μg S-protein without adjuvant (100×). (D) Few lesions are seen in the lung of a hamster vaccinated with 2 μg S-protein + AS01_B adjuvant (100×). (E) Mild focal lung lesions of alveolar bronchiolization in a hamster that was previously infected with SARS-CoV (hematoxylin, 400×). (F) A lack of viral antigen is seen here in mild lung lesions of a hamster that was previously infected with SARS-CoV (hematoxylin, 400×). Color images available online at www.liebertonline.com/vim.