Peptide and Non-peptide HIV Fusion Inhibitors

Current Pharmaceutical Design. 2002  |  Pubmed ID: 11945159

Fusion of the HIV envelope with the target cell membrane is a critical step of HIV entry into the target cell. The HIV envelope glycoprotein gp41 plays an important role in the fusion of viral and target cell membranes and serves as an attractive target for development of HIV fusion inhibitors. The extracellular domain of gp41 contains three important functional regions, i.e. fusion peptide (FP), N- and C-terminal heptad repeats (NHR and CHR, respectively). The FP region is composed of hydrophobic, glycine-rich residues that are essential for the initial penetration of the target cell membrane. NHR and CHR regions consist of hydrophobic residues, which have the tendency to form alpha-helical coiled coils. During the process of fusion of HIV or HIV-infected cells with uninfected cells, FP inserts into the target cell membrane and subsequently the NHR and CHR regions change conformations and associate with each other to form a fusion-active gp41 core. Peptides derived from NHR and CHR regions, designated N- and C-peptides, respectively, have potent inhibitory activity against HIV fusion by binding to the CHR and NHR regions, respectively, to prevent the formation of the fusion-active gp41 core. C-peptide may also bind to FP, thereby blocking its insertion into the target cell membrane. One of the C-peptides, T-20, which is in the phase III clinical trials, has potent in vivo activity against HIV infection and is expected to become the first peptide HIV fusion inhibitory drug in the near future. However, this peptide HIV fusion inhibitor lacks oral availability and is sensitive to the proteolytic digestion. Therefore, it is essential to develop small molecular non-peptide HIV fusion inhibitors having a mechanism of action similar to the C-peptides. One of the approaches in identifying the inhibitors is to use an immunological assay to screen chemical libraries for the compounds that potentially block the interaction between the NHR and CHR regions to form a fusion-active gp41 core. In combination with computer-aided molecular docking techniques, the first active non-peptide HIV fusion inhibitor targeting gp41, ADS-J1, was identified. Other potential candidates of non-peptide HIV fusion inhibitors have also been identified using different approaches. It is expected that both peptide and non-peptide HIV fusion inhibitors will be developed as new classes of anti-HIV drugs, which will be used alone or in combination with HIV reverse transcriptase and protease inhibitors, for the treatment of HIV infection and AIDS.

Anti-HIV-1 Activity of Cellulose Acetate Phthalate: Synergy with Soluble CD4 and Induction of "dead-end" Gp41 Six-helix Bundles

BMC Infectious Diseases. Apr, 2002  |  Pubmed ID: 11983022

Cellulose acetate phthalate (CAP), a promising candidate microbicide for prevention of sexual transmission of the human immunodeficiency virus type 1 (HIV-1) and other sexually transmitted disease (STD) pathogens, was shown to inactivate HIV-1 and to block the coreceptor binding site on the virus envelope glycoprotein gp120. It did not interfere with virus binding to CD4. Since CD4 is the primary cellular receptor for HIV-1, it was of interest to study CAP binding to HIV-1 complexes with soluble CD4 (sCD4) and its consequences, including changes in the conformation of the envelope glycoprotein gp41 within virus particles.

Identification of Inhibitors of the HIV-1 Gp41 Six-helix Bundle Formation from Extracts of Chinese Medicinal Herbs Prunella Vulgaris and Rhizoma Cibotte

Life Sciences. Aug, 2002  |  Pubmed ID: 12151056

An increasing portion of patients with HIV infection and/or AIDS cannot use currently FDA-approved anti-HIV drugs, including the reverse transcriptase and protease inhibitors, due to the adverse effects and the emergence of drug resistance. Thus, it is essential to develop new anti-HIV agents with a target different from the HIV reverse transcriptase and protease. Using a conformation-specific monoclonal antibody NC-1, we previously established a high throughput screening assay for identification of small molecular organic compounds that disrupt the HIV-1 gp41 six-helix bundle formation, a critical step of membrane fusion between the HIV and the target cell. In the present study, we used this assay to screen for inhibitors of the gp41 six-helix bundle formation from aqueous extracts of nine Chinese medicinal herbs with antiviral activity. We found that the extracts of two herbs, Prunella vulgaris and Rhizoma cibotte, showed potent inhibitory activity. The inhibitory activity of these two herb extracts significantly decreased after they were passed through polyamide resin mini-columns, which are able to bind polyphenols including tannin, an HIV-1 inhibitor with multiple mechanisms of action. The bound polyphenols were eluted from the polyamide columns and also showed potent inhibitory activity on the gp41 six-helix bundle formation. Tannin purchased from different commercial sources inhibited the gp41 six-helix bundle formation in a manner similar to the polyphenols isolated from the herb extracts. These results suggest that tannin may be one of major inhibitors of the HIV-1 gp41 six-helix bundle formation in the herb extracts and that tannin may inhibit HIV-1 entry by disrupting the gp41 six-helix bundle formation.

XTT Formazan Widely Used to Detect Cell Viability Inhibits HIV Type 1 Infection in Vitro by Targeting Gp41

AIDS Research and Human Retroviruses. Sep, 2002  |  Pubmed ID: 12396451

XTT can be metabolically reduced by mitochondrial dehydrogenase in viable cells to a water-soluble formazan product. Thus XTT has been widely used to evaluate cell viability and to screen anti-HIV agents and the cytotoxicity of these agents. The present studies demonstrated that XTT formazan derived from XTT in cell culture significantly inhibits the fusion of HIV-1-infected cells with uninfected cells. Synthetic XTT formazan effectively inhibited the replication of laboratory-adapted and primary HIV-1 isolates and cell-to-cell fusion with low cytotoxicity. It blocks the six-helix bundle formation between peptides derived from the N- and C-terminal heptad repeat regions of the gp41 ectodomain (designated N- and C-peptides, respectively). Analysis by a computer-aided docking program indicates that XTT formazan may bind to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled coil of gp41. These results suggest that XTT formazan inhibits HIV-1 entry by targeting the alpha-helical coiled-coil domain of gp41. This small molecular nonpeptide antiviral compound can be used as a lead for designing more effective HIV-1 entry inhibitors targeting the fusion stage of HIV-1 infection. But because XTT formazan itself has anti-HIV-1 activity, caution should be exercised when XTT is used to evaluate HIV-1 infectivity.

The in Vitro and in Vivo Protective Activity of Monoclonal Antibodies Directed Against Hantaan Virus: Potential Application for Immunotherapy and Passive Immunization

Biochemical and Biophysical Research Communications. Nov, 2002  |  Pubmed ID: 12408987

Hantaan virus (HTNV), a member of the genus Hantavirus, family Bunyaviridae, is an etiologic agent causing a serious human disease, hemorrhagic fever with renal syndrome (HFRS), with a mortality as high as 15% and is also a potential bioterrorism agent. It is urgently needed to develop anti-HTNV-neutralizing monoclonal antibodies (MAbs) for treatment and prevention of HTNV infection. In the present study, 18 murine MAbs directed against HTNV strain Chen were generated and characterized. Among these MAbs, 13 were directed against viral nucleocapsid protein (NP), four recognized the viral envelope glycoprotein G2 and one reacted with both NP and G2. Only those MAbs that recognize the epitopes on G2 were positive in hemagglutination inhibition (HI) test and had in vitro virus-neutralizing activity and in vivo protective activity against HTNV infection of susceptible mice. Since all the mice were protected by administration of the virus-neutralizing MAbs one day before and two days after HTNV challenge, these neutralizing MAbs are potentially useful for pre- and post-exposure prophylaxis and for immunotherapy of HTNV infection. Phase II clinical trials of these neutralizing MAbs for emergent treatment of patients with HTNV infection in early stages of HRFS are carried out in endemic areas in China.

Design of a Protein Surface Antagonist Based on Alpha-helix Mimicry: Inhibition of Gp41 Assembly and Viral Fusion

Angewandte Chemie (International Ed. in English). Jan, 2002  |  Pubmed ID: 12491408

Automatic Quantitation of HIV-1 Mediated Cell-to-cell Fusion with a Digital Image Analysis System (DIAS): Application for Rapid Screening of HIV-1 Fusion Inhibitors

Journal of Virological Methods. Feb, 2003  |  Pubmed ID: 12505629

Human immunodeficiency viruses type 1 (HIV-1) mediated cell-to-cell fusion plays an important role in HIV-1 spread from infected cells to uninfected cells and in HIV-1 cytopathogenesis. In the present study, we developed a convenient cell fusion assay using a computer-controlled digital image analysis system (DIAS) for automatic quantitation. Compared with a manual quantitative method, DIAS automatic method is less laborious, and more rapid. Furthermore, it is more objective and less dependent on the researchers' experience. This method has great potential to be developed further as a high-throughput screening assay for rapid screening of HIV-1 fusion inhibitors, for evaluating the activity of HIV-1 entry inhibitors and for studying the mechanism of action of anti-HIV-1 agents.

Role of the Fusion Peptide and Membrane-proximal Domain in HIV-1 Envelope Glycoprotein-mediated Membrane Fusion

Biochemistry. Dec, 2003  |  Pubmed ID: 14640682

The N-terminal fusion peptide and the interfacial sequence preceding the transmembrane anchor of HIV-1 gp41 are required for viral fusion. Studies with synthetic peptides indicated that these regions function by destabilizing membranes, which is regarded as a crucial step in the membrane fusion reaction. However, it is not clear whether membrane destabilization is induced by these sequences in the intact gp41. We address this question by examining fusion and destabilization of membranes expressing HIV-1(IIIB) wild-type Env and two mutant Envs. (1) A Glu residue at position 2 of the gp41 fusion peptide is substituted for Val (V2E) to produce one mutant. (2) Residues 665-682 in the membrane-proximal domain are deleted to form the other. The process of membrane destabilization was monitored by the influx of Sytox, an impermeant fluorescent dye, into the Env-expressing cells following the interaction with CD4-CXCR4 complexes, and fusion was monitored by observing dye transfer between Env-expressing cells and appropriate target cells. We also monitored the conformational changes in the Envs following their interactions with CD4 and CXCR4 by immunofluorescence using an anti-gp41 mAb that reacts with the six-helix bundle. In contrast to the wild type, both Env mutants did not mediate cell fusion. The V2E Env did not mediate membrane destabilization. However, the Env with an unmodified fusion peptide but with a deletion of residues 665-682 in the membrane-proximal domain did mediate membrane destabilization. The wild type and both mutant Envs undergo conformational changes detected by the anti-gp41 six-helix bundle mAbs. Our results suggest that in intact HIV-1 Env the membrane-proximal domain is not required for membrane perturbations, but rather enables the bending of gp41 that is required for viral and target membranes to come together. Moreover, the observation that the Delta665-683 Env self-inserts its fusion peptide but does not cause fusion suggests that self-insertion of the fusion peptide is not sufficient for HIV-1 Env-mediated fusion.

Development of a Cell-based Enzyme-linked Immunosorbent Assay for High-throughput Screening of HIV Type 1 Entry Inhibitors Targeting the Coreceptor CXCR4

AIDS Research and Human Retroviruses. Nov, 2003  |  Pubmed ID: 14678601

CXCR4, a coreceptor for human immunodeficiency virus type 1 (HIV-1) X4 virus, plays an important role in virus entry into the target cells. Antiviral agents that bind to CXCR4 are expected to inhibit HIV-1 entry. A cell-based enzyme-linked immunosorbent assay (ELISA) was developed utilizing an anti-CXCR4 monoclonal antibody, 12G5, and cells expressing CD4 and CXCR4, U373-MAGI-CXCR4(CEM) cells. Using this assay, we demonstrated that 12G5 specifically binds to the CXCR4-expressing cells, but not to CCR5-expressing cells and cells without CXCR4 and CCR5, consistent with the results obtained by using flow cytometry. The well-characterized CXCR4 antagonists, T22, T14012 (a downsized analog of T-22), and AMD3100, effectively inhibited 12G5 binding to CXCR4-expressing cells, while HIV-1 entry inhibitors targeting CD4 and gp41 as well as HIV-1 reverse transcriptase and protease inhibitors did not block the binding of 12G5 to U373-MAGI-CXCR4(CEM) cells. The prepared plates containing the fixed cells could be stored at -80 degrees C for at least 5 months without affecting the cell reactivity with 12G5, which enhances the convenience of this method. This suggests that the cell-based ELISA is specific, sensitive, convenient, rapid, and economic. With a robotic sample processing system, this assay can be used for high-throughput screening of HIV-1 entry inhibitors targeted to the HIV-1 coreceptor CXCR4.

Determination of the HIV-1 Gp41 Fusogenic Core Conformation Modeled by Synthetic Peptides: Applicable for Identification of HIV-1 Fusion Inhibitors

Peptides. Sep, 2003  |  Pubmed ID: 14706544

Triggered by receptor binding of gp120, the human immunodeficiency virus type 1 (HIV-1) gp41 changes its conformation to a fusogenic six-helix bundle structure. In the present study, this core conformation modeled by the peptides derived from the gp41 N- and C-terminal heptad repeat regions was determined by fluorescence native polyacrylamide gel electrophoresis and size exclusion high-performance liquid chromatography (HPLC). Two previously described small molecule HIV-1 fusion inhibitors significantly blocked the six-helix bundle formation. It suggests that these biophysical techniques can be used in a novel way to study the conformational change of gp41 during virus entry into cells and to identify HIV-1 fusion inhibitors.

Rapid and Automated Fluorescence-linked Immunosorbent Assay for High-throughput Screening of HIV-1 Fusion Inhibitors Targeting Gp41

Journal of Biomolecular Screening. Dec, 2003  |  Pubmed ID: 14711394

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 plays an important role in the virus entry. During the process of fusion between the viral and target cell membranes, the N- and C-terminal heptad repeat (HR) regions of the gp41 extracellular domain associate to form a 6-helical bundle, corresponding to the fusion-active gp41 core. Any compound that blocks the gp41 6-helix bundle formation between the N- and C-peptides, which are derived from the N- and C-terminal HR regions, respectively, may inhibit HIV-1 mediated membrane fusion. Based on this principle, we previously established a sandwich enzyme-linked immunosorbent assay (ELISA) for drug screening by using the N-peptide N36 and the C-peptide C34 and a monoclonal antibody (NC-1) which specifically recognizes the gp41 6-helix bundle. In the present study, a fluorescence-linked immunosorbent assay (FLISA) was developed by using fluorescein isothiocyanate (FITC)-conjugated C34 to replace C34 and by directly detecting fluorescence intensity instead of more complicated enzymatic reaction. Compared with the sandwich ELISA, this FLISA has similar sensitivity and specificity, but it is much more rapid, economic and convenient. Using an Integrated Robotic Sample Processing System, this assay has been applied for high-throughput screening of organic compounds on a large scale for HIV-1 fusion inhibitors targeting gp41.

Binding of the 2F5 Monoclonal Antibody to Native and Fusion-intermediate Forms of Human Immunodeficiency Virus Type 1 Gp41: Implications for Fusion-inducing Conformational Changes

Journal of Virology. Mar, 2004  |  Pubmed ID: 14963170

We investigated how the broadly neutralizing monoclonal antibody 2F5 affects the human immunodeficiency virus type 1 envelope glycoprotein as it undergoes receptor-induced conformational changes and show that 2F5 binds both native and fusion-intermediate conformations, suggesting inhibition of a late step in virus entry. We also demonstrate conformational changes in the C heptad of gp41.

Synthesis and Anti-HIV-1 Activity of 4-[4-(4,6-bisphenylamino-triazin-2-ylamino)-5-methoxy-2-methylphenylazo]-5-hydroxynaphthalene-2,7-disulfonic Acid and Its Derivatives

Bioorganic & Medicinal Chemistry. Mar, 2004  |  Pubmed ID: 14980633

A structure-based design approach has been used to optimize a lead HIV-1 entry inhibitor targeted to the envelope glycoprotein gp41. The docking study on this lead compound revealed important structural requirements that need to be preserved as well as structural non-requirements that could be eliminated to substantially reduce the molecular size of the lead compound. Based on the results from docking study, a limited number of analogues were designed and synthesized. This approach yielded a new analogue (compound 4) that retained the anti-HIV-1 activity with reduced molecular size approaching towards more drug-like character.

Interaction Between Heptad Repeat 1 and 2 Regions in Spike Protein of SARS-associated Coronavirus: Implications for Virus Fusogenic Mechanism and Identification of Fusion Inhibitors

Lancet. Mar, 2004  |  Pubmed ID: 15043961

Studies on the fusion-inhibitory peptides derived from the heptad repeat 1 and 2 (HR1 and HR2) regions of the HIV-1 envelope glycoprotein gp41 provided crucial information on the viral fusogenic mechanism. We used a similar approach to study the fusogenic mechanism of severe-acute-respiratory-syndrome-associated coronavirus (SARS-CoV).

Organ Distribution of Severe Acute Respiratory Syndrome (SARS) Associated Coronavirus (SARS-CoV) in SARS Patients: Implications for Pathogenesis and Virus Transmission Pathways

The Journal of Pathology. Jun, 2004  |  Pubmed ID: 15141376

We previously identified the major pathological changes in the respiratory and immune systems of patients who died of severe acute respiratory syndrome (SARS) but gained little information on the organ distribution of SARS-associated coronavirus (SARS-CoV). In the present study, we used a murine monoclonal antibody specific for SARS-CoV nucleoprotein, and probes specific for a SARS-CoV RNA polymerase gene fragment, for immunohistochemistry and in situ hybridization, respectively, to detect SARS-CoV systematically in tissues from patients who died of SARS. SARS-CoV was found in lung, trachea/bronchus, stomach, small intestine, distal convoluted renal tubule, sweat gland, parathyroid, pituitary, pancreas, adrenal gland, liver and cerebrum, but was not detected in oesophagus, spleen, lymph node, bone marrow, heart, aorta, cerebellum, thyroid, testis, ovary, uterus or muscle. These results suggest that, in addition to the respiratory system, the gastrointestinal tract and other organs with detectable SARS-CoV may also be targets of SARS-CoV infection. The pathological changes in these organs may be caused directly by the cytopathic effect mediated by local replication of the SARS-CoV; or indirectly as a result of systemic responses to respiratory failure or the harmful immune response induced by viral infection. In addition to viral spread through a respiratory route, SARS-CoV in the intestinal tract, kidney and sweat glands may be excreted via faeces, urine and sweat, thereby leading to virus transmission. This study provides important information for understanding the pathogenesis of SARS-CoV infection and sheds light on possible virus transmission pathways. This data will be useful for designing new strategies for prevention and treatment of SARS.

High Throughput Screening and Characterization of HIV-1 Entry Inhibitors Targeting Gp41: Theories and Techniques

Current Pharmaceutical Design. 2004  |  Pubmed ID: 15180543

The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein plays an important role in HIV-1 entry and severs as an attractive target for development of HIV-1 entry inhibitors, a new class of anti-HIV drugs. Triggered by gp120 binding to CD4 and a coreceptor, gp41 undergoes a conformation shift from a native prefusogenic state to a fusogenic state, in which the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) associate to form a six-helix bundle, representing the fusion-active gp41 core. Any compound that disrupts the gp41 six-helix bundle formation may inhibit the gp41-mediated membrane fusion, thereby blocking HIV-1 entry into target cells. Peptides derived from the gp41 NHR and CHR regions, designated N- and C-peptides, can interact with the counterpart regions in gp41 and interfere with the six-helix bundle formation between the viral NHR and CHR region, thus inhibiting fusion of the virus with the target cell. One of the C-peptides, T-20 (brand name: Fuzeon), was recently approved by the US FDA as the first HIV entry inhibitor which can be used for treatment of AIDS patients who fail to respond to the current antiretroviral drugs, e.g., the reverse transcriptase inhibitors and protease inhibitors. The limitations of T-20 include lack of oral availability and high cost of production. Thus it is essential to develop small molecule HIV-1 entry inhibitors targeting gp41. This review summarizes the newly developed techniques for high throughput screening (HTS) and characterization of the HIV-1 entry inhibitors targeting gp41. The theories behind these techniques are also discussed. It is expected that the "drug-like" compounds with potent HIV-1 fusion inhibitory activity will be identified in the near future and used as leads for development of the low molecular weight HIV-1 entry inhibitors for the chemotherapy of HIV-1 infection and AIDS.

Identification of Immunodominant Sites on the Spike Protein of Severe Acute Respiratory Syndrome (SARS) Coronavirus: Implication for Developing SARS Diagnostics and Vaccines

Journal of Immunology (Baltimore, Md. : 1950). Sep, 2004  |  Pubmed ID: 15356154

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is not only responsible for receptor binding and virus fusion, but also a major Ag among the SARS-CoV proteins that induces protective Ab responses. In this study, we showed that the S protein of SARS-CoV is highly immunogenic during infection and immunizations, and contains five linear immunodominant sites (sites I to V) as determined by Pepscan analysis with a set of synthetic peptides overlapping the entire S protein sequence against the convalescent sera from SARS patients and antisera from small animals immunized with inactivated SARS-CoV. Site IV located in the middle region of the S protein (residues 528-635) is a major immunodominant epitope. The synthetic peptide S(603-634), which overlaps the site IV sequence reacted with all the convalescent sera from 42 SARS patient, but none of the 30 serum samples from healthy blood donors, suggesting its potential application as an Ag for developing SARS diagnostics. This study also provides information useful for designing SARS vaccines and understanding the SARS pathogenesis.

Receptor-binding Domain of SARS-CoV Spike Protein Induces Highly Potent Neutralizing Antibodies: Implication for Developing Subunit Vaccine

Biochemical and Biophysical Research Communications. Nov, 2004  |  Pubmed ID: 15474494

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS.

N-substituted Pyrrole Derivatives As Novel Human Immunodeficiency Virus Type 1 Entry Inhibitors That Interfere with the Gp41 Six-helix Bundle Formation and Block Virus Fusion

Antimicrobial Agents and Chemotherapy. Nov, 2004  |  Pubmed ID: 15504864

A recently approved peptidic human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, T-20 (Fuzeon; Trimeris Inc.), has shown significant promise in clinical application for treating HIV-1-infected individuals who have failed to respond to the currently available antiretroviral drugs. However, T-20 must be injected twice daily and is too expensive. Therefore, it is essential to develop orally available small molecule HIV-1 fusion inhibitors. By screening a chemical library consisting of "drug-like" compounds, we identified two N-substituted pyrroles, designated NB-2 and NB-64, that inhibited HIV-1 replication at a low micromolar range. The absence of the COOH group in NB-2 and NB-64 resulted in a loss of anti-HIV-1 activity, suggesting that this acid group plays an important role in mediating the antiviral activity. NB-2 and NB-64 inhibited HIV-1 fusion and entry by interfering with the gp41 six-helix bundle formation and disrupting the alpha-helical conformation. They blocked a d-peptide binding to the hydrophobic pocket on surface of the gp41 internal trimeric coiled-coil domain. Computer-aided molecular docking analysis has shown that they fit inside the hydrophobic pocket and that their COOH group interacts with a positively charged residue (K574) around the pocket to form a salt bridge. These results suggest that NB-2 and NB-64 may bind to the gp41 hydrophobic pocket through hydrophobic and ionic interactions and block the formation of the fusion-active gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry. Therefore, NB-2 and NB-64 can be used as lead compounds toward designing and developing more potent small molecule HIV-1 fusion inhibitors targeting gp41.

Mapping of Antigenic Sites on the Nucleocapsid Protein of the Severe Acute Respiratory Syndrome Coronavirus

Journal of Clinical Microbiology. Nov, 2004  |  Pubmed ID: 15528730

Antigenic sites on the nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) were mapped by Pepscan analysis with overlapping peptides that span the N protein sequence. Two major immunodominant epitopes located in the C-terminal region (amino acids [aa] 362 to 412) and middle region (aa 153 to 178) reacted with more than 75% of sera from SARS patients. Several minor immunodominant epitopes were reactive with about 50% of the SARS sera. Antisera from mice immunized with inactivated SARS-CoV recognized the two major immunodominant epitopes and one antigenic site located adjacent to the N-terminal region (aa 76 to 101), which did not react with the sera from SARS patients. Several monoclonal antibodies against SARS-CoV bound to the N- or C-terminal antigenic sites. These results suggest that the above antigenic sites on the N protein are important in eliciting humoral immune response against SARS-CoV in humans and animals and can be used as antigens for developing diagnostic tests.

Inactivated SARS-CoV Vaccine Elicits High Titers of Spike Protein-specific Antibodies That Block Receptor Binding and Virus Entry

Biochemical and Biophysical Research Communications. Dec, 2004  |  Pubmed ID: 15530413

The only severe acute respiratory syndrome (SARS) vaccine currently being tested in clinical trial consists of inactivated severe acute respiratory syndrome-associate coronavirus (SARS-CoV). However, limited information is available about host immune responses induced by the inactivated SARS vaccine. In this study, we demonstrated that SARS-CoV inactivated by beta-propiolactone elicited high titers of antibodies in the immunized mice and rabbits that recognize the spike (S) protein, especially the receptor-binding domain (RBD) in the S1 region. The antisera from the immunized animals efficiently bound to the RBD and blocked binding of RBD to angiotensin-converting enzyme 2, the functional receptor on the susceptible cells for SARS-CoV. With a sensitive and quantitative single-cycle infection assay using pseudovirus bearing the SARS-CoV S protein, we demonstrated that mouse and rabbit antisera significantly inhibited S protein-mediated virus entry with mean 50% inhibitory titers of 1:7393 and 1:2060, respectively. These data suggest that the RBD of S protein is a major neutralization determinant in the inactivated SARS vaccine which can induce potent neutralizing antibodies to block SARS-CoV entry. However, caution should be taken in using the inactivated SARS-CoV as a vaccine since it may also cause harmful immune and/or inflammatory responses.

Different from the HIV Fusion Inhibitor C34, the Anti-HIV Drug Fuzeon (T-20) Inhibits HIV-1 Entry by Targeting Multiple Sites in Gp41 and Gp120

The Journal of Biological Chemistry. Mar, 2005  |  Pubmed ID: 15640162

Fuzeon (also known as T-20 or enfuvirtide), one of the C-peptides derived from the HIV-1 envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (CHR) region, is the first member of a new class of anti-HIV drugs known as HIV fusion inhibitors. It has been widely believed that T-20 shares the same mechanism of action with C34, another C-peptide. The C34 is known to compete with the CHR of gp41 to form a stable 6-helix bundle (6-HB) with the gp41 N-terminal heptad repeat (NHR) and prevent the formation of the fusogenic gp41 core between viral gp41 NHR and CHR, thereby inhibiting fusion between viral and target cell membranes. Here we present data to demonstrate that, contrary to this belief, T-20 cannot form stable 6-HB with N-peptides derived from the NHR region, nor can it inhibit the 6-HB formation of the fusogenic core. Instead, it may interact with N-peptides to form unstable or insoluble complexes. Our data suggest that T-20 has a different mechanism of action from C34. The interaction of T-20 with viral NHR region alone may not prevent the formation of the fusion active gp41 core. We also demonstrate that the T-20-mediated anti-HIV activity can be significantly abrogated by peptides derived from the membrane-spanning domain in gp41 and coreceptor binding site in gp120. These new findings imply that T-20 inhibits HIV-1 entry by targeting multiple sites in gp41 and gp120. Further elucidation of the mechanism of action of T-20 will provide new target(s) for development of novel HIV entry inhibitors.

Identification of a Critical Neutralization Determinant of Severe Acute Respiratory Syndrome (SARS)-associated Coronavirus: Importance for Designing SARS Vaccines

Virology. Mar, 2005  |  Pubmed ID: 15749124

The spike (S) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is not only responsible for receptor binding, but also a major antigenic determinant capable of inducing protective immunity. In this study, we demonstrated that the receptor-binding domain (RBD) of S protein is an important immunogenic site in patients with SARS and rabbits immunized with inactivated SARS-CoV. Serum samples from convalescent SARS patients and immunized rabbits had potent neutralizing activities against infection by pseudovirus expressing SARS-CoV S protein. Depletion of RBD-specific antibodies from patient or rabbit immune sera by immunoadsorption significantly reduced serum-mediated neutralizing activity, while affinity-purified anti-RBD antibodies had relatively higher potency neutralizing infectivity of SARS pseudovirus, indicating that the RBD of S protein is a critical neutralization determinant of SARS-CoV during viral infection and immunization. Two monoclonal antibodies (1A5 and 2C5) targeting at the RBD of S protein were isolated from mice immunized with inactivated SARS-CoV. Both 1A5 and 2C5 possessed potent neutralizing activities, although they directed against distinct conformation-dependant epitopes as shown by ELISA and binding competition assay. We further demonstrated that 2C5, but not 1A5, was able to block binding of the RBD to angiotensin-converting enzyme 2 (ACE2), the functional receptor on targeted cells. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV and for designing SARS vaccines.

Receptor-binding Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein Contains Multiple Conformation-dependent Epitopes That Induce Highly Potent Neutralizing Antibodies

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2005  |  Pubmed ID: 15814718

The spike (S) protein of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is a major antigenic determinant capable of inducing protective immunity. Recently, a small fragment on the SARS-CoV S protein (residues 318-510) was characterized as a minimal receptor-binding domain (RBD), which mediates virus binding to angiotensin-converting enzyme 2, the functional receptor on susceptible cells. In this study, we demonstrated that a fusion protein containing RBD linked to human IgG1 Fc fragment (designated RBD-Fc) induced high titer of RBD-specific Abs in the immunized mice. The mouse antisera effectively neutralized infection by both SARS-CoV and SARS pseudovirus with mean 50% neutralization titers of 1/15,360 and 1/24,737, respectively. The neutralization determinants on the RBD of S protein were characterized by a panel of 27 mAbs isolated from the immunized mice. Six groups of conformation-dependent epitopes, designated as Conf I-VI, and two adjacent linear epitopes were identified by ELISA and binding competition assays. The Conf IV and Conf V mAbs significantly blocked RBD-Fc binding to angiotensin-converting enzyme 2, suggesting that their epitopes overlap with the receptor-binding sites in the S protein. Most of the mAbs (23 of 25) that recognized the conformational epitopes possessed potent neutralizing activities against SARS pseudovirus with 50% neutralizing dose ranging from 0.005 to 6.569 microg/ml. Therefore, the RBD of SARS S protein contains multiple conformational epitopes capable of inducing potent neutralizing Ab responses, and is an important target site for developing vaccines and immunotherapeutics.

Theaflavin Derivatives in Black Tea and Catechin Derivatives in Green Tea Inhibit HIV-1 Entry by Targeting Gp41

Biochimica Et Biophysica Acta. May, 2005  |  Pubmed ID: 15823507

Theaflavin derivatives and catechin derivatives are the major polyphenols in black tea and green tea, respectively. Several tea polyphenols, especially those with galloyl moiety, can inhibit HIV-1 replication with multiple mechanisms of action. Here we showed that the theaflavin derivatives had more potent anti-HIV-1 activity than catechin derivatives. These tea polyphenols could inhibit HIV-1 entry into target cells by blocking HIV-1 envelope glycoprotein-mediated membrane fusion. The fusion inhibitory activity of the tea polyphenols was correlated with their ability to block the formation of the gp41 six-helix bundle, a fusion-active core conformation. Computer-aided molecular docking analyses indicate that these tea polyphenols, theaflavin-3,3'-digallate (TF3) as an example, may bind to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled coil formed by the N-terminal heptad repeats of gp41. These results indicate that tea, especially black tea, may be used as a source of anti-HIV agents and theaflavin derivatives may be applied as lead compounds for developing HIV-1 entry inhibitors targeting gp41.

ROv-ASP-1, a Recombinant Secreted Protein of the Helminth Onchocercavolvulus, is a Potent Adjuvant for Inducing Antibodies to Ovalbumin, HIV-1 Polypeptide and SARS-CoV Peptide Antigens

Vaccine. May, 2005  |  Pubmed ID: 15837368

We studied the adjuvanticity of recombinant Onchocerca volvulus activation associated protein-1 (rOv-ASP-1) for ovalbumin (OVA) in mice. After a single immunization and one boost, rOv-ASP-1 exceeded the efficacy of alum or MPL + TDM adjuvants in terms of end-point total IgG or IgG1 and IgG2a anti-OVA titres. Using the helminth-derived adjuvant, IgG isotype responses to OVA were of a mixed Th1/Th2 profile and spleen cell cytokines exclusively Th1-type. The potent adjuvanticity of rOv-ASP-1 was confirmed in mice vaccinated with a 37-mer peptide from the S protein of SARS-CoV and an HIV-1 gp120-CD4 chimeric polypeptide antigen. Unusually for a helminth product, the rOv-ASP-1 adjuvant augmented not only Th2 but also Th1 responses, the latter property being of potential utility in stimulating anti-viral immune responses.

Combination of Candidate Microbicides Cellulose Acetate 1,2-benzenedicarboxylate and UC781 Has Synergistic and Complementary Effects Against Human Immunodeficiency Virus Type 1 Infection

Antimicrobial Agents and Chemotherapy. May, 2005  |  Pubmed ID: 15855503

The combination of two candidate microbicides, cellulose acetate 1,2-benzenedicarboxylate (CAP), a polymer that blocks human immunodeficiency virus type 1 (HIV-1) entry by targeting gp120 and gp41, and UC781, a tight-binding HIV-1 reverse transcriptase inhibitor (RTI), resulted in effective synergy for inhibition of MT-2 cell infection by HIV-1(IIIB), a laboratory-adapted virus strain. The 95% effective concentration values for the combination were reduced about 15- to 20-fold compared with those corresponding to the single compounds. The combination of CAP and UC781 is also synergistic in inhibiting infection of peripheral blood mononuclear cells by a primary HIV-1 isolate, 92US657. Combinations of CAP with other RTIs, such as efavirenz or zidovudine, also had significant synergistic effects on the inhibition of HIV-1 infection. In addition, CAP and UC781 had complementary effects against HIV-1 infection since (i) CAP inhibited infection by the UC781-resistant strain HIV-1(IIIB) A17 and (ii) pretreatment of MT-2 cells with UC781, but not CAP, abolished subsequent infection after removal of the compound. This suggests that the combination of CAP and UC781 represents a promising candidate microbicide for prevention of sexual transmission of HIV-1.

Chemoenzymatic Synthesis of HIV-1 Gp41 Glycopeptides: Effects of Glycosylation on the Anti-HIV Activity and Alpha-helix Bundle-forming Ability of Peptide C34

Chembiochem : a European Journal of Chemical Biology. Jun, 2005  |  Pubmed ID: 15883971

C34 is a 34-mer peptide derived from the C-terminal ectodomain of HIV-1 envelope glycoprotein, gp41. The C34 region in native gp41 carries a conserved N-glycan at Asn637 and the sequence is directly involved in the virus-host membrane fusion, an essential step for HIV-1 infection. This paper describes the synthesis of glycoforms of C34 which carry a monosaccharide, a disaccharide, and a native oligosaccharide moiety. The synthesis of the glycopeptide which carries a native high-mannose type N-glycan was achieved by a chemoenzymatic approach by using an endoglycosidase-catalyzed oligosaccharide transfer as the key step. The effects of glycosylation on the inhibitory activity and the helix-bundle forming ability of C34 were investigated. It was found that glycosylation moderately decreases the anti-HIV activity of C34 and, in comparison with C34, glyco-C34 forms less compact six-helix bundles with the corresponding N-terminal peptide, N36. This study suggests that conserved glycosylation modulates the anti-HIV activity and conformations of the gp41 C-peptide, C34.

Identification of N-phenyl-N'-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamides As a New Class of HIV-1 Entry Inhibitors That Prevent Gp120 Binding to CD4

Virology. Sep, 2005  |  Pubmed ID: 15996703

We have identified two N-phenyl-N'-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamide analogs as a novel class of human immunodeficiency virus type 1 (HIV-1) entry inhibitors that block the gp120-CD4 interaction, using database screening techniques. The lead compounds, NBD-556 and NBD-557, are small molecule organic compounds with drug-like properties. These compounds showed potent cell fusion and virus-cell fusion inhibitory activity at low micromolar levels. A systematic study showed that these compounds target viral entry by inhibiting the binding of HIV-1 envelope glycoprotein gp120 to the cellular receptor CD4 but did not inhibit reverse transcriptase, integrase, or protease, indicating that they do not target the later stages of the HIV-1 life cycle to inhibit HIV-1 infection. These compounds were equally potent inhibitors of both X4 and R5 viruses tested in CXCR4 and CCR5 expressing cell lines, respectively, indicating that their anti-HIV-1 activity is not dependent on the coreceptor tropism of the virus. A surface plasmon resonance study, which measures binding affinity, clearly demonstrated that these compounds bind to unliganded HIV-1 gp120 but not to the cellular receptor CD4. NBD-556 and NBD-557 were active against HIV-1 laboratory-adapted strains including an AZT-resistant strain and HIV-1 primary isolates, indicating that these compounds can potentially be further modified to become potent HIV-1 entry inhibitors.

SARS Vaccine Development

Emerging Infectious Diseases. Jul, 2005  |  Pubmed ID: 16022774

Developing effective and safe vaccines is urgently needed to prevent infection by severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). The inactivated SARS-CoV vaccine may be the first one available for clinical use because it is easy to generate; however, safety is the main concern. The spike (S) protein of SARS-CoV is the major inducer of neutralizing antibodies, and the receptor-binding domain (RBD) in the S1 subunit of S protein contains multiple conformational neutralizing epitopes. This suggests that recombinant proteins containing RBD and vectors encoding the RBD sequence can be used to develop safe and effective SARS vaccines.

Vaccine Design for Severe Acute Respiratory Syndrome Coronavirus

Viral Immunology. 2005  |  Pubmed ID: 16035944

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a new coronavirus (SARS-CoV). Recent studies suggest that SARS-CoV is zoonotic and may have a broad host range besides humans. Although the global outbreak of SARS has been contained, there are serious concerns over its re-emergence and bioterrorism potential. As a part of preparedness, development of a safe and effective vaccine is one of the highest priorities in fighting SARS. A number of candidate vaccines, using a variety of approaches, are under development. The first vaccine tested in clinical trial is made from the inactivated form of SARS-CoV. Several live attenuated, genetically engineered or vector vaccines encoding the SARS-CoV spike (S) protein have been in pre-clinical studies. These vaccine candidates are effective in terms of eliciting protective immunity in the vaccinated animals. However, caution should be taken with the safety of whole virus or full-length S protein-based immunogens in humans because they may induce harmful immune or inflammatory responses. We propose to use the receptor-binding domain (RBD) of SARS-CoV S protein (residues 318--510) for developing a safe and effective subunit SARS vaccine, as it is not only a functional domain that mediates virus-receptor binding but also a major neutralization determinant of SARSCoV. It has been demonstrated that the RBD of SARS-CoV S protein contains multiple conformational epitopes capable of inducing highly potent neutralizing antibody responses and protective immunity.

Identification of Immunodominant Epitopes on the Membrane Protein of the Severe Acute Respiratory Syndrome-associated Coronavirus

Journal of Clinical Microbiology. Aug, 2005  |  Pubmed ID: 16081901

Similar to other coronaviruses, the membrane (M) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a major transmembrane glycoprotein with multiple biological functions. To date, limited information is available about its antigenic properties. In this study, we identified two major immunodominant epitopes on the M protein located in the extreme N-terminal region (residues 1 to 31) and the interior C-terminal region (residues 132 to 161), respectively, by Pepscan analyses against convalescent-phase sera from SARS patients and antisera from virus-immunized mice and rabbits. Synthetic peptides M1-31 derived from the N-terminal epitope and M132-161 derived from the C-terminal epitope were highly reactive with all of the convalescent-phase sera from 40 SARS patients but not with 30 control serum samples from healthy blood donors, suggesting their potential application for serologic diagnosis of SARS. We showed that both peptides (M1-31 and M132-161) were able to induce high titers of antibody responses in the immunized rabbits, highlighting their antigenicity and immunogenicity. These findings provide important information for developing SARS diagnostics and vaccines.

Neutralization of HIV-1 Primary Isolate by ELDKWA-specific Murine Monoclonal Antibodies

Immunobiology. 2005  |  Pubmed ID: 16323702

ELDKWA on HIV-1 gp41 is a conserved epitope recognized by one broadly neutralizing monoclonal antibody 2F5, which is a promising candidate target for vaccine design. Here we report two ELDKWA-specific monoclonal antibodies (mAbs), 18F11 and 7E10, that were screened from the splenocytes of mice immunized by recombinant GST-(ELDKWA)4 protein. In further evaluation, these mAbs exhibited appreciable neutralizing activities against HIV-1 primary isolate 92US675 (clade B) with IC50 (50% inhibition concentration) of 6.84 +/- 0.36 microg/ml and 10.66 +/- 1.69 microg/ml, respectively. Unexpectedly, neither of these two murine mAbs could neutralize laboratory-adapted strain HIV-1 IIIB (clade B). As a control, human mAb 2F5 neutralized both primary and laboratory-adapted strains. These data strongly suggest that ELDKWA-specific antibodies induced by different antigenic formats show different neutralizing activities against HIV-1, which implies another complication in the development of effective vaccines.

The Ribosomal Protein L32-2 (RPL32-2) of S. Pombe Exhibits a Novel Extraribosomal Function by Acting As a Potential Transcriptional Regulator

FEBS Letters. Mar, 2006  |  Pubmed ID: 16516201

Ribosomal proteins play important roles in stabilizing the rRNA structure to facilitate protein synthesis in ribosome. In the present study, we analyzed the potential extraribosomal function of the ribosomal protein L32-2 (RPL32-2), which was expressed by a gene clone isolated from a cDNA library of Schizosaccharomyces pombe (S. pombe). RPL32-2 fused with the GAL4 DNA-bind domain or the GAL4 transcriptional activating domain could, respectively, activate transcriptions of reporter genes in yeast strain AH109. The RPL32-2 mutants with truncation of either the N- or the C-terminal domain resulted in abolishment of this regulatory effect. The DNA binding site for RPL32-2 of S. pombe was identified by using a random oligonucleotide selection strategy and gel motility shift assay and Western blotting confirmed its binding specificity. Moreover, we found RPL32-2 was also able to interact with a to-be-identified AT sequence binding protein. These data suggest that RPL32-2 of S. pombe, besides its ribosomal function, may also act as a potential transcriptional regulator in nucleus.

Phosphorothioate Oligonucleotides Inhibit Human Immunodeficiency Virus Type 1 Fusion by Blocking Gp41 Core Formation

Antimicrobial Agents and Chemotherapy. Apr, 2006  |  Pubmed ID: 16569857

Several studies have shown that phosphorothioate oligodeoxynucleotides (PS-ONs) have a sequence-independent antiviral activity against human immunodeficiency virus type 1 (HIV-1). It has also been suggested that PS-ONs inhibit HIV-1 by acting as attachment inhibitors that bind to the V3 loop of gp120 and prevent the gp120-CD4 interaction. Here we show that PS-ONs (and their fully 2'-O-methylated derivatives) are potent inhibitors of HIV-1-mediated membrane fusion and HIV-1 replication in a size-dependent, phosphorothioation-dependent manner. PS-ONs interact with a peptide derived from the N-terminal heptad repeat region of gp41, and the HIV-1 fusion-inhibitory activity of PS-ONs is closely correlated with their ability to block gp41 six-helix bundle formation, a critical step during the process of HIV-1 fusion with the target cell. These results suggest that the increased hydrophobicity of PS-ONs may contribute to their inhibitory activity against HIV-1 fusion and entry, because longer PS-ONs (>or=30 bases) which have a greater hydrophobicity are more potent in blocking the hydrophobic interactions involved in the gp41 six-helix bundle formation and inhibiting the HIV-1-mediated cell-cell fusion than shorter PS-ONs (<30 bases). This novel antiviral mechanism of action of long PS-ONs has implications for therapy against infection by HIV-1 and other enveloped viruses with type I fusion proteins.

A Single Amino Acid Substitution (R441A) in the Receptor-binding Domain of SARS Coronavirus Spike Protein Disrupts the Antigenic Structure and Binding Activity

Biochemical and Biophysical Research Communications. May, 2006  |  Pubmed ID: 16615996

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318-510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbs as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.

Theta-defensins Prevent HIV-1 Env-mediated Fusion by Binding Gp41 and Blocking 6-helix Bundle Formation

The Journal of Biological Chemistry. Jul, 2006  |  Pubmed ID: 16648135

Retrocyclin-1, a -defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 microm) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.

Cross-neutralization of Human and Palm Civet Severe Acute Respiratory Syndrome Coronaviruses by Antibodies Targeting the Receptor-binding Domain of Spike Protein

Journal of Immunology (Baltimore, Md. : 1950). May, 2006  |  Pubmed ID: 16670317

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is considered as a protective Ag for vaccine design. We previously demonstrated that the receptor-binding domain (RBD) of S protein contains multiple conformational epitopes (Conf I-VI) that confer the major target of neutralizing Abs. Here we show that the recombinant RBDs derived from the S protein sequences of Tor2, GD03, and SZ3, the representative strains of human 2002-2003 and 2003-2004 SARS-CoV and palm civet SARS-CoV, respectively, induce in the immunized mice and rabbits high titers of cross-neutralizing Abs against pseudoviruses expressing S proteins of Tor2, GD03, and SZ3. We also demonstrate that the Tor2-RBD induced-Conf I-VI mAbs can potently neutralize both human SARS-CoV strains, Tor2 and GD03. However, only the Conf IV-VI, but not Conf I-III mAbs, neutralize civet SARS-CoV strain SZ3. All these mAbs reacted significantly with each of the three RBD variants (Tor2-RBD, GD03-RBD, and SZ3-RBD) that differ at several amino acids. Regardless, the Conf I-IV and VI epitopes were completely disrupted by single-point mutation of the conserved residues in the RBD (e.g., D429A, R441A, or D454A) and the Conf III epitope was significantly affected by E452A or D463A substitution. Interestingly, the Conf V epitope, which may overlap the receptor-binding motif and induce most potent neutralizing Abs, was conserved in these mutants. These data suggest that the major neutralizing epitopes of SARS-CoV have been apparently maintained during cross-species transmission, and that RBD-based vaccines may induce broad protection against both human and animal SARS-CoV variants.

Cellulose Acetate 1,2-benzenedicarboxylate Inhibits Infection by Cell-free and Cell-associated Primary HIV-1 Isolates

AIDS Research and Human Retroviruses. May, 2006  |  Pubmed ID: 16706617

Cellulose acetate 1,2-benzenedicarboxylate (CAP), a pharmaceutical excipient used for enteric film coating of capsules and tablets, was previously shown to have potent inhibitory activity against infection by human immunodeficiency virus type 1 (HIV-1) T cell line-adapted (TCLA) strains. In the present study, we determined the inhibitory activity of CAP against infection by cell-free and cell-associated primary HIV-1 isolates with distinct genotypes and biotypes in cervical explants, peripheral blood mononuclear cells (PBMCs), monocytederived macrophages (MDMs), and CEMx174 5.25M7 cells. CAP blocked infection by cell-free and cell-associated HIV-1 in cervical explants. It inhibited infection by cell-free primary HIV-1 isolates (clades A to G and group O) in PBMCs, MDMs, and CEMx174 5.25M7 cells and blocked transmissions of the cell-associated primary HIV-1 isolates from dendritic cells (DCs) to PBMCs, from MDMs to PBMCs, and from PBMCs to CEMx174 5.25M7 cells. The inhibitory activity of CAP on infection by the cell-free and cell-associated primary HIV-1 isolates is independent of viral subtypes and coreceptor usage. These data suggest that CAP is a good microbicide candidate that can be further developed for preventing sexual transmission of HIV-1.

Antigenic Properties of a Transport-competent Influenza HA/HIV Env Chimeric Protein

Virology. Aug, 2006  |  Pubmed ID: 16725170

The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.

Identification and Characterization of Novel Neutralizing Epitopes in the Receptor-binding Domain of SARS-CoV Spike Protein: Revealing the Critical Antigenic Determinants in Inactivated SARS-CoV Vaccine

Vaccine. Jun, 2006  |  Pubmed ID: 16725238

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is considered as a major antigen for vaccine design. We previously demonstrated that the receptor-binding domain (RBD: residues 318-510) of S protein contains multiple conformation-dependent neutralizing epitopes (Conf I to VI) and serves as a major target of SARS-CoV neutralization. Here, we further characterized the antigenic structure in the RBD by a panel of novel mAbs isolated from the mice immunized with an inactivated SARS-CoV vaccine. Ten of the RBD-specific mAbs were mapped to four distinct groups of conformational epitopes (designated Group A to D), and all of which had potent neutralizing activity against S protein-pseudotyped SARS viruses. Group A, B, C mAbs target the epitopes that may overlap with the previously characterized Conf I, III, and VI respectively, but they display different capacity to block the receptor binding. Group D mAb (S25) was directed against a unique epitope by its competitive binding. Two anti-RBD mAbs recognizing the linear epitopes (Group E) were mapped to the RBD residues 335-352 and 442-458, respectively, and none of them inhibited the receptor binding and virus entry. Surprisingly, most neutralizing epitopes (Groups A to C) could be completely disrupted by single amino acid substitutions (e.g., D429A, R441A or D454A) or by deletions of several amino acids at the N-terminal or C-terminal region of the RBD; however, the Group D epitope was not sensitive to the mutations, highlighting its importance for vaccine development. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV, and this panel of novel mAbs can be used as tools for studying the structure of S protein and for guiding SARS vaccine design.

Antigenic and Immunogenic Characterization of Recombinant Baculovirus-expressed Severe Acute Respiratory Syndrome Coronavirus Spike Protein: Implication for Vaccine Design

Journal of Virology. Jun, 2006  |  Pubmed ID: 16731915

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates the receptor interaction and immune recognition and is considered a major target for vaccine design. However, its antigenic and immunogenic properties remain to be elucidated. In this study, we immunized mice with full-length S protein (FL-S) or its extracellular domain (EC-S) expressed by recombinant baculoviruses in insect cells. We found that the immunized mice developed high titers of anti-S antibodies with potent neutralizing activities against SARS pseudoviruses constructed with the S proteins of Tor2, GD03T13, and SZ3, the representative strains of 2002 to 2003 and 2003 to 2004 human SARS-CoV and palm civet SARS-CoV, respectively. These data suggest that the recombinant baculovirus-expressed S protein vaccines possess excellent immunogenicity, thereby inducing highly potent neutralizing responses against human and animal SARS-CoV variants. The antigenic structure of the S protein was characterized by a panel of 38 monoclonal antibodies (MAbs) isolated from the immunized mice. The epitopes of most anti-S MAbs (32 of 38) were localized within the S1 domain, and those of the remaining 6 MAbs were mapped to the S2 domain. Among the anti-S1 MAbs, 17 MAbs targeted the N-terminal region (amino acids [aa] 12 to 327), 9 MAbs recognized the receptor-binding domain (RBD; aa 318 to 510), and 6 MAbs reacted with the C-terminal region of S1 domain that contains the major immunodominant site (aa 528 to 635). Strikingly, all of the RBD-specific MAbs had potent neutralizing activity, 6 of which efficiently blocked the receptor binding, confirming that the RBD contains the main neutralizing epitopes and that blockage of the receptor association is the major mechanism of SARS-CoV neutralization. Five MAbs specific for the S1 N-terminal region exhibited moderate neutralizing activity, but none of the MAbs reacting with the S2 domain and the major immunodominant site in S1 showed neutralizing activity. All of the neutralizing MAbs recognize conformational epitopes. These data provide important information for understanding the antigenicity and immunogenicity of S protein and for designing SARS vaccines. This panel of anti-S MAbs can be used as tools for studying the structure and function of the SARS-CoV S protein.

Recombinant Adeno-associated Virus Expressing the Receptor-binding Domain of Severe Acute Respiratory Syndrome Coronavirus S Protein Elicits Neutralizing Antibodies: Implication for Developing SARS Vaccines

Virology. Sep, 2006  |  Pubmed ID: 16793110

Development of an effective vaccine for severe acute respiratory syndrome (SARS) remains to be a priority to prevent possible re-emergence of SARS coronavirus (SARS-CoV). We previously demonstrated that the receptor-binding domain (RBD) of SARS-CoV S protein is a major target of neutralizing antibodies. This suggests that the RBD may serve as an ideal vaccine candidate. Recombinant adeno-associated virus (rAAV) has been proven to be an effective system for gene delivery and vaccine development. In this study, a novel vaccine against SARS-CoV was developed based on the rAAV delivery system. The gene encoding RBD was cloned into a pAAV-IRES-hrGFP plasmid. The immunogenicity induced by the resulting recombinant RBD-rAAV was evaluated in BALB/c mice. The results demonstrated that (1) a single dose of RBD-rAAV vaccination could induce sufficient neutralizing antibody against SARS-CoV infection; (2) two more repeated doses of the vaccination boosted the neutralizing antibody to about 5 times of the level achieved by a single dose of the immunization and (3) the level of the antibody continued to increase for the entire duration of the experiment of 5.5 months. These results suggested that RBD-rAAV is a promising SARS candidate vaccine.

Evaluation of "credit Card" Libraries for Inhibition of HIV-1 Gp41 Fusogenic Core Formation

Journal of Combinatorial Chemistry. Jul-Aug, 2006  |  Pubmed ID: 16827565

Protein-protein interactions are of critical importance in biological systems, and small molecule modulators of such protein recognition and intervention processes are of particular interest. To investigate this area of research, we have synthesized small-molecule libraries that can disrupt a number of biologically relevant protein-protein interactions. These library members are designed upon planar motif, appended with a variety of chemical functions, which we have termed "credit-card" structures. From two of our "credit-card" libraries, a series of molecules were uncovered which act as inhibitors against the HIV-1 gp41 fusogenic 6-helix bundle core formation, viral antigen p24 formation, and cell-cell fusion at low micromolar concentrations. From the high-throughput screening assays we utilized, a selective index (SI) value of 4.2 was uncovered for compound 2261, which bodes well for future structure activity investigations and the design of more potent gp41 inhibitors.

Identification of a D-amino Acid Decapeptide HIV-1 Entry Inhibitor

Biochemical and Biophysical Research Communications. Sep, 2006  |  Pubmed ID: 16854380

Entry of human immunodeficiency virus type 1 (HIV-1) virion into host cells involves three major steps, each being a potential target for the development of entry inhibitors: gp120 binding to CD4, gp120-CD4 complex interacting with a coreceptor, and gp41 refolding to form a six-helix bundle. Using a D-amino acid decapeptide combinatorial library, we identified peptide dC13 as having potent HIV-1 fusion inhibitory activity, and effectively inhibiting infection by several laboratory-adapted and primary HIV-1 strains. While dC13 did not block binding of gp120 to CD4, nor disrupt the gp41 six-helix bundle formation, it effectively blocked the binding of an anti-CXCR4 monoclonal antibody and chemokine SDF-1alpha to CXCR4-expressing cells. However, because R5-using primary viruses were also neutralized, the antiviral activity of dC13 implies additional mode(s) of action. These results suggest that dC13 is a useful HIV-1 coreceptor antagonist for CXCR4 and, due to its biostability and simplicity, may be of value for developing a new class of HIV-1 entry inhibitors.

Identification of the HIV-1 Gp41 Core-binding Motif--HXXNPF

FEBS Letters. Sep, 2006  |  Pubmed ID: 16904109

The HIV-1 gp41 core, a six-helix bundle formed between the N- and C-terminal heptad repeats, plays a critical role in fusion between the viral and target cell membranes. Using N36(L8)C34 as a model of the gp41 core to screen phage display peptide libraries, we identified a common motif, HXXNPF (X is any of the 20 natural amino acid residues). A selected positive phage clone L7.8 specifically bound to N36(L8)C34 and this binding could be blocked by a gp41 core-specific monoclonal antibody (NC-1). JCH-4, a peptide containing HXXNPF motif, effectively inhibited HIV-1 envelope glycoprotein-mediated syncytium-formation. The epitope of JCH-4 was proven to be linear and might locate in the NHR regions of the gp41 core. These data suggest that HXXNPF motif may be a gp41 core-binding sequence and HXXNPF motif-containing molecules can be used as probes for studying the role of the HIV-1 gp41 core in membrane fusion process.

Selection of a Novel Gp41-specific HIV-1 Neutralizing Human Antibody by Competitive Antigen Panning

Journal of Immunological Methods. Dec, 2006  |  Pubmed ID: 17078964

The HIV envelope glycoprotein (Env) is composed of two non-covalently associated subunits: gp120 and gp41. Panning of phage-displayed antibody libraries against Env-based antigens has resulted mostly in selection of anti-gp120 antibodies. Native gp41 in the absence of gp120 is unstable. The use of gp41 fragments as antigens has resulted in selection of antibodies with only relatively modest neutralizing activity. To enhance selection of antibodies specific for gp41 in the context of the whole Env we developed a methodology termed competitive antigen panning (CAP). Using CAP, we identified a novel gp41-specific human monoclonal antibody (hmAb), m48, from an immune library derived from long-term nonprogressors with high titers of broadly cross-reactive neutralizing antibodies (bcnAbs). Selection of m48 was only successful using CAP and not through the conventional pre-incubation methodology. In assays based on spreading infection in peripheral blood mononuclear cells (PBMCs) m48 neutralized a panel of HIV-1 primary isolates from different clades more potently than the well-characterized broadly cross-reactive HIV-1-neutralizing antibodies IgG1 4E10 and Fab Z13. These results may have implications for the selection of novel gp41-specific bcnAbs and other antibodies, and for the development of HIV-1 inhibitors and vaccine immunogens.

Microbicides: Stopping HIV at the Gate

Lancet. Aug, 2006  |  Pubmed ID: 16890813

China Needs Safe and Effective Microbicides for Preventing Sexual Transmission of HIV

The Lancet Infectious Diseases. Nov, 2006  |  Pubmed ID: 17067914

Receptor-binding Domain of SARS-CoV Spike Protein Induces Long-term Protective Immunity in an Animal Model

Vaccine. Apr, 2007  |  Pubmed ID: 17092615

Development of effective vaccines against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is still a priority in prevention of re-emergence of SARS. Our previous studies have shown that the receptor-binding domain (RBD) of SARS-CoV spike (S) protein elicits highly potent neutralizing antibody responses in the immunized animals. But it is unknown whether RBD can also induce protective immunity in an animal model, a key aspect for vaccine development. In this study, BALB/c mice were vaccinated intramuscularly (i.m.) with 10microg of RBD-Fc (RBD fused with human IgG1 Fc) and boosted twice at 3-week intervals and one more time at 12th month. Humoral immune responses of vaccinated mice were investigated for up to 12 months at a 1-month interval and the neutralizing titers of produced antibodies were reported at months 0, 3, 6 and 12 post-vaccination. Mice were challenged with the homologous strain of SARS-CoV 5 days after the last boost, and sacrificed 5 days after the challenge. Mouse lung tissues were collected for detection of viral load, virus replication and histopathological effects. Our results showed that RBD-Fc vaccination induced high titer of S-specific antibodies with long-term and potent SARS-CoV neutralizing activity. Four of five vaccinated mice were protected from subsequent SARS-CoV challenge because no significant virus replication, and no obvious histopathological changes were found in the lung tissues of the vaccinated mice challenged with SARS-CoV. Only one vaccinated mouse had mild alveolar damage in the lung tissues. In contrast, high copies of SARS-CoV RNA and virus replication were detected, and pathological changes were observed in the lung tissues of the control mice. In conclusion, our findings suggest that RBD, which can induce protective antibodies to SARS-CoV, may be further developed as a safe and effective SARS subunit vaccine.

Polymorphisms of Type I Interferon Receptor 1 Promoter and Their Effects on Chronic Hepatitis B Virus Infection

Journal of Hepatology. Feb, 2007  |  Pubmed ID: 17125879

Exposure to HBV leads to a distinct clinical course which is partially pertained to host genetic variability. We aimed to study polymorphisms of type I interferon receptor 1 (IFNAR1) promoter and their potential effects on chronic HBV infection.

Identification of the HIV-1 Gp41 Core-binding Motif in the Scaffolding Domain of Caveolin-1

The Journal of Biological Chemistry. Mar, 2007  |  Pubmed ID: 17197700

The human immunodeficiency virus, type 1 (HIV-1) gp41 core plays an important role in fusion between viral and target cell membranes. A single chain polypeptide, N36(L8)C34, which forms a six-helix bundle in physiological solution, can be used as a model of gp41 core. Here we identified from a 12-mer phage peptide library a positive phage clone displaying a peptide sequence with high binding activity to the HIV-1 gp41 core. The peptide sequence contains a putative gp41-binding motif, PhiXXXXPhiXPhi (X is any amino acid residue, and Phi is any one of the aromatic amino acid residues Trp, Phe, or Tyr). This motif also exists in the scaffolding domain of caveolin-1 (Cav-1), a known gp41-binding protein. Cav-1-(61-101) and Cav-1-(82-101), two recombinant fusion proteins containing the Cav-1 scaffolding domain, bound significantly to the gp41 expressed in mammalian cells and interacted with the polypeptide N36(L8)C34. These results suggest that the scaffolding domain of Cav-1 may bind to the gp41 core via the motif. This interaction may be essential for formation of fusion pore or endocytosis of HIV-1 and affect the pathogenesis of HIV-1 infection. Further characterization of the gp41 core-binding motifs may shed light on the alternative mechanism by which HIV-1 enters into the target cell.

The Mechanism by Which Molecules Containing the HIV Gp41 Core-binding Motif HXXNPF Inhibit HIV-1 Envelope Glycoprotein-mediated Syncytium Formation

The Biochemical Journal. May, 2007  |  Pubmed ID: 17223796

The HIV-1 gp41 (glycoprotein 41) core plays a critical role in fusion between the viral and target cell membranes. We previously identified a gp41 core-binding motif, HXXNPF, by screening the phage display peptide libraries. In the present study, we elucidated the mechanism of action of HXXNPF motif-containing molecules of different sizes, including the phage clone L7.8 (a selected positive phage clone), L7.8-g3p* (a 10-kDa fragment of the gene 3 protein) and JCH-4 (a peptide containing 13 residues of L7.8-g3p*), regarding their respective binding abilities to the six-helix bundle and inhibition on syncytium formation at different temperatures. We found that all of the HXXNPF motif-containing molecules could bind to the gp41 core, and that their binding sites may be located in the N-helix domain. L7.8-g3p* and JCH-4 effectively inhibited HIV-1 Env (envelope glycoprotein)-mediated syncytium formation at 37 degrees C, while the phage clone L7.8 showed no inhibition under the same conditions. However, at suboptimal temperature (31.5 degrees C), all of these HXXNPF motif-containing molecules were capable of inhibiting syncytium formation. These results suggest that these HXXNPF motif-containing molecules mainly bind to the gp41 core and stop the fusion process mediated by the fusion-active core, resulting in inhibition of HIV-1 fusion and entry. The HXXNPF motif-containing molecules may be used as probes for studying the role of the HIV-1 gp41 core in the late stage of the membrane-fusion process.

HIV Entry Inhibitors Targeting Gp41: from Polypeptides to Small-molecule Compounds

Current Pharmaceutical Design. 2007  |  Pubmed ID: 17269924

HIV envelope glycoprotein transmembrane subunit gp41 plays a critical role in the fusion between viral and target cell membranes. Upon gp120 binding to CD4 and a coreceptor (CCR5 or CXCR4), gp41 changes its conformation by forming N-helix trimer between N-heptad repeats (NHRs) and then six-helix bundle between the N-trimer and the C-heptad repeats (CHRs). Peptides derived from the NHR and CHR of gp41 extracellular region have demonstrated potent inhibitory activity on the HIV mediated cell fusion. One of these peptides, T-20, became the first success of a new class of anti-HIV agents, named HIV entry inhibitors. However, a relatively long peptide such as T-20 suffers from several limitations including lack of oral bioavailability and high cost of production. Great efforts have been made to develop alternative peptides and proteins with improved anti-HIV-1 activity, increased bioavailability and reduced cost of production. The most promising approach is the development of small molecule HIV entry inhibitors targeting gp41. Any molecule that blocks the process of NHR homotrimerization and the six-helix bundle formation by targeting the gp41 NHR, NHR trimer and CHR may inhibit HIV-mediated membrane fusion. The progress in development of those anti-HIV agents targeting gp41, from polypeptides to small-molecule compounds, is reviewed.

HIV Gp41 C-terminal Heptad Repeat Contains Multifunctional Domains. Relation to Mechanisms of Action of Anti-HIV Peptides

The Journal of Biological Chemistry. Mar, 2007  |  Pubmed ID: 17276993

T20 (Fuzeon), a novel anti-human immunodeficiency virus (HIV) drug, is a peptide derived from HIV-1 gp41 C-terminal heptad repeat (CHR). Its mechanism of action has not yet been defined. We applied Pepscan strategy to determine the relationship between functional domains and mechanisms of action of five 36-mer overlapping peptides with a shift of five amino acids (aa): CHR-1 (aa 623-658), C36 (aa 628-663), CHR-3 (aa 633-668), T20 (aa 638-673), and CHR-5 (aa 643-678). C36 is a peptide with addition of two aa to the N terminus of C34. Peptides CHR-1 and C36 contain N-terminal heptad repeat (NHR)- and pocket-binding domains. They inhibited HIV-1 fusion by interacting with gp41 NHR, forming stable six-helix bundles and blocking gp41 core formation. Peptide T20 containing partial NHR- and lipid-binding domains, but lacking pocket-binding domain, blocked viral fusion by binding its N- and C-terminal sequences with gp41 NHR and cell membrane, respectively. Peptide CHR-3, which is located in the middle between C36 and T20, overlaps >86% of the sequences of these two peptides, and lacks pocket- and lipid-binding domains, exhibited marginal anti-HIV-1 activity. These results suggest that T20 and C36 contain different functional domains, through which they inhibit HIV-1 entry with distinct mechanisms of action. The multiple functional domains in gp41 CHR and their binding partners may serve as targets for rational design of new anti-HIV-1 drugs and vaccines.

HIV-1 Gp41 Ectodomain Enhances Cryptococcus Neoformans Binding to HBMEC

Biochemical and Biophysical Research Communications. May, 2007  |  Pubmed ID: 17400192

Cryptococcus neoformans infection has significantly increased recently, particularly in AIDS patients and immunocompromised individuals. C. neoformans has a predilection to the brain, resulting in devastating meningoencephalitis. We have previously shown the invasion of C. neoformans into the human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. Here, we demonstrated that C. neoformans invasion of HBMEC was enhanced by HIV-1 gp41 protein. Peptide mapping defined its functional domain around the disulfide-bond linkage of gp41 molecule (a.a. 579-611). Recombinant protein gp41-I90 (a.a. 550-639) can also enhance the binding activity. The enhancement of C. neoformans binding to HBMEC is a strain-independent manner, suggesting that gp41 ectodomain peptide exerts its function directly on HBMEC. Importantly, the enhancement could be observed in mouse animal model. Our results suggest that HIV-1 gp41 ectodomain and C. neoformans may follow a similar invasion mechanism, possibly actin reorganization and/or membrane activation, during pathogen infections on HBMEC.

Discovery and Optimization of a Natural HIV-1 Entry Inhibitor Targeting the Gp41 Fusion Peptide

Cell. Apr, 2007  |  Pubmed ID: 17448989

A variety of molecules in human blood have been implicated in the inhibition of HIV-1. However, it remained elusive which circulating natural compounds are most effective in controlling viral replication in vivo. To identify natural HIV-1 inhibitors we screened a comprehensive peptide library generated from human hemofiltrate. The most potent fraction contained a 20-residue peptide, designated VIRUS-INHIBITORY PEPTIDE (VIRIP), corresponding to the C-proximal region of alpha1-antitrypsin, the most abundant circulating serine protease inhibitor. We found that VIRIP inhibits a wide variety of HIV-1 strains including those resistant to current antiretroviral drugs. Further analysis demonstrated that VIRIP blocks HIV-1 entry by interacting with the gp41 fusion peptide and showed that a few amino acid changes increase its antiretroviral potency by two orders of magnitude. Thus, as a highly specific natural inhibitor of the HIV-1 gp41 fusion peptide, VIRIP may lead to the development of another class of antiretroviral drugs.

Structure-based Identification of Small Molecule Compounds Targeting Cell Cyclophilin A with Anti-HIV-1 Activity

European Journal of Pharmacology. Jun, 2007  |  Pubmed ID: 17449029

Cyclophilin A acts as protein folding chaperones and intracellular transports in many cellular processes. Previous studies have shown that cyclophilin A can interact with HIV-1 (human immunodeficiency virus type 1) gag protein and enhance viral infectivity. Many cyclophilin A inhibitors such as cyclosporin A can inhibit HIV-1 replication in vitro. Here, we report a structure-based identification of novel non-peptidic cyclophilin A inhibitors as anti-HIV lead compounds. Following a computer-aided virtual screening and subsequent surface plasmon resonance (SPR) analysis, 12 low molecular weight cyclophilin A ligands were selected for further evaluation of their in vitro inhibition of peptidyl-prolyl cis-trans isomerase (PPIase) activity of cyclophilin A and HIV-1 replication. Five of these compounds (FD5, FD8, FD9, FD10 and FD12) exhibited inhibition against both PPIase activity and HIV-1 infection. These active compounds will be used as leads for structure and activity relationship (SAR) and optimization studies in order to design more effective anti-HIV-1 therapeutics, and as probes for investigating the effect of cyclophilins on HIV-1 replication.

Cleavage of Spike Protein of SARS Coronavirus by Protease Factor Xa is Associated with Viral Infectivity

Biochemical and Biophysical Research Communications. Jul, 2007  |  Pubmed ID: 17533109

The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection.

Conserved Residue Lys574 in the Cavity of HIV-1 Gp41 Coiled-coil Domain is Critical for Six-helix Bundle Stability and Virus Entry

The Journal of Biological Chemistry. Aug, 2007  |  Pubmed ID: 17616522

The fusion-active HIV-1 gp41 core structure is a stable six-helix bundle (6-HB) formed by its N- and C-terminal heptad-repeat sequences (NHR and CHR). A highly conserved, deep hydrophobic cavity on the surface of the N-helical trimer is important for stability of the 6-HB and serves as an ideal target for developing anti-human immunodeficiency virus (HIV) fusion inhibitors. We have recently identified several small molecule HIV-1 fusion inhibitors that bind to the gp41 cavity through hydrophobic and ionic interactions and block the gp41 6-HB formation. Molecular docking analysis reveals that these small molecules fit inside the hydrophobic cavity and interact with positively charged residue Lys574 to form a conserved salt bridge. In this study, the functionality of Lys574 has been finely characterized by mutational analysis and biophysical approaches. We found that substitutions of Lys574 with non-conserved residues (K574D, K574E, and K574V) could completely abolish virus infectivity. With a set of wild-type and mutant N36 peptides derived from the NHR sequence as a model, we demonstrated that non-conservative Lys574 substitutions severely impaired the stability and conformation of 6-HBs as detected by circular dichroism spectroscopy, native polyacrylamide gel electrophoresis, and enzyme-linked immunosorbent assay. The binding affinity of N36 mutants bearing non-conservative Lys574 substitutions to the peptide C34 derived from the CHR sequence dramatically decreased as measured by isothermal titration calorimetry. These substitutions also significantly reduced the potency of N-peptides to inhibit HIV-1 infection. Collectively, these data suggest that conserved Lys574 plays a critical role in 6-HB formation and HIV-1 infectivity, and may serve as an important target for designing anti-HIV drugs.

Potent Cross-reactive Neutralization of SARS Coronavirus Isolates by Human Monoclonal Antibodies

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2007  |  Pubmed ID: 17620608

The severe acute respiratory syndrome coronavirus (SARS-CoV) caused a worldwide epidemic in late 2002/early 2003 and a second outbreak in the winter of 2003/2004 by an independent animal-to-human transmission. The GD03 strain, which was isolated from an index patient of the second outbreak, was reported to resist neutralization by the human monoclonal antibodies (hmAbs) 80R and S3.1, which can potently neutralize isolates from the first outbreak. Here we report that two hmAbs, m396 and S230.15, potently neutralized GD03 and representative isolates from the first SARS outbreak (Urbani, Tor2) and from palm civets (SZ3, SZ16). These antibodies also protected mice challenged with the Urbani or recombinant viruses bearing the GD03 and SZ16 spike (S) glycoproteins. Both antibodies competed with the SARS-CoV receptor, ACE2, for binding to the receptor-binding domain (RBD), suggesting a mechanism of neutralization that involves interference with the SARS-CoV-ACE2 interaction. Two putative hot-spot residues in the RBD (Ile-489 and Tyr-491) were identified within the SARS-CoV spike that likely contribute to most of the m396-binding energy. Residues Ile-489 and Tyr-491 are highly conserved within the SARS-CoV spike, indicating a possible mechanism of the m396 cross-reactivity. Sequence analysis and mutagenesis data show that m396 might neutralize all zoonotic and epidemic SARS-CoV isolates with known sequences, except strains derived from bats. These antibodies exhibit cross-reactivity against isolates from the two SARS outbreaks and palm civets and could have potential applications for diagnosis, prophylaxis, and treatment of SARS-CoV infections.

The Function of Coreceptor As a Basis for the Kinetic Dissection of HIV Type 1 Envelope Protein-mediated Cell Fusion

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Apr, 2008  |  Pubmed ID: 18032634

The function of HIV-1 HXB2 envelope (Env) glycoprotein (gp) was investigated by surface plasmon resonance and fluorescence imaging techniques. Strikingly, it was found that gp120 shedding requires the presence of the X4 coreceptor. A similar coreceptor requirement was observed for the membrane mixing and the Env recruitment on the cell surface. However, exposure and membrane penetration of the fusion peptide do not require X4 and occur within the first minute after incubation of Env with CD4 and/or X4. Analogously X4 was not required but enhanced binding of the fusion inhibitor. In contrast, bundle formation of the gp41 ectodomain, as monitored by NC-1, was accelerated by the presence of X4. The kinetics of these key post-Env binding events as determined in real time by fluorescence microscopic imaging, coupled with the differential coreceptor requirement, led to the proposition that gp120 shedding, which takes place from 1 to 10 min after engagement of receptor and coreceptor to Env, is a primary function of the coreceptor. The shedding of the surface subunits is needed for the subsequent processes including hemifusion, full fusion, and Env recruitment. The temporal order of these fusogenic steps allows construction of a refined model on the Env-mediated cell fusion event.

Intranasal Vaccination of Recombinant Adeno-associated Virus Encoding Receptor-binding Domain of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Spike Protein Induces Strong Mucosal Immune Responses and Provides Long-term Protection Against SARS-CoV Infection

Journal of Immunology (Baltimore, Md. : 1950). Jan, 2008  |  Pubmed ID: 18178835

We have previously reported that a subunit protein vaccine based on the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein and a recombinant adeno-associated virus (rAAV)-based RBD (RBD-rAAV) vaccine could induce highly potent neutralizing Ab responses in immunized animals. In this study, systemic, mucosal, and cellular immune responses and long-term protective immunity induced by RBD-rAAV were further characterized in a BALB/c mouse model, with comparison of the i.m. and intranasal (i.n.) routes of administration. Our results demonstrated that: 1) the i.n. vaccination induced a systemic humoral immune response of comparable strength and shorter duration than the i.m. vaccination, but the local humoral immune response was much stronger; 2) the i.n. vaccination elicited stronger systemic and local specific cytotoxic T cell responses than the i.m. vaccination, as evidenced by higher prevalence of IL-2 and/or IFN-gamma-producing CD3+/CD8+ T cells in both lungs and spleen; 3) the i.n. vaccination induced similar protection as the i.m. vaccination against SARS-CoV challenge in mice; 4) higher titers of mucosal IgA and serum-neutralizing Ab were associated with lower viral load and less pulmonary pathological damage, while no Ab-mediated disease enhancement effect was observed; and 5) the vaccination could provide long-term protection against SARS-CoV infection. Taken together, our findings suggest that RBD-rAAV can be further developed into a vaccine candidate for prevention of SARS and that i.n. vaccination may be the preferred route of administration due to its ability to induce SARS-CoV-specific systemic and mucosal immune responses and its better safety profile.

Molecular Modeling Studies of N-substituted Pyrrole Derivatives--potential HIV-1 Gp41 Inhibitors

Bioorganic & Medicinal Chemistry. Mar, 2008  |  Pubmed ID: 18226912

2D-, 3D-QSAR and docking studies were carried out on 23 pyrrole derivatives, to model their HIV-1 gp41 inhibitory activities. The 2D, 3D-QSAR studies were performed using CODESSA software package and comparative molecular field analysis (CoMFA) technique, respectively. The CODESSA five-descriptor model has a correlation coefficient R(2)=0.825 and a standard deviation s(2)=0.132. The 3D-QSAR CoMFA study allowed to obtain a model showing a good correlative and predictive capability which statistical results, provided by a eight-component regression equation, are: R(2)=0.984, q(2)=0.463, s=0.119. Docking studies were employed to determine probable binding conformation of these analogues into the gp41 active site using the AutoDock program whose results were found complementary with thus of 2D- and 3D-QSAR analysis. These findings provide guidance for the design and structural modifications of these derivatives for better anti-HIV-1 activity which is important for the development of a new class of entry inhibitors.

Extracellular Matrix Protein Betaig-h3/TGFBI Promotes Metastasis of Colon Cancer by Enhancing Cell Extravasation

Genes & Development. Feb, 2008  |  Pubmed ID: 18245446

Metastasis, the major cause of cancer death, is a multistep process that requires interactions between cancer cells and stromal cells and between cancer cells and extracellular matrix. Molecular alterations of the extracellular matrix in the tumor microenvironment have a considerable impact on the metastatic process during tumorigenesis. Here we report that elevated expression of betaig-h3/TGFBI (transforming growth factor, beta-induced), an extracellular matrix protein secreted by colon cancer cells, is associated with high-grade human colon cancers. Ectopic expression of the betaig-h3 protein enhanced the aggressiveness and altered the metastatic properties of colon cancer cells in vivo. Inhibition of betaig-h3 expression dramatically reduced metastasis. Mechanistically, betaig-h3 appears to promote extravasation, a critical step in the metastatic dissemination of cancer cells, by inducing the dissociation of VE-cadherin junctions between endothelial cells via activation of the integrin alphavbeta5-Src signaling pathway. Thus, cancers associated with overexpression of betaig-h3 may have an increased metastatic potential, leading to poor prognosis in cancer patients.

Priming with RAAV Encoding RBD of SARS-CoV S Protein and Boosting with RBD-specific Peptides for T Cell Epitopes Elevated Humoral and Cellular Immune Responses Against SARS-CoV Infection

Vaccine. Mar, 2008  |  Pubmed ID: 18289745

Development of vaccines against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is crucial in the prevention of SARS reemergence. The receptor-binding domain (RBD) of SARS-CoV spike (S) protein is an important target in developing safe and effective SARS vaccines. Our previous study has demonstrated that vaccination with adeno-associated virus encoding RBD (RBD-rAAV) induces high titer of neutralizing antibodies. In this study, we further assessed the immune responses and protective effect of the immunization with RBD-rAAV prime/RBD-specific T cell peptide boost. Compared with the RBD-rAAV prime/boost vaccination, RBD-rAAV prime/RBD-peptide (RBD-Pep) boost induced similar levels of Th1 and neutralizing antibody responses that protected the vaccinated mice from subsequent SARS-CoV challenge, but stronger Th2 and CTL responses. No significant immune responses and protective effects were detected in mice vaccinated with RBD-Pep or blank AAV alone. Since T cell epitopes are highly conserved and boosting with peptides may induce the production of effector memory T cells, which may be effective against viruses with mutations in the neutralizing epitopes, our results suggest that the vaccination protocol used may be ideal for providing effective, broad and long-term protection against SARS-CoV infection.

Development of Subunit Vaccines Against Severe Acute Respiratory Syndrome

Drugs of Today (Barcelona, Spain : 1998). Jan, 2008  |  Pubmed ID: 18301805

Severe acute respiratory syndrome (SARS) is a novel infectious disease caused by SARS coronavirus (SARS-CoV). Although SARS appears to have been successfully contained, there is still a risk for its reemergence due to sporadic laboratory accidents or the presence of a natural reservoir for SARS-CoV-like virus. Therefore, the development of effective vaccines against SARS-CoV continues to be the current focus of SARS research. This review will first describe the rationale for developing safe and effective SARS vaccines, followed by elucidating viral antigens that could be used as potential vaccine components. After comparing current vaccine categories against SARS, this article will demonstrate the advantages of subunit vaccines, describe the current situation of developing subunit vaccines, and point out the possibility for further improvement of subunit SARS vaccines. This suggests that recombinant protein/peptide-based subunit vaccines containing the spike protein, especially the receptor-bind domain of SARS-CoV, could be developed as safe and effective SARS vaccines.

Design and Evaluation of Sifuvirtide, a Novel HIV-1 Fusion Inhibitor

The Journal of Biological Chemistry. Apr, 2008  |  Pubmed ID: 18303020

Enfuvirtide (T20) is the first and only HIV-1 fusion inhibitor approved for clinical use, but it can easily induce drug resistance limiting its practical application. A novel anti-HIV peptide, termed sifuvirtide, was designed based on the three-dimensional structure of the HIV-1 gp41 fusogenic core conformation. Here we report its in vitro anti-HIV potency, its mechanism of action, as well as the results from Phase Ia clinical studies. We demonstrated that sifuvirtide inhibited HIV-1-mediated cell-cell fusion in a dose-dependent manner and exhibited high potency against infections by a wide range of primary and laboratory-adapted HIV-1 isolates from multiple genotypes with R5 or X4 phenotypes. Notably, sifuvirtide was also highly effective against T20-resistant strains. Unlike T20, sifuvirtide could efficiently block six-helix bundle formation in a dominant negative fashion. These results suggest that sifuvirtide has a different mechanism of action from that of T20. Phase Ia clinical studies of sifuvirtide (FS0101) in 60 healthy individuals demonstrated good safety, tolerability, and pharmacokinetic profiles. A single dose regimen (5, 10, 20, 30, and 40 mg) by subcutaneous injection once daily at abdominal sites was well tolerated without serious adverse events. Pharmacokinetic studies of single and multiple administration of sifuvirtide showed that its decay half-lives were 20.0 +/- 8.6 h and 26.0 +/- 7.9 h, respectively. In summary, sifuvirtide has potential to become an ideal fusion inhibitor for treatment of HIV/AIDS patients, including those with HIV-1 strains resistant to T20.

Interaction of HIV-1 Gp41 Core with NPF Motif in Epsin: Implication in Endocytosis of HIV

The Journal of Biological Chemistry. May, 2008  |  Pubmed ID: 18375383

The human immunodeficiency virus, type 1 (HIV-1), gp41 core plays an important role in fusion between viral and target cell membranes. We previously identified an HIV-1 gp41 core-binding motif HXXNPF (where X is any amino acid residue). In this study, we found that Asn, Pro, and Phe were the key residues for gp41 core binding. There are two NPF motifs in Epsin-1-(470-499), a fragment of Epsin, which is an essential accessory factor of endocytosis that can dock to the plasma membrane by interacting with the lipid. Epsin-1-(470-499) bound significantly to the gp41 core formed by the polypeptide N36(L8)C34 and interacted with the recombinant soluble gp41 containing the core structure. A synthetic peptide containing the Epsin-1-(470-499) sequence could effectively block entry of HIV-1 virions into SupT1 T cells via the endocytosis pathway. These results suggest that interaction between Epsin and the gp41 core, which may be present in the target cell membrane, is probably essential for endocytosis of HIV-1, an alternative pathway of HIV-1 entry into the target cell.

Surface Exposure of the HIV-1 Env Cytoplasmic Tail LLP2 Domain During the Membrane Fusion Process: Interaction with Gp41 Fusion Core

The Journal of Biological Chemistry. Jun, 2008  |  Pubmed ID: 18408000

HIV-1 gp41 cytoplasmic tail (CT) is highly conserved among HIV-1 isolates, particularly the region designated lentivirus lytic peptide (LLP1-2), which includes two alpha-helical domains LLP1 and LLP2. Although the gp41 CT is recognized as a modulator of viral fusogenicity, little is known about the regulatory mechanism of this region in the viral fusion process. Here we report that anti-LLP1-2 and anti-LLP2 antibodies (IgG) inhibited HIV-1 Env-mediated cell fusion and bound to the interface between effector and target cells at a suboptimal temperature (31.5 degrees C), which slows down the fusion process and prolongs the fusion intermediate state. This suggests that LLP1-2, especially the LLP2 region located inside the viral membrane, is transiently exposed on the membrane surface during the fusion process. Synthetic LLP2 peptide could bind to the gp41 six-helix bundle core with high binding affinity. These results suggest that the gp41 CT may interact with the gp41 core, via the surface-exposed LLP2 domain, to regulate Env-mediated membrane fusion.

Identification of a Critical Motif for the Human Immunodeficiency Virus Type 1 (HIV-1) Gp41 Core Structure: Implications for Designing Novel Anti-HIV Fusion Inhibitors

Journal of Virology. Jul, 2008  |  Pubmed ID: 18417584

Human immunodeficiency virus type 1 (HIV-1) entry into the host cell involves a cascade of events and currently represents one of most attractive targets in the search for new antiviral drugs. The fusion-active gp41 core structure is a stable six-helix bundle (6-HB) folded by its trimeric N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR). Peptides derived from the CHR region of HIV-1 gp41 are potent fusion inhibitors that target the NHR to block viral and cellular membrane fusion in a dominant negative fashion. However, all CHR peptides reported to date are derived primarily from residues 628 to 673 of gp41; little attention has been paid to the upstream sequence of the pocket binding domain (PBD) in the CHR. Here, we have identified a motif ((621)QIWNNMT(627)) located at the upstream region of the gp41 CHR, immediately adjacent to the PBD ((628)WMEWEREI(635)). Biophysical characterization demonstrated that this motif is critical for the stabilization of the gp41 6-HB core. The peptide CP621-652, containing the (621)QIWNNMT(627) motif, was able to interact with T21, a counterpart peptide derived from the NHR, to form a typical 6-HB structure with a high thermostability (thermal unfolding transition [T(m)] value of 82 degrees C). In contrast, the 6-HB formed by the peptides N36 and C34, which has been considered to be a core structure of the fusion-active gp41, had a T(m) of 64 degrees C. Different from T-20 (brand name Fuseon), which is the first and only HIV-1 fusion inhibitor approved for clinical use, CP621-652 could efficiently block 6-HB formation in a dose-dependent manner. Significantly, CP621-652 had potent inhibitory activity against HIV-1-mediated cell-cell fusion and infection, especially against T-20- and C34-resistant virus. Therefore, our works provide important information for understanding the core structure of the fusion-active gp41 and for designing novel anti-HIV peptides.

Cross-reactive Human Immunodeficiency Virus Type 1-neutralizing Human Monoclonal Antibody That Recognizes a Novel Conformational Epitope on Gp41 and Lacks Reactivity Against Self-antigens

Journal of Virology. Jul, 2008  |  Pubmed ID: 18480433

Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.

Prolongation of Corneal Xenotransplant Survival by T-cell Vaccination-induced T-regulatory Cells

Xenotransplantation. May-Jun, 2008  |  Pubmed ID: 18611224

Corneal xenotransplantation is an alternative approach for overcoming shortage of allograft in clinics. However, the mechanism of acute corneal xenograft rejection and the method of prolonging xenograft survival have not been well defined.

Rationally Designed Anti-HIV Peptides Containing Multifunctional Domains As Molecule Probes for Studying the Mechanisms of Action of the First and Second Generation HIV Fusion Inhibitors

The Journal of Biological Chemistry. Oct, 2008  |  Pubmed ID: 18662985

We have previously shown that the first generation human immunodeficiency virus (HIV) fusion inhibitor T20 (Fuzeon) contains a critical lipid-binding domain (LBD), whereas C34, another anti-HIV peptide derived from the gp41 C-terminal heptad repeat, consists of an important pocket-binding domain (PBD), and both share a common 4-3 heptad repeat (HR) sequence (Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X., Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282, 9612-9620). T1249, the second generation HIV fusion inhibitor, has both LBD and PBD but a different HR sequence, suggesting that these three anti-HIV peptides may have distinct mechanisms of action. Here we rationally designed a set of peptides that contain multiple copies of a predicted HR sequence (5HR) or the HR sequence plus either LBD (4HR-LBD) or PBD (PBD-4HR) or both (PBD-3HR-LBD), and we compared their anti-HIV-1 activity and biophysical properties. We found that the peptide 5HR exhibited low-to-moderate inhibitory activity on HIV-1-mediated cell-cell fusion, whereas addition of LBD and/or PBD to the HR sequence resulted in a significant increase of the anti-HIV-1 activity. The peptides containing PBD, including PBD-4HR and PBD-3HR-LBD, could form a stable six-helix bundle with the N-peptide N46 and effectively blocked the gp41 core formation, whereas the peptides containing LBD, e.g. 4HR-LBD and PBD-3HR-LBD, could interact with the lipid vehicles. These results suggest that the HR sequence in these anti-HIV peptides acts as a structure domain and is responsible for its interaction with the HR sequence in N-terminal heptad repeat, whereas PBD and LBD are critical for interactions with their corresponding targets. T20, C34, and T1249 may function like 4HR-LBD, PBD-4HR, and PBD-3HR-LBD, respectively, to interact with different target sites for inhibiting HIV fusion and entry. Therefore, this study provides important information for understanding the mechanisms of action of the peptic HIV-1 fusion inhibitors and for rational design of novel antiviral peptides against HIV and other viruses with class I fusion proteins.

Evaluation of Recombinant Onchocerca Volvulus Activation Associated Protein-1 (ASP-1) As a Potent Th1-biased Adjuvant with a Panel of Protein or Peptide-based Antigens and Commercial Inactivated Vaccines

Vaccine. Sep, 2008  |  Pubmed ID: 18675867

Alum, the only adjuvant approved for clinical applications, can induce strong humoral (Th2) but weak cellular (Th1) immune responses. It is necessary to develop safe and effective adjuvants capable of inducing both humoral and cellular immune responses. We previously showed that activation-associated protein-1 (ASP-1) derived from Onchocerca volvulus has potent adjuvant activity. In this study, we have further evaluated the adjuvanticity of recombinant ASP-1 using a panel of recombinant proteins or synthetic peptide-based antigens, including ovalbumin (OVA), synthetic HIV peptide (HIV-p), recombinant HIV gp41 (rgp41) and HBV HBsAg, as well as three commercially available inactivated vaccines against haemorrhagic fever with renal syndrome (HFRS), Influenza and Rabies. Our results indicate that ASP-1 induced significantly higher IgG1 (Th2-associated) and IgG2a (Th1-associated) responses than alum adjuvant against OVA antigen, HIV-p, and rgp41. Consistently, it induced similar level of IgG1 responses as alum but higher level of IgG2a and IFN-gamma-producing T cell responses than alum adjuvant against HBsAg. Further, ASP-1 improved both IgG1 and IgG2a responses to three commercial inactivated vaccines when used separately or in combination. In conclusion, the recombinant ASP-1, unlike alum adjuvant, is able to induce both Th1 and Th2-associated humoral responses and Th1 cellular responses, suggesting that it can be further developed as a promising adjuvant for subunit-based and inactivated vaccines.

Novel Anti-HIV Peptides Containing Multiple Copies of Artificially Designed Heptad Repeat Motifs

Biochemical and Biophysical Research Communications. Oct, 2008  |  Pubmed ID: 18682248

The peptidic anti-HIV drug T20 (Fuzeon) and its analog C34 share a common heptad repeat (HR) sequence, but they have different functional domains, i.e., pocket- and lipid-binding domains (PBD and LBD, respectively). We hypothesize that novel anti-HIV peptides may be designed by using artificial sequences containing multiple copies of HR motifs plus zero, one or two functional domains. Surprisingly, we found that the peptides containing only the non-natural HR sequences could significantly inhibit HIV-1 infection, while addition of PBD and/or LBD to the peptides resulted in significant improvement of anti-HIV-1 activity. These results suggest that these artificial HR sequences, which may serve as structural domains, could be used as templates for the design of novel antiviral peptides against HIV and other viruses with class I fusion proteins.

Deletion of Fusion Peptide or Destabilization of Fusion Core of HIV Gp41 Enhances Antigenicity and Immunogenicity of 4E10 Epitope

Biochemical and Biophysical Research Communications. Nov, 2008  |  Pubmed ID: 18762167

The human monoclonal antibody 4E10 against the membrane-proximal external region (MPER) of HIV-1 gp41 demonstrates broad neutralizing activity across various strains, and makes its epitope an attractive target for HIV-1 vaccine development. Although the contiguous epitope of 4E10 has been identified, attempts to re-elicit 4E10-like antibodies have failed, possibly due to the lack of proper conformation of the 4E10 epitope. Here we used pIg-tail expression system to construct a panel of eukaryotic cell-surface expression plasmids encoding the extracellular domain of gp41 with deletion of fusion peptide and/or introduction of L568P mutation that may disrupt the gp41 six-helix bundle core conformation as DNA vaccines for immunization of mice. We found that these changes resulted in significant increase of the antigenicity and immunogenicity of 4E10 epitope. This information is thus useful for rational design of vaccines targeting the HIV-1 gp41 MPER.

Conserved Salt Bridge Between the N- and C-terminal Heptad Repeat Regions of the Human Immunodeficiency Virus Type 1 Gp41 Core Structure is Critical for Virus Entry and Inhibition

Journal of Virology. Nov, 2008  |  Pubmed ID: 18768964

The fusogenic human immunodeficiency virus type 1 (HIV-1) gp41 core structure is a stable six-helix bundle formed by its N- and C-terminal heptad repeat sequences. Notably, the negatively charged residue Asp(632) located at the pocket-binding motif in the C-terminal heptad repeat interacts with the positively charged residue Lys(574) in the pocket formation region of the N-terminal heptad repeat to form a salt bridge. We previously demonstrated that the residue Lys(574) plays an essential role in six-helix bundle formation and virus infectivity and is a key determinant of the target for anti-HIV fusion inhibitors. In this study, the functionality of residue Asp(632) has been specifically characterized by mutational analysis and biophysical approaches. We show that Asp(632) substitutions with positively charged residues (D632K and D632R) or a hydrophobic residue (D632V) could completely abolish Env-mediated viral entry, while a protein with a conserved substitution (D632E) retained its activity. Similar to the Lys(574) mutations, nonconserved substitutions of Asp(632) also severely impaired the alpha-helicity, stability, and conformation of six-helix bundles as shown by N36 and C34 peptides as a model system. Furthermore, nonconserved substitutions of Asp(632) significantly reduced the potency of C34 to sequestrate six-helix bundle formation and to inhibit HIV-1-mediated cell-cell fusion and infection, suggesting its importance for designing antiviral fusion inhibitors. Taken together, these data suggest that the salt bridge between the N- and C-terminal heptad repeat regions of the fusion-active HIV-1 gp41 core structure is critical for viral entry and inhibition.

Potent HIV Fusion Inhibitors Against Enfuvirtide-resistant HIV-1 Strains

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2008  |  Pubmed ID: 18852475

T20 (generic name: Enfuvirtide, brand name: Fuzeon) is the only FDA-approved HIV fusion inhibitor that is being used for treatment of HIV/AIDS patients who have failed to respond to current antiretroviral drugs. However, it rapidly induces drug resistance in vitro and in vivo. On the basis of the structural and functional information of anti-HIV peptides from a previous study, we designed an HIV fusion inhibitor named CP32M, a 32-mer synthetic peptide that is highly effective in inhibiting infection by a wide range of primary HIV-1 isolates from multiple genotypes with R5- or dual-tropic (R5X4) phenotype, including a group O virus (BCF02) that is resistant to T20 and C34 (another anti-HIV peptide). Strikingly, CP32M is exceptionally potent (at low picomolar level) against infection by a panel of HIV-1 mutants highly resistant to T20 and C34. These findings suggest that CP32M can be further developed as an antiviral therapeutic against multidrug resistant HIV-1.

Development and Preclinical Studies of Broad-spectrum Anti-HIV Agent (3'R,4'R)-3-cyanomethyl-4-methyl-3',4'-di-O-(S)-camphanoyl-(+)-cis-khellactone (3-cyanomethyl-4-methyl-DCK)

Journal of Medicinal Chemistry. Dec, 2008  |  Pubmed ID: 19053755

In prior investigation, we discovered that (3'R,4'R)-3-cyanomethyl-4-methyl-3',4'-di-O-(S)-camphanoyl-(+)-cis-khellactone (4, 3-cyanomethyl-4-methyl-DCK) showed promising anti-HIV activity. In these current studies, we developed and optimized successfully a practical 10-step synthesis for scale-up preparation to increase the overall yield of 4 from 7.8% to 32%. Furthermore, compound 4 exhibited broad-spectrum anti-HIV activity against wild-type and drug-resistant viral infection of CD4+ T cell lines as well as peripheral blood mononuclear cells by both laboratory-adapted and primary HIV-1 isolates with distinct subtypes and tropisms. Compound 4 was further subjected to in vitro and in vivo pharmacokinetic studies. These studies indicated that 4 has moderate cell permeability, moderate oral bioavailability, and low systemic clearance. These results suggest that 4 should be developed as a promising anti-HIV agent for development as a clinical trial candidate.

Design, Synthesis, and Biological Evaluation of N-carboxyphenylpyrrole Derivatives As Potent HIV Fusion Inhibitors Targeting Gp41

Journal of Medicinal Chemistry. Dec, 2008  |  Pubmed ID: 19053778

On the basis of the structures of small-molecule hits targeting the HIV-1 gp41, N-(4-carboxy-3-hydroxy)phenyl-2,5-dimethylpyrrole (2, NB-2), and N-(3-carboxy-4-chloro)phenylpyrrole (A(1), NB-64), 42 N-carboxyphenylpyrrole derivatives in two categories (A and B series) were designed and synthesized. We found that 11 compounds exhibited promising anti-HIV-1 activity at micromolar level and their antiviral activity was correlated with their inhibitory activity on gp41 six-helix bundle formation, suggesting that these compounds block HIV fusion and entry by disrupting gp41 core formation. The structure-activity relationship and molecular docking analysis revealed that the carboxyl group could interact with either Arg579 or Lys574 to form salt bridges and two methyl groups on the pyrrole ring were favorable for interaction with the residues in gp41 pocket. The most active compound, N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrrole (A(12)), partially occupied the deep hydrophobic pocket, suggesting that enlarging the molecular size of A(12) could improve its binding affinity and anti-HIV-1 activity for further development as a small-molecule HIV fusion and entry inhibitor.

Identification of Critical Antibody-binding Sites in the HIV-1 Gp41 Six-helix Bundle Core As Potential Targets for HIV-1 Fusion Inhibitors

Immunobiology. 2009  |  Pubmed ID: 19159827

Formation of the six-helix bundle (6-HB) core between the N- and C-terminal heptad repeats (NHR and CHR) regions of the HIV-1 envelope glycoprotein (Env) transmembrane subunit gp41 is a critical step during the process of virus and target cell membrane fusion. In the present study, we generated a panel of five monoclonal antibodies (mAbs) which specifically recognized the HIV-1 gp41 6-HB formed by the NHR-peptide N36 and CHR-peptide C34 mixture, but did not react with the isolated peptides N36 and C34. These mAbs did not block the HIV-1 Env-mediated cell-cell fusion at physiological temperature (37 degrees C), but inhibited the HIV-1 Env-mediated cell-cell fusion at suboptimal temperature (31.5 degrees C), under which condition the fusion process is slowed down and the viral 6-HB becomes accessible. The fusion inhibitory activity of the mAbs is correlated with their binding affinity with the 6-HB core. By screening 24 6-HB variants with single mutations at the b, c, and f positions in the helical wheels, we found that the critical binding sites of these mAbs were localized in the N-terminal region of the NHR and the C-terminal region of the CHR. These sites may serve as targets for design of small molecule HIV fusion inhibitors, e.g., organic compounds, peptides, and low molecular weight proteins.

The Spike Protein of SARS-CoV--a Target for Vaccine and Therapeutic Development

Nature Reviews. Microbiology. Mar, 2009  |  Pubmed ID: 19198616

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease caused by a novel coronavirus, SARS-coronavirus (SARS-CoV). The SARS-CoV spike (S) protein is composed of two subunits; the S1 subunit contains a receptor-binding domain that engages with the host cell receptor angiotensin-converting enzyme 2 and the S2 subunit mediates fusion between the viral and host cell membranes. The S protein plays key parts in the induction of neutralizing-antibody and T-cell responses, as well as protective immunity, during infection with SARS-CoV. In this Review, we highlight recent advances in the development of vaccines and therapeutics based on the S protein.

Synergistic Efficacy of Combination of Enfuvirtide and Sifuvirtide, the First- and Next-generation HIV-fusion Inhibitors

AIDS (London, England). Mar, 2009  |  Pubmed ID: 19242316

Enfuvirtide and sifuvirtide, the first- and next-generation HIV-fusion inhibitors, contain different functional domains and have distinct target sites. Here, we found that a combination of enfuvirtide and sifuvirtide resulted in potent synergism in inhibiting HIV-1-mediated cell-cell fusion and infection by X4 and R5 as well as enfuvirtide-resistant HIV-1 strains. These findings suggest that application of enfuvirtide and sifuvirtide in combination may improve their efficacy and resistant profile, leading to a reduction of the dosage and frequency of drug use.

Recombinant Ov-ASP-1, a Th1-biased Protein Adjuvant Derived from the Helminth Onchocerca Volvulus, Can Directly Bind and Activate Antigen-presenting Cells

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2009  |  Pubmed ID: 19299698

We previously reported that rOv-ASP-1, a recombinant Onchocerca volvulus activation associated protein-1, was a potent adjuvant for recombinant protein or synthetic peptide-based Ags. In this study, we further evaluated the adjuvanticity of rOv-ASP-1 and explored its mechanism of action. Consistently, recombinant full-length spike protein of SARS-CoV or its receptor-binding domain in the presence of rOv-ASP-1 could effectively induce a mixed but Th1-skewed immune response in immunized mice. It appears that rOv-ASP-1 primarily bound to the APCs among human PBMCs and triggered Th1-biased proinflammatory cytokine production probably via the activation of monocyte-derived dendritic cells and the TLR, TLR2, and TLR4, thus suggesting that rOv-ASP-1 is a novel potent innate adjuvant.

Antigenicity and Immunogenicity of SARS-CoV S Protein Receptor-binding Domain Stably Expressed in CHO Cells

Biochemical and Biophysical Research Communications. Jul, 2009  |  Pubmed ID: 19422787

The receptor-binding domain (RBD) of SARS coronavirus (SARS-CoV) spike (S) protein contains multiple conformation-dependent epitopes that induce neutralizing antibody responses. Here we used CHO-K1 cells to establish a cell line for stable expression of a 193-mer (residues 318-510) RBD (RBD193-CHO) and determined its antigenicity and immunogenicity. We found that RBD193-CHO reacted strongly with a panel of six monoclonal antibodies recognizing various conformational and linear epitopes in RBD, suggesting that this recombinant protein maintains intact conformation and good antigenicity. Immunization of mice with RBD193-CHO resulted in induction of high titers of RBD-specific neutralizing antibodies and potent IL-4-expressing T cell responses. RBD193-CHO induced immunity that protected a majority of the vaccinated mice from SARS-CoV challenge. These results suggest that the recombinant RBD produced in an established stable cell line maintains strong immunogenicity with high potential for use as an effective and economic subunit SARS vaccine.

Phenotypic and Genotypic Characterization of Human Immunodeficiency Virus Type 1 CRF07_BC Strains Circulating in the Xinjiang Province of China

Retrovirology. 2009  |  Pubmed ID: 19442296

HIV-1 CRF07_BC recombinant previously circulated mainly among the intravenous drug users (IDUs) in Xinjiang province of China and is currently spreading in the entire country. The aim of this study is to characterize the genotypic and phenotypic properties of HIV-1 CRF07_BC isolates in comparison with those of the subtype B' (Thailand B) which is prevalent in the former plasma donors (FPDs) in China.

High Genetic and Antigenic Similarity Between a Swine H3N2 Influenza A Virus and a Prior Human Influenza Vaccine Virus: a Possible Immune Pressure-driven Cross-species Transmission

Biochemical and Biophysical Research Communications. Jul, 2009  |  Pubmed ID: 19463787

In late April of 2009, a global outbreak of human influenza was reported. The causative agent is a highly unusual reassortant H1N1 influenza virus carrying genetic segments derived from swine, human and avian influenza viruses. In this study, we compared the HA, NA and other gene segments of a swine H3N2 influenza A virus, A/Swine/Guangdong/z5/2003, which was isolated from pigs in 2003 in Guangdong Province, China, to the predominant human and swine H3N2 viruses. We found that the similarity of gene segments of A/Swine/Guangdong/z5/2003 was closer to Moscow/99-like human H3N2 virus than Europe swine H3N2 viruses during 1999-2002. These results suggest that A/Swine/Guangdong/z5/2003 may be porcine in origin, possibly being driven by human immune pressure induced by either natural H3N2 virus infection or use of A/Moscow/10/99 (H3N2)-based human influenza vaccine. The results further confirm that swine may play a dual role as a "shelter" for hosting influenza virus from humans or birds and as a "mixing vessel" for generating reassortant influenza viruses, such as the one causing current influenza pandemic.

Drug-resistant Viruses May Repair Impaired Fitness by Mutations Outside the Drug Target Site

Future Microbiology. Jun, 2009  |  Pubmed ID: 19492961

Evaluation of: Ray N, Blackburn LA, Doms RW: HR-2 mutations in human immunodeficiency virus type 1 gp41 restore fusion kinetics delayed by HR-1 mutations that cause clinical resistance to enfuvirtide. J. Virol. 83(7), 2989-2995 (2009). During the treatment of HIV infection with viral fusion inhibitors derived from HIV-1 gp41 heptad repeat (HR)-2 regions, drug-induced mutations have been observed not only in the HR-1 region that contains the target sites for the inhibitors, but also the HR-2 region, which is outside the inhibitor target site. Using a kinetic cell-cell fusion assay, Ray et al. have demonstrated that the HR-1 mutation, which helps viruses to escape from treatment challenge, significantly delays fusion kinetics, leaving viruses more vulnerable to the host immune defense system, thus reducing the fitness of the virus. However, the mutations in the HR-2 region restore the delayed fusion kinetics close to its original level, partially repairing the impaired fitness caused by the mutations in the drug target site. The work sheds new light on the mechanism of HIV-1 drug resistance, which may be used for the development of new HIV fusion inhibitors with improved efficacy and drug resistance profiles.

Combinations of the First and Next Generations of Human Immunodeficiency Virus (HIV) Fusion Inhibitors Exhibit a Highly Potent Synergistic Effect Against Enfuvirtide- Sensitive and -resistant HIV Type 1 Strains

Journal of Virology. Aug, 2009  |  Pubmed ID: 19493996

T20 (generic name, enfuvirtide; brand name, Fuzeon) is a first-generation human immunodeficiency virus (HIV) fusion inhibitor approved for salvage therapy of HIV-infected patients refractory to current antiretroviral drugs. However, its clinical use is limited because of rapid emergence of T20-resistant viruses in T20-treated patients. Therefore, T1249 and T1144 are being developed as the second- and third-generation HIV fusion inhibitors, respectively, with improved efficacy and drug resistance profiles. Here, we found that combinations of T20 with T1249 and/or T1144 resulted in exceptionally potent synergism (combination index, <0.01) against HIV-1-mediated membrane fusion by 2 to 3 orders of magnitude in dose reduction. Highly potent synergistic antiviral efficacy was also achieved against infection by laboratory-adapted and primary HIV-1 strains, including T20-resistant variants. The mechanism underlying the synergistic effect could be attributed to the fact that T20, T1249, and T1144 all contain different functional domains and have different primary binding sites in gp41. As such, they may work cooperatively to inhibit gp41 six-helix bundle core formation, thereby suppressing virus-cell fusion. Therefore, these findings strongly imply that, rather than replacing T20, combining it with HIV fusion inhibitors of different generations might produce synergistic activity against both T20-sensitive and -resistant HIV-1 strains, suggesting a new therapeutic strategy for the treatment of HIV-1 infection/AIDS.

Discovery of Diarylpyridine Derivatives As Novel Non-nucleoside HIV-1 Reverse Transcriptase Inhibitors

Bioorganic & Medicinal Chemistry Letters. Sep, 2009  |  Pubmed ID: 19666220

Two series (4 and 5) of diarylpyridine derivatives were designed, synthesized, and evaluated for anti-HIV-1 activity. The most promising compound, 5e, inhibited HIV-1 IIIB, NL4-3, and RTMDR1 with low nanomolar EC50 values and selectivity indexes of >10,000. The results of this study indicate that diarylpyridine can be used as a novel scaffold to derive a new class of potent NNRTIs, active against both wild-type and drug-resistant HIV-1 strains.

Recombinant Receptor-binding Domain of SARS-CoV Spike Protein Expressed in Mammalian, Insect and E. Coli Cells Elicits Potent Neutralizing Antibody and Protective Immunity

Virology. Oct, 2009  |  Pubmed ID: 19683779

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease. The potential recurrence of the disease from animal reservoirs highlights the significance of development of safe and efficient vaccines to prevent a future SARS epidemic. In this study, we expressed the recombinant receptor-binding domain (rRBD) in mammalian (293T) cells, insect (Sf9) cells, and E. coli, respectively, and compared their immunogenicity and protection against SARS-CoV infection in an established mouse model. Our results show that all rRBD proteins expressed in the above systems maintained intact conformation, being able to induce highly potent neutralizing antibody responses and complete protective immunity against SARS-CoV challenge in mice, albeit the rRBD expressed in 293T cells elicited stronger humoral immune responses with significantly higher neutralizing activity (P<0.05) than those expressed in Sf9 and E. coli cells. These results suggest that all three rRBDs are effective in eliciting immune responses and protection against SARS-CoV and any of the above expression systems can be used for production of rRBD-based SARS subunit vaccines. Preference will be given to rRBD expressed in mammalian cells for future evaluation of the vaccine efficacy in a non-human primate model of SARS because of its ability to refold into a native conformation more readily and to induce higher level of neutralizing antibody responses than those expressed in E. coli and insect cells.

Design, Synthesis, and Structure-activity Relationship of a Novel Series of 2-aryl 5-(4-oxo-3-phenethyl-2-thioxothiazolidinylidenemethyl)furans As HIV-1 Entry Inhibitors

Journal of Medicinal Chemistry. Dec, 2009  |  Pubmed ID: 19746983

We previously identified two small molecules targeting the HIV-1 gp41, N-(4-carboxy-3-hydroxy)phenyl-2,5-dimethylpyrrole 12 (NB-2) and N-(3-carboxy-4-chloro)phenylpyrrole 13 (NB-64), that inhibit HIV-1 infection at low micromolar levels. On the basis of molecular docking analysis, we designed a series of 2-aryl 5-(4-oxo-3-phenethyl-2-thioxothiazolidinylidenemethyl)furans. Compared with 12 and 13, these compounds have bigger molecular size (437-515 Da) and could occupy more space in the deep hydrophobic pocket on the gp41 NHR trimer. Fifteen 2-aryl 5-(4-oxo-3-phenethyl-2-thioxothiazolidinylidenemethyl)furans (11a-o) were synthesized by Suzuki-Miyaura cross-coupling followed by a Knoevenagel condensation and tested for their anti-HIV-1 activity and cytotoxicity on MT-2 cells. We found that all 15 compounds had improved anti-HIV-1 activity and 3 of them (11a, 11b, and 11d) exhibited inhibitory activity against replication of HIV-1(IIIB) and 94UG103 at <100 nM range, more than 20-fold more potent than 12 and 13, suggesting that these compounds can serve as leads for development of novel small molecule HIV fusion inhibitors.

ADS-J1 Inhibits Human Immunodeficiency Virus Type 1 Entry by Interacting with the Gp41 Pocket Region and Blocking Fusion-active Gp41 Core Formation

Antimicrobial Agents and Chemotherapy. Dec, 2009  |  Pubmed ID: 19786602

We previously identified a small-molecule anti-human immunodeficiency virus type 1 (anti-HIV-1) compound, ADS-J1, using a computer-aided molecular docking technique for primary screening and a sandwich enzyme-linked immunosorbent assay (ELISA) as a secondary screening method. In the present study, we demonstrated that ADS-J1 is an HIV-1 entry inhibitor, as determined by a time-of-addition assay and an HIV-1-mediated cell fusion assay. Further mechanism studies confirmed that ADS-J1 does not block gp120-CD4 binding and exhibits a marginal interaction with the HIV-1 coreceptor CXCR4. However, ADS-J1 inhibited the fusion-active gp41 core formation mimicked by peptides derived from the viral gp41 N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), as determined by ELISA, native polyacrylamide gel electrophoresis, and circular dichroism analysis. Moreover, using a surface plasmon resonance assay, we found that ADS-J1 could bind directly to IQN17, a trimeric peptide containing the gp41 pocket region, resulting in the conformational change of IQN17 and the blockage of its interaction with a short D peptide, PIE7. The positively charged residue (K574) located in the gp41 pocket region is critical for the binding of ADS-J1 to NHR. These results suggest that ADS-J1 may bind to the viral gp41 NHR region through its hydrophobic and ionic interactions with the hydrophobic and positively charged resides located in the pocket region, subsequently blocking the association between the gp41 NHR and CHR regions to form the fusion-active gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry.

E14-F55 Combination in M2 Protein: a Putative Molecular Determinant Responsible for Swine-origin Influenza A Virus Transmission in Humans

PLoS Currents. 2009  |  Pubmed ID: 20029615

The species-specific signatures of the swine-origin influenza A (H1N1) virus (S-OIV), which caused the current influenza pandemic, have not been well defined. By comparing the protein sequences of S-OIVs with those of swine, avian and human influenza viruses, we found that that almost all human IAVs and those causing influenza pandemics, including the 2009 S-OIVs, had a combination of glutamic acid (E) residue at position 14 and phenylalanine (F) residue at position 55 in their M2 protein, while only 22% and 4% of the swine and avian IAVs had the E14-F55 combination. These finding suggests that E14-F55 combination in the M2 protein of S-OIV may be a molecular determinant associated with its human-to-human transmission.

Research and Development of Universal Influenza Vaccines

Microbes and Infection / Institut Pasteur. Apr, 2010  |  Pubmed ID: 20079871

The continuous threat of influenza pandemics determines the urgency and necessity to develop safe and effective vaccines against divergent influenza viruses. This review describes the advancements in the research and development of universal influenza vaccines based on the relatively conserved sequences of M2e, HA, and other proteins of influenza viruses.

An M2e-based Multiple Antigenic Peptide Vaccine Protects Mice from Lethal Challenge with Divergent H5N1 Influenza Viruses

Virology Journal. 2010  |  Pubmed ID: 20082709

A growing concern has raised regarding the pandemic potential of the highly pathogenic avian influenza (HPAI) H5N1 viruses. Consequently, there is an urgent need to develop an effective and safe vaccine against the divergent H5N1 influenza viruses. In the present study, we designed a tetra-branched multiple antigenic peptide (MAP)-based vaccine, designated M2e-MAP, which contains the sequence overlapping the highly conserved extracellular domain of matrix protein 2 (M2e) of a HPAI H5N1 virus, and investigated its immune responses and cross-protection against different clades of H5N1 viruses.

Association of Candidate Susceptible Loci with Chronic Infection with Hepatitis B Virus in a Chinese Population

Journal of Medical Virology. Mar, 2010  |  Pubmed ID: 20087947

A number of genetic loci have been proposed to be associated with persistent hepatitis B virus (HBV) infection. This study aimed to evaluate the association and interaction of susceptible genes with HBV persistence in a Chinese population. A total of 17 polymorphisms in 9 candidate genes were studied in 361 Chinese chronic hepatitis B patients and 304 patients who recovered spontaneously. Distributions of susceptible polymorphisms were examined in healthy Chinese and Caucasian populations. Gene-gene interactions were tested by the multifactor dimensionality reduction (MDR) method. The TNF -308 G/G genotype and G allele, IL-10RB codon 47 A allele, and MCP-1 -2518 G/G genotype and G allele were more frequent in patients than controls (P < 0.01, after multiple corrections Pc < 0.05), while the frequencies of TNF -308 A/G genotype and IL-10 -592 A/A genotype were significantly higher in controls than in the patient group (Pc < 0.05). The frequencies of the risk allele MCP-1 -2518 G and CTLA4 6230 G were much higher in Chinese than in the Caucasian groups (P < 0.001). An interaction between CCR5 -2459, TNFA -863, IL-10RB codon 47, and MCP-1 -2518 was detected by MDR (P = 0.001). The results indicate that genetic determinants may affect the outcome of HBV infection in both independent and synergic manners. J. Med. Virol. 82:371-378, 2010. (c) 2010 Wiley-Liss, Inc.

HIV Type 1 B'/C Recombinant (CRF07_BC) in Virologic Noncontrollers Elicits Neutralizing Antibodies Against Heterologous but Not Autologous Viruses

AIDS Research and Human Retroviruses. Feb, 2010  |  Pubmed ID: 20156107

3-hydroxyphthalic Anhydride-modified Chicken Ovalbumin Exhibits Potent and Broad Anti-HIV-1 Activity: a Potential Microbicide for Preventing Sexual Transmission of HIV-1

Antimicrobial Agents and Chemotherapy. May, 2010  |  Pubmed ID: 20194691

Heterosexual transmission is the primary route by which women acquire human immunodeficiency virus (HIV)/AIDS. Thus, development of woman-controlled topical microbicides for prevention of sexual transmission of HIV is urgently needed. Here we report that 3-hydroxyphthalic anhydride-modified chicken ovalbumin (HP-OVA) exhibits potent antiviral activity against a broad spectrum of human immunodeficiency virus type 1 (HIV-1) isolates with different genotypes and biotypes. Its antiviral activity is correlated with the percentages of the chemically modified and unmodified lysines and arginines in OVA. HP-OVA inhibits HIV-1 fusion and entry through multiple mechanisms of action, including (i) blocking gp120 binding to CD4 and (ii) interfering with gp41 six-helix bundle formation. Because of the widespread availability and established safety profile of OVA, HP-OVA has good potential to be developed as an effective, safe, and affordable microbicide for prevention of HIV sexual transmission.

HIV-1 Gp41 Fusion Intermediate: a Target for HIV Therapeutics

Journal of the Formosan Medical Association = Taiwan Yi Zhi. Feb, 2010  |  Pubmed ID: 20206833

Human immunodeficiency virus (HIV)-1 infection is initiated by the binding of gp120 envelope glyco-protein to its cell receptor (CD4) and a coreceptor (CXCR4 or CCR5), followed by a series of conformational changes in the gp41 transmembrane subunit. These changes include insertion of fusion peptide into the target cell membrane and association of C-heptad repeat (CHR) peptide with the N-heptad repeat (NHR) trimer, a pre-hairpin fusion intermediate. A stable six-helix bundle core is then formed, bringing the viral envelope and target cell membrane into close proximity for fusion. Peptides derived from the CHR region, such as T20 and C34, inhibit HIV-1 fusion by interacting with the gp41 fusion intermediate. A number of anti-HIV-1 peptides and small molecule compounds targeting the gp41 NHR-trimer have been identified. By combining HIV fusion/entry inhibitors targeting different sites in the gp41 fusion intermediate, a potent synergistic effect takes place, resulting in a potential new therapeutic strategy for the HIV infection/AIDS. Here, we present an overview of the current development of anti-HIV drugs, particularly those targeting the gp41 fusion intermediate.

Genomic Signature and Mutation Trend Analysis of Pandemic (H1N1) 2009 Influenza A Virus

PloS One. 2010  |  Pubmed ID: 20221396

A novel swine-origin pandemic influenza A(H1N1) virus (H1N1pdm, also referred to as S-OIV) was identified as the causative agent of the 21(st) century's first influenza pandemic, but molecular features conferring its ability of human-to-human transmission has not been identified. Here we compared the protein sequences of 2009 H1N1pdm strains with those causing other pandemics and the viruses isolated from humans, swines and avians, and then analyzed the mutation trend of the residues at the signature and non-signature positions, which are species- and non-species-associated, respectively, in the proteins of H1N1pdm during the pandemic of 2009. We confirmed that the host-specific genomic signatures of 2009 H1N1pdm, which are mainly swine-like, were highly identical to those of the 1918 H1N1pdm. During the short period of time when the pandemic alert level was raised from phase 4 to phase 6, one signature residue at the position of NP-100 mutated from valine to isoleucine. Four non-signature residues, at positions NA-91, NA-233, HA-206, and NS1-123, also changed during the epidemic in 2009. All these mutant residues, except that at NA-91, are located in the viral functional domains, suggesting that they may play roles in the human adaption and virulence of 2009 H1N1pdm.

Potent and Broad Neutralizing Activity of a Single Chain Antibody Fragment Against Cell-free and Cell-associated HIV-1

MAbs. May-Jun, 2010  |  Pubmed ID: 20305395

Several human monoclonal antibodies (hmAbs) exhibit relatively potent and broad neutralizing activity against HIV-1, but there has not been much success in using them as potential therapeutics. We have previously hypothesized and demonstrated that small engineered antibodies can target highly conserved epitopes that are not accessible by full-size antibodies. However, their potency has not been comparatively evaluated with known HIV-1-neutralizing hmAbs against large panels of primary isolates. We report here the inhibitory activity of an engineered single chain antibody fragment (scFv), m9, against several panels of primary HIV-1 isolates from group M (clades A-G) using cell-free and cell-associated virus in cell line-based assays. M9 was much more potent than scFv 17b, and more potent than or comparable to the best-characterized broadly neutralizing hmAbs IgG(1) b12, 2G12, 2F5 and 4E10. It also inhibited cell-to-cell transmission of HIV-1 with higher potency than enfuvirtide (T-20, Fuzeon). M9 competed with a sulfated CCR5 N-terminal peptide for binding to gp120-CD4 complex, suggesting an overlapping epitope with the coreceptor binding site. M9 did not react with phosphatidylserine (PS) and cardiolipin (CL), nor did it react with a panel of autoantigens in an antinuclear autoantibody (ANA) assay. We further found that escape mutants resistant to m9 did not emerge in an immune selection assay. These results suggest that m9 is a novel anti-HIV-1 candidate with potential therapeutic or prophylactic properties, and its epitope is a new target for drug or vaccine development.

A 219-mer CHO-expressing Receptor-binding Domain of SARS-CoV S Protein Induces Potent Immune Responses and Protective Immunity

Viral Immunology. Apr, 2010  |  Pubmed ID: 20374001

Development of vaccines is essential for the prevention of future recurrences of severe acute respiratory syndrome (SARS), caused by the SARS coronavirus (SARS-CoV). The spike (S) protein, especially receptor-binding domain (RBD) of SARS-CoV, plays important roles in the prevention of SARS infection, and is thus an important component in SARS vaccine development. In this study, we expressed a 219-mer (residues 318-536) RBD protein in Chinese hamster ovary (CHO)-K1 cells (RBD219-CHO), and tested its immune responses and protective immunity in a mouse model. The results showed that this recombinant protein was correctly folded, being able to maintain intact conformation and authentic antigenicity. It could induce strong humoral and cellular immune responses and high titers of neutralizing antibodies in the vaccinated mice. RBD219-CHO protein elicited potent protective immunity that protected all vaccinated mice from SARS-CoV challenge. These results suggest that the recombinant RBD219-CHO protein has great potential for the development of an effective and safe SARS subunit vaccine.

Maleic Anhydride-modified Chicken Ovalbumin As an Effective and Inexpensive Anti-HIV Microbicide Candidate for Prevention of HIV Sexual Transmission

Retrovirology. 2010  |  Pubmed ID: 20420669

Previous studies have shown that 3-hydroxyphthalic anhydride (HP)-modified bovine milk protein, beta-lactoglobulin (beta-LG), is a promising microbicide candidate. However, concerns regarding the potential risk of prion contamination in bovine products and carcinogenic potential of phthalate derivatives were raised. Here we sought to replace bovine protein with an animal protein of non-bovine origin and substitute HP with another anhydride for the development of anti-HIV microbicide for preventing HIV sexual transmission.

Diarylaniline Derivatives As a Distinct Class of HIV-1 Non-nucleoside Reverse Transcriptase Inhibitors

Journal of Medicinal Chemistry. Jul, 2010  |  Pubmed ID: 20527972

By using structure-based drug design and isosteric replacement, diarylaniline and 1,5-diarylbenzene-1,2-diamine derivatives were synthesized and evaluated against wild type HIV-1 and drug-resistant viral strains, resulting in the discovery of diarylaniline derivatives as a distinct class of next-generation HIV-1 non-nucleoside reverse transcriptase inhibitor (NNRTI) agents. The most promising compound 37 showed significant EC(50) values of 0.003-0.032 microM against HIV-1 wild-type strains and of 0.005-0.604 microM against several drug-resistant strains. Current results also revealed important structure-activity relationship (SAR) conclusions for diarylanilines and strongly support our hypothesis that an NH(2) group on the central benzene ring ortho to the aniline moiety is crucial for interaction with K101 of the NNRTI binding site in HIV-1 RT, likely by forming H-bonds with K101. Furthermore, molecular modeling studies with molecular mechanism/general Born surface area (MM/GBSA) technology demonstrated the rationality of our hypothesis.

Novel Recombinant Engineered Gp41 N-terminal Heptad Repeat Trimers and Their Potential As Anti-HIV-1 Therapeutics or Microbicides

The Journal of Biological Chemistry. Aug, 2010  |  Pubmed ID: 20538590

Peptides derived from N-terminal heptad repeat (NHR) of the HIV-1 gp41 are generally poor inhibitors of HIV-1 entry, because they tend to aggregate and do not form a trimeric coiled-coil. In this study, we have fused portions of gp41 NHR, e.g. N36 or N28, to the T4 fibritin trimerization domain, Foldon (Fd), thus constructing novel NHR trimers, designated N36Fd or N28Fd, which could be expressed in Escherichia coli cells. The purified N36Fd and N28Fd exhibited SDS-resistant trimeric coiled-coil conformation with improved alpha-helicity compared with the corresponding N-peptides. They could interact with a C-peptide (e.g. C34) to form stable six-helix bundle and possessed potent anti-HIV-1 activity against a broad spectrum of HIV-1 strains. N28Fd was effective against T20-resistant HIV-1 variants and more resistant to proteinase K compared with T20 (enfuvirtide), a C-peptide-based HIV fusion inhibitor. Therefore, N28Fd trimer has great potentials for further development as an affordable therapeutic or microbicide for treatment and prevention of HIV-1 infection.

Identification of a Gp41 Core-binding Molecule with Homologous Sequence of Human TNNI3K-like Protein As a Novel Human Immunodeficiency Virus Type 1 Entry Inhibitor

Journal of Virology. Sep, 2010  |  Pubmed ID: 20592080

Human immunodeficiency virus type 1 (HIV-1) gp41 plays a critical role in the viral fusion process, and its N- and C-terminal heptad repeat domains serve as important targets for developing anti-HIV-1 drugs, like T-20 (generic name, enfuvirtide; brand name, Fuzeon). Here, we conducted a yeast two-hybrid screening on a human bone marrow cDNA library using the recombinant soluble gp41 ectodomain as the bait and identified a novel gp41 core-binding molecule, designated P20. P20 showed no homology with a current HIV fusion inhibitor, T-20, but had sequence homology to a human protein, troponin I type 3 interacting kinase (TNNI3K)-like protein. While it could bind to the six-helix bundle core structure formed by the N- and C-terminal heptad repeats, P20 did not interrupt the formation of the six-helix bundle. P20 was effective in blocking HIV-1 Env-mediated syncytium formation and inhibiting infection by a broad spectrum of HIV-1 strains with distinct subtypes and coreceptor tropism, while it was ineffective against other enveloped viruses, such as vesicular stomatitis virus and influenza A virus. P20 exhibited no significant cytotoxicity to the CD4(+) cells that were used for testing antiviral activity. Among the 11 P20 mutants, four analogous peptides with a common motif (WGRLEGRRT) exhibited significantly reduced anti-HIV-1 activity, suggesting that this region is the critical active site of P20. Therefore, this peptide can be used as a lead for developing novel HIV fusion inhibitors and as a probe for studying the membrane-fusogenic mechanism of HIV.

Tricyclononene Carboxamide Derivatives As Novel Anti-HIV-1 Agents

European Journal of Medicinal Chemistry. Sep, 2010  |  Pubmed ID: 20598780

By modifying the chemical structure of anti-orthopoxvirus compound ST-246, we designed and synthesized a series of tricyclononene carboxamide derivatives and tested their anti-HIV-1 activity and cytotoxicity. We found that benzoimidazol-containing compound 7g was highly effective in inhibiting HIV-1 R5 infection with an IC(50) value of 0.41 microM and a selectivity index of 292, but it exhibited no significant inhibitory activity on HIV-1 reverse transcriptase, integrase and protease. CoMFA was used to analyze structure-activity relationships with good predictive power (r(2) = 0.921; q(2) = 0.582). Moreover, the CoMFA model showed that the length of the molecule, the amide, and the amine moieties all played crucial roles in anti-HIV activity. These results suggest that 7g may serve as a lead for the development of novel anti-HIV-1 therapies.

A Recombinant Mimetics of the HIV-1 Gp41 Prehairpin Fusion Intermediate Fused with Human IgG Fc Fragment Elicits Neutralizing Antibody Response in the Vaccinated Mice

Biochemical and Biophysical Research Communications. Jul, 2010  |  Pubmed ID: 20599765

HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

Development of a Safe and Convenient Neutralization Assay for Rapid Screening of Influenza HA-specific Neutralizing Monoclonal Antibodies

Biochemical and Biophysical Research Communications. Jul, 2010  |  Pubmed ID: 20617558

The worldwide outbreak of the swine-origin 2009 H1N1 influenza A virus (IAV) and an increasing number of influenza cases caused by a highly pathogenic avian influenza (HPAI) H5N1 have accelerated the need to develop vaccines and antiviral agents against IAVs. Among various antivirals, neutralizing monoclonal antibodies (mAbs) are considered important passive therapeutics having an immediate effect against viral pathogens. Here we report a pseudovirus neutralization assay for rapid screening of neutralizing mAbs targeting hemagglutinin (HA) of H5N1 and H1N1 IAV. In this study, we generated six pseudoviruses with an HIV-1 backbone, respectively, expressing HA of four clades of H5N1 IAV and the 2009 epidemic H1N1 IAV. The resulting pseudoviruses were able to infect a variety of human and non-human cells, with 293T cells from human kidney as the most susceptible target cells. Using the established pseudovirus neutralization assay, we showed that three of ten selected mAbs specific to HA could potently neutralize infection of a pseudovirus bearing HA from the homologous IAV A/VietNam/1194/2004(H5N1) strain. This was highly consistent with the result of a microneutralization assay testing the same strain of a live IAV. Since the pseudovirus neutralization assay does not involve an infectious virus and can be performed without the requirement of a biosafety-3 laboratory, it may be applied for safe and rapid screening of neutralizing mAbs and antiviral agents targeting HA of IAVs.

An H5N1 M2e-based Multiple Antigenic Peptide Vaccine Confers Heterosubtypic Protection from Lethal Infection with Pandemic 2009 H1N1 Virus

Virology Journal. 2010  |  Pubmed ID: 20624292

A 2009 global influenza pandemic caused by a novel swine-origin H1N1 influenza A virus has posted an increasing threat of a potential pandemic by the highly pathogenic avian influenza (HPAI) H5N1 virus, driving us to develop an influenza vaccine which confers cross-protection against both H5N1 and H1N1 viruses. Previously, we have shown that a tetra-branched multiple antigenic peptide (MAP) vaccine based on the extracellular domain of M2 protein (M2e) from H5N1 virus (H5N1-M2e-MAP) induced strong immune responses and cross-protection against different clades of HPAI H5N1 viruses. In this report, we investigated whether such M2e-MAP presenting the H5N1-M2e consensus sequence can afford heterosubtypic protection from lethal challenge with the pandemic 2009 H1N1 virus.

Development of a Safe and Convenient Neutralization Assay for Rapid Screening of Influenza HA-specific Neutralizing Monoclonal Antibodies

Biochemical and Biophysical Research Communications. Jun, 2010  |  Pubmed ID: 20685351

The worldwide outbreak of the swine-origin 2009 H1N1 influenza A virus (IAV) and an increasing number of influenza cases caused by a highly pathogenic avian influenza (HPAI) H5N1 have accelerated the need to develop vaccines and antiviral agents against IAVs. Among various antivirals, neutralizing monoclonal antibodies (mAbs) are considered important passive therapeutics having an immediate effect against viral pathogens. Here we report a pseudovirus neutralization assay for rapid screening of neutralizing mAbs targeting hemagglutinin (HA) of H5N1 and H1N1 IAV. In this study, we generated six pseudoviruses with an HIV-1 backbone respectively expressing HA of four clades of H5N1 IAV and the 2009 epidemic H1N1 IAV. The resulting pseudoviruses were able to infect a variety of human and non-human cells, with 293T cells from human kidney as the most susceptible target cells. Using the established pseudovirus neutralization assay, we showed that three of ten selected mAbs specific to HA could potently neutralize infection of a pseudovirus bearing HA from the homologous IAV A/VietNam/1194/2004(H5N1) strain. This was highly consistent with the result of a microneutralization assay testing the same strain of a live IAV. Since the pseudovirus neutralization assay does not involve an infectious virus and can be performed without the requirement of a biosafety-3 laboratory, it may be applied for safe and rapid screening of neutralizing mAbs and antiviral agents targeting HA of IAVs.

A Novel Strategy for Rapid Construction of Libraries of Full-length Antibodies Highly Expressed on Mammalian Cell Surfaces

Acta Biochimica Et Biophysica Sinica. Aug, 2010  |  Pubmed ID: 20705599

Development of a versatile mammalian display system is essential for the selection of functional human antibodies with high affinities. Here we described a novel strategy for rapid construction of full-length antibody libraries that could be efficiently expressed on mammalian cell surfaces. The universal vector pDGB-HC-TM was constructed by inserting multiple cloning site unique sequences recognized by restriction endonucleases BsmBI, SfiI, and BstXI for the pop-in and pop-out of genes of interest. Cytomegalovirus promoter, a commonly used promoter for high expression of proteins in a variety of mammalian cells, was used to drive expression of the inserted antibody genes and a transmembrane domain from platelet-derived growth factor receptor was fused in frame to the C-terminus of heavy chain consistent region to anchor the antibody expressed on the mammalian cell surface. Using this strategy, we constructed a full-length human antibody display library. DNA sequence analysis and expression analysis indicated that the library constructed had a combinatory expressible, detectable diversity of 6.58 x 10(10).

Induction of Protection Against Divergent H5N1 Influenza Viruses Using a Recombinant Fusion Protein Linking Influenza M2e to Onchocerca Volvulus Activation Associated Protein-1 (ASP-1) Adjuvant

Vaccine. Oct, 2010  |  Pubmed ID: 20732469

Our previous studies have shown the adjuvanticity of an Onchocerca volvulus recombinant protein, Ov-ASP-1 (ASP-1), when administered in an aqueous formulation with bystander vaccine antigens or commercial vaccines. In this study, we reported a novel formulation that took advantage of the protein nature of the ASP-1 adjuvant by creating recombinant fusion protein vaccines linking the highly conserved extracellular domain of M2 protein (M2e) consensus sequence of H5N1 influenza viruses with the ASP-1 adjuvant. Two recombinant fusion proteins designated M2e-ASP-1 and M2e3-ASP-1 were studied, in which ASP-1 was fused with one or three tandem copies of the M2e antigen. Our results show that these novel recombinant influenza vaccines, particularly M2e3-ASP-1, induced strong anti-M2e-specific humoral and cellular immune responses in the established mouse model. Furthermore, M2e3-ASP-1 was able to provide significant cross-clade protection against divergent H5N1 viruses. Consequently, this study has demonstrated a potential novel vaccine formulation that could provide a complementary prophylactic strategy in preventing the threat of future influenza outbreak resulting from rapid evolution of the H5N1 virus and co-circulation of multiple antigenic variants in various regions.

Development of Peptide and Small-molecule HIV-1 Fusion Inhibitors That Target Gp41

ChemMedChem. Nov, 2010  |  Pubmed ID: 20845360

It has been 25 years since the development of the first efficient HIV-1/AIDS treatment. Scientists now know more about the HIV-1 infection life cycle, and more than 30 antiretroviral drugs have been developed, including HIV-1 fusion inhibitors. Fundamental work was begun in the early 1990s and led to the development of a novel class of anti-HIV-1 drugs, culminating in a peptide known as T20, which is currently the only HIV-1 fusion inhibitor approved by the US Food and Drug Administration. However, more work needs to be done to perfect the development of peptide and small-molecule HIV fusion inhibitors, particularly those that target gp41. Herein we present a brief overview of the development of this class of anti-HIV-1 drug by focusing on the achievements, challenges, and lessons learned. We cite hallmark studies of the past and comment on future drug development.

Potent and Persistent Antibody Responses Against the Receptor-binding Domain of SARS-CoV Spike Protein in Recovered Patients

Virology Journal. 2010  |  Pubmed ID: 21047436

The spike (S) protein of SARS-CoV not only mediates receptor-binding but also induces neutralizing antibodies. We previously identified the receptor-binding domain (RBD) of S protein as a major target of neutralizing antibodies in animal models and thus proposed a RBD-based vaccine. However, the antigenicity and immunogenicity of RBD in humans need to be characterized.

Design, Synthesis, and Evaluation of Diarylpyridines and Diarylanilines As Potent Non-nucleoside HIV-1 Reverse Transcriptase Inhibitors

Journal of Medicinal Chemistry. Nov, 2010  |  Pubmed ID: 21049929

On the basis of the structures and activities of our previously identified non-nucleoside reverse transcriptase inhibitors (NNRTIs), we designed and synthesized two sets of derivatives, diarylpyridines (A) and diarylanilines (B), and tested their anti-HIV-1 activity against infection by HIV-1 NL4-3 and IIIB in TZM-bl and MT-2 cells, respectively. The results showed that most compounds exhibited potent anti-HIV-1 activity with low nanomolar EC(50) values, and some of them, such as 13m, 14c, and 14e, displayed high potency with subnanomolar EC(50) values, which were more potent than etravirine (TMC125, 1) in the same assays. Notably, these compounds were also highly effective against infection by multi-RTI-resistant strains, suggesting a high potential to further develop these compounds as a novel class of NNRTIs with improved antiviral efficacy and resistance profile.

Roles of the Hemagglutinin of Influenza A Virus in Viral Entry and Development of Antiviral Therapeutics and Vaccines

Protein & Cell. Apr, 2010  |  Pubmed ID: 21203946

Seasonal influenza epidemics and influenza pandemics caused by influenza A virus (IAV) has resulted in millions of deaths in the world. The development of anti-IAV vaccines and therapeutics is urgently needed for prevention and treatment of IAV infection and for controlling future influenza pandemics. Hemagglutinin (HA) of IAV plays a critical role in viral binding, fusion and entry, and contains the major neutralizing epitopes. Therefore, HA is an attractive target for developing anti-IAV drugs and vaccines. Here we have reviewed the recent progress in study of conformational changes of HA during viral fusion process and development of HA-based antiviral therapeutics and vaccines.

Structure-based Design, Synthesis and Biological Evaluation of New N-carboxyphenylpyrrole Derivatives As HIV Fusion Inhibitors Targeting Gp41

Bioorganic & Medicinal Chemistry Letters. Jan, 2010  |  Pubmed ID: 19932616

A new series of N-carboxyphenylpyrrole ligands were designed using GeometryFit based on an X-ray crystal structure of gp41. The synthesized ligands showed significant inhibitory activities against HIV gp41 6-helix bundle formation, HIV-1 mediated cell-cell fusion and HIV-1 replication.

Combinations of 3-hydroxyphthalic Anhydride-modified Ovalbumin with Antiretroviral Drug-based Microbicide Candidates Display Synergistic and Complementary Effects Against HIV-1 Infection

Journal of Acquired Immune Deficiency Syndromes (1999). Apr, 2011  |  Pubmed ID: 21239999

The development of a safe, effective, and affordable microbicide to prevent the sexual transmission of HIV combination is urgently needed. Our previous studies demonstrated that 3-hydroxyphthalic anhydride-modified chicken ovalbumin (HP-OVA) exhibited potent antiviral activity against a broad spectrum of HIV, simian immunodeficiency virus, and herpes simplex virus, making it a promising candidate as a component of combination microbicide. We intended to evaluate potential the synergistic anti-HIV-1 effect of HP-OVA in combination with antiretroviral drug (ARV)-based microbicide candidates.

Four-way Ligation for Construction of a Mammalian Cell-based Full-length Antibody Display Library

Acta Biochimica Et Biophysica Sinica. Mar, 2011  |  Pubmed ID: 21257624

A unique four-way ligation strategy was developed for rapid construction of a full-length antibody library. A mammalian expression vector was constructed that contained dual mammalian expression cassettes and sequences recognized by the unique restriction enzymes BsmBI, BstXI, and SfiI. Both full-length light-chain and variable domain of heavy-chain genes were inserted into the vector in one step by four-way ligation, and full-length bivalent antibodies were displayed on mammalian cell surfaces. Using this strategy, only 2 weeks were required to successfully construct high-quality, full-length human antibody libraries.

HIV-associated Dementia in the Era of Highly Active Antiretroviral Therapy (HAART)

Microbes and Infection / Institut Pasteur. May, 2011  |  Pubmed ID: 21262373

Neurological complications associated with HIV-1 are being recognized as a common disorder in AIDS patients, especially patients with HIV-associated dementia (HAD). However, our knowledge of the complicated pathogenesis and clinical symptoms of HAD is limited by an incomplete understanding of the biology of HIV-1 in the nervous system. Therefore, this review focuses on the pathogenesis of HAD in the context of novel highly active antiretroviral therapy (HARRT) regimens.

A Recombinant Vaccine of H5N1 HA1 Fused with Foldon and Human IgG Fc Induced Complete Cross-clade Protection Against Divergent H5N1 Viruses

PloS One. 2011  |  Pubmed ID: 21304591

Development of effective vaccines to prevent influenza, particularly highly pathogenic avian influenza (HPAI) caused by influenza A virus (IAV) subtype H5N1, is a challenging goal. In this study, we designed and constructed two recombinant influenza vaccine candidates by fusing hemagglutinin 1 (HA1) fragment of A/Anhui/1/2005(H5N1) to either Fc of human IgG (HA1-Fc) or foldon plus Fc (HA1-Fdc), and evaluated their immune responses and cross-protection against divergent strains of H5N1 virus. Results showed that these two recombinant vaccines induced strong immune responses in the vaccinated mice, which specifically reacted with HA1 proteins and an inactivated heterologous H5N1 virus. Both proteins were able to cross-neutralize infections by one homologous strain (clade 2.3) and four heterologous strains belonging to clades 0, 1, and 2.2 of H5N1 pseudoviruses as well as three heterologous strains (clades 0, 1, and 2.3.4) of H5N1 live virus. Importantly, immunization with these two vaccine candidates, especially HA1-Fdc, provided complete cross-clade protection against high-dose lethal challenge of different strains of H5N1 virus covering clade 0, 1, and 2.3.4 in the tested mouse model. This study suggests that the recombinant fusion proteins, particularly HA1-Fdc, could be developed into an efficacious universal H5N1 influenza vaccine, providing cross-protection against infections by divergent strains of highly pathogenic H5N1 virus.

Discovery of a Potent Peptidic Cyclophilin A Inhibitor Trp-Gly-Pro

European Journal of Medicinal Chemistry. May, 2011  |  Pubmed ID: 21396746

Through virtual screening of a rationally built database consisting of 40 peptides, we identified three short peptides. After testing these three synthetic peptides, we found that the peptide Trp-Gly-Pro (WGP) showed comparable inhibitory ability as positive control cyclosporine A (CsA) on CypA-mediated PPIase activity with IC50 values of 33.11 nM and 10.25 nM, respectively. The peptide WGP had same order of CypA-binding affinity as CsA with dissociation equilibrium constant KD of 3.41×10(-6) and 6.42×10(-6) M, respectively. This peptide could also inhibit HIV-1IIIB infection. This study provides a novel strategy for rational design and development of peptidic drugs.

Susceptibility of HIV-1 Subtypes B', CRF07_BC and CRF01_AE That Are Predominantly Circulating in China to HIV-1 Entry Inhibitors

PloS One. 2011  |  Pubmed ID: 21412427

The B', CRF07_BC and CRF01_AE are the predominant HIV-1 subtypes in China. It is essential to determine their baseline susceptibility to HIV entry inhibitors before these drugs are used in China.

Complement Inhibition Alleviates Paraquat-induced Acute Lung Injury

American Journal of Respiratory Cell and Molecular Biology. Oct, 2011  |  Pubmed ID: 21421909

The widely used herbicide, paraquat (PQ), is highly toxic and claims thousands of lives from both accidental and voluntary ingestion. The pathological mechanisms of PQ poisoning-induced acute lung injury (ALI) are not well understood, and the role of complement in PQ-induced ALI has not been elucidated. We developed and characterized a mouse model of PQ-induced ALI and studied the role of complement in the pathogenesis of PQ poisoning. Intraperitoneal administration of PQ caused dose- and time-dependent lung damage and mortality, with associated inflammatory response. Within 24 hours of PQ-induced ALI, there was significantly increased expression of the complement proteins, C1q and C3, in the lung. Expression of the anaphylatoxin receptors, C3aR and C5aR, was also increased. Compared with wild-type mice, C3-deficient mice survived significantly longer and displayed significantly reduced lung inflammation and pathology after PQ treatment. Similar reductions in PQ-induced inflammation, pathology, and mortality were recorded in mice treated with the C3 inhibitors, CR2-Crry, and alternative pathway specific CR2-fH. A similar therapeutic effect was also observed by treatment with either C3a receptor antagonist or a blocking C5a receptor monoclonal antibody. Together, these studies indicate that PQ-induced ALI is mediated through receptor signaling by the C3a and C5a complement activation products that are generated via the alternative complement pathway, and that complement inhibition may be an effective clinical intervention for postexposure treatment of PQ-induced ALI.

HIV-1 Gp41 Core with Exposed Membrane-proximal External Region Inducing Broad HIV-1 Neutralizing Antibodies

PloS One. 2011  |  Pubmed ID: 21483871

The membrane-proximal external region (MPER) of the HIV-1 gp41 consists of epitopes for the broadly cross-neutralizing monoclonal antibodies 2F5 and 4E10. However, antigens containing the linear sequence of these epitopes are unable to elicit potent and broad neutralizing antibody responses in vaccinated hosts, possibly because of inappropriate conformation of these epitopes. Here we designed a recombinant antigen, designated NCM, which comprises the N- and C-terminal heptad repeats that can form a six-helix bundle (6HB) core and the MPER domain of gp41. Two mutations (T569A and I675V) previously reported to expose the neutralization epitopes were introduced into NCM to generate mutants named NCM(TA), NCM(IV), and NCM(TAIV). Our results showed that NCM and its mutants could react with antibodies specific for 6HB and MPER of gp41, suggesting that these antigens are in the form of a trimer of heterodimer (i.e., 6HB) with three exposed MPER tails. Antigen with double mutations, NCM(TAIV), elicited much stronger antibody response in rabbits than immunogens with single mutation, NCM(TA) and NCM(IV), or no mutation, NCM. The purified MPER-specific antibodies induced by NCM(TAIV) exhibited broad neutralizing activity, while the purified 6HB-specific antibodies showed no detectable neutralizing activity. Our recombinant antigen design supported by an investigation of its underlying molecular mechanisms provides a strong scientific platform for the discovery of a gp41 MPER-based AIDS vaccine.

CL-385319 Inhibits H5N1 Avian Influenza A Virus Infection by Blocking Viral Entry

European Journal of Pharmacology. Jun, 2011  |  Pubmed ID: 21536025

CL-385319, an N-substituted piperidine, is effective in inhibiting infection of H1-, H2-, and to a lesser extent, H3-typed influenza A viruses by interfering with the fusogenic function of the viral hemagglutinin. Here we show that CL-385319 is effective in inhibiting infection of highly pathogenic H5N1 influenza A virus in Madin-Darby Canine Kidney (MDCK) cells with an IC50 of 27.03±2.54 μM. This compound with low cytotoxicity (CC50=1.48±0.01 mM) could also inhibit entry of pseudoviruses carrying hemagglutinins from H5N1 strains that were isolated from different places at different times, while it had no inhibitory activity on the entry of VSV-G pseudotyped particles. CL385319 could not inhibit N1-typed neuraminidase activity and the adsorption of H5-typed HA to chicken erythrocytes at the concentration as high as 1 mg/ml (2.8 mM). Computer-aid molecular docking analysis suggested that CL-385319 might bind to the cavity of HA2 stem region which was known to undergo significant rearrangement during membrane fusion. Pseudoviruses with M24A mutation in HA1 or F110S mutation in HA2 were resistant to CL-385319, indicating that these two residues in the cavity region may be critical for CL-385319 bindings. These findings suggest that CL-385319 can serve as a lead for development of novel virus entry inhibitors for preventing and treating H5N1 influenza A virus infection.

HIV-1 Gp41 Ectodomain Enhances Cryptococcus Neoformans Binding to Human Brain Microvascular Endothelial Cells Via Gp41 Core-induced Membrane Activities

The Biochemical Journal. Sep, 2011  |  Pubmed ID: 21668410

Cryptococcus neoformans causes life-threatening meningoencephalitis, particularly prevalent in AIDS patients. The interrelationship between C. neoformans and HIV-1 is intriguing, as both pathogens elicit severe neuropathological complications. We have previously demonstrated that the HIV-1 gp41 ectodomain fragments gp41-I33 (amino acids 579-611) and gp41-I90 (amino acids 550-639) can enhance C. neoformans binding to HBMECs (human brain microvascular endothelial cells). Both peptides contain the loop region of gp41. In the present study, we used immunofluorescence microscopy and transmission and scanning electron microscopy to explore the underlying mechanisms. Our findings indicated that both C. neoformans and gp41-I90 up-regulated ICAM-1 (intercellular adhesion molecule 1) on the HBMECs and elicited membrane ruffling on the surface of HBMECs. The HIV-1 gp41 ectodomain could also induce CD44 and β-actin redistribution to the membrane lipid rafts, but it could not enhance PKCα (protein kinase Cα) phosphorylation like C. neoformans. Instead, gp41-I90 was able to induce syncytium formation on HBMECs. The results of the present study suggest HIV-1 gp41-enhanced C. neoformans binding to HBMECs via gp41 core domain-induced membrane activities, revealing a potential mechanism of invasion for this pathogenic fungus into the brain tissues of HIV-1-infected patients.

An Amphiphilic Conjugate Approach Toward the Design and Synthesis of Betulinic Acid-polyphenol Conjugates As Inhibitors of the HIV-1 Gp41 Fusion Core Formation

ChemMedChem. Sep, 2011  |  Pubmed ID: 21688394

Exploration of potent inhibitors of the HIV-1 gp41 fusion core formation is a promising strategy to discover small-molecule HIV-1 entry inhibitors for the treatment of HIV-1 infection. In this paper, a series of novel betulinic acid-polyphenol conjugates was designed, guided by molecular modeling of the binding of betulinic acid (BA) and phenolic galloyl/caffeoyl groups in the groove on the gp41 N-terminal heptad repeat (NHR) trimeric coiled coil. These conjugates were synthesized via conjugation of galloyl and caffeoyl groups with BA at the C-28 position. Their inhibitory activities of HIV gp41 six-helix bundle (6-HB) formation between the NHR peptide N36 and the C-terminal heptad repeat (CHR) peptide C34 were evaluated with size-exclusion HPLC. Conjugates bearing a galloyl group were found to exhibit four to sixfold higher inhibitory activities than that of parent compound BA, suggesting that they may be exploitable as HIV-1 fusion/entry inhibitors targeting gp41. The docking study on BA and its derivatives suggests that hydrophobic and hydrogen-bonding pockets exist in the groove of the gp41 NHR trimeric coiled coil and that a potent inhibitor should have amphiphilic structures to cooperatively interact with both pockets. This possibility was explored by incorporating both lipophilic and hydrophilic groups into the conjugates in a well-defined orientation to bind with both pockets in the gp41 NHR-trimer.

A Novel Chimeric Protein-based HIV-1 Fusion Inhibitor Targeting Gp41 Glycoprotein with High Potency and Stability

The Journal of Biological Chemistry. Aug, 2011  |  Pubmed ID: 21690094

T20 (enfuvirtide, Fuzeon) is the first generation HIV-1 fusion inhibitor approved for salvage therapy of HIV-1-infected patients refractory to current antiretroviral drugs. However, its application is limited by the high cost of peptide synthesis, rapid proteolysis, and poor efficacy against emerging drug-resistant strains. Here we reported the design of a novel chimera protein-based fusion inhibitor targeting gp41, TLT35, that uses a flexible 35-mer linker to couple T20 and T1144, the first and next generation HIV-1 fusion inhibitors, respectively. TLT35, which was expressed in Escherichia coli with good yield, showed low nm activity against HIV-1-mediated cell-cell fusion and infection by laboratory-adapted HIV-1 strains (X4 or R5), including T20-resistant variants and primary HIV-1 isolates of clades A to G and group O (R5 or X4R5). TLT35 was stable in human sera and in peripheral blood mononuclear cell culture and was more resistant to proteolysis than either T20 or T1144 alone. Circular dichroism spectra showed that TLT35 folded into a thermally stable conformation with high α-helical content and T(m) value in aqueous solution. It formed a highly stable complex with gp41 N-terminal heptad repeat peptide and blocked formation of the gp41 six-helix-bundle core. These merits combined with an anticipated low production cost for expression of TLT35 in E. coli make this novel protein-based fusion inhibitor a promising candidate for further development as an anti-HIV-1 microbicide or therapeutic for the prevention and treatment of HIV-1 infection.

Heteromeric Assembled Polypeptidic Artificial Hydrolases with a Six-helical Bundle Scaffold

Chembiochem : a European Journal of Chemical Biology. Nov, 2011  |  Pubmed ID: 21957084

Enzyme efficiency results from the cooperation of functional groups in the catalytic site. In order to mimic a natural enzyme, a definite 3D scaffold must be carefully designed so that the functional groups can work cooperatively. During the HIV-1 fusion process, the gp41 N- and C-terminal heptad repeat regions form a coiled-coil six-helical bundle (6HB) that brings the viral and target cell membranes into close proximity for fusion. We used 6HB as the molecular model for a novel scaffold for the design of an artificial enzyme, in which the modified C34 and N36 peptides formed a unique 6HB structure through specific molecular recognition, and the position and orientation of the side-chain groups on this scaffold were predictable. The histidine modified 6HB C34(H13/20)/N36(H15/22) showed enzyme-like hydrolytic activity towards p-nitrophenyl acetate (PNPA; k(cat)/K(M) =3.66 M(-1) s(-1)) through the cooperation of several inter- or intrahelical imidazole groups. Since the catalytic activity of 6HB depends on the C- and N-peptide assembly, either HIV fusion inhibitors that can compete with the formation of catalytic 6HB or denaturants that can destroy the ordered structure were able to modulate its activity. Further engineering of the solvent-exposing face with Glu(-)-Lys(+) salt bridges enhanced the helicity and the stability of 6HB. As a result, the population and stability of cooperative catalytic units increased. In addition, the Glu(-)-Lys(+) -stabilized 6HB SC35(H13/20)/N36(H15/22) had increased catalytic efficiency (k(cat)/K(M) =6.30 M(-1) s(-1)). A unique 6HB system was specifically assembled and provided a scaffold sufficiently stable to mimic the function of enzymes or other biomolecules.

Design, Synthesis, and Biological Activity of a Novel Series of 2,5-disubstituted Furans/pyrroles As HIV-1 Fusion Inhibitors Targeting Gp41

Bioorganic & Medicinal Chemistry Letters. Nov, 2011  |  Pubmed ID: 21978673

Based on molecular docking analysis of earlier results, we designed a series of 2,5-disubstituted furans/pyrroles (5a-h) as HIV-1 entry inhibitors. Compounds were synthesized by Suzuki-Miyaura cross coupling, followed by a Knoevenagel condensation or Wittig reaction. Four of these compounds were found to be effective in inhibiting HIV-1 infection, with the best compounds being 5f and 5h, which exhibited significant inhibition on HIV-1(IIIB) infection at micromolar levels with low cytotoxicity. These compounds are also effective in blocking HIV-1 mediated cell-cell fusion and the gp41 six-helix bundle formation, suggesting that they are also HIV-1 fusion inhibitors targeting gp41 and have potential to be developed as a new class of anti-HIV-1 agents.

In Vitro Selection and Characterization of HIV-1 Variants with Increased Resistance to Sifuvirtide, a Novel HIV-1 Fusion Inhibitor

The Journal of Biological Chemistry. Feb, 2011  |  Pubmed ID: 21098485

Sifuvirtide, a novel fusion inhibitor against human immunodeficiency virus type I (HIV-1), which is more potent than enfuvirtide (T20) in cell culture, is currently under clinical investigation for the treatment of HIV-1 infection. We now report that in vitro selection of HIV-1 variants resistant to sifuvirtide in the presence of increasing concentrations of sifuvirtide has led to several specific mutations in the gp41 region that had not been previously reported. Many of these substitutions were confined to the N-terminal heptad repeat region at positions 37, 38, 41, and 43, either singly or in combination. A downstream substitution at position 126 (N126K) in the C-terminal heptad repeat region was also found. Site-directed mutagenesis studies have further identified the critical amino acid substitutions and combinations thereof in conferring the resistant genotypes. Furthermore, the mutant viruses demonstrated variable degrees of cross-resistance to enfuvirtide, some of which are preferentially more resistant to sifuvirtide. Impaired infectivity was also found for many of the mutant viruses. Biophysical and structural analyses of the key substitutions have revealed several potential novel mechanisms against sifuvirtide. Our results may help to predict potential resistant patterns in vivo and facilitate the further clinical development and therapeutic utility of sifuvirtide.

Design, Synthesis, and Biological Activity of Novel 5-((arylfuran/1H-pyrrol-2-yl)methylene)-2-thioxo-3-(3-(trifluoromethyl)phenyl)thiazolidin-4-ones As HIV-1 Fusion Inhibitors Targeting Gp41

Journal of Medicinal Chemistry. Jan, 2011  |  Pubmed ID: 21190369

On the basis of our earlier molecular docking analysis, we designed and synthesized 5-((arylfuran/1H-pyrrol-2-yl)methylene)-2-thioxo-3-(3-(trifluoromethyl)phenyl)thiazolidin-4-ones (12a-o) as HIV-1 entry inhibitors. Compounds 12a-o effectively inhibited infection by both laboratory-adapted and primary HIV-1 strains and blocked HIV-1 mediated cell-cell fusion and gp41 six-helix bundle formation. Molecular docking analyses on two highly active inhibitors, 12b, containing a carboxylic acid group, and 12m, containing a tetrazole group, indicated that they both fit snugly into the hydrophobic cavity of HIV-1 gp41 from which each has important ionic interactions with lysine 574 (K574). By contrast, molecular docking of 12i, a less active compound containing a pyrrole instead of a furan ring, indicated a completely different orientation from 12b and 12m and missed critical interactions.

Design, Synthesis and Biological Evaluation of 3-substituted 2,5-dimethyl-N-(3-(1H-tetrazol-5-yl)phenyl)pyrroles As Novel Potential HIV-1 Gp41 Inhibitors

Bioorganic & Medicinal Chemistry. Nov, 2011  |  Pubmed ID: 22014749

Based on the structure of HIV-1 gp41 binding site for small-molecule inhibitors, optimization of lead 2 resulted in the discovery of a new series of 2,5-dimethyl-3-(5-(N-phenylrhodaninyl)methylene)-N-(3-(1H-tetrazol-5-yl)phenyl)pyrrole compounds with improved anti-HIV-1 activity. The most active compounds 13a and 13j exhibited significant potency against gp41 6-HB formation with IC(50) values of 4.4 and 4.6 μM and against HIV-1 replication in the MT-2 cells with EC(50) values of 3.2 and 2.2 μM, respectively, thus providing a new starting point to develop highly potent small-molecule HIV fusion inhibitors targeting gp41.

HIV-1 Glycoprotein 41 Ectodomain Induces Activation of the CD74 Protein-mediated Extracellular Signal-regulated Kinase/mitogen-activated Protein Kinase Pathway to Enhance Viral Infection

The Journal of Biological Chemistry. Dec, 2011  |  Pubmed ID: 22039051

Besides mediating the viral entry process, the human immunodeficiency virus (HIV-1) envelope protein gp41 can bind to many host cell components and regulate cell functions. Using a yeast two-hybrid system, we screened a human bone marrow cDNA library and identified a novel gp41-binding protein, CD74 (the MHC class II-associated invariant chain). Here, we report possible biological effects mediated by interaction between gp41 and CD74. We found that HIV-1 gp41 could bind directly to host CD74 in HIV-1-infected cells, and the peptide 6358 derived from gp41 loop region (aa 597-611) could effectively block the gp41-CD74 interaction. As a result of this binding, recombinant soluble gp41 and gp41 peptide 6358 activated the CD74-mediated ERK/MAPK pathway and significantly enhanced HIV-1 infection in vitro. Conversely, the enhancing effect could be suppressed by the recombinant CD74 extracellular domain. These results reveal a novel mechanism underlying gp41 mediation of HIV-1 infection and replication.

Interactions Between Different Generation HIV-1 Fusion Inhibitors and the Putative Mechanism Underlying the Synergistic Anti-HIV-1 Effect Resulting from Their Combination

The FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Nov, 2011  |  Pubmed ID: 22085645

We previously reported that the combinatorial use of T20 and T1144, the first and next generations of HIV fusion inhibitors, containing different functional domains resulted in synergistic anti-HIV-1 effect, but this effect diminished when T20 and T1144 were covalently linked together. To elucidate the mechanism underlying this synergistic anti-HIV-1 effect, we studied the interactions between T20 and T1144 either in a mixture state or in a covalently linked state. T20 alone in solution was largely featureless, while T1144 alone was in α-helical trimeric conformation. When mixed in solution, T20 and T1144 showed a loose and transient interaction, with a moderate 10% α-helical content increase, but this interaction was greatly enhanced in the linked state, and T20 and T1144 showed ∼100% α-helical content. These results suggested that the loose and transient interaction between T20 and T1144 may destabilize the T1144 trimer, which makes its otherwise shielded binding sites more accessible to N-terminal heptad repeat (NHR) and increases its associating rate, thus increasing its anti-HIV-1 potency against the temporarily exposed target in NHR and causing the synergistic anti-HIV-1 effect. However, the strong interaction between T20 and T1144 in the covalently linked state may shield their NHR-binding sites, resulting in reduction of the synergistic effect.-Cai, L., Pan, C., Xu, L., Shui, Y., Liu, K., Jiang, S. Interactions between different generation HIV-1 fusion inhibitors and the putative mechanism underlying the synergistic anti-HIV-1 effect resulting from their combination.

Identification of HBsAg-specific Antibodies from a Mammalian Cell Displayed Full-length Human Antibody Library of Healthy Immunized Donor

Cellular & Molecular Immunology. Dec, 2011  |  Pubmed ID: 22179672

Hepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the Flp recombinase-mediated integration (Flp-In) system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting (FACS), eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA) were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.Cellular & Molecular Immunology advance online publication, 19 December 2011; doi:10.1038/cmi.2011.55.

Design, Synthesis and Biological Evaluation of 1-[(2-benzyloxyl/alkoxyl) Methyl]-5-halo-6-aryluracils As Potent HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors with Improved Drug Resistance Profile

Journal of Medicinal Chemistry. Jan, 2012  |  Pubmed ID: 22283377

Since the emergence of drug-resistant mutants has limited the efficacy of non-nucleoside reverse transcriptase inhibitors (NNRTIs), it is essential to develop new antivirals with better drug-resistance and pharmacokinetic profiles. Here we designed and synthesized a series of 1-[(2-benzyloxyl/alkoxyl)methyl]-5-halo-6-aryluracils, the HEPT analogues, and evaluated their biological activity using Nevirapine and 18 (TNK-651) as reference compounds. Most of these compounds, especially 6b, 7b, 9b, 11b and 7c, exhibited highly potent anti-HIV-1 activity against both wild-type and NNRTI-resistant HIV-1 strains. The compound 7b, that had the highest selectivity index (SI = 38,215), is more potent than Nevirapine and 18. These results suggest that introduction of halogen at the C-5 position may contribute to the effectiveness of these compounds against RTI-resistant variants. In addition, m-substituents on the C-6 aromatic moiety could significantly enhance activity against NNRTI-resistant HIV-1 strains. These compounds can be further developed as next-generation NNRTIs with improved antiviral efficacy and drug-resistance profile.

What Can Academia Learn from XMRV Studies?

Nature Reviews. Urology. Feb, 2012  |  Pubmed ID: 22331091

A Natural Theaflavins Preparation Inhibits HIV-1 Infection by Targeting the Entry Step: Potential Applications for Preventing HIV-1 Infection

Fitoterapia. Mar, 2012  |  Pubmed ID: 22155187

Theaflavins are the major components of tea polyphenols in brewed black tea. We previously reported that theaflavin derivatives, such as TF3, inhibited HIV-1 entry by targeting gp41. However, it is difficult to purify the individual theaflavins and the purified compounds are highly unstable. To develop theaflavins as affordable anti-HIV-1 microbide for preventing HIV sexual transmission, we intended to use an economic natural preparation containing 90% of theaflavins (TFmix). Its antiviral activity against HIV-1 strains was evaluated in vitro using p24 production and luciferase assays. The mechanism by which TFmix inhibits HIV-1 infection was investigated using time-of-addition, cell-cell fusion and biophysical assays. The data suggested TFmix exhibited potent anti-HIV-1 activity on lab-adapted and primary HIV-1 strains with IC(50) less than 1.20μM. It also effectively inhibited infection by T-20 resistant HIV-1 strains. The mechanism studies suggest that TFmix mainly inhibit the HIV-1 entry by targeting gp41 since it is effective in inhibiting gp41 six-helix bundle (6-HB) formation and HIV-1 envelope protein-mediated cell-cell fusion. TFmix could also inhibit HIV-1 reverse transcriptase (RT) activity, but the IC(50) is about 8-fold higher than that for inhibiting gp41 6-HB formation, suggesting RT is not a major target for TFmix. In conclusion, TFmix is an economic natural product preparation containing high content of theaflavins with potent anti-HIV-1 activity by targeting the viral entry step through the disruption of gp41 6-HB core structure. It has a potential to be developed as a safe and affordable topical microbicide for preventing sexual transmission of HIV.

Mutations of Gln64 in the HIV-1 Gp41 N-terminal Heptad Repeat Render Viruses Resistant to Peptide HIV Fusion Inhibitors Targeting the Gp41 Pocket

Journal of Virology. Jan, 2012  |  Pubmed ID: 22013063

To prove that the peptidic HIV-1 fusion inhibitors containing the pocket-binding domain (PBD) mainly target the hydrophobic pocket in the gp41 N-terminal heptad repeat (NHR), we constructed pseudoviruses by replacement of Q64 in the gp41 pocket region with Ala (Q64A) or Leu (Q64L). These viruses were highly resistant to C34 and CP32M containing the PBD, while they were susceptible to T20 (enfuvirtide) lacking the PBD but containing the GIV-motif-binding domain (GBD) and lipid-binding domain (LBD). They were also sensitive to C52L, which contains the PBD, GBD, and LBD. Those mutations may disrupt the hydrophilic interaction between Q64 in the NHR and N113 in the peptides containing the PBD. This report provides insights into the mechanisms of drug resistance, with implications for the design of novel HIV fusion and entry inhibitors.