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1、VIROLOGICA SINICA, October 2007, 22 (5):360-365 CLC number: R511 Document code: A Article ID: 1674-0769(2007)05-0360-06 ? Received: 2007-03-15, Accepted: 2007-06-26 * Foundation item: Joint funds of National Natural Science Foundation of China (U0632010); Program of Guangdong Provincial Key Lab of B
2、ioengineering Medicine (51207026). * Corresponding author. Tel: +86-20-85223426, E-mail: Immune Responses and Histopathological Changes in Rabbits Immunized with Inactivated SARS Coronavirus* Chuan-hai ZHANG1,2, Xin-jian LIU1,3, Yi-fei WANG1*, Jia-hai LU4, Huan-ying ZHENG5, Sheng XIONG1, Mei-ying ZH
3、ANG1?and Qiu-ying LIU1 (1. Biomedicine Research 2. Department of Chemistry and Biology, West Anhui University, Lu-an 237012, China; 3. The First Peoples Hospital, Shanghai Jiaotong University, Shanghai 200080, China; 4. School of Public Health, Sun Yat-sen University, Guangzhou 510080, China; 5. Cen
4、ter for Diseases Control and Prevention of Guangdong Province, Guangzhou 510300, China ) Abstract: To evaluate the immunogenicity of inactivated SARS coronavirus (SARS-CoV), three groups of rabbits were immunized three times at 2-week intervals with inactivated vaccine + adjuvant, adjuvant, and norm
5、al saline respectively. Eight batchs of serum were sampled from the auricular vein at day 7 to day 51, and specific IgG antibody titers and neutralizing antibody titers were detected by indirect ELISA and micro-cytopathic effect neutralizing test. Antibody specificity was identified by proteinchip a
6、ssay. Histopathological changes were detected by H Humoral immune response; Histopathological change Severe acute respiratory syndrome (SARS) is a novel viral epidemic which spread to more than thirty countries in 2003 and severely threatened worldwide public health. The clinical symptom of SARS is
7、characterized by fever, dyspnoea, lymphopenia, and rapidly progressing changes observed by radiography. Its pathogen has been affirmed to be a novel coronavirus, SARS coronavirus (SARS-CoV), which ZHANG et al. Immune Responses and Histopathological Changes in Rabbits Immunized with SARS-CoV 361 esta
8、blished a solid bases for effective control and ultimate eradication of the disease (3,4,8,9). The genomes of SARS-CoV has been mapped and some important structural and functional proteins have been determined, but many features regarding the noso- genesis, the antigenicity and the immuogenicity of
9、SARS-CoV still remain indistinct (2,7,10,17). Vaccines play a crucial role in the control of infectious diseases. Previous experiences on human antiviral vaccines indicated that inactivated vaccines are much easier to develop and hold more potential. To investigate a SARS inactivated vaccine, effec-
10、 tiveness and safety of vaccine are two important aspects. Our efforts have focused on the investigation of the immunogenicity and safety of inactivated SARS-CoV on animals such as mice, rabbits, pigs, and horses (15,16). This paper reports the specific antibody kinetics and the histopathological ch
11、anges in rabbits immunized with inactivated SARS-CoV, which a view to gaining some insight towards the develop- ment of SARS inactivated vaccine. MATERIALS AND METHODS SARS-CoV strain SARS-CoV F69 strain (GenBank AY313906) was isolated from the samples of a onset of SARS patient in Guangdong provinc
12、e, China in 2003, and was screened out as the vaccine strain (5,18). Vero E6 cells were cultivated routinely with MEM medium con- taining no bovine serum, followed by infection with F69 strain virus. After the cytopathic effect (CPE) reached more than 75%, the cell suspension was frozen and thawed t
13、hree times, and stored at 7?. The titer of virus suspension was 106.7 TCID50/mL determined with micro-plate CPE method. Vaccine preparation Large-scale cultivated F69 virus suspension was inactivated with 0.4% formaldehyde (v/v) for 24 h at 37?, and the inactivation efficiency (100%) was precisely i
14、dentified (6). After centrifugation at 4 000 g for 30 min to remove precipitate, the virus supernatant was collected and purified by concentrating and gel permeation chromatography, then stored at 70? as the immunogen for animal immunization. The protein concentration of the prepared vaccine was 0.8
15、 mg/mL. Animal immunization Adult New Zealand rabbits (male, 2.0-2.5 kg) were randomly divided into 3 groups (n=4): the experi- mental group (G1, immunized with vaccine + adjuvant), the adjuvant group (G2, immunized just with adjuvant), and the control group (G3, injected with 0.9% NaCl sulution). R
16、abbits all were inoculated 3 times. The immune protocol was shown in Table 1. Eight batches of blood were sampled from the auricular vein at day 7, 14, 21, 28, 35, 38, 42 and 51. Serum was separated by centrifugation at 2 500g for 10 min and stored at 20?. Enzyme-linked immunosorbent assay The titer of serum specific IgG antibody was measured Table 1. Rabbit immunization protocol Immunizatio
