SARS vaccine protective in mice.

To the Editor: Less than a year after the identification of the severe acute respiratory syndrome coronavirus (SARS-CoV) (1), 3 independent laboratories reported protection from SARS-CoV challenge in animal models using a DNA vaccine or recombinant forms of the modified vaccinia Ankara or a parainfluenza virus, encoding the spike gene (2-4). Their protective efficacies are encouraging because they provide proof that a SARS-CoV vaccine is feasible. However, vaccines based on those technologies are not licensed for human use, and recommendation and licensing will likely take many years. We have developed an inactivated virus vaccine that induces neutralizing antibodies and protects against SARS-CoV challenge.

The vaccine was produced as described elsewhere (5). Briefly, the SARS-CoV (strain FRA, GenBank accession no. AY310120) was grown in Vero cells, inactivated with [beta]-propiolactone (BPL), and complete inactivation was confirmed by 2 consecutive passages on Vero cells. Inactivated virus was purified by column chromatography followed by sucrose gradient centrifugation. The fraction containing virus was dialyzed against phosphate-buffered saline pH 7.2, and total protein content was determined by using the Micro BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA). Immunogenicity of the vaccine was tested by immunizing BALB/c mice at 0, 2, and 4 weeks with 5 [micro]g of inactivated virus combined with the adjuvant MF59, an oil squalene-in-water emulsion (6) approved for human use in Europe for an influenza vaccine. Ten days after the third immunization, serum samples were tested for the presence of SARS-CoV spike protein-specific antibodies by using enzyme-linked immunosorbent assay, and high titers (1-3 x [10.sup.4]) of anti-SARS-CoV spike immunoglobulin (Ig) G antibodies were detected. IgG subclass determination indicated a predominant Th2-type immune response similar to that observed in BALB/c mice vaccinated with a UV-inactivated SARS-CoV plus alum (7).

Efficacy of the inactivated virus vaccine was also assessed in a BALB/c mouse model of SARS-CoV infection (8). Animal studies were approved by the National Institutes of Health Animal Care and Use Committee and were conducted in an animal biosafety level 3 facility. SARS-CoV replicates in the respiratory tract of BALB/c mice following intranasal infection with [10.sup.4] 50% tissue culture infectious doses ([TCID.sub.50]) of virus. Generally, virus titers peak within 2 days after infection and are cleared within 7 days (8). BALB/c mice were immunized at 0, 2, and 4 weeks with 5 [micro]g of BPL-inactivated SARS virus with or without the MF59 adjuvant. Mice were also immunized with 4 different control preparations: phosphate-buffered saline, adjuvant MF59 alone, and 5 [micro]g of BPL inactivated influenza A virus vaccine with or without MF59. Serum samples were collected 2 weeks after each dose, and assayed for their ability to neutralize SARS-CoV (8). After 2 vaccine doses, SARS-CoV neutralizing antibodies were detected only in the group of mice immunized with the BPL-inactivated SARS virus vaccine plus MF59 adjuvant (1:91). Two weeks after the third dose, the BPL-inactivated SARS virus vaccine without MF59 induced neutralizing titers of 1:64, while the adjuvanted vaccine elicited neutralizing titers >1:600 (Table).

Mice were challenged intranasally at this point with [10.sup.4] [TCID.sub.50] SARS-CoV (Urbani strain, GenBank accession no. AY278741). Nasal turbinates and lung tissues were analyzed for infectious virus 2 days later (Table). SARS-CoV titers in mice from the control groups were [approximately equal to] [10.sup.6] [TCID.sub.50] virus/g of lung tissue and[approximately equal to] [10.sup.3] [TCID.sub.50] virus/g of nasal turbinate tissue. Complete protection from virus replication was observed in mice that received the MF59 adjuvanted SARS-CoV vaccine. Immunization with the nonadjuvanted vaccine resulted in complete protection of the upper respiratory tract and a significant reduction (30,000-fold) of viral titers in the lower respiratory tract compared to the control groups. The incomplete protection of this group was attributed to a single animal that contained detectable infectious virus in the lung.

Accelerated or enhanced virus replication or disease in immunized persons is a concern in developing any vaccine. This may be particularly true for SARS-CoV vaccines since adverse effects have been reported for one animal coronavirus vaccine, feline infectious peritonitis virus (9). Additionally, some in vitro experiments were performed with pseudo-typed lentiviruses that expressed the spike glycoprotein derived from SARS-like virus isolated from civets. In these experiments, the presence of antibodies that neutralized most human isolates of SARS-CoV demonstrated enhanced entry into renal epithelial cells (10). In our studies, we did not find enhanced virus replication in the respiratory tract of vaccinated mice upon SARS-CoV challenge. However, since mice are a model of SARS-CoV infection but not disease, the issue of disease enhancement will have to be carefully evaluated if and when an appropriate animal model in which this phenomenon can be demonstrated becomes available.

In summary, an inactivated SARS-CoV vaccine, produced with a technology that has a safety record established by immunizing hundreds of millions of persons, protects mice from challenge with SARS-CoV. The vaccine adjuvanted with MF59 elicits neutralizing antibodies (titer 1:91) after only 2 doses. We conclude that the vaccine described here has desirable properties, and our data support further development and plans for clinical trials.

Table. Immunogenicity and efficacy of [beta]-propiolactone
(BPL)-inactivated severe acute respiratory syndrome coronavirus
(SARS-CoV) vaccine in mice against subsequent challenge with live
SARS-CoV

                                    Neutralization titer ([dagger])

                                   2 wk post   2 wk post   2 wk post
Immunogen *                        1st dose    2nd dose    3rd dose

PBS                                  <1:8        <1:8        <1:8
MF59                                 <1:8        <1:8        <1:8
Influenza A (5 [micro]g)             <1:8        <1:8        <1:8
Influenza A (5 [micro]g) + MF59      <1:8        <1:8        <1:8
BPL-SARS-CoV (5 [micro]g)            <1:8        <1:8        1:64
BPL-SARS-CoV (5 [micro]g) + MF59     <1:8        1:91        1:645

                                       Virus replication upon
                                     challenge ([double dagger])

                                                Lungs

                                                   Mean ([+ or -]
                                   No. infected/   SE) virus titer
Immunogen *                         no. tested       ([section])

PBS                                    4/4         6.3 [+ or -] 0.3
MF59                                   4/4         6.1 [+ or -] 0.13
Influenza A (5 [micro]g)               4/4         6.3 [+ or -] 0.07
Influenza A (5 [micro]g) + MF59        4/4         6.0 [+ or -] 0.19
BPL-SARS-CoV (5 [micro]g)              1/4         2.0 [+ or -] 0.0
                                                     ([paragraph]) (#)
BPL-SARS-CoV (5 [micro]g) + MF59       0/4         [less than or equal
                                                     to] 1.5 [+ or -] 0
                                                     ([paragraph]) **

                                       Virus replication upon
                                     challenge ([double dagger])

                                             Nasal turbinates

                                                   Mean ([+ or -]
                                   No. infected    SE) virus titer
Immunogen *                         no. tested       ([section])

PBS                                    3/4        2.8 [+ or -] 0.35
MF59                                   3/4        3.0 [+ or -] 0.58
Influenza A (5 [micro]g)               3/4        2.9 [+ or -] 0.36
Influenza A (5 [micro]g) + MF59        4/4        3.0 [+ or -] 0.11
BPL-SARS-CoV (5 [micro]g)              0/4        [less than or equal
                                                    to] 1.8 [+ or -] 0
                                                    ** ([dagger]
                                                    [dagger])
BPL-SARS-CoV (5 [micro]g) + MF59       0/4        [less than or equal
                                                    to] 1.8 [+ or -] 0
                                                    ** ([dagger]
                                                    [dagger]])

* The indicated immunogens or control preparations were administered
to mice by subcutaneous injection on 3 occasions 2 weeks apart; PBS,
phosphate-buffered saline.

([dagger]) Neutralization titers were determined as described (8).

([double dagger]) Mice were challenged with [10.sup.4] 50% tissue
culture infectious doses ([TCID.sub.50]) SARS-CoV intranasally.

([section]) Virus liters are expressed as [log.sub.10]
[TCID.sub.50]/g of tissue.

([paragraph]) p<0.00001 in a 2-tailed Student t test, compared to
titers seen in mice that were immunized with PBS.

(#) Indicates the titer of a single animal. The remaining 3 mice had
no detectable levels of virus.

** Virus not detected; the lower limit of detection of infectious
virus was 1.5 [log.sub.10] [TCID.sub.50]/g in a 10% wt/vol
suspension of lung homogenate and 1.8 [log.sub.10]
[TCID.sub.50]/g in a 5% wt/vol suspension of nasal turbinates.

([dagger][dagger]) p = 0.025 in a 2-tailed Student t test, compared
to titers seen in mice that were immunized with PBS.

Acknowledgments

This study was supported by the Fonds der Chemischen Industrie, the 6th Framework Program of the European Commission (FP6-511065), and Chiron Vaccines.

(1) These authors contributed equally to this work.

References

(1.) Ksiazek TG, Erdman D. Goldsmith CS, Zaki SR, Peret T, Emery S, et al. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med. 2003;348:1953-66.

(2.) Yang ZY, Kong WP, Huang Y, Roberts A, Murphy BR, Subbarao K, et al. DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature. 2004;428:561-4.

(3.) Bisht H, Roberts A, Vogel L, Bukreyev A, Collins PL, Murphy BR, et al. Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice. Proc Natl Acad Sci U S A. 2004:101:6641-6.

(4.) Bukreyev A, Lamirande EW, Buchholz UJ, Vogel LN, Elkins WR, St Claire M, et al. Mucosal immunisation of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS. Lancet. 2004;363:2122-7.

(5.) Song HC, Seo M-Y, Stadler K, Yoo BJ, Choo Q-L, Coates S, et al. Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein. J Virol. 2004;78:10328-35.

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(7.) Takasuka N, Fujii H, Takahashi Y, Kasai M, Morikawa S. Itamura S. et al. A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice. Int Immunol. 2004;16:1423-30.

(8.) Subbarao K, McAuliffe J. Vogel L, Fahle G, Fischer S, Tatti K, et al. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice. J Virol. 2004;78:3572-7.

(9.) Vennema H, de Groot RJ, Harbour DA, Dalderup M. Gruffydd-Jones T, Horzinek MC, et al. Early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization. J Virol. 1990;64:1407-9.

(10.) Yang ZY, Werner HC, Kong WP, Leung K, Traggiai E, Lanzavecchia A, et al. Evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses. Proc Natl Acad Sci U S A. 2005; 102:797-801.

Konrad Stadler, * (1) Anjeanette Roberts, ([dagger])(1) Stephan Becker, ([double dagger])(1) Leatrice Vogel, ([dagger]) Markus Eickmann, ([double dagger]) Larissa Kolesnikova,([double dagger]) Hans-Dieter Klenk,([double dagger]) Brian Murphy, ([dagger]) Rino Rappuoli, * Sergio Abrignani, * and Kanta Subbarao ([dagger])

* Chiron Vaccines, Siena, Italy; ([dagger]) National Institutes of Health, Bethesda, Maryland, USA; and ([double dagger]) Institut fur Virologie, Marburg, Germany

Addresses for correspondence: Sergio Abrignani, Chiron Vaccines, Via Fiorentina 1. 53100 Siena, Italy; fax: [phone]; email: [e-mail]