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author:("Jiang, shiba")
2.  TRAIL-R1 Is a Negative Regulator of Pro-Inflammatory Responses and Modulates Long-Term Sequelae Resulting from Chlamydia trachomatis Infections in Humans 
PLoS ONE  2014;9(4):e93939.
The immune system eliminates Chlamydia trachomatis infection through inflammation. However, uncontrolled inflammation can enhance pathology. In mice, TNF-related apoptosis-inducing ligand receptor (TRAIL-R), known for its effects on apoptosis, also regulates inflammation. In humans, the four homologues of TRAIL-R had never been investigated for effects on inflammation. Here, we examined whether TRAIL-R regulates inflammation during chlamydial infection. We examined TRAIL-R1 single nucleotide polymorphisms (SNPs) in an Ecuadorian cohort with and without C. trachomatis infections. There was a highly significant association for the TRAIL+626 homozygous mutant GG for infection vs no infection in this population. To confirm the results observed in the human population, primary lung fibroblasts and bone marrow-derived macrophages (BMDMs) were isolated from wildtype (WT) and TRAIL-R-deficient mice, and TRAIL-R1 levels in human cervical epithelial cells were depleted by RNA interference. Infection of BMDMs and primary lung fibroblasts with C. trachomatis strain L2, or the murine pathogen C. muridarum, led to higher levels of MIP2 mRNA expression or IL-1β secretion from TRAIL-R-deficient cells than WT cells. Similarly, depletion of TRAIL-R1 expression in human epithelial cells resulted in a higher level of IL-8 mRNA expression and protein secretion during C. trachomatis infection. We conclude that human TRAIL-R1 SNPs and murine TRAIL-R modulate the innate immune response against chlamydial infection. This is the first evidence that human TRAIL-R1 is a negative regulator of inflammation and plays a role in modulating Chlamydia pathogenesis.
PMCID: PMC3973638  PMID: 24695582
3.  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.
PMCID: PMC3968029  PMID: 24676272
4.  Different Frequencies of Drug Resistance Mutations among HIV-1 Subtypes Circulating in China: A Comprehensive Study 
PLoS ONE  2014;9(3):e91803.
The rapid spreading of HIV drug resistance is threatening the overall success of free HAART in China. Much work has been done on drug-resistant mutations, however, most of which were based on subtype B. Due to different genetic background, subtypes difference would have an effect on the development of drug-resistant mutations, which has already been proved by more and more studies. In China, the main epidemic subtypes are CRF07_BC, CRF08_BC, Thai B and CRF01_AE. The depiction of drug resistance mutations in those subtypes will be helpful for the selection of regimens for Chinese. In this study, the distributions difference of amino acids at sites related to HIV drug resistance were compared among subtype B, CRF01_AE, CRF07_BC and CRF08_BC strains prevalent in China. The amino acid composition of sequences belonging to different subtypes, which were obtained from untreated and treated individuals separately, were also compared. The amino acids proportions of 19 sites in RT among subtype B, CRF01_AE and CRF08_BC have significant difference in drug resistance groups (chi-square test, p<0.05). Genetic barriers analysis revealed that sites 69, 138, 181, 215 and 238 were significantly different among subtypes (Kruskal Wallis test, p<0.05). All subtypes shared three highest prevalent drug resistance sites 103, 181 and 184 in common. Many drug resistant sites in protease show surprising high proportions in almost all subtypes in drug-naïve patients. This is the first comprehensive study in China on different development of drug resistance among different subtypes. The detailed data will lay a foundation for HIV treatment regimens design and improve HIV therapy in China.
PMCID: PMC3963863  PMID: 24663120
5.  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.
PMCID: PMC3754113  PMID: 23824801
6.  Computational Prediction of Neutralization Epitopes Targeted by Human Anti-V3 HIV Monoclonal Antibodies 
PLoS ONE  2014;9(2):e89987.
The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs) can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs). Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design.
PMCID: PMC3934971  PMID: 24587168
7.  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.
PMCID: PMC3891527
8.  A Replicating Modified Vaccinia Tiantan Strain Expressing an Avian-Derived Influenza H5N1 Hemagglutinin Induce Broadly Neutralizing Antibodies and Cross-Clade Protective Immunity in Mice 
PLoS ONE  2013;8(12):e83274.
To combat the possibility of a zoonotic H5N1 pandemic in a timely fashion, it is necessary to develop a vaccine that would confer protection against homologous and heterologous human H5N1 influenza viruses. Using a replicating modified vaccinia virus Tian Tan strain (MVTT) as a vaccine vector, we constructed MVTTHA-QH and MVTTHA-AH, which expresses the H5 gene of a goose-derived Qinghai strain A/Bar-headed Goose/Qinghai/1/2005 or human-derived Anhui Strain A/Anhui/1/2005. The immunogenicity profiles of both vaccine candidates were evaluated. Vaccination with MVTTHA-QH induced a significant level of neutralizing antibodies (Nabs) against a homologous strain and a wide range of H5N1 pseudoviruses (clades 1, 2.1, 2.2, 2.3.2, and 2.3.4). Neutralization tests (NT) and Haemagglutination inhibition (HI) antibodies inhibit the live autologous virus as well as a homologous A/Xingjiang/1/2006 and a heterologous A/Vietnam/1194/2004, representing two human isolates from clade 2.2 and clade 1, respectively. Importantly, mice vaccinated with intranasal MVTTHA-QH were completely protected from challenge with lethal dosages of A/Bar-headed Goose/Qinghai/1/2005 and the A/Viet Nam/1194/2004, respectively, but not control mice that received a mock MVTTS vaccine. However, MVTTHA-AH induced much lower levels of NT against its autologous strain. Our results suggest that it is feasible to use the H5 gene from A/Bar-headed Goose/Qinghai/1/2005 to construct an effective vaccine, when using MVTT as a vector, to prevent infections against homologous and genetically divergent human H5N1 influenza viruses.
PMCID: PMC3866202  PMID: 24358269
9.  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.
PMCID: PMC3852489  PMID: 24324708
10.  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.
PMCID: PMC3880874
receptor binding; transmission; H5N1; mammal; influenza A virus
11.  Construction and Characterization of Highly Infectious Full-Length Molecular Clones of a HIV-1 CRF07_BC Isolate from Xinjiang, China 
PLoS ONE  2013;8(11):e79177.
Among the various subtypes of the M group of human immunodeficiency virus type 1 (HIV-1), clade CRF07_BC is the most prevalent in China. To date, no strong replicable CRF07_BC infectious clone has been constructed. Here we report on the construction and characterization of highly replicable infectious molecular clones from the isolate XJDC6291 of this HIV-1 subtype. Four full-length clones pXJDC2-7, pXJDC3-7, pXJDC2-6 and pXJDC3-6 were successfully produced, but only pXJDC2-7 presented detectable infectivity and replication capability. To improve the replication capability of pXJDC2-7, a 4.8 kb region spanning from the pol Integrase to nef gene of the clone was replaced by PCR products of the corresponding fragments from the original isolate XJDC6291, which produced two clones pXJDC13 and pXJDC17 that exhibited strong replication capability. The viral stocks obtained by pXJDC-13 and pXJDC-17 transfection into 293T cells replicated efficiently in human PBMCs, human primary CD4+ T cells and displayed CCR5 tropism. Sequence alignment between pXJDC13, pXJDC17 and pXJDC2-7 suggested that polymorphisms in the V1V2 region may influence infectivity, and reverse genetic experiment showed that V1V2 polymorphisms may influence the infectivity of the clones but did not affect the replication capability at a significant level. pXJDC13 and pXJDC17 displayed strong replication capability and are the first full-length infectious clones of HIV-1 CRF07_BC clade in the world. The availability of CRF07_BC infectious clones provides a useful tool for a wide range of studies, including antiretroviral drug and vaccine research as related to this HIV subtype.
PMCID: PMC3850540  PMID: 24324545
12.  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.
PMCID: PMC3823846  PMID: 24244593
13.  Adjuvants and Immunization Strategies to Induce Influenza Virus Hemagglutinin Stalk Antibodies 
PLoS ONE  2013;8(11):e79194.
The global population remains vulnerable in the face of the next pandemic influenza virus outbreak, and reformulated vaccinations are administered annually to manage seasonal epidemics. Therefore, development of a new generation of vaccines is needed to generate broad and persistent immunity to influenza viruses. Here, we describe three adjuvants that enhance the induction of stalk-directed antibodies against heterologous and heterosubtypic influenza viruses when administered with chimeric HA proteins. Addavax, an MF59-like nanoemulsion, poly(I:C), and an RNA hairpin derived from Sendai virus (SeV) Cantell were efficacious intramuscularly. The SeV RNA and poly(I:C) also proved to be effective respiratory mucosal adjuvants. Although the quantity and quality of antibodies induced by the adjuvants varied, immunized mice demonstrated comparable levels of protection against challenge with influenza A viruses on the basis of HA stalk reactivity. Finally, we present that intranasally, but not intramuscularly, administered chimeric HA proteins induce mucosal IgA antibodies directed at the HA stalk.
PMCID: PMC3819267  PMID: 24223176
14.  Potential strategies and biosafety protocols used for dual-use research on highly pathogenic influenza viruses 
Reviews in medical virology  2012;22(6):412-419.
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.
PMCID: PMC3580078  PMID: 22987727
15.  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.
PMCID: PMC3586247  PMID: 23252385
receptor-binding domain; recombinant vaccine; SARS; severe acute respiratory syndrome; spike (S) protein
16.  A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV 
Virology Journal  2013;10:266.
Evidence points to the emergence of a novel human coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), which causes a severe acute respiratory syndrome (SARS)-like disease. In response, the development of effective vaccines and therapeutics remains a clinical priority. To accomplish this, it is necessary to evaluate neutralizing antibodies and screen for MERS-CoV entry inhibitors.
In this study, we produced a pseudovirus bearing the full-length spike (S) protein of MERS-CoV in the Env-defective, luciferase-expressing HIV-1 backbone. We then established a pseudovirus-based inhibition assay to detect neutralizing antibodies and anti-MERS-CoV entry inhibitors.
Our results demonstrated that the generated MERS-CoV pseudovirus allows for single-cycle infection of a variety of cells expressing dipeptidyl peptidase-4 (DPP4), the confirmed receptor for MERS-CoV. Consistent with the results from a live MERS-CoV-based inhibition assay, the antisera of mice vaccinated with a recombinant protein containing receptor-binding domain (RBD, residues 377–662) of MERS-CoV S fused with Fc of human IgG exhibited neutralizing antibody response against infection of MERS-CoV pseudovirus. Furthermore, one small molecule HIV entry inhibitor targeting gp41 (ADS-J1) and the 3-hydroxyphthalic anhydride-modified human serum albumin (HP-HSA) could significantly inhibit MERS-CoV pseudovirus infection.
Taken together, the established MERS-CoV inhibition assay is a safe and convenient pseudovirus-based alternative to BSL-3 live-virus restrictions and can be used to rapidly screen MERS-CoV entry inhibitors, as well as evaluate vaccine-induced neutralizing antibodies against the highly pathogenic MERS-CoV.
PMCID: PMC3765664  PMID: 23978242
Novel human coronavirus; MERS-CoV; Spike protein; Pseudovirus; Neutralizing antibodies; Antiviral therapeutics
17.  Identification and Structural Characterization of a Broadly Neutralizing Antibody Targeting a Novel Conserved Epitope on the Influenza Virus H5N1 Hemagglutinin 
Journal of Virology  2013;87(4):2215-2225.
The unabated circulation of the highly pathogenic avian influenza A virus/H5N1 continues to be a serious threat to public health worldwide. Because of the high frequency of naturally occurring mutations, the emergence of H5N1 variants with high virulence has raised great concerns about the potential transmissibility of the virus in humans. Recent studies have shown that laboratory-mutated or reassortant H5N1 viruses could be efficiently transmitted among mammals, particularly ferrets, the best animal model for humans. Thus, it is critical to establish effective strategies to combat future H5N1 pandemics. In this study, we identified a broadly neutralizing monoclonal antibody (MAb), HA-7, that potently neutralized all tested strains of H5N1 covering clades 0, 1, 2.2, 2.3.4, and and completely protected mice against lethal challenges of H5N1 viruses from clades 1 and 2.3.4. HA-7 specifically targeted the globular head of the H5N1 virus hemagglutinin (HA). Using electron microscopy technology with three-dimensional reconstruction (3D-EM), we discovered that HA-7 bound to a novel and highly conserved conformational epitope that was centered on residues 81 to 83 and 117 to 122 of HA1 (H5 numbering). We further demonstrated that HA-7 inhibited viral entry during postattachment events but not at the receptor-binding step, which is fully consistent with the 3D-EM result. Taken together, we propose that HA-7 could be humanized as an effective passive immunotherapeutic agent for antiviral stockpiling for future influenza pandemics caused by emerging unpredictable H5N1 strains. Our study also provides a sound foundation for the rational design of vaccines capable of inducing broad-spectrum immunity against H5N1.
PMCID: PMC3571485  PMID: 23221567
18.  Receptor-binding domain as a target for developing SARS vaccines 
Journal of Thoracic Disease  2013;5(Suppl 2):S142-S148.
A decade ago, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a global pandemic with a mortality rate of 10%. Reports of recent outbreaks of a SARS-like disease caused by Middle East respiratory syndrome coronavirus (MERS-CoV) have raised serious concerns of a possible reemergence of SARS-CoV, either by laboratory escape or the presence of a natural reservoir. Therefore, the development of effective and safe SARS vaccines is still needed. Based on our previous studies, we believe that the receptor-binding domain (RBD) in the S1 subunit of the SARS-CoV spike (S) protein is the most important target for developing a SARS vaccine. In particular, RBD of S protein contains the critical neutralizing domain (CND), which is able to induce highly potent neutralizing antibody response and cross-protection against divergent SARS-CoV strains. Furthermore, a RBD-based subunit vaccine is expected to be safer than other vaccines that may induce Th2-type immunopathology. This review will discuss key advances in the development of RBD-based SARS vaccines and the possibility of using a similar strategy to develop vaccines against MERS-CoV.
PMCID: PMC3747534  PMID: 23977435
Virus; severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV); receptor-binding domain (RBD); spike protein; vaccine
19.  P2X4 Assembles with P2X7 and Pannexin-1 in Gingival Epithelial Cells and Modulates ATP-induced Reactive Oxygen Species Production and Inflammasome Activation 
PLoS ONE  2013;8(7):e70210.
We have previously reported that Porphyromonas gingivalis infection of gingival epithelial cells (GEC) requires an exogenous danger signal such as ATP to activate an inflammasome and caspase-1, thereby inducing secretion of interleukin (IL)-1β. Stimulation with extracellular ATP also stimulates production of reactive oxygen species (ROS) in GEC. However, the mechanism by which ROS is generated in response to ATP, and the role that different purinergic receptors may play in inflammasome activation, is still unclear. In this study, we revealed that the purinergic receptor P2X4 is assembled with the receptor P2X7 and its associated pore, pannexin-1. ATP induces ROS production through a complex consisting of the P2X4, P2X7, and pannexin-1. P2X7−mediated ROS production can activate the NLRP3 inflammasome and caspase-1. Furthermore, separate depletion or inhibition of P2X4, P2X7, or pannexin-1 complex blocks IL-1β secretion in P. gingivalis-infected GEC following ATP treatment. However, activation via P2X4 alone induces ROS generation but not inflammasome activation. These results suggest that ROS is generated through stimulation of a P2X4/P2X7/pannexin-1 complex, and reveal an unexpected role for P2X4, which acts as a positive regulator of inflammasome activation during microbial infection.
PMCID: PMC3723664  PMID: 23936165
20.  Identification of Critical Regions in Human SAMHD1 Required for Nuclear Localization and Vpx-Mediated Degradation 
PLoS ONE  2013;8(7):e66201.
The sterile alpha motif (SAM) and HD domain-containing protein-1 (SAMHD1) inhibits the infection of resting CD4+ T cells and myeloid cells by human and related simian immunodeficiency viruses (HIV and SIV). Vpx inactivates SAMHD1 by promoting its proteasome-dependent degradation through an interaction with CRL4 (DCAF1) E3 ubiquitin ligase and the C-terminal region of SAMHD1. However, the determinants in SAMHD1 that are required for Vpx-mediated degradation have not been well characterized. SAMHD1 contains a classical nuclear localization signal (NLS), and NLS point mutants are cytoplasmic and resistant to Vpx-mediated degradation. Here, we demonstrate that NLS-mutant SAMHD1 K11A can be rescued by wild-type SAMHD1, restoring its nuclear localization; consequently, SAMHD1 K11A became sensitive to Vpx-mediated degradation in the presence of wild-type SAMHD1. Surprisingly, deletion of N-terminal regions of SAMHD1, including the classical NLS, generated mutant SAMHD1 proteins that were again sensitive to Vpx-mediated degradation. Unlike SAMHD1 K11A, these deletion mutants could be detected in the nucleus. Interestingly, NLS-defective SAMHD1 could still bind to karyopherin-β1 and other nuclear proteins. We also determined that the linker region between the SAM and HD domain and the HD domain itself is important for Vpx-mediated degradation but not Vpx interaction. Thus, SAMHD1 contains an additional nuclear targeting mechanism in addition to the classical NLS. Our data indicate that multiple regions in SAMHD1 are critical for Vpx-mediated nuclear degradation and that association with Vpx is not sufficient for Vpx-mediated degradation of SAMHD1. Since the linker region and HD domain may be involved in SAMHD1 multimerization, our results suggest that SAMHD1 multimerization may be required for Vpx-mediation degradation.
PMCID: PMC3708934  PMID: 23874389
21.  Comparative Immunogenicity of Evolved V1V2-Deleted HIV-1 Envelope Glycoprotein Trimers 
PLoS ONE  2013;8(6):e67484.
Despite almost 30 years of research, no effective vaccine has yet been developed against HIV-1. Probably such a vaccine would need to induce both an effective T cell and antibody response. Any vaccine component focused on inducing humoral immunity requires the HIV-1 envelope (Env) glycoprotein complex as it is the only viral protein exposed on the virion surface. HIV-1 has evolved several mechanisms to evade broadly reactive neutralizing antibodies. One such a mechanism involves variable loop domains, which are highly flexible structures that shield the underlying conserved epitopes. We hypothesized that removal of such loops would increase the exposure and immunogenicity of these conserved regions. Env variable loop deletion however often leads to protein misfolding and aggregation because hydrophobic patches becoming solvent accessible. We have therefore previously used virus evolution to acquire functional Env proteins lacking the V1V2 loop. We then expressed them in soluble (uncleaved) gp140 forms. Three mutants were found to perform optimally in terms of protein expression, stability, trimerization and folding. In this study, we characterized the immune responses to these antigens in rabbits. The V1V2 deletion mutant ΔV1V2.9.VK induced a prominent response directed to epitopes that are not fully available on the other Env proteins tested but that effectively bound and neutralized the ΔV1V2 Env virus. This Env variant also induced more efficient neutralization of the tier 1 virus SF162. The immune refocusing effect was lost after booster immunization with a full-length gp140 protein with intact V1V2 loops. Collectively, this result suggests that deletion of variable domains could alter the specificity of the humoral immune response, but did not result in broad neutralization of neutralization-resistant virus isolates.
PMCID: PMC3694020  PMID: 23840716
22.  An HIV-1 Envelope Glycoprotein Trimer with an Embedded IL-21 Domain Activates Human B Cells 
PLoS ONE  2013;8(6):e67309.
Broadly neutralizing antibodies (bNAbs) that target the HIV-1 envelope glycoproteins (Env) can prevent virus acquisition, but several Env properties limit its ability to induce an antibody response that is of sufficient quantity and quality. The immunogenicity of Env can be increased by fusion to co-stimulatory molecules and here we describe novel soluble Env trimers with embedded interleukin-4 (IL-4) or interleukin-21 (IL-21) domains, designed to activate B cells that recognize Env. In particular, the chimeric EnvIL-21 molecule activated B cells efficiently and induced the differentiation of antibody secreting plasmablast-like cells. We studied whether we could increase the activity of the embedded IL-21 by designing a chimeric IL-21/IL-4 (ChimIL-21/4) molecule and by introducing amino acid substitutions in the receptor binding domain of IL-21 that were predicted to enhance its binding. In addition, we incorporated IL-21 into a cleavable Env trimer and found that insertion of IL-21 did not impair Env cleavage, while Env cleavage did not impair IL-21 activity. These studies should guide the further design of chimeric proteins and EnvIL-21 may prove useful in improving antibody responses against HIV-1.
PMCID: PMC3691133  PMID: 23826263
23.  Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases 
PLoS ONE  2013;8(6):e66276.
The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs).
Methodology/Principal Findings
The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA.
Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.
PMCID: PMC3688872  PMID: 23824680
24.  Epitope Mapping of M36, a Human Antibody Domain with Potent and Broad HIV-1 Inhibitory Activity 
PLoS ONE  2013;8(6):e66638.
M36 is the first member of a novel class of potent HIV-1 entry inhibitors based on human engineered antibody domains (eAds). It exhibits broad inhibitory activity suggesting that its CD4-induced epitope is highly conserved. Here, we describe fine mapping of its epitope by using several approaches. First, a panel of mimotopes was affinity-selected from a random peptide library and potential m36-binding residues were computationally predicted. Second, homology modeling of m36 and molecular docking of m36 onto gp120 revealed potentially important residues in gp120-m36 interactions. Third, the predicted contact residues were verified by site-directed mutagenesis. Taken together, m36 epitope comprising three discontinuous sites including six key gp120 residues (Site C1: Thr123 and Pro124; Site C3: Glu370 and Ile371; Site C4: Met426 and Trp427) were identified. In the 3D structure of gp120, the sites C1 and C4 are located in the bridging sheet and the site C3 is within the β15-α3 excursion, which play essential roles for the receptor- and coreceptor-binding and are major targets of neutralizing antibodies. Based on these results we propose a precise localization of the m36 epitope and suggest a mechanism of its broad inhibitory activity which could help in the development of novel HIV-1 therapeutics based on eAds.
PMCID: PMC3679054  PMID: 23776690
25.  Identification of a Human Protein-Derived HIV-1 Fusion Inhibitor Targeting the gp41 Fusion Core Structure 
PLoS ONE  2013;8(5):e66156.
The HIV-1 envelope glycoprotein (Env) gp41 plays a crucial role in the viral fusion process. The peptides derived from the C-terminal heptad repeat (CHR) of gp41 are potent HIV fusion inhibitors. However, the activity of these anti-HIV-1 peptides in vivo may be attenuated by their induction of anti-gp41 antibodies. Thus, it is essential to identify antiviral peptides or proteins with low, or no, immunogenicity to humans. Here, we found that the C-terminal fragment (aa 462–521) of the human POB1 (the partner of RalBP1), designated C60, is an HIV-1 fusion inhibitor. It bound to N36, the peptide derived from the N-terminal heptad repeat (NHR) of gp41, and to the six-helix bundle (6-HB) formed by N36 and C34, a CHR-peptide, but it did not bind to C34. Unlike the CHR-peptides, C60 did not block gp41 6-HB formation. Rather, results suggest that C60 inhibits HIV-1 fusion by binding to the 6-HB, in particular, the residues in the gp41 NHR domain that are exposed on the surface of 6-HB. Since 6-HB plays a crucial role in the late stage of fusion between the viral envelope and endosomal membrane during the endocytic process of HIV-1, C60 may serve as a host restriction factor to suppress HIV-1 entry into CD4+ T lymphocytes. Taken together, it can be concluded from these results that C60 can be used as a lead for the development of anti-HIV-1 therapeutics or microbicides for the treatment and prevention of HIV-1 infection, as well as a molecular probe to study the fusogenic mechanism of HIV-1.
PMCID: PMC3669275  PMID: 23741527

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