Comparison of transfection efficiency of polymer-based and lipid-based transfection reagents.

OBJECTIVES: In this study, the optimal dose of Lipofectamine 3000 and Turbofect to transfect adherent cell lines such as CHO-K1 and HEK293 cells in comparison with non-adherent H9T-cells with pEGFP-N1 and pCDH was identified. BACKGROUND: Lipofectamine 3000 is a new transfection reagent which is claimed to be more efficient than other transfection reagents like Turbofect. Transfection efficiency could be affected by the nature of target cell line and vector. METHODS: Transfection efficiency and cytotoxicity of each reagent was identified by using flow cytometry and XTT assay, respectively. RESULTS: Lipofectamine 3000 was more efficient in transfecting pCDH, while Turbofect was more efficient in separate transfection of CHO-K1 and HEK293 with pEGFP-N1. Lipofectamine 3000 could be cytotoxic in transfecting H9T-cells with pCDH. Also, H9T-cells were not sufficiently transfected with each plasmid vector by using each Lipofectamine 3000 and Turbofect. Turbofect had less cytotoxicity effect on all three cell lines than Lipofectamine 3000.Transfection of suspended cells like H9T-cells by using Lipofectamine 3000 and Turbofect would not result in sufficient transfection. CONCLUSION: Lipofectamine 3000 is the best choice for transfection of CHO-K1 and HEK293 with pCDH while Turbofect is preferably used in transfecting these cell lines with pEGFP-N1 (Tab. 1, Fig. 2, Ref. 26).

[Production and evaluation of immunologic characteristics of mzNLA-3, a non-infectious HIV-1 clone with a large deletion in the pol sequence].

Inactivation ofintegrase and reverse transcriptase can revoke the replication of HIV virions, and non-infectious HIV particles are desirable virus-like particle (VLP) vaccine candidates. Here, we produced inactive in replication HIV-1 particles fit for vaccine and virological purposes by introducing a mutation into the pol sequence. Proviral DNA (pNLA-3) was cut at two points in the pol region using the Bal I restriction enzyme and then religated. HEK 293T cells were transfected with the resultant plasmid (pmzNL4-3) to produce mutated virions. To confirm a production of VLPs and evaluate their biological activity the p24 load and syncytium formation (MT2 cells) were analyzed. The assay indicated that mzNL4-3 virions were assembled and contained functional envelope glycoproteins (ENV). In addition, mzNL4-3 virions were not able to infect MT2 and HEK 293T cells. Furthermore, the immunogenicity of VLPs was investigated in a mouse model. According to the data on vaccinated mice, the titer of ENV-specific antibodies rose rapidly after a boosting injection. Moreover, lymphoid cells extracted from these mice proliferated after exposure to the antigen. The mzNL4-3 virus particles possessed immunogenic antigens of HIV and can effectively trigger humoral and CD4 immune responses. Non-infectious mzNL4-3 virions may also be used in biomedical experiments to improve the biological safety conditions. Moreover, the mzNL4-3 seems to be a promising candidate for further HIV-1 vaccine investigations.

Design, synthesis and docking studies of new 4-hydroxyquinoline-3-carbohydrazide derivatives as anti-HIV-1 agents.

A new class of 4-hydroxyquinoline-3-carbohydrazide derivatives was prepared and evaluated for its anti-HIV activity. The primary bioassay results indicated that most of tested compounds possess moderate inhibitory properties against HIV-1 virus (NL4-3) in Hela cells cultures. Our results also indicated that compounds 6d and 7e were the most potent anti-HIV agents among the synthesized compounds with inhibition rate of 32 and 28% at concentration of 100 µM, respectively. A docking study using the later crystallographic data available for PFV integrase including its complexes with Mg2+ and raltegravir, showed that the designed compounds bind into the active site of integrase such that carboxylic and hydroxyl groups of 4-hydroxyquinoline-3-carbohydrazide chelate the Mg2 + ion. Interestingly, all of the synthesized compounds were found to present no significant cytotoxicity at concentration of 100 µM. Therefore, these compounds can provide a very good basis for the development of new anti-HIV-1 agents.

Evaluation of the in vitro antiretroviral potential of some Biginelli-type pyrimidines.

Despite the success of highly active antiretroviral therapy, AIDS still remains as one of the most important world health problems. Toxicity of current available drugs and inevitable emergence of multi-drug resistant strains makes things worse. In the present study a series of novel Biginelli-type pyrimidine compounds were evaluated as potential anti-human immunodeficiency virus (HIV)-1 agents using green fluorescence protein (GFP) reporter single round HIV-1 infection assay. The rate of infected cells was monitored by flowcytometry. The effect of compounds on the cellular proliferation was considered as the cyotoxicity. The anti-HIV-1 active compounds were selected for HIV-1 replication and syncytium formation assays. The antiretroviral activity of compounds was measured against luciferase reporter A murine leukemia virus (AMLV) virions as the retrovirus control. Compounds 2, 5, 6, 8, 11, 12, 13, 17, 18, 20, and 21 were the most potent against HIV-1. Compound 8 had the 50% inhibitory concentration (IC50) of 100 nmol/l for inhibiting HIV-1 replication and 50% cytotoxic concentration (CC50) was up to 100 µmol/l (therapeutic index (TI) >1000). Results show that the active compounds were able to inhibit the retrovirus control as well. Analysis of structure of the studied compounds proved relationships with their anti-HIV-1 effects. Some of the studied compounds seem to be promising anti-HIV-1 drug candidates. Structural manipulation based on the well-defined structure-activity relationships might propose some new leads for anti-HIV-1 drug discovery programs.

Development of single-cycle replicable human immunodeficiency virus 1 mutants.

Non-infectious but antigenic human immunodeficiency virus 1 (HIV-1) particles are essential tool for the research on many topics associated with this virus. Here we report the construction of plasmid containing the HIV-1 genome mutated in the pol gene, which was co-transfected with plasmids expressing the pol gene products reverse transcriptase (RT) and integrase (IN), and the glycoprotein G of vesicular stomatitis virus (VSV-G). The virions produced in HEK 293 T cells were antigenic, but able to replicate only for one cycle, e.g. first generation single-cycle replicable (SCR) virions. The presence of VSV-G in the envelope of these virions had to ensure a wider spectrum of susceptible cell types for the replication of SCR. Replication of the first generation SCR virions in HEK 293T, MT-2, and mouse spleen cells was examined by p24-capture ELISA, syncytium formation assay, and electron microscopy (EM). HEK 293T and MT-2 cell lines showed a similar replication capacity, while primary cultures of mouse spleen cells were much less effective. The infection of MT-2 cells with the first generation of SCR virions yielded the second generation SCR virions, which were non-infectious. Summing up, the HIV-1 SCR virions represent the useful tool for HIV-1 research facilitating a better biological safety. Moreover, considering their antigenic composition and limited replication, SCR virions may be a promising candidate for the vaccine studies.

HIV-1 Gag p24-Nef fusion peptide induces cellular and humoral immune response in a mouse model.

UNLABELLED: Many Human immunodeficiency virus (HIV) candidate vaccines have been tested in clinical trials, but none was sufficiently effective in the prevention of HIV infection. A HIV vaccine should induce humoral as well as cell-mediated response, the latter including the cytotoxic CD8+ T lymphocyte (CTL) response. In this study, we immunized BALB/c mice with a purified fusion peptide Gag p24-Nef and evaluated immune responses. As for the cellular responses, the adjuvanted fusion peptide induced lymphocyte proliferation, CTL response, and cytokines IFN-gamma and IL-4 in the Th1 pattern. Humoral immune response to the adjuvanted fusion peptide included an increase in IgG antibodies of more IgG2a than IgG1 subtype. These results indicate that the employed HIV-1 peptide construct can elicit both cellular and humoral immune responses in mice. Further studies aimed at memory T cells and other aspects of immune responses are needed before a comprehensive assessment of this candidate vaccine could be provided. KEYWORDS: epitopes; fusion peptide; HIV-1 p24-Nef; immune response.