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author:("Jiang, shiba")
1.  Optimization of antiviral potency and lipophilicity of halogenated 2,6-diarylpyridinamines (DAPAs) as a novel class of HIV-1 NNRTIs 
ChemMedChem  2014;9(7):1546-1555.
Nineteen new halogenated diarylpyridinamine (DAPA) analogues (6a-n and 8a-e) modified on the phenoxy C-ring were synthesized and evaluated for anti-HIV activity and certain drug-like properties. Ten compounds showed high anti-HIV activity (EC50 < 10 nM). Particularly, (E)-6-(2”-bromo-4”-cyanovinyl-6“-methoxy)phenoxy-N2-(4′-cyanophenyl)pyridin-2,3-diamine (8c) displayed low nanomolar antiviral potency (3–7 nM) against wild-type and resistant viral strains with E138K or K101E mutation, associated with resistance to rilvipirine (1b). Compound 8c exhibited much lower resistance fold changes (RFC 1.1–2.1) than 1b (RFC 11.8–13.0). Compound 8c also exhibited better metabolic stability (in vitro half-life) than 1b in human liver microsomes (HLM), possessed low lipophilicity (clog D: 3.29; measured log P: 3.31), and had desirable lipophilic efficiency indices (LE > 0.3, LLE >5, LELP <10). With balanced potency and drug-like properties, 8c merits further development as an anti-HIV drug candidate.
doi:10.1002/cmdc.201400075
PMCID: PMC4085996  PMID: 24895029
anti-HIV activity; antiviral agents; diarylpyridinamine; drug-like properties; lipophiliicity
3.  Current advancements and potential strategies in the development of MERS-CoV vaccines 
Expert review of vaccines  2014;13(6):761-774.
Middle East respiratory syndrome (MERS) is a newly emerging infectious disease caused by a novel coronavirus, MERS-coronavirus (MERS-CoV), a new member in the lineage C of β-coronavirus (β-CoV). The increased human cases and high mortality rate of MERS-CoV infection make it essential to develop safe and effective vaccines. In this review, the current advancements and potential strategies in the development of MERS vaccines, particularly subunit vaccines based on MERS-CoV spike (S) protein and its receptor-binding domain (RBD), are discussed. How to improve the efficacy of subunit vaccines through novel adjuvant formulations and routes of administration as well as currently available animal models for evaluating the in vivo efficacy of MERS-CoV vaccines are also addressed. Overall, these strategies may have important implications for the development of effective and safe vaccines for MERS-CoV in the future.
doi:10.1586/14760584.2014.912134
PMCID: PMC4241375  PMID: 24766432
adjuvants; administration routes; coronavirus; MERS-CoV; Middle East respiratory syndrome; receptor-binding domain; spike protein; subunit vaccines; vaccines
4.  HIV enhancing activity of semen impairs the antiviral efficacy of microbicides 
Science translational medicine  2014;6(262):262ra157.
Topically applied microbicides potently inhibit HIV in vitro but have largely failed to exert protective effects in clinical trials. One possible reason for this discrepancy is that the preclinical testing of microbicides does not faithfully reflect the conditions of HIV sexual transmission. Here, we report that candidate microbicides that target HIV components show greatly reduced antiviral efficacy in the presence of semen, the main vector for HIV transmission. This diminished antiviral activity was dependent on the ability of amyloid fibrils in semen to enhance the infectivity of HIV. Thus, the anti-HIV efficacy of microbicides determined in the absence of semen greatly underestimated the drug concentrations needed to block semen-exposed virus. One notable exception was Maraviroc. This HIV entry inhibitor targets the host cell CCR5 coreceptor and was highly active against both untreated and semen-exposed HIV. These data help explain why microbicides have failed to protect against HIV in clinical trials and suggest that antiviral compounds targeting host factors hold promise for further development. These findings also suggest that the in vitro efficacy of candidate microbicides should be determined in the presence of semen to identify the best candidates for the prevention of HIV sexual transmission.
doi:10.1126/scitranslmed.3009634
PMCID: PMC4372245  PMID: 25391483
5.  Intranasal vaccination with recombinant receptor-binding domain of MERS-CoV spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: Implication for designing novel mucosal MERS vaccines 
Vaccine  2014;32(18):2100-2108.
Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) was originally identified in Saudi Arabia in 2012. It has caused MERS outbreaks with high mortality in the Middle East and Europe, raising a serious concern about its pandemic potential. Therefore, development of effective vaccines is crucial for preventing its further spread and future pandemic. Our previous study has shown that subcutaneous (s.c.) vaccination of a recombinant protein containing receptor-binding domain (RBD) of MERS-CoV S fused with Fc of human IgG (RBD-Fc) induced strong systemic neutralizing antibody responses in vaccinated mice. Here, we compared local and systemic immune responses induced by RBD-Fc via intranasal (i.n.) and s.c. immunization pathways. We found that i.n. vaccination of MERS-CoV RBD-Fc induced systemic humoral immune responses comparable to those induced by s.c. vaccination, including neutralizing antibodies, but more robust systemic cellular immune responses and significantly higher local mucosal immune responses in mouse lungs. This study suggests the potential of developing MERS-CoV RBD protein into an effective and safe mucosal candidate vaccine for prevention of respiratory tract infections caused by MERS-CoV.
doi:10.1016/j.vaccine.2014.02.004
PMCID: PMC4194189  PMID: 24560617
MERS-CoV; Spike protein; Receptor-binding domain; Mucosal immune response; Systemic immune response; Neutralizing antibody
7.  Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate 
Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318–510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318–536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development.
doi:10.4161/hv.27464
PMCID: PMC4130269  PMID: 24355931
SARS-CoV; receptor-binding domain; vaccine; deglycosylation; yeast expression
8.  Intranasal Administration of Maleic Anhydride-Modified Human Serum Albumin for Pre-Exposure Prophylaxis of Respiratory Syncytial Virus Infection 
Viruses  2015;7(2):798-819.
Respiratory syncytial virus (RSV) is the leading cause of pediatric viral respiratory tract infections. Neither vaccine nor effective antiviral therapy is available to prevent and treat RSV infection. Palivizumab, a humanized monoclonal antibody, is the only product approved to prevent serious RSV infection, but its high cost is prohibitive in low-income countries. Here, we aimed to identify an effective, safe, and affordable antiviral agent for pre-exposure prophylaxis (PrEP) of RSV infection in children at high risk. We found that maleic anhydride (ML)-modified human serum albumin (HSA), designated ML-HSA, exhibited potent antiviral activity against RSV and that the percentages of the modified lysines and arginies in ML- are correlated with such anti-RSV activity. ML-HSA inhibited RSV entry and replication by interacting with viral G protein and blocking RSV attachment to the target cells, while ML-HAS neither bound to F protein, nor inhibited F protein-mediated membrane fusion. Intranasal administration of ML-HSA before RSV infection resulted in significant decrease of the viral titers in the lungs of mice. ML-HSA shows promise for further development into an effective, safe, affordable, and easy-to-use intranasal regimen for pre-exposure prophylaxis of RSV infection in children at high risk in both low- and high-income countries.
doi:10.3390/v7020798
PMCID: PMC4353917  PMID: 25690799
human serum albumin (HSA); pre-exposure prophylaxis (PrEP); respiratory syncytial virus (RSV); entry inhibitor; antiviral
9.  Receptor Binding Domain Based HIV Vaccines 
BioMed Research International  2015;2015:594109.
This paper analyzes the main trend of the development of acquired immunodeficiency syndrome (AIDS) vaccines in recent years. Designing an HIV-1 vaccine that provides robust protection from HIV-1 infection remains a challenge despite many years of effort. Therefore, we describe the receptor binding domain of gp120 as a target for developing AIDS vaccines. And we recommend some measures that could induce efficiently and produce cross-reactive neutralizing antibodies with high binding affinity. Those measures may offer a new way of the research and development of the potent and broad AIDS vaccines.
doi:10.1155/2015/594109
PMCID: PMC4312573  PMID: 25667925
10.  Exceptionally Potent Neutralization of Middle East Respiratory Syndrome Coronavirus by Human Monoclonal Antibodies 
Journal of Virology  2014;88(14):7796-7805.
ABSTRACT
The recently discovered Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans, with high mortality. Specific, highly effective therapeutics and vaccines against the MERS-CoV are urgently needed to save human lives and address the pandemic concerns. We identified three human monoclonal antibodies (MAbs), m336, m337, and m338, targeting the receptor (CD26/DPP4) binding domain (RBD) of the MERS-CoV spike glycoprotein from a very large naïve-antibody library (containing ∼1011 antibodies). They bound with high affinity: equilibrium dissociation constants for the three MAbs were equal to 4.2, 9.3, and 15 nM, respectively, as measured by Biacore for Fabs binding to RBD. The avidity for IgG1 m336, m337, and m338 was even higher: 99, 820, and 560 pM, respectively. The antibodies bound to overlapping epitopes that overlap the receptor binding site on the RBD as suggested by competition experiments and further supported by site-directed mutagenesis of the RBD and a docking model of the m336-RBD complex. The highest-affinity MAb, m336, neutralized both pseudotyped and live MERS-CoV with exceptional potency, 50% neutralization at 0.005 and 0.07 μg/ml, respectively, likely by competing with DPP4 for binding to the S glycoprotein. The exceptionally high neutralization activity of these antibodies and especially m336 suggests that they have great potential for prophylaxis and therapy of MERS-CoV infection in humans and as a tool for development of vaccine immunogens. The rapid identification (within several weeks) of potent MAbs suggests a possibility to use the new large antibody library and related methodology for a quick response to the public threat resulting from emerging coronaviruses.
IMPORTANCE A novel human coronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV), was found to infect humans with a high mortality rate in 2012, just 1 decade after the appearance of the first highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV). There are no effective therapeutics available. It is highly desirable to find an approach for rapidly developing potent therapeutics against MERS-CoV, which not only can be implemented for MERS treatment but also can help to develop a platform strategy to combat future emerging coronaviruses. We report here the identification of human monoclonal antibodies (MAbs) from a large nonimmune antibody library that target MERS-CoV. One of the antibodies, m336, neutralized the virus with exceptional potency. It therefore may have great potential as a candidate therapeutic and as a reagent to facilitate the development of vaccines against MERS-CoV.
doi:10.1128/JVI.00912-14
PMCID: PMC4097770  PMID: 24789777
11.  A Conformation-Dependent Neutralizing Monoclonal Antibody Specifically Targeting Receptor-Binding Domain in Middle East Respiratory Syndrome Coronavirus Spike Protein 
Journal of Virology  2014;88(12):7045-7053.
ABSTRACT
Prophylactic and therapeutic strategies are urgently needed to combat infections caused by the newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have developed a neutralizing monoclonal antibody (MAb), designated Mersmab1, which potently blocks MERS-CoV entry into human cells. Biochemical assays reveal that Mersmab1 specifically binds to the receptor-binding domain (RBD) of the MERS-CoV spike protein and thereby competitively blocks the binding of the RBD to its cellular receptor, dipeptidyl peptidase 4 (DPP4). Furthermore, alanine scanning of the RBD has identified several residues at the DPP4-binding surface that serve as neutralizing epitopes for Mersmab1. These results suggest that if humanized, Mersmab1 could potentially function as a therapeutic antibody for treating and preventing MERS-CoV infections. Additionally, Mersmab1 may facilitate studies of the conformation and antigenicity of MERS-CoV RBD and thus will guide rational design of MERS-CoV subunit vaccines.
IMPORTANCE MERS-CoV is spreading in the human population and causing severe respiratory diseases with over 40% fatality. No vaccine is currently available to prevent MERS-CoV infections. Here, we have produced a neutralizing monoclonal antibody with the capacity to effectively block MERS-CoV entry into permissive human cells. If humanized, this antibody may be used as a prophylactic and therapeutic agent against MERS-CoV infections. Specifically, when given to a person (e.g., a patient's family member or a health care worker) either before or after exposure to MERS-CoV, the humanized antibody may prevent or inhibit MERS-CoV infection, thereby stopping the spread of MERS-CoV in humans. This antibody can also serve as a useful tool to guide the design of effective MERS-CoV vaccines.
doi:10.1128/JVI.00433-14
PMCID: PMC4054355  PMID: 24719424
12.  3-Hydroxyphthalic Anhydride-Modified Human Serum Albumin as a Microbicide Candidate Against HIV Type 1 Entry by Targeting Both Viral Envelope Glycoprotein gp120 and Cellular Receptor CD4 
AIDS Research and Human Retroviruses  2013;29(11):1455-1464.
Abstract
We previously reported that 3-hydroxyphthalic anhydride-modified human serum albumin (HP-HSA) as an anti-HIV microbicide could potently inhibit infection by a broad spectrum of HIV-1 strains; however, its mechanism of action is still elusive. Here, we aimed to identify the target(s) of HP-HSA. HIV-1 envelope glycoprotein (Env)-mediated cell–cell fusion assays were conducted using noninfectious CHO-WT cells or infectious HIV-1IIIB-infected H9 cells as effector cells and MT-2 as target cells. The cell-to-cell transmission and single-round HIV-1 infection assays were performed by measuring luciferase activity. Binding of HP-HSA to CD4 or gp120 was determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, while binding of HP-HSA to the coreceptor CXCR4 or CCR5 was detected by cell-based ELISA. HP-HSA strongly inhibited HIV-1 Env-mediated cell–cell fusion and blocked infection by HIV-1 pseudoviruses bearing Env of HIV-1HXB2 (X4 strain) or HIV-1SF162 (R5 strain). HP-HSA was also effective in blocking HIV-1BaL transmission from infected to uninfected cells. HP-HSA could strongly bind to HIV-1 Env gp120 and cellular receptor CD4. These results suggest that HP-HSA inhibits HIV-1 entry into the target cell by interacting with viral Env gp120 and/or the cellular CD4 receptor, making it a promising microbicide candidate for preventing HIV-1 sexual transmission.
doi:10.1089/aid.2013.0043
PMCID: PMC3809378  PMID: 23711095
13.  Highly Pathogenic Avian Influenza A(H5N1) Mutants Transmissible by Air Are Susceptible to Human and Animal Neutralizing Antibodies 
The Journal of Infectious Diseases  2013;208(8):1315-1319.
A laboratory-generated reassortant H5 hemagglutinin (HA)/influenza A(H1N1) strain containing 4 mutations in influenza A(H5N1) HA has become transmissible by air among mammals. Here, we constructed 15 influenza A(H5N1) pseudoviruses containing a single mutation or a combination of mutations and showed that the pseudoviruses were susceptible to neutralizing antibodies from patients with influenza A(H5N1) infection and from mice immunized with a vaccine containing the conserved HA1 sequence of influenza A(H5N1). These results indicate that antibodies in patients currently infected by influenza A(H5N1) and antibodies induced by vaccines containing conserved sequences in HA1 of wild-type influenza A(H5N1) are highly effective in cross-neutralizing future influenza A(H5N1) mutants with airborne transmissibility, suggesting that human influenza pandemics caused by these influenza A(H5N1) variants can be prevented.
doi:10.1093/infdis/jit323
PMCID: PMC3778968  PMID: 23868877
Patient serum specimens; Neutralizing antibodies; H5N1 influenza virus; Air-transmissibility
14.  Studying the Nucleated Mammalian Cell Membrane by Single Molecule Approaches 
PLoS ONE  2014;9(5):e91595.
The cell membrane plays a key role in compartmentalization, nutrient transportation and signal transduction, while the pattern of protein distribution at both cytoplasmic and ectoplasmic sides of the cell membrane remains elusive. Using a combination of single-molecule techniques, including atomic force microscopy (AFM), single molecule force spectroscopy (SMFS) and stochastic optical reconstruction microscopy (STORM), to study the structure of nucleated cell membranes, we found that (1) proteins at the ectoplasmic side of the cell membrane form a dense protein layer (4 nm) on top of a lipid bilayer; (2) proteins aggregate to form islands evenly dispersed at the cytoplasmic side of the cell membrane with a height of about 10–12 nm; (3) cholesterol-enriched domains exist within the cell membrane; (4) carbohydrates stay in microdomains at the ectoplasmic side; and (5) exposed amino groups are asymmetrically distributed on both sides. Based on these observations, we proposed a Protein Layer-Lipid-Protein Island (PLLPI) model, to provide a better understanding of cell membrane structure, membrane trafficking and viral fusion mechanisms.
doi:10.1371/journal.pone.0091595
PMCID: PMC4012985  PMID: 24806512
16.  Identification of the Critical Sites of NNRTI-Resistance in Reverse Transcriptase of HIV-1 CRF_BC Strains 
PLoS ONE  2014;9(4):e93804.
Background
The polymorphisms involved in drug resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) in HIV-1 CRF_BC, the most prevalent HIV-1 strain in China, have been poorly characterized.
Results
To reveal the drug resistance mutations, we compared the gene sequences of pol region of HIV-1 CRF_BC from 631 treatment-naïve and 363 treatment-experienced patients using the selection pressure-based method. We calculated an individual Ka/Ks value for each specific amino acid mutation. Result showed that eight polymorphic mutations (W88C, K101Q, I132L, R135L, T139K/R, H221Y and L228R) in RT for treatment-experienced patients were identified, while they, except for R135L, were completely absent in those from treatment-naïve patients. The I132L and T139K/R mutants exhibited high-level resistance to DLV and NVP and moderate resistance to TMC-125 and EFV, while the K101Q and H221Y mutants exhibited an increased resistance to all four NNRTIs tested. The W88C, R135L, and L228R may be RTI-induced adaptive mutations. Y181C+K101Q mutant showed a 2.5-, 4.4-, and 4.7-fold higher resistance to TMC-125, NVP and EFV, respectively, than Y181C alone mutant, while Y181C+H221Y or K103N+H221Y mutants had significantly higher resistance to all four NNRTIs than Y181C or K103N mutants. K103N+T139K and G190A+T139K mutant induce higher resistance (2.0∼14.2-fold and 1.5∼7.2-fold, respectively) to all four NNRTIs than K103N or G190A alone mutation.
Conclusions
I132L and T139K/R are rare but critical mutations associated with NNRTI-resistance for some NNRTIs. K101Q, H221Y and T139K can enhance K103N/Y181C/G190A-assocated NNRTI-resistance. Monitoring these mutations will provide useful information for rational design of the NNRTI-based antiretroviral regimen for HIV-1 CRF_BC-infected patients.
doi:10.1371/journal.pone.0093804
PMCID: PMC3990534  PMID: 24743727
17.  Modulation of HBV replication by microRNA-15b through targeting hepatocyte nuclear factor 1α 
Nucleic Acids Research  2014;42(10):6578-6590.
Hepatitis B virus (HBV) infection remains a major health problem worldwide. The role played by microRNAs (miRNAs) in HBV replication and pathogenesis is being increasingly recognized. In this study, we found that miR-15b, an important miRNA during HBV infection and hepatocellular carcinoma development, directly binds hepatocyte nuclear factor 1α (HNF1α) mRNA, a negative regulator of HBV Enhancer I, to attenuate HNF1α expression, resulting in transactivation of HBV Enhancer I, in turn causing the enhancement of HBV replication and expression of HBV antigens, including HBx protein, finally leading to the down-regulated expression of miR-15b in both cell lines and mice in a long cascade of events. Our research showed that miR-15b promotes HBV replication by augmenting HBV Enhancer I activity via direct targeting HNF1α, while HBV replication and antigens expression, particularly the HBx protein, then repress the expression of miR-15b. The reciprocal regulation between miR-15b and HBV controls the level of HBV replication and might play a role in persistent HBV infection. This work adds to the body of knowledge concerning the complex interactions between HBV and host miRNAs.
doi:10.1093/nar/gku260
PMCID: PMC4041434  PMID: 24705650
18.  Differences in the Pathogenicity and Inflammatory Responses Induced by Avian Influenza A/H7N9 Virus Infection in BALB/c and C57BL/6 Mouse Models 
PLoS ONE  2014;9(3):e92987.
Avian influenza A/H7N9 virus infection causes pneumonia in humans with a high case fatality rate. However, virus-induced modulation of immune responses is being recognized increasingly as a factor in the pathogenesis of this disease. In this study, we compared the pathogenicity of A/H7N9 infection in BALB/c and C57BL/6 mouse models, and investigated the putative involvement of proinflammatory cytokines in lung injury and viral clearance. In both mouse strains, A/Anhui/1/2013(H7N9) infection with 106 TCID50 resulted in viral replication in lung, severe body weight loss and acute lung injury. During the early infection stage, infected C57BL/6 mice exhibited more severe lung injury, slower recovery from lung damage, less effective viral clearance, higher levels of interlukine (IL)-6, monocyte chemotactic protein (MCP)-1, and IL-1β, and lower levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ than infected BALB/c mice. These results suggest that TNF-α and IFN-γ may help suppress viral gene expression and increase viral clearance, and that IL-6 and MCP-1 may contribute to lung injury in A/H7N9-infected individuals. In addition, lung damage and the distribution of virus antigen in tissues were similar in young and middle-aged mice. These results suggest that the more serious lung injury in middle-aged or older H7N9 cases is not mainly caused by differences in viral replication in the lung but probably by a dysregulated immune response induced by underlying comorbidities. These results indicate that the extent of dysregulation of the host immune response after H7N9 virus infection most probably determines the outcome of H7N9 virus infection.
doi:10.1371/journal.pone.0092987
PMCID: PMC3968029  PMID: 24676272
19.  Identification of a Receptor-Binding Domain in the S Protein of the Novel Human Coronavirus Middle East Respiratory Syndrome Coronavirus as an Essential Target for Vaccine Development 
Journal of Virology  2013;87(17):9939-9942.
A novel human Middle East respiratory syndrome coronavirus (MERS-CoV) caused outbreaks of severe acute respiratory syndrome (SARS)-like illness with a high mortality rate, raising concerns of its pandemic potential. Dipeptidyl peptidase-4 (DPP4) was recently identified as its receptor. Here we showed that residues 377 to 662 in the S protein of MERS-CoV specifically bound to DPP4-expressing cells and soluble DPP4 protein and induced significant neutralizing antibody responses, suggesting that this region contains the receptor-binding domain (RBD), which has a potential to be developed as a MERS-CoV vaccine.
doi:10.1128/JVI.01048-13
PMCID: PMC3754113  PMID: 23824801
20.  Correction: HIV-1 Fusion Is Blocked through Binding of GB Virus C E2D Peptides to the HIV-1 gp41 Disulfide Loop 
PLoS ONE  2014;9(1):10.1371/annotation/b46459e1-73ff-4b75-8f24-264cbcf4a5a9.
doi:10.1371/annotation/b46459e1-73ff-4b75-8f24-264cbcf4a5a9
PMCID: PMC3891527
21.  A Truncated Receptor-Binding Domain of MERS-CoV Spike Protein Potently Inhibits MERS-CoV Infection and Induces Strong Neutralizing Antibody Responses: Implication for Developing Therapeutics and Vaccines 
PLoS ONE  2013;8(12):e81587.
An emerging respiratory infectious disease with high mortality, Middle East respiratory syndrome (MERS), is caused by a novel coronavirus (MERS-CoV). It was first reported in 2012 in Saudi Arabia and has now spread to eight countries. Development of effective therapeutics and vaccines is crucial to save lives and halt the spread of MERS-CoV. Here, we show that a recombinant protein containing a 212-amino acid fragment (residues 377-588) in the truncated receptor-binding domain (RBD: residues 367–606) of MERS-CoV spike (S) protein fused with human IgG Fc fragment (S377-588-Fc) is highly expressed in the culture supernatant of transfected 293T cells. The purified S377-588-Fc protein efficiently binds to dipeptidyl peptidase 4 (DPP4), the receptor of MERS-CoV, and potently inhibited MERS-CoV infection, suggesting its potential to be further developed as a therapeutic modality for treating MERS-CoV infection and saving the patients’ lives. The recombinant S377-588-Fc is able to induce in the vaccinated mice strong MERS-CoV S-specific antibodies, which blocks the binding of RBD to DPP4 receptor and effectively neutralizes MERS-CoV infection. These findings indicate that this truncated RBD protein shows promise for further development as an effective and safe vaccine for the prevention of MERS-CoV infection.
doi:10.1371/journal.pone.0081587
PMCID: PMC3852489  PMID: 24324708
22.  Receptor binding and transmission studies of H5N1 influenza virus in mammals 
The H5N1 influenza A virus that is currently circulating in Asia, Africa and Europe has resulted in persistent outbreaks in poultry with sporadic transmission to humans. Thus far, it is believed that H5N1 does not possess sufficient ability for human-to-human transmission and subsequent pandemic infection. Both receptor binding specificity and virus infectivity are key factors in determining whether influenza A virus becomes pandemic. The use of human viral isolates in various studies has helped to illustrate the changes in receptor binding specificity and virulence as a result of adaptation in humans. In this review, we highlight the important amino acids and domains of viral proteins related to receptor binding specificity that have been reported for humans and avians using mammalian models. Thus, this review will consolidate findings from studies that have shed light on the receptor binding and transmission characteristics of the H5N1 influenza virus, with the goal of improving our ability to predict the transmission efficiency or pandemic potential of new viral strains.
doi:10.1038/emi.2013.89
PMCID: PMC3880874  PMID: 26038448
receptor binding; transmission; H5N1; mammal; influenza A virus
23.  Simultaneous Expression of Displayed and Secreted Antibodies for Antibody Screen 
PLoS ONE  2013;8(11):e80005.
The display of full-length antibody on the cell surface was achieved by fusing a transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the C-terminus of the heavy chain constant region. We also incorporated a furin cleavage site between the constant region and PDGFR transmembrane domain to obtain secreted antibodies. As a result, antibodies can be expressed simultaneously on the cell surface in a membrane-anchored version for screening and selecting through fluorescence-activated cell sorting (FACS) analysis, as well as in conditioned medium in a secreted version for function analysis.
doi:10.1371/journal.pone.0080005
PMCID: PMC3823846  PMID: 24244593
24.  Potential strategies and biosafety protocols used for dual-use research on highly pathogenic influenza viruses 
Reviews in medical virology  2012;22(6):412-419.
Summary
Influenza A viruses (IAVs), particularly the highly pathogenic avian influenza (HPAI) H5N1, have posed a substantial threat to public health worldwide. Although the laboratory generation of the mutant influenza virus H5N1 with airborne transmissibility among mammals, which has been considered as a dual-use research, may benefit the development of effective vaccines and therapeutics against the emerging infectious agents, it may also pose threats to national biosecurity, laboratory biosafety, and/or public health. This review introduces the classification and characterization of IAVs, pinpoints historic pandemics and epidemics caused by IAVs, emphasizes the significance and necessity of biosafety, summarizes currently established biosafety-related protocols for IAV research, and provides potential strategies to improve biosafety protocols for dual-use research on the highly pathogenic avian influenza viruses and other emerging infectious agents.
doi:10.1002/rmv.1729
PMCID: PMC3580078  PMID: 22987727
25.  Roadmap to developing a recombinant coronavirus S protein receptor-binding domain vaccine for severe acute respiratory syndrome 
Expert review of vaccines  2012;11(12):1405-1413.
A subunit vaccine, RBD-S, is under development to prevent severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV), which is classified by the US NIH as a category C pathogen. This vaccine is comprised of a recombinant receptor-binding domain (RBD) of the SARS-CoV spike (S) protein and formulated on alum, together with a synthetic glucopyranosyl lipid A. The vaccine would induce neutralizing antibodies without causing Th2-type immunopathology. Vaccine development is being led by the nonprofit product development partnership; Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development in collaboration with two academic partners (the New York Blood Center and University of Texas Medical Branch); an industrial partner (Immune Design Corporation); and Walter Reed Army Institute of Research. A roadmap for the product development of the RBD-S SARS vaccine is outlined with a goal to manufacture the vaccine for clinical testing within the next 5 years.
doi:10.1586/erv.12.126
PMCID: PMC3586247  PMID: 23252385
receptor-binding domain; recombinant vaccine; SARS; severe acute respiratory syndrome; spike (S) protein

Results 1-25 (101)