Designing a non-virulent HIV-1 strain: potential implications for vaccine

<strong>BACKGROUND</strong>: Culturing and working with wild type HIV virions may produce contamination and infection. The main<br /> objective of this study was to design a non-infective HIV-1 strain that can be used safely in research laboratories for<br /> various research topics on HIV life cycle and pathogenesis.<br /> <strong>METHODS</strong>: Non-infective HIV-1 strain (mzNL4-3) was designed by deleting a 2 Kb sequence of HIV-1 (NL4-3 strain)<br /> genome that codes reverse transcriptase (RT) and integrase (IN) enzymes.<br /> <strong>RESULTS</strong>: The deletion removed 95% of RT and 34% of IN peptide sequences and abolished the functions of these enzymes<br /> totally. This deletion didn’t produce any other alteration in HIV genome, not only in mRNA level but also in<br /> transcription or translation levels. We named this strain, mutated z NL4-3 (mzNL4-3).<br /> <strong>CONCLUSIONS</strong>: Our mutated HIV-1 virions can be produced by transforming any mammalian cell line with mzNL4-3<br /> vector (pmzNL4-3) but these virions can not replicate in any competent target cell. Hence, it can be used for many of<br /> the HIV-1 researches in a level 2 lab. mzNL4-3 has also the major antigen markers of HIV-1, NY5 and LAV strains,<br /> which are the most common strains of HIV-1. Therefore, mzNL4-3 can also be considered as a choice for HIV-1 vaccine<br /> investigation. Probably mzNL4-3 could be used for lab research and vaccine investigation.<br /> <strong>KEYWORDS</strong>: HIV-1, mzNL4-3, non-infectious, mutant, safe research, vaccine

Inhibition of HIV and HSV infection by vaginal lactobacilli in vitro and in vivo

Background and the purpose of the study The cervico-vaginal mucosa which is populated with microflora (mostly includes lactobacilli) is the portal of entry for sexually transmitted pathogens.MethodsThe in vitro anti-viral effect of vaginal and non-vaginal lactobacillus was evaluated using single cycle HIV-1 replication and HSV-2 plaque reduction assays. The XTT proliferation assay was used to monitor the cellular toxicity. The in vivo anti-HSV-1 activity was evaluated in BALB/c mouse model by monitoring skin lesion and immune response development. Results and major conclusion DMEM culture supernatant of L. Gasseri and L. fermentum (PH 7.3) did not show toxic effect but inhibited 50% of HIV replication at 12 and 31% concentrations, respectively. Coculture of L. gasseri (1000 CFU/ target cell) showed mild cytotoxicity but inhibited 68% of HIV replication. The supernatant of L. crispatus inhibited 50% of HSV replication at 4% and also co-culture of L. gasseri, L. rhamnosus and L. crispatus revokes almost all of the HSV multiplication. Culture supernatants of L. gasseri and L. crispatus had significant virucidal effect against the HIV and HSV and inhibited HSV infection in a stage before viral entry to the target cells. Alive L. gasseri cells showed high potential for inhibiting HSV-1 infection in vivo condition. Current data indicates that lactobacilli supernatant encompasses components with neutralizing activity against HIV and HSV and it would be a determinant factor for viral diseases transmission and promising lead for anti-viral probiotic design

Toward the Development of a Single-Round Infection Assay Based on EGFP Reporting for Anti-HIV-1 Drug Discovery

Background: The rapid increase of HIV-1 strains resistant to current antiretroviral drugs is a challenge for successful AIDS therapy. This necessitates the development of novel drugs, and to this end, availability of screening systems for in vitro drug discovery is a priority. Herein, we report the modification of a previously developed system for increased sensitivity, ease of use, and cost-efficiency, based on the application of the EGFP marker. Methods: A PCR-amplified gfp gene (gfp) was cloned into pmzNL4-3, the plasmid already designed to produce single-cycle replicable virions, in frame with the reverse-transcriptase gene to construct the pmzNL4-3/GFP plasmid. GFP-mzNL4-3 pseudo-typed virions, as the first progeny viruses, were recovered from the culture supernatant of HEK293T cells co-transfected with pmzNL4-3/GFP and the helper plasmids pSPAX2 and pMD2G, which respectively encode HIV-1 Gag-Pol and vesicular stomatitis virus glycoprotein. Single-cycle replication and virion production were assessed by syncytia formation, p24 antigen assays, and electron and fluorescence microscopy. Results: The incorporation of EGFP into the viral particles allowed their quantification by fluorometry, flow-cytometry, and fluorescence microscopy; however, this modification did not affect the single-round infectivity or production rate of the GFP fluorescence-emitting virions. Conclusions: Our results certify the development of a rapid, inexpensive, and safe GFP-reporting single-cycle replicable system for anti-HIV drug discovery. Further experiments are needed to measure the validity and robustness of the assay

Synthesis of Novel 3-(5-(Alkyl/arylthio)-1,3,4-Oxadiazol-2-yl)-8-Phenylquinolin-4(1<i>H</i>)-One Derivatives as Anti-HIV Agents

<p>Novel quinolone derivatives featuring an 1,3,4-oxadiazole ring as a metal-chelating component and a benzyl group base on HIV-1 integrase inhibitors pharmacophore were designed and synthesized. An antiviral assay revealed that most analogues inhibited HIV-1 replication in the cell culture. Our results showed that compounds bearing small alkyl groups as R group were inactive in anti-HIV-1 assay, whereas compounds possessing benzyl or substituted benzyl at the same position showed good anti-HIV activity with the range of 20–57% at 100 μM concentration. Among them, 3-(5-((2-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4-(1<i>H</i>)-one (compound <b>13</b>) showed reasonable cell-based antiviral activity (EC₅₀ = 50 μM) with no considerable cytotoxicity (CC₅₀ > 100 μM) in the cell viability assay, suggesting that it may be amenable to further development for identifying new anti-HIV-1 agents. Docking studies using the later crystallographic data available for PFV integrase corroborate favorable binding to the active site of HIV integrase, providing a basis for the design of more potent analogues.</p

Synthesis, Molecular Modelling and Biological Studies of 3-hydroxy-pyrane-4-one and 3-hydroxy-pyridine-4-one Derivatives as HIV-1 Integrase Inhibitors

Background: Despite the progress in the discovery of antiretroviral compounds for treating HIV-1 infection by targeting HIV integrase (IN), a promising and well-known drug target against HIV-1, there is a growing need to increase the armamentarium against HIV, for avoiding the drug resistance issue. Objective: To develop novel HIV-1 IN inhibitors, a series of 3-hydroxy-pyrane-4-one (HP) and 3- hydroxy-pyridine-4-one (HPO) derivatives have been rationally designed and synthesized. Methods: To provide a significant characterization of the novel compounds, in-depth computational analysis was performed using a novel HIV-1 IN/DNA binary 3D-model for investigating the binding mode of the newly conceived molecules in complex with IN. The 3D-model was generated using the proto-type foamy virus (PFV) DNA as a structural template, positioning the viral polydesoxyribonucleic chain into the HIV-1 IN homology model. Moreover, a series of in vitro tests were performed including HIV-1 activity inhibition, HIV-1 IN activity inhibition, HIV-1 IN strand transfer activity inhibition and cellular toxicity. Results: Bioassay results indicated that most of HP analogues including HPa, HPb, HPc, HPd, HPe and HPg, showed favorable inhibitory activities against HIV-1-IN in the low micromolar range. Particularly halogenated derivatives (HPb and HPd) offered the best biological activities in terms of reduced toxicity and optimum inhibitory activities against HIV-1 IN and HIV-1 in cell culture. Conclusion: Halogenated derivatives, HPb and HPd, displayed the most promising anti-HIV profile, paving the way to the optimization of the presented scaffolds for developing new effective antiviral agents