Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell–cell fusion but not interaction with M protein

Virology
Volume 405, Issue 1, 15 September 2010, Pages 139-148

Corrin E., McBride Carolyn, E. Machamer

Abstract

Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell–cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.

Keywords

coronavirus, SARS-CoV, spike, palmitoylation, fusion, detergent-resistant membranes, trafficking

Synthesis, docking studies, and evaluation of pyrimidines as inhibitors of SARS-CoV 3CL protease

Bioorganic & Medicinal Chemistry Letters
Volume 20, Issue 12, 15 June 2010, Pages 3569-3572

R. Ramajayam, Kian- Pin Tan, Hun- Ge Liu, Po- Huang Liang

Abstract

A series of 2-(benzylthio)-6-oxo-4-phenyl-1,6-dihydropyrimidine as SARS-CoV 3CL protease inhibitors were developed and their potency was evaluated by in vitro protease inhibitory assays. Two candidates had encouraging results for the development of new anti-SARS compounds.

Keywords

SARS-CoV, Pyrimidines

Synthesis, docking studies, and evaluation of pyrimidines as inhibitors of SARS-CoV 3CL protease

Bioorganic & Medicinal Chemistry Letters
Volume 20, Issue 12, 15 June 2010, Pages 3569-3572

R. Ramajayam, Kian-Pin, Tan, Hun-GeLiu, Po-HuangLiang

Abstract

A series of 2-(benzylthio)-6-oxo-4-phenyl-1,6-dihydropyrimidine as SARS-CoV 3CL protease inhibitors were developed and their potency was evaluated by in vitro protease inhibitory assays. Two candidates had encouraging results for the development of new anti-SARS compounds.

Keywords

SARS-CoV, Pyrimidines

TACE antagonists blocking ACE2 shedding caused by the spike protein of SARS-CoV are candidate antiviral compounds

Antiviral Research
Volume 85, Issue 3, March 2010, Pages 551-555

Shiori Haga, Noriyo Nagata, Tadashi Okamura, Norio Yamamoto, Tetsutaro Sata, Naoki Yamamoto, Takehiko Sasazuki, Yukihito Ishizaka

Abstract

Because outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV) might reemerge, identifying antiviral compounds is of key importance. Previously, we showed that the cellular factor TNF-α converting enzyme (TACE), activated by the spike protein of SARS-CoV (SARS-S protein), was positively involved in viral entry, implying that TACE is a possible target for developing antiviral compounds. To demonstrate this possibility, we here tested the effects of TACE inhibitors on viral entry. In vitro and in vivo data revealed that the TACE inhibitor TAPI-2 attenuated entry of both pseudotyped virus expressing the SARS-S protein in a lentiviral vector backbone and infectious SARS-CoV. TAPI-2 blocked both the SARS-S protein-induced shedding of angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV, and TNF-α production in lung tissues. Since the downregulation of ACE2 by SARS-S protein was proposed as an etiological event in the severe clinical manifestations, our data suggest that TACE antagonists block SARS-CoV infection and also attenuate its severe clinical outcome.

Keywords

SARS-CoV, TACE, ACE2, Shedding

A new mouse-adapted strain of SARS-CoV as a lethal model for evaluating antiviral agents in vitro and in vivo

Virology
Volume 395, Issue 2, 20 December 2009, Pages 210-222

Craig W. Day, Ralph Baric, Sui Xiong Cai, Matt Frieman, Yohichi Kumaki John D. Morrey, Donald F. Smee, Dale L. Barnard.

Abstract

Severe acute respiratory syndrome (SARS) is a highly lethal emerging disease caused by coronavirus SARS-CoV. New lethal animal models for SARS were needed to facilitate antiviral research. We adapted and characterized a new strain of SARS-CoV (strain v2163) that was highly lethal in 5- to 6-week-old BALB/c mice. It had nine mutations affecting 10 amino acid residues. Strain v2163 increased IL-1α, IL-6, MIP-1α, MCP-1, and RANTES in mice, and high IL-6 expression correlated with mortality. The infection largely mimicked human disease, but lung pathology lacked hyaline membrane formation. In vitro efficacy against v2163 was shown with known inhibitors of SARS-CoV replication. In v2163-infected mice, Ampligen™ was fully protective, stinging nettle lectin (UDA) was partially protective, ribavirin was disputable and possibly exacerbated disease, and EP128533 was inactive. Ribavirin, UDA, and Ampligen™ decreased IL-6 expression. Strain v2163 provided a valuable model for anti-SARS research.

Keywords

SARS-CoV, Lethal, IL-6, Ribavirin, Ampligen™, UDA, Protease inhibitor, Cytokine, Chemokine, Mouse

Antibody-mediated synergy and interference in the neutralization of SARS-CoV at an epitope cluster on the spike protein

Biochemical and Biophysical Research Communications
Volume 390, Issue 3, 18 December 2009, Pages 1056-1060

Lilin Zhong, Lia Haynes, Evi Budo Struble, Azaibi Tamin, Maria Luisa Virata-Theimer, Pei Zhang.

Abstract

Incomplete neutralization of virus, especially when it occurs in the presence of excess neutralizing antibody, represents a biological phenomenon that impacts greatly on antibody-mediated immune prophylaxis of viral infection and on successful vaccine design. To understand the mechanism by which a virus escapes from antibody-mediated neutralization, we have investigated the interactions of non-neutralizing and neutralizing antibodies at an epitope cluster on the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV). The epitope cluster was mapped at the C-terminus of the spike protein; it consists of structurally intertwined epitopes recognized by two neutralizing monoclonal antibodies (mAbs), 341C and 540C, and a non-neutralizing mAb, 240C. While mAb 341C binds to a mostly linear epitope composed of residues 507PAT509 and V349, mAb 240C binds to an epitope that partially overlaps the former by at least two residues (P507 and A508). The epitope corresponding to mAb 540C is a conformational one, involving residues L504 and N505. In neutralization assays, non-neutralizing 240C disrupted virus neutralization by mAb 341C and/or mAb 540C, whereas a combination of mAbs 341C and 540C blocked virus infectivity synergistically. These findings indicate that the epitope cluster on the spike protein may serve as an evolutionarily conserved platform at which a dynamic interplay between neutralizing and non-neutralizing antibodies occurs, thereby determining the outcome of SARS-CoV infection.

Keywords

SARS-CoV, Monoclonal antibody, Neutralization, Epitope

Identification of a New Region of SARS-CoV S Protein Critical for Viral Entry

Journal of Molecular Biology
Volume 394, Issue 4, 11 December 2009, Pages 600-605

Ying Guo, Jennifer Tisoncik, Susanna McReynolds, Michael Farzan, Bellur S. Prabhakar, Thomas Gallagher, Lijun Rong, Michael Caffrey.

Abstract

Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) is initiated by specific interactions between the SARS-CoV spike (S) protein and its receptor ACE2. In this report, we screened a peptide library representing the SARS-CoV S protein sequence using a human immunodeficiency virus-based pseudotyping system to identify specific regions that affect viral entry. One of the 169 peptides screened, peptide 9626 (S residues 217–234), inhibited SARS-CoV S-mediated entry of the pseudotyped virions in 293T cells expressing a functional SARS-CoV receptor (human angiotensin-converting enzyme 2) in a dose-dependent manner (IC50 ∼ 11 μM). Alanine scanning mutagenesis was performed to assess the roles of individual residues within this region of S, which was previously uncharacterized. The effects included significant reductions in expression (K223A), viral incorporation (L218A, I230A, and N232A), and reduced viral entry (L224A, L226A, I228A, T231A, and F233A). Taken together, these results reveal a new region of the S protein that is crucial for SARS-CoV entry.

Keywords

viral entry, SARS-CoV, envelope, mutagenesis, spike

Dual effect of nitric oxide on SARS-CoV replication: Viral RNA production and palmitoylation of the S protein are affected

Virology
Volume 395, Issue 1, 5 December 2009, Pages 1-9

Sara Åkerström, Vithiagaran Gunalan, Choong Tat Keng, Yee-Joo Tan, Ali Mirazimi

Abstract

Nitric oxide is an important molecule playing a key role in a broad range of biological process such as neurotransmission, vasodilatation and immune responses. While the anti-microbiological properties of nitric oxide-derived reactive nitrogen intermediates (RNI) such as peroxynitrite, are known, the mechanism of these effects are as yet poorly studied. Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) belongs to the family Coronaviridae, was first identified during 2002-2003. Mortality in SARS patients ranges from between 6 to 55%. We have previously shown that nitric oxide inhibits the replication cycle of SARS-CoV in vitro by an unknown mechanism. In this study, we have further investigated the mechanism of the inhibition process of nitric oxide against SARS-CoV. We found that peroxynitrite, an intermediate product of nitric oxide in solution formed by the reaction of NO with superoxide, has no effect on the replication cycle of SARS-CoV, suggesting that the inhibition is either directly effected by NO or a derivative other than peroxynitrite. Most interestingly, we found that NO inhibits the replication of SARS-CoV by two distinct mechanisms. Firstly, NO or its derivatives cause a reduction in the palmitoylation of nascently expressed spike (S) protein which affects the fusion between the S protein and its cognate receptor, angiotensin converting enzyme 2. Secondly, NO or its derivatives cause a reduction in viral RNA production in the early steps of viral replication, and this could possibly be due to an effect on one or both of the cysteine proteases encoded in Orf1a of SARS-CoV.

Keywords

SARS-CoV, Palmitoylation, Nitric oxide, Spike protein

Investigation of the pharmacophore space of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) NTPase/helicase by dihydroxychromone derivatives

Bioorganic & Medicinal Chemistry Letters
Volume 19, Issue 16, 15 August 2009, Pages 4538-4541

Chaewoon Lee, Jin Moo Lee, Na- Ra Lee, Dong-Eun Kim, Yong-Joo Jeong, You hoon Chong.

Abstract

Aryl diketoacids have been identified as the first SARS-CoV NTPase/helicase inhibitors with a distinct pharmacophore featuring an arylmethyl group attached to a diketoacid. In order to search for the pharmacophore space around the diketoacid core, three classes of dihydroxychromone derivatives were prepared. Based on SAR study, an extended feature of the pharmacophore model of SARS-CoV NTPase/helicase was proposed which is constituted of a diketoacid core, a hydrophobic arylmethyl substituent, and a free catechol unit.

Keywords

SARS (Severe Acute Respiratory Syndrome), NTPase/helicase, Dihydroxychromone, Pharmacophore

SARS-CoV 3CLpro inhibitory effects of quinone-methide triterpenes from Tripterygium regelii

Bioorganic & Medicinal Chemistry Letters

Volume 20, Issue 6, 15 March 2010, Pages 1873-1876

Young Bae Ryu, Su-Jin Park, Young Min Kim, Ju-YeonLee, Woo Duck Seo, Jong SunChang, Ki Hun Park, Mun-Chual Rho, Woo Song Lee

Abstract

Quinone-methide triterpenes, celastrol (1), pristimerin (2), tingenone (3), and iguesterin (4) were isolated from Triterygium regelii and dihydrocelastrol (5) was synthesized by hydrogenation under palladium catalyst. Isolated quinone-methide triterpenes (1–4) and 5 were evaluated for SARS-CoV 3CLpro inhibitory activities and showed potent inhibitory activities with IC50 values of 10.3, 5.5, 9.9, and 2.6 μM, respectively, whereas the corresponding 5 having phenol moiety was observed in low activity (IC50 = 21.7 μM). As a result, quinone-methide moiety in A-ring and more hydrophobic E-ring assist to exhibit potent activity. Also, all quinone-methide triterpenes 1–4 have proven to be competitive by the kinetic analysis.

Keyword

SARS-CoV, 3CLpro, Tripterygium regelii, Quinone-methide, Celastrol, Iguesterin

High-throughput assay using a GFP-expressing replicon for SARS-CoV drug discovery

Antiviral Research
Volume 80, Issue 2, November 2008, Pages 107-113

Feng Ge, Sheng Xiong, Fu-Sen Lin, Zhi-Ping Zhang, Xian-En Zhang

Abstract

The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The development of rapid screening assays is essential for antiviral drug discovery. By using a cell line expressing a SARS-CoV subgenomic replicon, we developed a high-throughput assay and used it to screen small molecule compounds for inhibitors of SARS-CoV replication in the absence of live virus. The assay system involves minimal manipulation after assay set-up, facilitates automated read-out and minimizes risks associated with hazardous viruses. Based on this assay system, we screened 7035 small molecule compounds from which we identified 7 compounds with anti-SARS-CoV activity. We demonstrate that the compounds inhibited SARS-CoV replication-dependent GFP expression in the replicon cells and reduced SARS-CoV viral protein accumulation and viral RNA copy number in the replicon cells. In a SARS-CoV plaque reduction assay, these compounds were confirmed to have antiviral activity. The target of one of the hit compounds, C12344, was validated by the generation of resistant replicon cells and the identification of the mutations conferring the resistant phenotype. These compounds should be valuable for developing anti-SARS therapeutic drugs as well as research tools to study the mechanism of SARS-CoV replication.

Keywords

SARS-CoV replicon, High-throughput screen (HTS)

An immunosuppressed Syrian golden hamster model for SARS-CoV infection

Virology
Volume 380, Issue 2, 25 October 2008, Pages 312-321

Scott R. Schaecher, Jennifer Stabenow, Christina Oberle, Jill Schriewer, R. Mark Buller, John E. Sagartz, Andrew Pekosz.

Abstract

Several small animal models have been developed for the study of severe acute respiratory syndrome coronavirus (SARS-CoV) replication and pathogenesis. Syrian golden hamsters are among the best small animal models, though little clinical illness and no mortality are observed after virus infection. Cyclophosphamide was used to immunosuppress hamsters leading to a prolonged disease course and higher mortality after SARS-CoV infection. In addition, there was a significant weight loss, expanded tissue tropism, and increased viral pathology in the lung, heart, kidney, and nasal turbinate tissues. Infection with recombinant SARS-CoV viruses bearing disruptions in the gene 7 coding region showed no significant change in replication kinetics, tissue tropism, morbidity, or mortality suggesting that the ORF7a (7a) and ORF7b (7b) proteins are not required for virus replication in immunosuppressed hamsters. This modified hamster model may provide a useful tool for SARS-CoV pathogenesis studies, evaluation of antiviral therapy, and analysis of additional SARS-CoV mutants.

Keywords

SARS-CoV, Coronavirus, Cyclophosphamide, ORF7a, ORF7b, Hamster, Accessory gene, Pathogenesis

A chimeric multi-epitope DNA vaccine elicited specific antibody response against severe acute respiratory syndrome-associated coronavirus which attenuated the virulence of SARS-CoV in vitro

Immunology Letters
Volume 119, Issues 1–2, 15 August 2008, Pages 71-77

Xiaohua Wang, Wei Xu, Deyan Tong, Jing Ni, Haifeng Gao, Ying Wang, Yiwei Chu, Pingping Li, Xiaoming Yang, Sidong Xiong.

Abstract

Epitope-based vaccines designed to induce antibody responses specific for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) are being developed as a means for increasing vaccine potency. In this study, we identified four B cell epitopes from the spike (S) and membrane (M) protein through bioinformatics analysis and constructed a multi-epitope DNA vaccine. Intramuscular immunization of mice with this vaccine was sufficient to induce specific prime as well as a long-term memory humoral immune response to at least two candidate epitopes, S437–459 and M1–20. A DNA prime–protein boost strategy greatly enhanced the antibody generation and the immune sera not only reacted with the lysates of SARS-CoV-infected Vero cells but also neutralized the cytopathic effect of SARS by 75% at 1:160 dilution. The novel immunogenic S protein peptide revealed in this study provides new target for SARS vaccine design; and our work indicated multi-epitope DNA vaccine as an effective means for eliciting polyvalent humoral immune response against SARS-CoV.

Keywords

SARS-CoV, Epitope, DNA vaccine, Antibody, Prime–boost

Lipid rafts are involved in SARS-CoV entry into Vero E6 cells

Biochemical and Biophysical Research Communications
Volume 369, Issue 2, 2 May 2008, Pages 344-349

Yanning Lu, Ding Xiang Liu, James P. Tam.

Abstract

Lipid rafts often serve as an entry site for certain viruses. Here, we report that lipid rafts in Vero E6 cells are involved in the entry of severe acute respiratory syndrome coronavirus (SARS-CoV). Infectivity assay showed the integrity of lipid rafts was required for productive infection of pseudotyped SARS-CoV. Depletion of plasma membrane cholesterol with MβCD relocalized raft-resident marker caveolin-1 as well as SARS-CoV receptor ACE2 to a nonraft environment, but did not significantly change the surface expression of ACE2. MβCD-treatment inhibited infectivity of pseudotyped SARS-CoV by 90%. Biochemical fractionation and confocal imaging confirmed that ACE2 colocalized with raft-resident markers. Furthermore, an ectodomain of SARS-CoV S protein (S1188HA) could associate with lipid rafts after binding to its receptor, and colocalize with raft-resident marker ganglioside GM1. The binding of S1188HA was not affected by depleting plasma membrane cholesterol. Taken together, our results support that lipid rafts serve as an entry port for SARS-CoV.

Keywords

Lipid rafts, SARS-CoV, Vero E6, ACE2, Spike protein, Entry

SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium

Virus Research
Volume 133, Issue 1, April 2008, Pages 33-44

Amy C. Sims, Susan E. Burkett, Boyd Yount, Raymond J. Pickles.

Abstract

SARS coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The severe and sudden onset of symptoms, resulting in an atypical pneumonia with dry cough and persistent high fever in cases of severe acute respiratory virus brought to light the importance of coronaviruses as potentially lethal human pathogens and the identification of several zoonotic reservoirs has made the reemergence of new strains and future epidemics all the more possible. In this chapter, we describe the pathology of SARS-CoV infection in humans and explore the use of two models of the human conducting airway to develop a better understanding of the replication and pathogenesis of SARS-CoV in relevant in vitro systems. The first culture model is a human bronchial epithelial cell line Calu-3 that can be inoculated by viruses either as a non-polarized monolayer of cells or polarized cells with tight junctions and microvilli. The second model system, derived from primary cells isolated from human airway epithelium and grown on Transwells, form a pseudostratified mucociliary epithelium that recapitulates the morphological and physiological features of the human conducting airway in vivo. Experimental results using these lung epithelial cell models demonstrate that in contrast to the pathology reported in late stage cases SARS-CoV replicates to high titers in epithelial cells of the conducting airway. The SARS-CoV receptor, human angiotensin 1 converting enzyme 2 (hACE2), was detected exclusively on the apical surface of cells in polarized Calu-3 cells and human airway epithelial cultures (HAE), indicating that hACE2 was accessible by SARS-CoV after lumenal airway delivery. Furthermore, in HAE, hACE2 was exclusively localized to ciliated airway epithelial cells. In support of the hACE2 localization data, the most productive route of inoculation and progeny virion egress in both polarized Calu-3 and ciliated cells of HAE was the apical surface suggesting mechanisms to release large quantities of virus into the lumen of the human lung. Preincubation of the apical surface of cultures with antisera directed against hACE2 reduced viral titers by two logs while antisera against DC-SIGN/DC-SIGNR did not reduce viral replication levels suggesting that hACE2 is the primary receptor for entry of SARS-CoV into the ciliated cells of HAE cultures. To assess infectivity in ciliated airway cultures derived from susceptible animal species we generated a recombinant SARS-CoV by deletion of open reading frame 7a/7b (ORF 7a/7b) and insertion of the green fluorescent protein (GFP) resulting in SARS-CoV GFP. SARS-CoV GFP replicated to similar titers as wild type viruses in Vero E6, MA104, and CaCo2 cells. In addition, SARS-CoV replication in airway epithelial cultures generated from Golden Syrian hamster tracheas reached similar titers to the human cultures by 72 h post-infection. Efficient SARS-CoV infection of ciliated cell-types in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis.

Keywords

Human airway epithelia, SARS-CoV, Coronavirus replication, SARS-CoV GFP, Coronavirus pathogenesis

Priming with SARS CoV S DNA and boosting with SARS CoV S epitopes specific for CD4+ and CD8+ T cells promote cellular immune responses

Vaccine
Volume 25, Issues 39–40, 28 September 2007, Pages 6981-6991

Jun Huang, Yingnan Cao, Jiali Du, Xianzhang Bu, Rui Ma, Changyou Wu.

Abstract

Cellular immune response plays an important role in antiviral immunity. In our previous study, immunization of mice with severe acute respiratory syndrome coronavirus (SARS CoV) spike (S) DNA vaccine could induce both humoral and cellular immunity in response to a pool of entire overlapping S peptides. Identification of functional dominant epitopes in SARS CoV S protein for T cells is crucial for further understanding of cellular immune responses elicited by SARS CoV S DNA vaccine. In present study, mice were immunized with SARS CoV S DNA vaccine. Subsequently, a pool of 17–19 mers overlapped SARS CoV S peptides, which served as immunogens, were scanned to identify the specific epitopes for T cells. Two H-2d restricted CD4+ T epitopes, N60 (S435–444) and P152 (S1111–1127), and two H-2d restricted CD8+ T cell epitopes, N50 (S365–374) and P141 (S1031–1047) were identified by three different methods, enzyme-linked immunosorbent assay (ELISA), enzyme linked immunospot assay (ELISPOT) and fluorescence activated cell sorter (FACS). The dominant CD4+ T cell epitope (N60) and CD8+ T cell epitope (N50) located in the receptor-binding domain (RBD) of SARS CoV S protein, which mediated virus combining and fusing to susceptible cells. Importantly, our novel finding is that mice primed with SARS S DNA vaccine and boosted with T cell epitopes (N50 and N60) could promote antigen specific CD4+ and CD8+ T cell immune responses. Our study provides valuable information for the design of vaccine for SARS study.

Keywords

SARS CoV, T cells, Epitope, Vaccines

Towards our understanding of SARS-CoV, an emerging and devastating but quickly conquered virus

Comparative Immunology, Microbiology and Infectious Diseases
Volume 30, Issues 5–6, September 2007, Pages 309-327

Youjun Feng, George F. Gao.

Abstract

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease caused by a novel coronavirus (SARS-CoV), which has overwhelmed more than 30 countries claiming nearly 8400 cases with over 800 fatalities. Thanks to the unprecedented international collaboration, the whole-genomes of SARS-CoVs were successfully deciphered shortly after the identification of the causative pathogen for outbreak of SARS in southern China, in 2003. Hitherto, the SARS-CoV, as a viral paradigm of emerging infectious entities, has been extensively studied that has ranged from epidemiology, molecular virology/immunology to structural genomics. Also, several lines of breakthroughs have been record-brokenly obtained, that included the finding of ACE2, a functional receptor for the SARS-CoV, solution of the 3CLpro structure, a first crystal structure of SARS-related macromolecules, revealing of bats as natural reservoirs for SARS-like viruses and the possible involvement of civet cats in the SARS emergence. This review intends to outline the major progress in the journey of SARS-related exploration, by emphasizing those inaugurated studies with milestone-like significance contributed by Chinese research groups.

Keywords

Severe acute respiratory syndrome (SARS), Coronavirus (CoV), SARS-CoV, Molecular epidemiology, Natural reservoir, Receptor, T cell immunity, CTL epitopes, Structural genomics

Isolation and identification of an scFv antibody against nucleocapsid protein of SARS-CoV

Microbes and Infection
Volume 9, Issue 8, July 2007, Pages 1026-1033

Aizhi Zhao, Weijun Qin, Yueheng Han, Weihong Wen, Wenhong Zhang, Zhonghui Lian, Gang Chen, Zhuoli Zhang, Jianqiang Peng, He Wang, Yinglu Guo.

Abstract

To develop reagents for early diagnosis and therapeutic drugs against SARS-associated coronavirus (SARS-CoV), a large (3 × 109) immunized human antibody library was constructed from peripheral blood mononuclear cells from six SARS convalescent patients. A single chain variable fragment antibody (N18) with high affinity against N protein of SARS-CoV was isolated. Sequence analysis revealed that the VL gene was composed of VL3 h (V lambda subgroup) and JL2 regions and the VH gene was composed of VH1-69 (VH1 subgroup), D2-15, D3-22 and JH6 regions. Soluble N18 antibody was expressed in Escherichia coli HB2151, purified by Ni–NTA affinity chromatography and verified by SDS-PAGE and Western blot. The potential application for early diagnosis was evaluated using N protein capture ELISA in which N18 antibody demonstrated high sensitive activity in detecting N protein of SARS-CoV. Finally, the potential usefulness of the N18 antibody in prophylaxis, vaccine design and therapy of SARS is discussed.

Keywords

SARS-CoV, scFv, Nucleocapsid, Phage display

Utility of the aged BALB/c mouse model to demonstrate prevention and control strategies for Severe Acute Respiratory Syndrome coronavirus (SARS-CoV)

Vaccine
Volume 25, Issue 12, 8 March 2007, Pages 2173-2179

Leatrice N. Vogel, Anjeanette Roberts, Christopher D. Paddock, Gillian L. Genrich, Elaine W. Lamirande, Sagar U. Kapadia, John K. Rose, Sherif R. Zaki, Kanta Subbarao.

Abstract

The causative agent of Severe Acute Respiratory Syndrome (SARS) was identified as a coronavirus (CoV) following the outbreak of 2002–2003. There are currently no licensed vaccines or treatments for SARS-CoV infections. Potential prevention and control strategies that show promise in vitro must be evaluated in animal models. The aged BALB/c mouse model for SARS supports a high level of viral replication in association with clinical illness and disease that mimics SARS in the elderly. We tested two preventive strategies, vaccination and passive transfer of serum antibody, to determine the extent of protection achieved against SARS-CoV challenge in this model. These approaches were able to achieve or induce antibody titers sufficient to reduce viral load, protect from weight loss and reduce or eliminate histopathologic changes in the lungs of aged mice. This study validates the utility of the aged BALB/c mouse model for evaluation of the efficacy of vaccines and immunoprophylaxis.

Keywords

SARS-CoV, Aged mouse model, Prophylaxis

Antibodies against trimeric S glycoprotein protect hamsters against SARS-CoV challenge despite their capacity to mediate FcγRII-dependent entry into B cells in vitro

Vaccine
Volume 25, Issue 4, 8 January 2007, Pages 729-740

Yiu Wing Kam, François Kien, Anjeanette Roberts, Yan Chung Cheung, Elaine W. Lamirande, Leatrice Vogel, Shui Ling Chu, Jane Tse, Jeannette Guarner, Sherif R. Zaki, Kanta Subbarao, Malik Peiris, Béatrice Nal, Ralf Altmeyere.

Abstract

Vaccine-induced antibodies can prevent or, in the case of feline infectious peritonitis virus, aggravate infections by coronaviruses. We investigated whether a recombinant native full-length S-protein trimer (triSpike) of severe acute respiratory syndrome coronavirus (SARS-CoV) was able to elicit a neutralizing and protective immune response in animals and analyzed the capacity of anti-S antibodies to mediate antibody-dependent enhancement (ADE) of virus entry in vitro and enhancement of replication in vivo. SARS-CoV-specific serum and mucosal immunoglobulins were readily detected in immunized animals. Serum IgG blocked binding of the S-protein to the ACE2 receptor and neutralized SARS-CoV infection in vitro. Entry into human B cell lines occurred in a FcγRII-dependent and ACE2-independent fashion indicating that ADE of virus entry is a novel cell entry mechanism of SARS-CoV. Vaccinated animals showed no signs of enhanced lung pathology or hepatitis and viral load was undetectable or greatly reduced in lungs following challenge with SARS-CoV. Altogether our results indicate that a recombinant trimeric S protein was able to elicit an efficacious protective immune response in vivo and warrant concern in the safety evaluation of a human vaccine against SARS-CoV.

Keywords

SARS-CoV, Protection, ADE