Inhibitors of SARS-CoV entry – Identification using an internally-controlled dual envelope pseudovirion assay

Antiviral Research
Volume 92, Issue 2, November 2011, Pages 187-194

Yanchen Zhou, Juliet Agudelo, KaiLu, David H.Goetz Elizabeth Hansell, Yen Ting Chen William R.Roush, James McKerrow, Charles S.Craik, Sean M.Amberg, Graham Simmons

Abstract

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, dual envelope pseudovirion (DEP) assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes.

Keywords

Inhibitors of SARS-CoV entry, Antiviral, HTS, High-throughput screening, Dual envelope pseudovirion assay, Pseudovirus

SARS-CoV S glycoprotein and influenza matrix 1 efficiently form virus-like particles (VLPs) that protect mice against challenge with SARS-CoV

Vaccine
Volume 29, Issue 38, 2 September 2011, Pages 6606-6613

Ye V. Liu, Michael J. Massare, Dale L.Barnard, Thomas Kort, Margret Nathana, Lei Wang, Gale Smith

Abstract

SARS-CoV was the cause of the global pandemic in 2003 that infected over 8000 people in 8 months. Vaccines against SARS are still not available. We developed a novel method to produce high levels of a recombinant SARS virus-like particles (VLPs) vaccine containing the SARS spike (S) protein and the influenza M1 protein using the baculovirus insect cell expression system. These chimeric SARS VLPs have a similar size and morphology to the wild type SARS-CoV. We tested the immunogenicity and protective efficacy of purified chimeric SARS VLPs and full length SARS S protein vaccines in a mouse lethal challenge model. The SARS VLP vaccine, containing 0.8 μg of SARS S protein, completely protected mice from death when administered intramuscular (IM) or intranasal (IN) routes in the absence of an adjuvant. Likewise, the SARS VLP vaccine, containing 4 μg of S protein without adjuvant, reduced lung virus titer to below detectable level, protected mice from weight loss, and elicited a high level of neutralizing antibodies against SARS-CoV. Sf9 cell-produced full length purified SARS S protein was also an effective vaccine against SARS-CoV but only when co-administered IM with aluminum hydroxide. SARS-CoV VLPs are highly immunogenic and induce neutralizing antibodies and provide protection against lethal challenge. Sf9 cell-based VLP vaccines are a potential tool to provide protection against novel pandemic agents.

Keywords

Severe acute respiratory syndrome, Virus like particles, Lung virus titer, Neutralizing antibody, Baculovirus, Influenza

The immune responses of HLA-A*0201 restricted SARS-CoV S peptide-specific CD8+ T cells are augmented in varying degrees by CpG ODN, PolyI:C and R848

Vaccine
Volume 29, Issue 38, 2 September 2011, Pages 6670-6678

Kai Zhao, Hui Wang Chang youWu

Abstract

The induction of antigen specific memory CD8+ T cells in vivo is very important to new vaccines against infectious diseases. In the present study, we aimed to evaluate the immune responses of peptide-specific CD8+ T cells induced by HLA-A*0201 restricted severe acute respiratory syndrome-associated coronavirus (SARS-CoV) S epitopes plus CpG oligodeoxynucleotide (CpG ODN), PolyI:C and R848 as adjuvants. Furthermore, the generation, distribution and phenotype of long-lasting peptide-specific memory CD8+ T cells were assessed by ELISA, ELISPOT and flow cytometry. Our results showed that antigen specific CD8+ T cells were elicited by HLA-A*0201 restricted SARS-CoV S epitopes. Furthermore, the frequency of peptide-specific CD8+ T cells was dramatically increased after both prime and boost immunization with peptides plus CpG ODN, whereas slight enhancements were induced following boost vaccination with peptides plus PolyI:C or R848. SARS-CoV S peptide-specific IFN-γ+CD8+ T cells were distributed throughout the lymphoid and non-lymphoid tissues. Results also demonstrated that the HLA-A*0201 restricted peptide-specific CD8+ T cells induced by peptides plus CpG ODN carried a memory cell phenotype with CD45RB+ and CD62L− and possessed long-term survival ability in vivo. Taken together, our results implied that HLA-A*0201 restricted SARS-CoV S epitopes plus CpG ODN might be the superior candidates for SARS vaccine.

Keywords

SARS-CoV, Epitope, Adjuvant, T cell, Vaccine

Virucidal activity of a scorpion venom peptide variant mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses

Peptides
Volume 32, Issue 7, July 2011, Pages 1518-1525

QiaoliLi, Zhenhuan Zhao Dihan Zhou Yaoqing Chenab WeiHong LuyangCao JingyiYang YanZhang Wei Shi ZhijianCao, YingliangWu HuiminYana WenxinLi

Abstract

Outbreaks of SARS-CoV, influenza A (H5N1, H1N1) and measles viruses in recent years have raised serious concerns about the measures available to control emerging and re-emerging infectious viral diseases. Effective antiviral agents are lacking that specifically target RNA viruses such as measles, SARS-CoV and influenza H5N1 viruses, and available vaccinations have demonstrated variable efficacy. Therefore, the development of novel antiviral agents is needed to close the vaccination gap and silence outbreaks. We previously indentified mucroporin, a cationic host defense peptide from scorpion venom, which can effectively inhibit standard bacteria. The optimized mucroporin-M1 can inhibit gram-positive bacteria at low concentrations and antibiotic-resistant pathogens. In this investigation, we further tested mucroporin and the optimized mucroporin-M1 for their antiviral activity. Surprisingly, we found that the antiviral activities of mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses were notably increased with an EC50 of 7.15 μg/ml (3.52 μM) and a CC50 of 70.46 μg/ml (34.70 μM) against measles virus, an EC50 of 14.46 μg/ml (7.12 μM) against SARS-CoV and an EC50 of 2.10 μg/ml (1.03 μM) against H5N1, while the original peptide mucroporin showed no antiviral activity against any of these three viruses. The inhibition model could be via a direct interaction with the virus envelope, thereby decreasing the infectivity of virus. This report provides evidence that host defense peptides from scorpion venom can be modified for antiviral activity by rational design and represents a practical approach for developing broad-spectrum antiviral agents, especially against RNA viruses.

Keywords

Mucroporin-M1, Scorpion venom, Measles, SARS-CoV, H5N1, Antiviral

Inhibition of severe acute respiratory syndrome coronavirus replication in a lethalSARS-CoV BALB/c mouse model by stinging nettle lectin,Urtica dioicaagglutinin

Antiviral Research
Volume 90, Issue 1, April 2011, Pages 22-32

Yohichi Kumaki, Miles K.Wandersee, Aaron J.Smith, Yanchen Zhou, Graham Simmons, Nathan M.Nelson, Kevin W.Bailey, Zachary G. Vest, Joseph K.-K.Li, Paul Kay-SheungChan, Donald F. Smee, Dale L.Barnarda

Abstract

Urtica dioicaagglutinin (UDA) is a small plant monomeric lectin, 8.7kDa in size, with anN-acetylglucosamine specificity that inhibits viruses fromNidovirales in vitro. In the current study, we firstexamined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhib-ited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at1.1±0.4g/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/cmouse model. BALB/c mice were infected with two LD50(575PFU) of virus for 4h before the mice weretreated intraperitoneally with UDA at 20, 10, 5 or 0mg/kg/day for 4 days. Treatment with UDA at 5mg/kgsignificantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p<0.001), butdid not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were alsosignificantlyprotectedagainstweightloss(p<0.001).UDAalsoeffectivelyreducedlungpathologyscores.At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than didthe placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to asubstantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode ofaction of UDAin vitrowas further investigated using live SARS-CoV Urbani strain virus and retroviral par-ticles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV orSARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested thatUDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorp-tion or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumablyby binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for the inhibition of virusreplication was partially characterized. When UDA was exposed toN-acetylglucosamine and then UDAwas added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data supportthe conclusion that UDA might bind toN-acetylglucosamine-like residues present on the glycosylatedenvelope glycoproteins, thereby preventing virus attachment to cells.

Keywords:

BALB/c mouse, SARS-CoVUrtica, dioicaagglutinin (UDA)