A lipopeptide-based HIV-1/2 fusion inhibitor, known as LP-19 and formed by adding a fatty acid group (palmitic acid C16) to the C-terminus of 2P23, was designed to target the highly conserved pocket site of gp41with the M-T hook structure. Its sequence is EMTWEEWEKKVEELEKKIEELLK-PEG8-K(C16) (11). The C34 peptide sequence is WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL, and T-20 peptide sequence is YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF. The peptides were synthesized by Beijing ZhongkeYaguang Biotechnology Co., Ltd. LP-19, C34, and T-20 were melted at concentrations of 20 μM, 100 μM, and 200 μM and stored in a refrigerator below -20°C for use and diluted when conducting experiments.

MT-2, HEK 293T, and TZM-bl cell lines and laboratory-adapted HIV strains (NL4-3) were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. MT-2, TZM-bl, and HEK 293T were respectively prepared in RPMI 1640 and DMEM medium, supplemented with 10% fetal bovine serum, 100 ug/mL penicillin and streptomycin. HIV clinical isolates used in the antiviral activities assay were isolated and replicated from HIV infected people in China from the European Research Infrastructures for Poverty Related Diseases project. Informed consent was obtained and signed before sample collection. This study was reviewed by the Institutional Research Ethics Communication of Chinese Center for Disease Control and Prevention (No. X150129355). In the study, 47 culture supernatants of HIV clinical isolates were selected from Anhui, Beijing, Guangxi, and Sichuan respectively. HIV isolates of BJ2015EU14, BJ2015EU16, GX2016EU02, GX2016EU08, XC2014EU09 were national standard strains of pathogenic microorganisms (www.nprc.org.cn).

50% cells culture infectious dose (TCID₅₀) was expressed as viral titration. HIV titration is determined by setting up serial dilutions of HIV NL4-3 strains or HIV clinical isolates in TZM-bl cell line. One virus of HIV stock was thawed and placed into the first row of the plate in three replicates in 96-well plates, where a 5-fold dilution was firstly made and then continuous dilution for 11 times in triplicate. To each well, 1×10⁴ cells of supplemented DMEM medium were added to 200 μL, and the concentration of DEAE-dextran was 15 μg/mL. And then the plates were incubated at 37°C and 5% CO₂ for 48 hours. Britelite™ plus Reporter Gene Assay System (PerkinElmer) detected fluorescence, and the viral TCID₅₀ was calculated by the Reed-Muench method.

HIV NL4-3 strains (200 TCID₅₀/well), 1×10⁴ cells (TZM-bl), and LP-19 or C34 or T-20 serial dilutions were added to the 96-well plate, and the final concentration of DEAE-dextran in each well was 15 μg/mL; the experiment was repeated three times. After incubation at 37°C and 5% CO₂ for 48 hours, the fluorescence value of TZM-bl was detected. The inhibitory percentage calculation is as follows: (average fluorescence value of virus control wells - fluorescence value of LP-19 or C34 or T-20 wells)/(average fluorescence value of virus control wells - average fluorescence value of cell control wells) × 100%. The 50% inhibitory concentration (IC₅₀) and 90% inhibitory concentration (IC₉₀) of LP-19 lipopeptide or C34 or T-20 was calculated according GraphPad Prism software.

In vitro selection for HIV resistance to LP-19 was performed. 1×10⁴ MT-2 cells were quantitatively infected with NL4-3 strain and inoculated into wells of a 12-well plate. A duplicate well was set up, and positive and negative control wells were set up at the same time (15). 200 TCLD₅₀/mL NL4-3 virus was used to infect the MT-2 cells with the addition of 1-fold IC₅₀ of LP-19 during cell culture. The cells were incubated at 37°C and 5% CO₂. The viral replication was monitored by observing the formation of syncytia by optical microscopy. When viruses appeared as a massive syncytium formation, the culture supernatant was harvested at regular time intervals, and the inhibitor concentration was doubled. The culture cells and supernatant were harvested and stored at -80°C. Meanwhile C34 was used as the parallel control of inhibitor.

Viral RNA was extracted and purified from infected cells using the QIAamp Viral RNA mini kit (Qiagen). The HIV gp41 region was amplified using an in-house-designed polymerase chain reaction (PCR) system using first round specific primers (gp41 F1: 5’- AGAGCAGTGGGAATAGGAGCTTTG -3’, gp41 R1: 5’- TGACCACTTGCCACCCATCTTATAGCAA -3’). Second round specific primers (gp41 F2: 5’- TCTTGGGAGCAGCAGGAAGCACTAT -3’; gp41 R2: 5’- GCCCTGTCTTATTCTTCTAGGTATGTGGCG -3’) were used to amplify the HIV-1 gp41 region (sequenced by TianyiHuiyuan Biotechnology Co., Ltd.). The sequencing results were compared with the pNL4-3 (GenBank: AF324493.2) sequence of NCBI, and the drug resistance-related sites of viral mutations under continuous drug pressure were found.

The QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) introduces the mutations encoding A243V into the NL4-3 plasmid, and contains the primers and sequences constructed by the mutant site viruses: A243V-F: 5’- GTGAACGGATCCTTGGTACTTATCTGGGACGATC -3’, A243V-R: 5’- GATCGTCCCAGATAAGTACCAAGGATCCGTTCAC -3’. Plasmids containing mutation sites were correctly sequenced (sequenced by TianyiHuiyuan Biotechnology Co., Ltd.).

This was carried out based on the instructions of Lipofectamine™ 3000 Reagent (Invitrogen), a cationic lipid-based transfection reagent. 1×10⁶ HEK 293T was inoculated per well in a 6-well plate, and 2 mL DMEM complete medium was added to each well,. The culture was kept overnight in an incubator at a constant temperature of 37°C and 5% CO₂. When the HEK 293T density reached about 50-70%, the serum-free and antibiotic-free DMEM medium was replaced before the experiment. 125 µL Opti-DMEM and 3.75 µL of Lipofectamine™ 3000 Reagent were added to a 1.5 mL sterilized EP tube and mixed thoroughly. Then 125 µL Opti-DMEM, 5 µL P3000™ Reagent, and 5 µg pNL4-3 were added to another 1.5 mL sterilized EP tube and mixed thoroughly. The mixture in the second tube was added to the first tube, mixed well, and was allowed to stand at room temperature for 15 minutes. The mixture was then evenly and carefully dropped into a six-well plate and incubated at a constant temperature incubator of 37°C and 5% CO₂ for 4 to 6 hours. Then the complete DMEM medium should be replaced. The supernatants of the virus were harvested 48 hours after transfection, then divided and stored at -80°C in the refrigerator.

To evaluate the antiviral activity of LP-19, especially in direct comparison with well-established entry inhibitors T-20 and C34, we utilized TZM-bl reporter assay, whose antiviral effects can be directly measured by the decrease in luciferase signal. The TZM-bl reporter assay revealed that compared with T-20 and C34, LP-19 a 441-fold and 7-fold increased viral inhibitory effect, and the IC₅₀ of LP-19, C34, and T-20 against HIV NL4-3 strains was 0.15 ± 0.01 nM, 1.02 ± 0.19 nM, and 66.19 ± 20.73 nM, respectively ( Figure 1 ). This result suggested that LP-19 may possess greater antiviral activity than currently available fusion inhibitors.

When HIV antiviral drugs are used as a monotherapy, drug-resistant variants inevitably appear. In addition, when a particular HIV drug is used in a multidrug therapy, it is critical to determine the types of drug-resistant mutants that may have evolved or whether these mutations are more resistant or affect inhibitory activity to its antiviral drug (18). Indeed, almost all clinically significant drug resistance mutation arise because of selective drug pressure. Therefore, in vitro selection of viruses with resistance to LP-19 was performed ( Figures 3A, B ). HIV NL4-3 virus was cultured in MT-2 cells at an initial concentration of 1-fold IC₅₀, when massive syncytia formation was observed, and LP-19 increased as double concentration of IC₅₀ in a culture supernatant and cultured continuously for 39 weeks.

When LP-19 concentrations reached 8-fold and 16-fold over the initial concentrations at culture of 12 weeks and 18 weeks, D230N and N239S mutation sites were observed in CP in the gp41 region, but mutation recovery occurred at drug concentrations of 32-fold (22 weeks), 64-fold (28 weeks), 128-fold (32 weeks), 256-fold (38 weeks), and 512-fold (39 weeks). The A243V site was identified at 64-fold, 128-fold, 256-fold and 512-fold drug concentration (cultured for 28, 32, 38, and 39 weeks). The concentration was 256-fold of the initial concentration at 38 weeks, and the G183S site (TM region of gp41) appeared. Until 39 weeks, when the drug concentration reached 512-fold of the initial concentration, no mutations in the NHR gp41 region were found ( Figure 3C ).

In order to identify if the A243V mutant site is related to LP-19 resistance, NL4-3/A243V viruses containing the mutant site were produced. The titers of NL4-3 strains and mutant NL4-3/A243V strains were 18,275 TCID₅₀/mL and 13,975 TCID₅₀/mL, respectively ( Figure 4 ). The wild NL4-3 and mutant NL4-3/A243V viruses were detected for viral sensitivity to LP-19. The results showed that LP-19 in the mutant NL4-3/A243V viruses remained at the same level of antiviral activity compared to wild NL4-3 viruses, even is a 1.3-fold activity than before the mutation ( Figure 4C ).