key: cord-0033662-k38xlki3 authors: Zhang, Chuan-hai; Liu, Xin-jian; Wang, Yi-fei; Lu, Jia-hai; Zheng, Huan-ying; Xiong, Sheng; Zhang, Mei-ying; Liu, Qiu-ying title: Immune responses and histopathological changes in rabbits immunized with inactivated SARS coronavirus date: 2008-01-13 journal: Virol Sin DOI: 10.1007/s12250-007-0033-0 sha: 22294046de643fbde83f3767f493b38bbfe69478 doc_id: 33662 cord_uid: k38xlki3 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 normal 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 assay. Histopathological changes were detected by H&E staining. The results showed that, rabbits in the experimental group immunized with inactivated SARS-CoV all generated specific IgG antibodies with neutralizing activity, which suggested the inactivated SARS-CoV could preserve its antigenicity well and elicit an effective humoral immune responses. The peak titer value of specific IgG antibody and neutralizing antibody reached 1:40960 and 1:2560 respectively. In the experimental group, no obvious histopathological changes was detected in the H&E stained slides of heart, spleen, kidney and testis samples, but the livers had slight histopathological changes, and the lungs presented remarkable histopathological changes. These findings are of importance for SARS-CoV inactivated vaccine development. established 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 nosogenesis, the antigenicity and the immuogenicity of 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, effectiveness 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 changes in rabbits immunized with inactivated SARS-CoV, which a view to gaining some insight towards the development of SARS inactivated vaccine. SARS-CoV F69 strain (GenBank AY313906) was isolated from the samples of a onset of SARS patient in Guangdong province, China in 2003, and was screened out as the vaccine strain (5, 18) . Vero E6 cells were cultivated routinely with MEM medium containing 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 three times, and stored at -7 . The titer of virus suspension was 10 6.7 TCID 50 /mL determined with micro-plate CPE method. 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 identified (6) . After centrifugation at 4 000× g for 30 min to remove precipitate, the virus supernatant 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 2 500×g for 10 min and stored at -20 . The titer of serum specific IgG antibody was measured On day 51 after the first vaccination, all the rabbits were sacrified, then autopsied to gain the lung, liver, heart, spleen, kidney and testis. Tissues of the sampled organs were fixed in 10% neutral-buffered formalin and routinely processed for paraffin embedding. Serial 4 m sections were cut from each block. The tissue slices were stained with hemotoxylin and eosin dyes (H&E), and detected with photomicrography. Antibody titers of eight batches of serum samples from day 7 to day 51 were measured by indirect ELISA (Fig.1) and control group (G3), the sera all were negative. The inactivated SARS-CoV antigen elicited potent neutralizing antibody responses (Fig. 2) . On day 14, neutralizing antibody was detected in the experimental In experimental group (G1), no obvious histopathological changes were detected in H&E stained tissue slides of the heart, spleen, kidney, testis, but the liver tissues had slight histopathological changes, while the Crossreactivity of antibody against SARS-coronavirus nucleocapsid protein with IL-11 Molecular evolution of the SARS coronavirus during the course of the SARS epidemic in China Identification of a novel coronavirus in patients with severe acute respiratory syndrome A novel coronavirus associated with severe acute respiratory syndrome Establishment of SARS virus vaccine line Study on the function in inactivating antigenicity of SARS coronavirus by formaldehyde The genome sequence of the SARS-associated coronavirus Coronavirus as a possible cause of severe acute respiratory syndrome Characterization of a novel coronavirus associated with severe acute respiratory syndrome Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection Shanghai Health Digit Co Glycan arrays lead to the discovery of autoimmunogenic activity of SARS-CoV Manual for the virological investigation of polio CD4 and CD8 T cells have redundant but not identical roles in virus-induced demyelination Immunogencity of SARS inactivated vaccine in BALB/c mice Immune responses in Balb/c mice induced by a candidate SARS-CoV inactivated vaccine prepared from F69 strain Recent progress in the studies on SARS and its pathogeny Isolation, identification and variance of a coronavirus from a imputing SARS case