Plasma Rna And Pbmcs Dna Hiv-1 Drug-resistant Quasispecies Polymorphism And Molecular Evolutionary Studies

Human immunodeficiency virus (HIV) is highly variable human pathogen. Under the pressure of the antiviral drugs, HIV can rapidly make drug resistance mutations, resulting in the failure of antiviral therapy (ART). Resistant HIV strains can be disseminated so that they can present in antiretroviral treatment–Na?¨ve populations and compromise initial antiretroviral therapy. Drug resistance has become a very important public health problem, and has become the mostly obstacle of AIDS (acquired immunodeficiency syndrome) prevention and cure. Finding out the molecular evolution rule of HIV drug resistance and comprehending the effect of new drug resistant mutations are bases of comprehensive study of the resistance molecular mechanism, and this can provide a scientific basis for effective prevention and restraint of HIV drug-resistant strains.To illustrate the HIV-1 resistance molecular evolution of plasma and peripheral blood lymphocytes (PBMCs) and clarify the contribution of new drug resistant mutations, we developed a series of study on the base of AIDS patients'cohort receiving free ART in countryside of Henan province. First of all, we investigated the development and evolvement of HIV drug resistance among the AIDS patients in countryside of Henan province, part of them instead of second-line antiretroviral regimen, then isolated primary HIV-1 strains from several patients and identified the genotype drug resistance. Afterwards on the basis of plasma and PBMCs samples from partial patients followed up long-term, we revealed the resistant evolutionary relationship of virus quasispecies between plasma and PBMCs through the large number of clone sequencing and genotype resistance analysis. Finally, aim at H221Y, which emerged frequently and had not yet definite phenotype explanation, we elucidated the contributions to drug resistance of H221Y and united with other mutations by constructing serial strains with different drug resistant mutations and phenotypic identification.PartⅠDrug resistance study of second-line antiretroviral regimen from AIDS patients receiving ART in countryside of Henan provinceThe countryside of Henan province was one of the regions receiving free ART earliest. The patients took the same antiretroviral regimen at the same time and supervised by doctor in rural. Our lab has monitored the HIV drug resistance of this region for 7 years since September 2003. From February 2010, partial patients replaced the first-line antiretroviral regimen with second-line, according to clinical symptoms, viral loads, the numbers of CD4+T lymphocyte, and the results of HIV drug resistance survey.77 patients among the ART cohort followed up twice in October 2009 and June 2010, and 154 person-times were investigated and collected plasma. 43 of the 77 patients replaced the antiretroviral regimen with second-line regimen in February to March in 2010; the rest 34 patients still took first-line regimen. Questionnaires related to ART including clinical manifestations, anti-viral management, medication compliance and other socio-demographic characteristics were collected every follow-up, at the same time plasma were collected to detect VL and CD4+T lymphocyte number, in addition genotypes of drug resistance were analyzed. Main results and conclusions include:1. VL of regimen changed group declined remarkably (P=0.005) three months later. After replacement, the viral inhibit rate ascended to 65.80% (27/41) from 46.34% (19/41) before replacement. VL of regimen unchanged group declined remarkably too (P<0.005), and the viral inhibit rate ascended to 40.63% (13/32) of second follow-up from 21.86% (7/32) of first follow-up.2. The CD4+T lymphocyte number of regimen changed group altered invisibly (P=0.279). 39.53% (17/43) patients'CD4+T lymphocyte numbers exceeded 350cells/μl before regimen transformation, and 44.19% (19/43) after regimen replacement which increased appreciably compared to before regimen instead. The CD4+T lymphocyte number of regimen unchanged group changed unclearly (P=0.608) too. In the first follow-up, 82.35% (28/34) patients'CD4+T lymphocyte number exceeded 350cells/μl before regimen instead, and 70.59% (24/34) in the second follow-up.3. Drug resistance rate declined little in second follow-up among both regimen changed and unchanged group. After regimen replacement, drug resistance rate of changed group descended to 6/11 from 7/9 of before regimen instead. As to regimen unchanged group, during second follow-up, drug resistance rate descended to 7/14 from12/20 during first follow-up. PartⅡThe polymorphism and molecular evolution of HIV-1 drug resistant quasispecies in plasma RNA and PBMCs DNAHIV exists as complex quasispecies in the HIV infection individual. Under different types and intensities of drug selection pressures, HIV-1 drug resistance develops over time and gradually increases as well as the virus composition and distribution of quasispecies are always in the process of dynamic change. The drug resistance populations consist of major and minor resistant HIV strains, and the low-abundance HIV drug resistant variants can out-compete other virus in presence of drug pressure and become the major resistant population. In the process of HIV infection, various changes in the virus genome are continuously recorded in the latent integrate provirus. At the same time, the drug resistant viruses continuously become well established in long-term reservoirs that allow drug resistance to persist. The evolutions of resistant strains are independent in different organizations, and it has been reported that resistant quasispecies distributions are significantly different in plasma and peripheral blood lymphocytes (PBMCs), lymph and nerve tissues. Generally, plasma is considered to reflect instantly the status in vivo and is the appointed sample of drug resistance detection and supervision by World Health Organization. Although molecular evolution of resistant strains in the plasma has been studied more profoundly, evolutionary characteristics of resistant virus quasispecies in plasma and peripheral blood lymphocytes(PBMCs) of long-term treatment patients and their dynamic evolution relations has no systematic research.The objective of this study was to illustrate the molecular evolution rule of HIV-1 drug resistance quasispecies in plasma and peripheral blood lymphocytes (PBMCs) and to reveal the resistance evolutionary relationship between plasma and PBMCs virus quasispecies under the special rural anti-viral treatment mode through the large number of clone sequencing, genotype resistance analysis from AIDS patients receiving long-term ART in rural parts of Henan province. We investigated 6 AIDS patients from the drug resistance supervision cohort in the rural part of Henan province who had received ART for a long time. Follow-up times of these patients were at least 8 times, while the most was 10 times and cumulative follow-up was 54 times covering 5 years. Questionnaires related to ART including clinical manifestations, anti-viral management, medication compliance and other socio-demographic characteristics were collected every follow-up. 2162bp gene fragments (2147 ~ 4308) containing HIV-1 protease and reverse transcriptase regions of plasma and PBMCs were obtained by RT-PCR or direct PCR respectively, and then cloned and sequenced. 20-30 clones selected from each sample were sequenced. 1588 sequences were obtained in which 967 sequences from plasma samples and the others from PBMCs. Drug resistant mutations and evolution were analyzed. The head results and conclusions as follow:1. The distribution of drug resistant quasispecies in plasma and PBMCs. The varieties of PIs (protease inhibitors), NRTIs (nucleoside reverse transcriptase inhibitors) mutations and NNRTIs (non-nucleoside reverse transcriptase inhibitors) mutations in plasma are more than those in PBMCs. In most patients, drug resistant mutations coexisting in plasma and PBMCs often occur with high-frequency, which reflects that predominant drug resistant mutations of two types of tissue are similar.2. The evolvement of drug resistant quasispecies in plasma and PBMCs. In most patients, PI mutations, NRTI mutations and NNRTI mutations in plasma arises earlier than those in PBMCs. In few patients, HIV-1 drug resistant evolution in plasma and PBMCs are obviously differential. Evolution of HIV-1 NRTIs and NNRTIs drug resistance-associated mutations patterns of the 6 patients shows gradually accumulative process along with long-term treatment, which leads to further drug resistance.3. The characteristics of NRTIs drug resistant patterns in plasma and PBMCs. T215Y appears solely or with other mutations at the beginning of follow-up in all samples. The combination of T215Y and M41L occurs early and stays the whole course of supervision. L210W comes into the NRTI mutation patterns at medium or terminal follow-up. Other piled mutations include D67N, K70R and E44D (A). In very few patients, T215Y tends to be replaced by T215F. The drug resistant degree of NRTI mutation patterns relates with mutation category of the patterns, that is, patterns with more mutations always show higher drug resistance.4. The characteristics of NNRTIs drug resistant patterns in plasma and PBMCs. By and large, NNRTIs mutation patterns are simpler than NRTIs mutation patterns, and frequently include fewer mutations. Y181C exists in all samples except plasma of patient 2 & 4, moreover, the mutation can stay steadily the entire course once emergence. K103N, as the other important NNRTI mutation, occurs in patient 2, 4, 5 and 6, remarkably it appears transitorily in plasma of patient 4 and PBMCs of patient 5, but within other samples, the mutation can stay stably till the end of follow-up in case appearance. H221Y emerges in almost all samples except PBMCs of patient 5, however, the occurrence time of the mutation varies from sample to sample, and same to Y181C, the mutation can stay steadily the course once emergence. NNRTI mutations occur earlier and always show higher drug resistance to NVP, especially, single mutation can lead to high drug resistance to NVP.5. The characteristics of MDR (multidrug resistance) drug resistant patterns in plasma and PBMCs. Multi-drug resistant patterns of plasma are more complicated than those of PBMCs, the kinds of each multi-drug resistant patterns of plasma are more complex than those of PBMCs too. NRTI+NNRTI double drug resistant patterns are the most primary form, which happen in all samples. In the next place, PI+NRTI+NNRTI trinal drug resistant patterns appear in all plasma of the six patients. Multi-drug resistant patterns often show higher drug resistant degree.6. The mean distance of MDR (multidrug resistance) drug resistant patterns in plasma and PBMCs. Mean distance of plasma quasispecies is higher than that of PBMCs in patient 1 and 5, and it is lower in patient 3, and it is no statistical difference in patient 4. However, in these patients, drug resistant patterns of plasmas are more intricate than that of PBMCs, which indicates that mean distance can not represent the complexity degree of the drug resistance.PartⅢEvolvement of HIV-1 Drug Resistant mutations in vitro without drug pressureHIV-1 drug resistant quasispecies show complex dynamic transformation and evolvement in vivo under the pressure of drugs. In the process of infecting cells in vitro, virus quasispecies with various of drug resistant mutations grow in target cells, and the virus change complicatedly without the pressure of drugs, at some times they can revert to wild type or compete each other among the quasispecies, ultimately the virus with best replication capacity and fitness can vegetate richly and become exist stably, thus movement reflects the trauma degree of fitness because of different mutations.To reveal the impact of drug resistant mutations on virus replication capacity and fitness, we observed replication dynamics of the drug resistant HIV-1 isolates and evolvement tendency of the drug resistant mutations in vitro without drug pressure through culturing strains in vitro, which isolated from AIDS patients receiving ART. PBMCs from 15 AIDS patients receiving ART (3TC+D4T+NVP) were collected, and the primary HIV-1 stains were separated utilizing co-cultivated with PBMCs from normal people. Virus growth and the evolution of the drug resistant mutations in vitro without drug pressure were observed. Primary results include:1. Eight strong positive strains were isolated successfully from 8/15 AIDS patients with viral loads higher than 1000 copies/ml, and the replication dynamics were analyzed. Three strains grew rapidly/highly and the other five strains grew slowly/highly.2. Two of them were drug resistant strains. Drug resistant mutations of the two strains were respectively K103N/K238T and M184V/K103N/Y181C/H221Y which show high-level resistance to NVP and 3TC/NVP, respectively. Strains with K103N shows superior fitness and can exist steadily without drug pressure. Strains with M184V and K103N/Y181C/H221Y can also replicate stably in vitro without drug pressure. NNRTIs mutation K238T reproduces astatically and reverts gradually to K238.PartⅣThe impact of H221Y mutations and combined with other mutations on NVP drug resistanceH221Y was found to be a NNRTIs mutation in recent years which conferring resistance to NVP, and had wild prevalence in our country. All the 6 patients occurred H221Y in partⅡof this paper, and the occurrence frequency within quasispecies always exceeded 30%, especially, at follow-up anaphase, the frequency could exceed 90%. Remarkably, H221Y often appeared together with Y181C in this part. In addition, one primary HIV-1 drug resistant stain in partⅢcarried K103N/Y181C/H221Y mutation combination, and the strain can replicate stably in vitro in absence of drugs. Now, the impact of H221Y on drug resistance is still faint, and the interaction of H221Y with other mutations is yet indistinct.Objective of this part is to clarify the impact of H221Y by itself and combination with other mutations on drug resistance. 646bp HIV-1 pol gene fragments were embedded pNL4-3 bone plasmid, and H221Y and (/or) Y181C were reverted to wild type by site-directed mutagenesis, then strains with various mutations were packed, in the end phenotype analysis was analyzed on TZM-bl cells. The results as follows:1. 20 strains with different drug resistant mutation combinations were constructed, these mutation combinations include K101Q/Y181C/H221Y (1-1), K101Q/Y181C (1-2), K101Q/H221Y (1-3), K101Q (1-4), K101E/Y181C/H221Y (2-1), K101E/Y181C (2-2), K101E/H221Y (2-3), K101E (2-4), V179E/Y181C/H221Y (3-1), V179E/Y181C (3-2), V179E/H221Y (3-3), V179E (3-4), V179D/Y181C/H221Y (4-1), V179D/Y181C (4-2), V179D/H221Y (4-3), V179D (4-4), K103N/Y181C/H221Y (5-1), K103N/Y181C (5-2), K103N /H221Y (5-3), K103N (5-4).2. TCID50 and IC50 to NVP of 12 strains were detected. The IC50 of strain 1-1, 1-2, 1-3, 1-4, 4-1, 4-2, 4-3, 4-4, 5-1, 5-2, 5-3 and 5-4 to NVP were separately 108.150±1.909μM, 47.807±18.131μM, 0.141±0.052μM, 0.037±0.010μM, 162.633±47.497μM, 44.980±12.763μM, 0.415±0.014μM, 0.142±0.022μM, 191.100±91.358μM, 89.180±22.656μM, 4.408±0.915μM, 2.013±0.360μM and 0.031±0.005μM.3. Y181C conferred extraordinary resistance to NVP. As to mutation combinations such as K101Q/H221Y, K101Q, V179D/H221Y, V179D, K103N/H221Y, K103N, the IC50 to NVP improved by Y181C were respectively 759.144±41.903, 1296.979±289.108, 390.039±101.646, 312.469±45.255, 41.879±8.403 and 47.878±4.197 times.4. H221Y could improve the resistance degree of all mutation combinations to NVP to a certain extent. As to mutation combinations such as K101Q/Y181C, K101Q, V179D/Y181C, V179D, K103N/Y181C, K103N, the IC50 to NVP improved by H221Y were respectively 2.237±0.910, 3.238±0.317, 3.644±0.469, 2.969±0.435, 2.080±0.496 and 2.183±0.091 times.5. According to K101Q, V179D and K103N, times of IC50 to NVP improved by Y181C/H221Y were respectively 3444.646±834.542, 1132.624±180.368 and 100.621±32.478.