The Drug Resistance And Mechanisms Of PEGylated HIV-1 Fusion Inhibitors

Background and objectiveAIDS is a chronic infectious disease caused by human immunodeficiency virus(HIV).No effective HIV-1 vaccines have been successfully developed,and anti-HIV-1 drugs are still the primary means of preventing and treating AIDS.Traditional anti-HIV-1 drugs are mainly small molecule inhibitors targeting virus reverse transcriptases,proteases or integrases,and they would take action after entering the cells.With the gradual elucidation of the molecular mechanism of HIV-1 invading host cells,HIV-1 fusion inhibitors that prevent viruses from entering cells have become a hot spot.As the only HIV-1 fusion inhibitor approved by the US FDA,enfuvirtide's clinical application had been severely restricted due to its short half-life and high injection frequency.Long-acting HIV fusion inhibitors have better pharmacokinetic properties,which not only could reduce the frequency of injection and economic burden of patients,but also could prevent high-risk populations from HIV-1 infection.However,with the widespread use of antiviral drugs,some amino acid mutates have been appeared in HIV-1 genome under the pressure of drug selection,causing the viruses to be insensitive to drugs.Polyethylene glycol modification could prolong the half-life of polypeptides by improving pharmacokinetics,and many PEGylated polypeptide drugs are now being clinically used.In this project,we cooperated with the Institute of Microbiology,Chinese Academy of Science.PEGylated C34 was synthesized and used as the research object.On the basis of analysis of their drug sensitivities on HIV-1 clinical viruses isolated in China,we further explored their mechanisms of action and resistance,which would provide new ideas for the design of drugs in the future.Methods1.Replication and preparation of HIV-1 strains47 HIV-1 clinical isolates were collected from Sichuan,Guangxi,Beijing,and Anhui.The near full-length genome was amplified,and phylogenetic trees were constructed to analyze the subtypes.In order to replicate the 47 clinical strains,peripheral blood mononuclear cells were isolated by density gradient force centrifugation.pNL4-3 was transfected into HEK293T cells to obtain laboratory adapted strain NL4-3,then TCID50 and p24 were determined on TZM-bl cells.2.The inhibitory activities of HIV-1 fusion inhibitorsThe synthesis and preparation of PEGylated HIV-1 fusion inhibitors were carried out in collaboration with the Institute of Microbiology,Chinese Academy of Sciences.Polypeptides T20,C34 and PEGylated C34(PEG2kC34 and PEG5kC34)were serially diluted and then co-cultured with HIV-1 envelope protein,the HIV-1 laboratory adaptation strain NL4-3 or HIV-1 clinical isolates on TZM-bl cells for 48 h.The inhibitory activities of PEGylated HIV-1 fusion inhibitors were tested by detecting the relative fluorescence units of TZM-bl cells.3.The inhibitory activities of PEGylated C34 on the formation of HIV-1 gp41 six-helix structuresThe changes of the molar ratio of the PEGylated HIV-1 fusion inhibitors from 195 nm to 270 nm and from 20 ? to 98 ? at 222 nm were determined by circular dichroism.The binding capacities of the HIV-1 fusion inhibitors and N36 were determined by calculating the percentage of the a-helix and the Tm.The N36 with a final concentration of 100 ?M and the HIV-1 fusion inhibitors at different dilutions were incubated at 37 ? for 30 min to test the abilities of them to form six-helix structures by native polyacrylamide gel electrophoresis.4.The determination of PEGylated C34 half-life in rats12 Sprague-Dawley rats were randomly divided into 3 groups.Blood was collected from the tail vein before and after subcutaneous injection of HIV-1 fusion inhibitors,and the diluted plasma was co-cultured with NL4-3 on TZM-bl cells for 48 h.The serum dilution to inhibit 50%virus infection was calculated by detecting the relative fluorescence units.5.Screening and identification of drug resistance sites of PEGylated HIV-1 fusion inhibitors in vitroMT2 cells were passaged every 3 to 4 days and viral replication was monitored by observing the formation of syncytia.At each viral breakthrough point,the concentration of the inhibitors was doubled.Nested polymerase chain reactions were used to amplify the gp41 genes of HIV-1 at different drug concentration.The mutation sites were obtained by aligning the gp41 gene sequences.Using plasmid pNL4-3 as a backbone,plasmids containing mutation sites were constructed by molecular cloning.And then the plasmids were transfected into HEK293T cells,and the TCID50 and p24 of viruses were determined.The phenotypic resistance assay was taken to confirm the sites.6.Next generation sequencing to detect resistance sites of viruses under the pressure of HIV-1 fusion inhibitors in vitroThe virus culture supernatant was collected,and the RNA of the 8th,9th and 10th generation virus was extracted.Then HIV-1 gp41 genes was amplified by one-step nested PCR.Next generation sequencing was taken by the Institute of Infectious Diseases,Beijing Ditan Hospital.7.Virus fitness assayEqual amount of the mutation sites containing virus and the wild virus were co-cultured with PM1 cells.Half of the cells were collected at day 3,4,5 and 6,then stained with anti-Thy1.1 and anti-Thy1.2 antibodies.The proportion of infected cells was detected by flow cytometry,and the relative replication fitness of the strains was determined.Results1.PEGylated HIV-1 fusion inhibitors could effectively inhibit the replication of the HIV-1 subtypes circulating in China,showing a better broad-spectrumTwo PEGylated C34 were successfully synthesized.Their average molecular weights were 6,500 Da and 9,300 Da.The purities of them were proved to be higher than 98%by liquid chromatography analysis.PEGylated HIV-1 fusion inhibitors could effectively inhibit HIV-1 envelope protein-mediated cell-cell fusion and the replication of laboratory-adapted strain NL4-3 as well as the HIV-1 clinical isolates of China's circulating subtypes,especially for the CRF01_AE subtype which had the highest proportion in China with an average EC50 of 19.34 nM and 19.64 nM.PEGylated C34 had lower cytotoxicity in vitro with the CC50 greater than 100?M.2.PEGylated C34 could inhibit the fusion of viral envelope proteins with cell membranes by binding to NHR to form a stable six-helix structure and exhibited a longer half-lifeCircular dichroism results showed that both PEGylated fusion inhibitors could bind to the corresponding sequences of HIV-1 gp41 to form high a-helical complexes.The a-helix or melting temperatures of the composites N36/PEG2kC34 and N36/PEG5kC34 were 93.90%and 96.58%or 57.03 0 C and 55.04?.Similar to C34,PEGylated C34 could also bind to N36 to form a stable helical structure,and this binding has a dose-dependent relationship with N36 in a certain range.And the half-life of PEGylated C34 were significantly prolonged in SD rats.3.The appearance of L44V and N126K sites increased the drug resistance of PEGylated C34The final concentrations of the polypeptides C34,PEG2kC34 and PEG5kC34 in the drug resistance experiments were 4,096,1,024 and 512-fold,respectively,of their initial concentrations.Three HIV-1 resistant strains were obtained after 7 months of screening.The mutations occurred in 44,126 or 250 sites.In the presence of C34,the previously unreported L44V mutation began to appear in the 9th generation,and the N126K mutation in the 5th generation.With regard to PEG2kC34,the L44V and N126K mutations appeared simultaneously in the 4th generation.However,as for PEG5kC34,only R250Q mutation was detected in the 7th generation.L44V or N126K mutation could reduce the drug sensitivities of PEG2kC34 and PEG5kC34 by 4.2-fold and 2.3-fold or 1.2-fold and 1.6-fold,while L44V and N126K double mutations could increase the resistance by 12.4 or 9.8-fold.Neutralization assay results of 10E8 antibody on virus containing mutant sites showed that drug resistance sites had a certain effect on gp41 structure.Therefore,we hypothesize that PEGylated C34 resistance maintenance requires both L44V and N126K.4.The L33S and R271K sites increased the drug resistance of PEGylated T20 in vitroWhen the L33S mutation presented in gp41,the EC50 of T20,PEG2kT20 and PEG5kT20 had increased 12,83 and 28-fold,respectively.When the R271K mutation presented in gp120,the EC50 of T20,PEG2kT20 and PEG5kT20 had increased less than 3-fold.When the L33S and R271K sites simultaneously mutated,the EC50 of them had increased more than 83-fold.That is to say,R271K in gp120 could increase the resistance of L33S,which has not been reported before.The cross-resistance of T20 to PEGylate T20 showed that T20 resistance was different from PEG2kT20 and PEG5kT20.NL4-3-V38A,NL4-3-V38E-N42S,NL4-3-V38A-N42D and NL4-3-V38A-N42T showed different degrees of resistance to T20,PEG2kT20 and PEG5kT20,While NL4-3-N43K showed a certain degree of resistance to T20 and PEG5kT20 but not observed resistance to PEG2kT20 in this study.Multiple nucleotide polymorphism sites were detected in next generation sequencing,and the drug resistance of PEGylated C34 and T20 was different.5.N126K mutation could significantly decrease the relative replication fitness of the virusThe replication fitness of AT1 and AT2 derivatives that containing the resistance sites were detected by flow cytometric.The results showed that L44V,N126K and R250Q could reduce the relative replication fitness of the virus in different degrees.And the N126K mutation could significantly affect the relative replication fitness of the virus.ConclusionPEGylated HIV-1 fusion inhibitors could effectively inhibit HIV-1 env-mediated cell-cell fusion and replication of laboratory-adapted strain NL4-3,and had a better broad-spectrum inhibition against HIV-1 clinical isolates of China's circulating subtypes,their half-life in SD rats were significantly prolonged.Drug pressure assay showed that N126K and L44V sites appeared simultaneously could increase the resistance to PEGylated C34,while N126K mutation could significantly decrease the relative replication fitness of the virus.Unlike PEGylated C34,the main drug resistance site of PEGylated T20 was L33S.When L33S was simultaneously appearance with R271K,it could improve the virus drug resistance.This study provided theoretical basis for the study of drug resistance and mechanisms of long-acting HIV-1 fusion inhibitor.