HIV (human immunodeficiency virus) microbicide and application thereof
A microbicide, HIV-1 technology, applied in the field of HIV infection prevention, can solve problems such as increasing HIV infection
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Embodiment 1
[0039] Example 1-Sifuvirtide Gel Cell Level Anti-HIV-1 Pseudovirus Activity Test
[0040] Sifuvirtide was dissolved in sterile PBS solution (pH=7.4), or dissolved in 0.0015% hydroxyethylcellulose (HEC) gel. Four HIV-1 Env-pseudoviruses were produced in 293T cells by co-transfection of different subtypes of HIV env expression plasmids with the HIV-1 backbone plasmid pNL4-3Δenv (including all HIV-1 genomes except Env) Among them, the HIV env expression plasmids of four different subtypes are encoding HIV-1 SVPB16 (B subtype), SVPC12 (C subtype), 32-72 (CRF07_BC) and SH188.6 (CRF01_AE) viral strain envelopes albumen. Pseudoviruses were collected and stored at -80°C after titration.
[0041] The specific method is as follows, on a 96-well plate, about 1×10 per well 4 TZM-bl cells were plated and cultured at 37°C for one day in DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% bovine fetoprotein and penicillin-streptomycin. Pseudovirus (per hole 100TCID 50 ) and th...
Embodiment 2
[0043] Example 2-Sifuvirtide Gel In Vitro Safety Evaluation
[0044] In vitro safety evaluation using cell models. Three different concentrations of SFT (3 nM, 30 nM and 300 nM) in 0.015% HEC gel and PBS solutions were prepared. The effect of the above samples on the viability of Caco-2 rectal epithelial cells or macrophage RAW264.7 was detected by MTT colorimetry.
[0045] Follow the method of Denizot and Lang, in simple terms, 5×10 4 ml -1 The cells were seeded in 96-well plates in 0.015% HEC gel containing different concentrations (3nM, 30nM and 300nM) of SFT or SFT in PBS solution, 37°C, humidified 5% CO 2 After culturing for 12 hours under the condition, 50 μl of cells were collected and stored at -80° C., pending further determination of cytokine content. After the remaining samples were cultured for 48 hours, 10 μl of MTT (5 mg / ml) was added to the culture plate and incubated at 37° C. for 4 hours. Then, the medium was removed and formazan was dissolved in acidifie...
Embodiment 3
[0048] Example 3-Safety test of sifuvirtide in mice
[0049] The BALB / c female mice without pathogens of 6-8 weeks were randomly divided into 8 groups, 5 in each group, and 40 μ l of pH 4.5 containing 1.5% HEC gel (placebo control), respectively containing three 1.5% HEC gel, 1% TFV, 3% carrageenan, 6% CS, 1% N-9 gel with various concentrations of SFT (0.03mM, 0.3mM, 3mM). Continuous vaginal administration for 3 days, once every 12 hours. Twelve hours after the last administration, rinse the cervix and vagina with 200 μl sterile normal saline and collect the lavage fluid, then treat the lavage fluid with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche), and centrifuge at 200×g for 10 minutes at 4°C , the supernatant was collected and stored at -80°C for use in the determination of cytokines. After the mice were sacrificed, the vaginal tissues were collected for further mononuclear cell isolation or histological examination.
[0050] Mouse reproductive tract...
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