A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice

Abstract

The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-gamma and TNF-alpha) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.

Fig. 1.

The level of SARS-specific IgG in subcutaneously vaccinated mice. Mice were subcutaneously primed with 10 μg of UV-inactivated SARS-CoV virion (B), or virion with 2 mg of alum (C), or alum alone or none (A) and boosted with the same dose in their footpads at 7 weeks after priming. Serum was collected at the indicated time point and subjected to ELISA to detect SARS-specific IgG using SARS-CoV-infected Vero cell lysates as a coating antigen. Circles and bars represent the amount of IgG antibody in the serum of each mouse and the mean, respectively. The amount of IgG was arbitrarily calculated based on the concentration of hyper-immune sera. A representative result of two independent experiments is shown. (D) Mice were vaccinated with 10 μg of UV-inactivated SARS-CoV virion subcutaneously into their backs. Serum was collected from individual mice at the indicated time point and subjected to ELISA to detect SARS-specific IgG.

Fig. 2.

The number of SARS-specific IgG₁ plasma cells in BM. Mice were primed and boosted by subcutaneous injection into their back with 10 μg of UV-inactivated SARS-CoV virion with 2 mg of alum (VA). BMs were collected at 10 days after boost and subjected to ELISPOT to detect SARS-specific IgG₁ plasma cells. Bars represent the number of plasma cells specific to N1–49 and N340–390 antigen in SARS-vaccinated and control mice, respectively. Data are means of triplicate cultures. The number of spots from control mice was below the detection limit (i.e. <1 ASC/9 × 10⁵ cells: dashed line). A representative result of two independent experiments is shown. N.D.: not detected.

Fig. 3.

IgG subclass of immunized serum. Mice were subcutaneously primed and boosted by injection in their footpads with 10 μg of UV-inactivated SARS-CoV virion (V), or virion with 2 mg of alum (VA). Serum was collected from individual mice at 1 week after boost and subjected to ELISA to detect SARS-specific IgG₁, IgG_2a, IgG_2b and IgG₃ titer. The Y value is the reciprocal serum dilution number where the OD490nm ≥ 0.2 in each ELISA. Circles and bars represent the titer for each mouse and the mean, respectively; results are representative of two separate experiments.

Fig. 4.

Specificity of the serum antibodies. (A) Purified UV-inactivated SARS-CoV virion (0.5 μg) was fractionated by SDS–PAGE and subjected to western blotting. Diluted pooled sera (1:1000) from mice primed and boosted with virion/alum were exploited to detect virus proteins. Upper and lower arrows indicate the predicted band of S (spike protein) and N (nucleocapside protein) of SARS-CoV, respectively. The size of molecular weight markers (kDa) is shown on the left. (B) S protein- or N protein-specific ELISA. ELISA plates were coated at the indicated dilution with 1% NP40 lysates of chick embryo fibroblasts that had been infected with S protein-expressing vaccinia virus (circle), N protein-expressing vaccinia virus (triangle) or uninfected (mock; square). Diluted serum (1:1000) from mice prime and boost immunized with virion/alum, was exploited for detection of virus proteins.

Fig. 5.

In vitro responses of SARS-CoV-specific T cells taken from mice vaccinated with inactivated SARS-CoV. Mice were subcutaneously primed with 10 μg of UV-inactivated SARS-CoV virion, or virion with 2 mg of alum, or none, and then boosted with the same dose in their footpads at 7 weeks after priming. Draining lymph nodes and spleens were isolated at 1 week after boost and stimulated with T-cell depleted splenocytes that had been pulsed with the indicated concentration of UV-inactivated SARS-CoV virion. These cells were cultured for 2–4 days and [³H]thymidine was added 8 h prior to the harvest. The peak response on day 4 after cultivation is shown in (A). (B) Culture supernatant was collected at day 2–4 post cultivation and the level of IL-2, IFN-γ, IL-4, IL-5 and TNF-α was determined by CBA kit. The maximum cytokine production at day 4 is shown. Results are representative of two separate experiments.