Monoclonal antibodies to SARS-associated coronavirus (SARS-CoV): Identification of neutralizing and antibodies reactive to S, N, M and E viral proteins

Journal of Virological Methods
Volume 128, Issues 1–2, September 2005, Pages 21-28

Ralph A. Tripp, Lia M. Haynes, Deborah Moore, Barbara Anderson, Azaibi Tamin, Brian H. Harcourt, Les P. Jones, Mamadi Yilla, Gregory J. Babcock, Thomas Greenough, Donna M. Ambrosino, Rene Alvareza, Justin Callaway, Sheana Cavitt, Kurt Kamrud, Harold Alterson, Jonathan Smith, Jennifer L. Harcourt…Larry J. Anderson.

Abstract

Monoclonal antibodies (Mabs) against the Urbani strain of the SARS-associated coronavirus (SARS-CoV) were developed and characterized for reactivity to SARS-CoV and SARS-CoV S, N, M, and E proteins using enzyme-linked immunoabsorbent (ELISA), radioimmunoprecipitation, immunofluorescence, Western Blot and microneutralization assays. Twenty-six mAbs were reactive to SARS-CoV by ELISA, and nine were chosen for detailed characterization. Five mAbs reacted against the S protein, two against the M protein, and one each against the N and E proteins. Two of five S protein mAbs neutralized SARS-CoV infection of Vero E6 cells and reacted to an epitope within amino acids 490–510 in the S protein. While two of the three non-neutralizing antibodies recognized at second epitope within amino acids 270–350. The mAbs characterized should prove useful for developing SARS-CoV diagnostic assays and for studying the biology of infection and pathogenesis of disease.

Keywords

SARS-coronavirus, Monoclonal antibody, Immunoassay, Epitope, Neutralizing

The design and application of DNA chips for early detection of SARS-CoV from clinical samples

Journal of Clinical Virology
Volume 33, Issue 2, June 2005, Pages 123-131

Yi-ming Zhou, Ren-quan Yang, Sheng-ce Tao, Ze Li, Qiong Zhang, Hua-fang Gao, Zhi-wei Zhang, Jian-yu Du, Pei-xuan Zhu, Li-li Ren, Liang Zhang, Dong Wang, Lan Guo, Yan-bin Wang, Yong Guo, Yan Zhang, Chuan-zan Zhao, Can Wang…Jing Cheng

Abstract

Background:

SARS coronavirus has been identified as the cause of severe acute respiratory syndrome (SARS). Few tests allow confirmation or exclusion of SARS within the first few days of infection. A gene chip is a useful tool for the study of microbial infections mainly for its capability of performing multi-target analysis in a single test.

Objectives:

Investigate the possibility of early detection of SARS virus from clinical samples using the gene chip-based method.

Study design:

We purified RNA from SARS-CoV obtained from routinely collected peripheral blood and sputum samples of 34 patients who had been identified as probable SARS patients by following the interim U.S. case definition. Four segments of the SARS-CoV were amplified using reverse transcription-nested PCR and the products examined using the 70-mer gene chips for SARS-CoV detection.

Results:

A blind-test of both peripheral blood and sputum specimens lead to the positive detection of SARS-CoV in 31 out of 34 patients. SARS-CoV was not found in peripheral blood or sputum specimens from three patients. Two of the 34 patients were only 3 days post-onset of symptoms and were subsequently confirmed to be SARS positive. Our results indicate that the gene chip-based molecular test is specific for SARS-CoV and allows early detection of patients with SARS with detection rate about 8% higher than the single PCR test when the sputum sample is available.

Keywords

SARS, Coronavirus, SARS-CoV, Early detection, Gene chip

Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of SARS-CoV

Virology
Volume 334, Issue 1, 30 March 2005, Pages 134-143

Te-hui W. Chou, Shixia Wang, Pavlo V. Sakhatskyy, Innocent Mboudoudjeck, John M. Lawrence, Song Huang, Scott Coley, Baoan Yang, Jiaming Li, Qingyu Zhu, Shan Lu

Abstract

Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12–798) and one on S2 subunit (aa 797–1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310–535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV.

Keywords

SARS-CoV, Monoclonal antibody, Epitope mapping, Inactivated vaccine

Inhibition of SARS-CoV replication by siRNA

Antiviral Research
Volume 65, Issue 1, January 2005, Pages 45-48

Chang-Jer Wu, Hui-Wen Huang, Chiu-Yi Liu, Cheng-Fong Hong, Yi-LinChan

Abstract

Serious outbreaks of severe acute respiratory syndrome (SARS), caused by the newly discovered coronavirus SARS-CoV, occurred between late 2002 and early 2003 and there is an urgent need for effective antiviral agents. RNA interference in animals and post-transcriptional gene silencing plants is mediated by small double-stranded RNA molecules named small interfering RNA (siRNA). Recently, siRNA-induced RNA interference(RNAi) may provide a new approach to therapy for pathogenic viruses, e.g. HIV and HCV. In this study, the silencing potential of seven synthetic siRNAs against SARS-CoV leader, TRS, 3′-UTR and Spike coding sequence have been applied to explore the possibility for prevention of SARS-CoV infection. We demonstrate that siRNAs directed against Spike sequences and the 3′-UTR can inhibit the replication of SARS-CoV in Vero-E6 cells, and holds out promise for the development of an effective antiviral agent against SARS-CoV.

Keywords

SARS-CoV, siRNA, Spike protein, Antiviral agent

Intranasal immunization with inactivated SARS-CoV (SARS-associated coronavirus) induced local and serum antibodies in mice

Vaccine
Volume 23, Issue 7, 4 January 2005, Pages 924-931

Di Qu, Bojian Zheng, Xin Yao, Yi Guan, Zheng-Hong Yuan, Nan-Shan Zhong, Li-Wei Lu, Jian-Ping Xie, Yu-Mei Wen

Abstract

SARS-CoV (severe acute respiratory syndrome-associated coronavirus) strain GZ50 was partially purified and inactivated with 1:2000 formaldehyde. In cell culture the inactivated virus blocked the replication of live virus by decreasing the TCID5.0 of the live virus 103.6 to 104.6 times. Inactivated GZ50 was used to immunize mice intranasally either alone, or after precipitation with polyethylene glycol (PEG), or with CpG, or CTB as an adjuvant. The titer of serum neutralizing antibodies was up to 1:640. In mice immunized with adjuvants or PEG precipitated GZ50, specific IgA was detected in tracheal-lung wash fluid by immunofluorescence. Though serum antibodies were detected, no anti-SARS-IgA could be detected in mice immunized only with inactivated GZ50. The roles of adjuvants in intranasal immunization with inactivated. SARS-CoV is discussed.

Keywords

SARS-CoV, Intranasal immunization, Inactivated vaccine