Research On Regularity Of Molecular Evolution Of Drug Resistance For HIV-1 From Countryside In Central China

So far, cross-sectional monitoring of HIV-1 resistance has been widely performed in China, however further study of cohort and evolutional regularity is deficient. There is not enough apprehension for regularity of development of HIV-1 resistance and biological characteristics of resistant strains. A major blindness exists in application of antiretroviral drugs and clinical treatment. This research elucidates prevalence, development and evolutional regularity of HIV-1 resistance after antiretroviral therapy (ART) and its effect on efficacy of ART through cohort study of patients who has received normative ART in HeNan Province.Part one : cohort study on the development and evolution of HIV-1 drug resistance in countryside of central ChinaSeventy-six AIDS patients receiving free ART in HeNan province were investigated through prospective cohort study from March in 2004. The antiretroviral regimen included two nucleoside reverse transcriptase inhibitors (AZT+ddI) and one non-nucleoside transcriptase inhibitor (NVP). There were six times of follow-up during treatment over 2.5 years. Questionnaire related to ART was collected and peripheral venous blood was sampled to detect CD4+T cell count and viral load and perform genotypic resistance assay every follow-up. 427 questionnaires with EDTA-anticoagulant venous blood were collected during the six times of follow-up. Characteristic of demography, side effect of drugs and adherence were obtained from questionnaires; the status of viral inhibition and rehabilitation of immune function was analyzed from CD4 count and viral load; characteristic of resistance mutations and susceptibility to drugs were obtained from genotypic resistance assay. Prevalence, development and evolutional regularity of HIV-1 resistance were elucidated. Results are as follow:A. Proportion of patients who was resistant to at least one drug was up to 78.9% during follow-up. The resistance began to emerge mainly during 18 months after initiating ART.B. Prevalence of resistance to nucleotide reverse transcriptase inhibitors (NRTIs) and non-nucleotide reverse transcriptase inhibitors (NNRTIs) was respectively between 6.6-23.7% and 13.2-42.1% through cohort study. Tendency of resistance prevalence of the two kinds of drugs generally ascended.C. There were seventeen resistance mutations associated with NRTIs including TAMs (M41L, D67N, K70R, L210W, T215Y, K219Q), Q151M complex (V75I, F77L, F116Y and O151M), T69S-SS insert mutation, mutations related to ddI (K65E, L74V, M184V/I) and some minor mutations (E44D, V118I). TAMs that globally ascended during the whole treatment were the main mutations leading to resistance to NRTIs. Incidence of the other mutations was low and duration was short. There were fifteen resistance mutations associated with NNRTIs and the main three mutations were K103N, Y181C and G190A which had a high incidence with continuous increase.D. Population with resistance was mainly intermediately/highly resistant to AZT, lowly resistant to ddI and highly resistant to NVP. Patents with resistance to AZT and ddI were cross-resistant to ABC, d4T and TDF simultaneously. Patients with mutation V184V/I was lowly resistant to 3TC and FTC. Patients who were highly resistant to NVP also had high-level resistance to DLV and EFV. 28.9% patients had multi-drug resistance mutations to NRTIs and NNRTIs.E. Adherence was the main influencing factor of resistance development. The relation between adherence and incidence of resistance in the prophase and latter phase of treatment presented different patterns. In the prophase of treatment (3-12 months), it presented 'bell shape' curve, as meant that patients with good adherence (>90%) and bad adherence (<50%) had lower incidence of resistance and that with intermediate adherence (75-90%) had highest incidence of resistance. In the latter phase of treatment (18-30 months), patients with bad adherence still had lower incidence of resistance, but patients with good/intermediate adherence had higher incidence of resistance.F. Average CD4 count of all patients in the thirtieth month of treatment decreased by 215/ml than that in third month of treatment. Average viral load decreased by 0.21og copies/ml. Mean of CD4 count between susceptible and resistant patients didn't have significant difference during whole treatment. Mean of log in viral load in susceptible patients was lower than that in resistant patients significantly.G. Average HIV RT gene divergence of resistant patients was higher than that of susceptible and treatment-discontinued patients significantly. More mutations lead to higher gene divergence.Part two: research on selective kinetics of HIV-1 drug resistanceFour patients with good adherence in cohort study of resistance, possessing wild-type virus at the beginning of treatment and gradually becoming resistant to NRTIs and NNRTIs during treatment were selected as research subjects. RT gene from every patient's blood sampled during 4-5 times of follow-up was cloned and 150-250 clones were selected from each sample to sequence. Sequences from 855 clones were compared in HIV-1 resistance database of Stanford University to obtain genotypic resistance mutations. Characteristics of selective kinetics of developing resistance mutations associated with NRTIst and NNRTIs were elucidated through analyzing feature of genotypic resistance for every patient in different phase of treatment and evolutional regularity of resistant strain in China was revealed. Results are as follow:A. Resistance mutations to NRTIs were a gradually accumulative process and they displayed three different evolutional pathways in the four patients selected. The first was TAMs-1 pattern. Typical resistance mutations of TAMs were generated accumulatively one by one from wild-type strain. TAMs mainly including M41L, L210W, T215Y were formed first and followed by minor resistance mutations with consecutive accumulation. At last, mutations with six TAMs-1s were formed at the thirtieth month of treatment. The second was TAMs-2 pattern. Other resistance mutations were accumulated at the basis of D67N and K70R from the third month of treatment. Typical mutation combination of TAMs-2 including D67N, K70R, K219Q was formed at the twenty-fourth month of treatment. Later, more complex TAMs-2 pattern was formed with other mutation's accumulation. The third was the pattern of combining TAMs-1 and TAMs-2. TAMs-1 including M41L, T215Y and TAMs-2 including D67N, K70R were respectively formed at the twelfth month of treatment. The pattern of combining TAMs-1 and TAMs-2 including M41L, T215Y, D67N, K70R was formed at the thirtieth month of treatment.B. Evolutional regularity of resistance to NNRTIs was that mutation pattern developed first was the final form. Mutation G190A. G190A appeared in the third month of treatment. The combination of G190A and F227L was formed in the eighteenth month and after that three mutations including G190A, F227L, P236V were generated. Mutation Y188C. Y188C mutation was formed in the third month of treatment and maintained at the end of follow-up. Mutations V179D and Y181C. V179D and Y181C mutations appeared in the third month of treatment and evolved into V179D, Y181C, P236V at the end of follow-up.Wild type before the twenty-fourth month of treatment. Mutations mainly including K103N and M230L or K103N and Y181C emerged in the late treatment. In short, development of resistance mutations during the long-term ART is a gradual process from simple to complex mutations. The main mutations associated with NNRTIs developed earlier than that related to NRTIs. The former was simple but persistent.Part three: research on Competitive fitness of HIV-1 resistant strains3TC-resistant and NVP-resistant strains were respectively induced using susceptible strain to investigate influence of resistance mutations to fitness of viral replication. Both of the two resistant strains were co-cultured with wild-type strain respectively containing a drug or not. Four generations were obtained. RNA copies of resistant strain and susceptible one from every generation were detected by real-time quantitive RT-PCR using Taqman MGB probe. Fitness of replication of resistant and susceptible strains was evaluated through variable tendency with time. Results are as follow:A. The first 3TC-resistant and NVP-resistant strains in China were induced successfully in our lab using two different strategies. IC₅₀ of 3TC-resistant strain was greater than 2mM, 1.1×10⁵ times higher than that of wild-type strain; IC₅₀ of NVP-resistant strain was greater than 1mM, 3×10⁴ times higher that that of wild-type strain.B. Equivalent resistant and susceptible strains were co-cultured in drug-contained culture system for four generations. From the first generation to the fourth generation, proportion of RNA copies of 3TC-resistant strain increased from 76.2% to 98.8% and that of wild-type strain decreased from 23.8% to 1.2%; Proportion of RNA copies of NVP-resistant strain increased from 62.0% to 90.5% and that of wild-type strain decreased from 38.0% to 9.5%. The results indicated that fitness of replication for the two resistant strains in drug-contained culture system were better than that of wild-type strain.C. Equivalent resistant and susceptible strains were co-cultured in culture system without drugs for four generations. From first generation to fourth generation, proportion of RNA copies of 3TC-resistant strain decreased from 35.2% to 5.3% and that of wild-type strain increased from 64.8% to 94.7%, as indicated that fitness of replication of 3TC-resistant strain was lower that that of wild-type strain; Proportion of RNA copies of NVP-resistant strain increased from 53.9% to 75.0% and that of wild-type strain decreased from 46.1% to 25.0%, as indicated that fitness of replication of NVP-resistant strain was higher that that of wild-type strain.These results above could partially explain the phenomenon that prevalence of resistance mutations to NNRTIs was high and persistent in the part one.Part four research on phenotypic resistance of HIV-1 subtype-B in ChinaPhenotypic resistance assay directly assesses HIV-1 resistance by analyzing susceptibility to drugs in vitro. Genotypic resistance assay is widely adopted in China at present and phenotypic resistance of clinical HIV-1 isolates has never been investigated, so is the concordance between results of phenotypic and genotypic resistance assays. In the study, eight HIV-1 clinical isolates were selected to perform genotypic and phenotypic resistance assays. Results are as follow:A. IC₅₀ of AZT, 3TC, NVP, IDV to test isolates was respectively between 0.0006-0.1300μmol/l, 0.0016-0.0390μmol/l, 0.0104-0.4234μmol/l and 0.0163 - 0.1142μmol/l through phenotypic resistance assay. Fold change by comparingtest isolates to that of susceptible ones was respectively between 0.1-26.0, 0.5—11.5, 2.3—94.1 and 0.8—5.4.B. There was a high concordance between results of genotypic and phenotypic resistance assays. Genotypic resistance assay could be substituted for phenotypic resistance assay to predict HIV-1 resistance in China.