Antiviral Activity of Dual-acting Hydrocarbon-stapled Peptides against HIV-1 Predominantly Circulating in China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>New rationally designed i,i+7-hydrocarbon-stapled peptides that target both HIV-1 assembly and entry have been shown to have antiviral activity against HIV-1 subtypes circulating in Europe and North America. Here, we aimed to evaluate the antiviral activity of these peptides against HIV-1 subtypes predominantly circulating in China.</p><p><b>METHODS</b>The antiviral activity of three i,i+7-hydrocarbon-stapled peptides, NYAD-36, NYAD-67, and NYAD-66, against primary HIV-1 CRF07_BC and CRF01_AE isolates was evaluated in peripheral blood mononuclear cells (PBMCs). The activity against the CRF07_BC and CRF01_AE Env-pseudotyped viruses was analyzed in TZM-bl cells.</p><p><b>RESULTS</b>We found that all the stapled peptides were effective in inhibiting infection by all the primary HIV-1 isolates tested, with 50% inhibitory concentration toward viral replication (IC50) in the low micromolar range. NYAD-36 and NYAD-67 showed better antiviral activity than NYAD-66 did. We further evaluated the sensitivity of CRF01_AE and CRF07_BC Env-pseudotyped viruses to these stapled peptides in a single-cycle virus infectivity assay. As observed with the primary isolates, the IC50s were in the low micromolar range, and NYAD-66 was less effective than NYAD-36 and NYAD-67.</p><p><b>CONCLUSION</b>Hydrocarbon-stapled peptides appear to have broad antiviral activity against the predominant HIV-1 viruses in China. This finding may provide the impetus to the rational design of peptides for future antiviral therapy.</p>

Distinctive Drug-resistant Mutation Profiles and Interpretations of HIV-1 Proviral DNA Revealed by Deep Sequencing in Reverse Transcriptase / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate distinctive features in drug-resistant mutations (DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients.</p><p><b>METHODS</b>Forty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA.</p><p><b>RESULTS</b>Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184I and M230I were more prevalent in proviral DNA than in viral RNA (Fisher's exact test, P<0.05). Considering 'majority resistant variants', 15 samples (19.48%) showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering 'minority resistant variants', 22 samples (28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA.</p><p><b>CONCLUSION</b>Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.</p>

Development of peptidic MERS-CoV entry inhibitors / 药学学报

In 2012, a new SARS-like coronavirus emerged in the Middle East, namely the Middle East respiratory syndrome coronavirus (MERS-CoV). It has caused outbreaks with high mortality. During infection of target cell, MERS-CoV S protein S1 subunit binds to the cellular receptor (DPP4), and its S2 subunit HR1 and HR2 regions intact with each other to form a stable six-helix bundle to mediate the fusion between virus and target cell membranes. Hence, blocking the process of six-helix bundle formation can effectively inhibit MERS-CoV entry into the target cells. This review focuses on the recent advance in the development of peptidic entry inhibitors targeting the MERS-CoV S2 subunit.

Small molecular agents against MERS-CoV infection / 药学学报

Middle East respiratory syndrome coronavirus (MERS-CoV) has caused outbreaks of SARS-like disease with 35% case-fatality rate, mainly in the Middle East. A more severe outbreak of MERS occurred recently in the Republic of Korea, where 186 people contracted the infections, causing great concern worldwide. So far, there has been no clinically available drug for the treatment of MERS-CoV infection. The potential drugs against MERS-CoV mainly consist of monoclonal antibodies, peptides and small molecular agents. Small molecular agents have an advantage of easier synthesis, lower cost in production and relatively higher stability. There is better chance for those candidates to gain a quick development. This article reviews the progress of developing small molecular MERS-CoV agents.

Tactics used by HIV-1 to evade host innate, adaptive, and intrinsic immunities / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To review the mechanisms by which HIV evades different components of the host immune system.</p><p><b>DATA SOURCES</b>This review is based on data obtained from published articles from 1991 to 2012. To perform the PubMed literature search, the following key words were input: HIV and immune evasion.</p><p><b>STUDY SELECTION</b>Articles containing information related to HIV immune evasion were selected.</p><p><b>RESULTS</b>Although HIV is able to induce vigorous antiviral immune responses, viral replication cannot be fully controlled, and neither pre-existing infected cells nor latent HIV infection can be completely eradicated. Like many other enveloped viruses, HIV can escape recognition by the innate and adaptive immune systems. Recent findings have demonstrated that HIV can also successfully evade host restriction factors, the components of intrinsic immune system, such as APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G), TRIM5α (tripartite motif 5-α), tetherin, and SAMHD1 (SAM-domain HD-domain containing protein).</p><p><b>CONCLUSIONS</b>HIV immune evasion plays an important role in HIV pathogenesis. Fully understanding the tactics deployed by HIV to evade various components of the host immune systems will allow for the development of novel strategies aimed toward the prevention and cure of HIV/AIDS.</p>

Rapid construction of a mammalian cell surface display library of full-length human antibodies / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a mammalian cell surface display library of full-length human antibodies.</p><p><b>METHODS</b>The total RNA was isolated from human peripheral blood mononuclear cells (PBMCs), and the genes encoding the heavy chain variable regions and kappa light chains (VH and Cκ) of the antibodies were amplified by RT-PCR. The amplified VH and Cκ gene sequences were separately inserted into the vector pDGB-HC-TM. The ligation mixtures were transformed into competent E.coli DH5α cells to construct the antibody libraries, and the library sizes and diversity were analyzed. The library DNAs were transfected into CHO cells and the expression of the full-length human antibodies on the surface of CHO cells was analyzed by flow cytometry.</p><p><b>RESULTS</b>The heavy chain gene library constructed showed a diversity of 2.6 × 10(5), and the kappa light chain gene library had a diversity of 2.0 × 10(5). Sequence analysis of 10 clones randomly selected from the constructed heavy chain gene library and 10 from the light chain gene library showed that 8 heavy chain clones and all 10 light chain clones contained correct open reading frames. Flow cytometry demonstrated that all the 18 clones expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces was detected with the positive cells reaching 31.5.</p><p><b>CONCLUSIONS</b>A full-length human mammalian display antibody library with a combinatory diversity of 5.2 × 10(10) can be constructed in two weeks, which allows the display of full-length antibodies on mammalian cell surface.</p>

Construction of rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries.</p><p><b>METHODS</b>Peripheral blood lymphocytes were isolated from patients with rheumatoid arthritis. The repertoires of kappa light chain (LCκ) and heavy chain variable region (VH) of the antibodies were amplified by RT-PCR. The amplified LCκ and VH genes were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO-10 strain to construct the rheumatoid arthritis-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length fully human antibody expressed on the surface of 293T cells were analyzed by flow cytometry.</p><p><b>RESULTS</b>The libraries of rheumatoid arthritis-specific antibody LCκ and heavy chain (IgG1) were constructed. The expression of full-length fully human antibody on the surface of 293T cells was confirmed by flow cytometry. With the rates of correct LCκ and heavy chain sequence insertion reaching 80% and 60%, respectively, as shown by DNA sequence analysis of the randomly selected clones, the libraries showed an expressible combinatory diversity of 6.13×10(10).</p><p><b>CONCLUSION</b>The constructed libraries provide a useful platform for screening rheumatoid arthritis-specific antibodies.</p>

Study of the mechanism of caffeoyl glucopyranoses in inhibiting HIV-1 entry using pseudotyped virus system / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the inhibitory activities of caffeoyl glucopyranoses purified from Balanophora japonica Makino on HIV entry and their mechanism.</p><p><b>METHODS</b>HIV-1 Env pseudovirus was used to evaluate the anti-HIV-1 activity of those compounds. ELISA and molecular docking were used to study the mechanism of the actions of the active compounds.</p><p><b>RESULTS</b>We used the HIV-1 Env pseudovirus to test the anti-HIV-1 activity of the six phenolic compounds (final concentration 25 microg/ml), and found that only 1,2,6-Tri-O-caffeoyl-beta-D-glucopyranose (TCGP) and 1,3-Di-O-caffeoyl-4-O-galloyl-beta-D- glucopyranose (DCGGP) could effectively inhibit the entry of HIV-1 Env pseudovirus into the target cells in a dose-dependent manner, with IC(50) values of 5.5-/+0.2 and 5.3-/+0.1 microg/ml, respectively. These two compounds could also blocked the gp41 six-helix bundle formation. Molecular docking analysis suggested that they might bind to the hydrophobic cavity of the gp41 N-trimeric coiled-coil.</p><p><b>CONCLUSION</b>TCGP and DCGGP are potent HIV-1 entry inhibitors targeting gp41 and can serve as lead compounds for developing novel anti-HIV-1 microbicides for prevention of sexual HIV-1 transmission.</p>

Directed shift of vaginal flora after topical application of sucrose gel in a phase III clinical trial: a novel treatment for bacterial vaginosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Bacterial vaginosis (BV) is one of the most common infectious diseases among sexually active women and is associated with the increased acquisition of a variety of sexually transmitted diseases. This study aimed to compare the efficacy of a non-antibiotic sucrose gel against an antibiotic metronidazole gel for the treatment of BV.</p><p><b>METHODS</b>A randomized, double-blinded, multi-center, parallel-group, placebo-controlled phase III clinical trial was conducted at eight hospitals in China. A total of 560 subjects with clinically diagnosed BV were randomly assigned into three groups for vaginally receiving sucrose, metronidazole, and placebo gels, respectively, twice daily for five consecutive days. The efficacy of therapeutic cure, defined as an achievement of both microbiologic cure (a Nugent score of 3 or less) and clinical cure (a resolution of the clinical findings from the baseline visit), was evaluated at the 1st and 2nd test-of-cure (TOC) visits at 7-10 and 21-35 days after the start of treatment, respectively.</p><p><b>RESULTS</b>Therapeutic cure rates for sucrose, metronidazole, and placebo gel groups were 83.13%, 71.30% and 0.92%, at the 1st TOC, and 61.04%, 66.67% and 7.34%, at the 2nd TOC, respectively. While there was no significant difference between the sucrose and metronidazole gel groups at the 2nd TOC (P = 0.305), and sucrose gel was more effective than metronidazole gel at the 1st TOC (P = 0.009).</p><p><b>CONCLUSION</b>These findings suggest that sucrose gel restores normal vaginal flora more rapidly than metronidazole gel and can be used as a novel treatment for BV.</p>

A non-infectious and quantitative cell-based bioassay for screening HIV entry inhibitors targeting HIV envelope proteins / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop an objective bioassay for quantitative detection of HIV-induced cell-cell fusion for screening HIV entry inhibitors.</p><p><b>METHODS</b>HL2/3 cells expressing HIV envelope proteins gp120/gp41, Tat, and other HIV proteins were co-cultured with HeLa-CD4-LTR-beta-gal cells expressing CD4 receptor and HIV LTR triggered reporter gene beta-galactosidase. The enzyme activities of beta-galactosidase were detected by a chromogenic substrate, chlorophenol red-beta-galactopyranoside (CPRG). Specific HIV entry inhibitors were used to validate the established detecting method.</p><p><b>RESULTS</b>No syncytium was formed by mixing HL2/3 and HeLa-CD4-LTR-beta-gal cells. However, the membrane could be fused and the Tat expressed by HL2/3 cells could bind to HIV LTR on HeLa-CD4-LTR-beta-gal cells and trigger the expression of beta-galactosidase. CPRG allowed quantitative and sensitive detection of the activity of beta-galactosidase. Further studies showed that HIV entry inhibitors could inhibit the activity of beta-galactosidase in a dose-dependent manner.</p><p><b>CONCLUSION</b>We have developed a simple, cheap, objective and quantitative non-infectious cell-cell fusion bioassay that can be used to screen for anti-HIV agents targeting the virus entry from natural and synthetic compound libraries.</p>

VIR576 inhibits antigen-specific T cell activation by binding to the transmembrane domain of T cell receptor / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the mechanism underlying the inhibitory effect of the anti-HIV peptide VIR576 on antigen-specific T cell activation.</p><p><b>METHODS</b>CCK-8 assay was used to investigate the effect of VIR576 on the proliferation of splenocytes of OVA-specific DO11.10 Tg mice in response to chicken OVA. Hemolysis test, hemolysis inhibition assay and fluorescence binding assay were used to investigate the interaction of VIR576 with the transmembrane domain (TMD) of the T cell receptor (TCR).</p><p><b>RESULTS</b>VIR576 inhibited HIV glycoprotein gp41 fusion peptide-mediated antigen specific T cell activation, and VIR576 itself also inhibited splenocyte proliferation in responses to OVA (P<0.05). Hemolysis test, hemolysis inhibition assay and fluorescence binding assay demonstrated that VIR576 suppressed TCR-TMD-mediated hemolysis and competitively inhibited Rho-VIR576 binding to TCR-TMD peptide.</p><p><b>CONCLUSION</b>VIR576 is effective in suppressing the antigen-specific T cell activation via TCR and can interact with TCR-TMD. VIR576 may serve as a potent microbicide candidate to block sexual transmission of HIV due to of its inhibitory effect on both HIV entry and antigen-specific T cell activation.</p>

Design, synthesis and biologic evaluation of diarylbenzimidazole derivatives as novel HIV-1 non-nucleoside reverse transcriptase inhibitors / 药学学报

Twenty seven new diarylbenzimidazole derivatives (A1-A21, B1-B6) were designed, synthesized, and evaluated in MT-2 cell line as potential HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) agents with a new skeleton based on molecular modeling technique and hit 1,2-diarylbenzimidazole A1 (EC50 69.9 miromol x L(-1)). Hence, 1,2-diarylbenzimidazoles A6 and B3, and 1,6-diarylbenzimidazole B6 showed obvious potency against HIV-1 replication in MT-2 cell line with EC50 values of 15.33, 9.81 and 1.37 micromol x L(-1) respectively. All target compounds were synthesized commonly from substituted 2-nitroanilines by 1-3 steps under mild reaction conditions. Current studies provided preliminary SAR, thus indicating that 1,6-diaryl substitution on the benzimidazole ring would be a right direction for further modification. Furthermore, the docking studies demonstrated that B6 could fit well into the HIV-1 NNRTI binding pocket with a similar binding orientation and conformation to that of TMC278, a promising NNRTI candidate inclinical trial III, Therefore, active compound B6 could serve as a new starting point to develop a series of 1,6-diarylbenzimidazole derivatives as HIV-1 NNRTI agents with a novel skeleton.

HIV/AIDS and ocular complications / 国际眼科杂志(Guoji Yanke Zazhi)

The introduction of highly active antiretroviral therapy (HAART) has greatly changed the pattern and natural history of ocular diseases of HIV-infected patients, resulting from the immune recovery and reduction of opportunistic infections. However, ophthalmic complica-tion continues to be concern in AIDS even in the HAART era, especially in developing areas, where absolute majority of HIV-positive patients live. Lack of test facilities and experience, poor conditions of hygiene, different microbiological environment, absence of effective treatment etc., characterize the ophthalmic manifestation of HIV-infected patients in developing countries from that in developed regions and thus pose a great challenge to the ophthalmic treatment in developing area. Not only varied from region to region, ocular complications are distinctive between adults and children. At the same time, the side effects due to the application of HAART pose their own risks of ocular complication and should, therefore, be given more research attention.

1,2,6-tri-O-galloyl-beta-D-glucopyranose inhibits gp41-mediated HIV envelope fusion with target cell membrane / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the inhibitory effect of 1,2,6-Tri-O-galloyl-beta-D-glucopyranose (TGGP) from Balanophora japonica Makino on human immunodeficiency virus (HIV) entry into the host cells and explore the mechanisms.</p><p><b>METHODS</b>TGGP was purified from Balanophora japonica Makino by n-hexane and ethyl acetate extraction and column chromatography. The inhibitory activity of TGGP on HIV gp41 six-helix bundle formation was measured with ELISA, N-PAGE and SE-HPLC, and the inhibitory effect of TGGP on HIV envelope grlycoprotein-induced cell-cell fusion was detected using a non-infectious cell-based assay.</p><p><b>RESULTS</b>TGGP inhibited HIV gp41 six-helix bundle formation, with an IC50 of 1.37-/+0.19 microg/ml as determined by ELISA, and this activity was further confirmed by N-PAGE and SE-HPLC. TGGP at 25 microg/ml significantly inhibited syncytium formation between the effector (CHO-WT) and the target (MT-2) cells.</p><p><b>CONCLUSION</b>The HIV transmembrane subunit gp41 mediates the entry of HIV into the target cells. TGGP can inhibit HIV fusion and entry into the target cells by inhibiting the formation of gp41 six-helix bundles, suggesting the potential of TGGP as a microbicide to prevent sexual transmission of HIV.</p>