Pretreatment HIV-1 drug resistance in Argentina: results from a surveillance study performed according to WHO-proposed new methodology in 2014-15.

BACKGROUND: In Argentina, current national guidelines recommend starting with NNRTI-based regimens. Recently, there have been some local reports regarding concerning levels of NNRTI-transmitted resistance, but surveillance has never been carried out at a national level. OBJECTIVES: To determine the prevalence of HIV drug resistance in people starting ART in Argentina using a WHO-proposed methodology. METHODS: This was a cross-sectional, nationally representative study. Twenty-five antiretroviral-dispensing sites throughout the country were randomly chosen to enrol at least 330 persons starting ART, to generate a point prevalence estimate of resistance-associated mutations (RAMs) with a 5% CI (for the total population and for those without antiretroviral exposure). All consecutive patients older than 18 years starting or restarting ART in the chosen clinics were eligible. Samples were processed with Trugene and analysed using the Stanford algorithm. RESULTS: Between August 2014 and March 2015, we obtained 330 samples from people starting ART. The mean ±â€ŠSD age was 35 ±â€Š11 years, 63.4% were male, 16.6% had prior antiretroviral exposure and the median (IQR) CD4 count was 275 cells/mm3 (106-461). The prevalence of RAMs found was 14% (±4%) for the whole population (3% NRTI-RAMs; 11% NNRTI-RAMs and 2% PI-RAMs) and 13% (±4%) for those without prior antiretroviral exposure (3%, 10% and 2%, respectively). The most common mutation was K103N. CONCLUSIONS: This surveillance study showed concerning levels of HIV drug resistance in Argentina, especially to NNRTIs. Due to this finding, Argentina's Ministry of Health guidelines will change, recommending performing a resistance test for everyone before starting ART. If this is taken up properly, it also might function as a continuing surveillance tool.

Improved strategies for HIV diagnosis among men who have sex with men (MSM) in Buenos Aires, Argentina, a population with a high prevalence and incidence of HIV infection.

BACKGROUND: In Argentina, HIV diagnosis in adults is made using one or two enzyme immunoassay tests and a confirmatory test. These strategies may fail to identify infected individuals during early primary infection, which represents an important public health problem among groups with a high HIV incidence, such as men who have sex with men (MSM) (6.3% persons/year). The general objective of this study was to contribute to reducing HIV transmission among MSM through the identification of antibody-negative, nucleic acid-positive individuals. FINDINGS: A total of 1549 MSM were recruited for an HIV seroprevalence study. A total of 161 (10.4%) MSM were HIV-positive and 14 (0.9%) were indeterminate. Among the 1374 negative individuals, 16 (1.2%) exhibited reactive results in the screening assay. Indeterminate Western blot (WB) samples and negative WB samples (with discordant results in the screening) were analysed to detect HIV nucleic acid by viral load testing. Up to 23.1% of HIV-indeterminate WB samples and 7.1% of HIV-negative WB samples with discordant results in the screening assays had detectable nucleic acid. Overall, 14.8% of the samples with discordant or indeterminate results were identified as HIV-positive using direct diagnosis. With the identification of four new cases using the nucleic acid detection test, the HIV prevalence in MSM increased by 0.3% (from 10.4 to 10.7%). CONCLUSIONS: The results of this study suggest the importance of including nucleic acid detection in the HIV algorithm for MSM with HIV-indeterminate WB results and those with HIV-negative WB results and discordant results in screening assays, in order to decrease HIV transmission among this population with a high HIV prevalence and incidence.

Bioinformatic analysis of post-transmission viral readaptation in Argentine patients with acute HIV-1 infection.

During the acute phase of HIV-1 infection, a strong readaptation occurs in the viral population. Our objective was to analyze the post-transmission mutations associated with escape to the cytotoxic immune response and its relationship with the progression of the infection. In this study, a total of 17 patients were enrolled during acute/early primary HIV infection and 8 subjects that were the HIV positive partner resulting in 8 transmission pairs. Genotyping of the genetic polymorphisms of HLA class I A and B was performed using PCR-SSOP. Viral RNA extraction was from plasma. 570 single Gag-gene amplifications were obtained by limiting-dilution RT-PCR. Epitope prediction was performed with NetMHC CBS prediction server for the 19 HLA-A and B alleles. Cytotoxic response prediction was performed by using the IEDB Analysis Resource. From our results, we deduce that the transmitted CTL / gag escape frequency in the founder virus was at least double compared to the post-transmission events. Additionally, by means of an algorithm that combines these frequencies, we observed that the founder viruses better adapted to the HLA A / B alleles of the recipient could contribute to a greater progression of the infection. Our results suggest that there is a large adaptation of HIV-1 to the HLA A / B alleles prevalent in our population. However, despite this adaptive advantage, the virus needs to make "readjustments" through new escape and compensatory mutations. Interestingly, according to our results, this readaptation could have a role in the progression of the infection.

Enhanced fidelity of 3TC-selected mutant HIV-1 reverse transcriptase.

Monotherapy with (-)2',3'-dideoxy-3'-thiacytidine (3TC) leads to the appearance of a drug-resistant variant of human immunodeficiency virus-type 1 (HIV-1) with the methionine-184 --> valine (M184V) substitution in the reverse transcriptase (RT). Despite resulting drug resistance, treatment for more than 48 weeks is associated with a lower plasma viral burden than that at baseline. Studies to investigate this apparent contradiction revealed the following. (i) Titers of HIV-neutralizing antibodies remained stable in 3TC-treated individuals in contrast to rapid declines in those treated with azidothymidine (AZT). (ii) Unlike wild-type HIV, growth of M184V HIV in cell culture in the presence of d4T, AZT, Nevirapine, Delavirdine, or Saquinavir did not select for variants displaying drug resistance. (iii) There was an increase in fidelity of nucleotide insertion by the M184V mutant compared with wild-type enzyme.

Hepatitis B virus genotype distribution and its lamivudine-resistant mutants in HIV-coinfected patients with chronic and occult hepatitis B.

Hepatitis B virus (HBV) genotypes were examined in HIV-infected patients with chronic and occult HBV infection. From a total population of 593 HIV-infected patients, 22 individuals (prevalence 3.7%) were found to be HBsAg while 72 (12.1%) were found to be anti-HBc alone. From them, 20 and 4 were HBV DNA positive, respectively. These last four patients are therefore considered to be HBV infected in an occult form. The genotypes could be determined in all 24 HBV-infected patients. HBV-A was the most common (20/24; 83.3%), followed by HBV-D (2/24; 8.3%) and HBV-F (1/24; 4.2%). The remaining sample exhibited mixed infection involving genotypes A and D as pure ones, thus also forming part of three intergenotypic recombinant forms exhibiting different mosaic S gene patterns. The sexual route of transmission was predominant among HBV genotype A-infected patients. Among the 24 HBV DNA-positive patients, point mutations related to lamivudine resistance were found in four strains. These viral strains showed a methionine-to-valine substitution at codon 204 (rtM204V) in association with an upstream B-domain change at rtL180M. Additionally, two of them exhibited the additional rtV173L mutation. The value of HBV molecular monitoring including both HBV viral genomic characterization and genotypic resistance profile in HIV-HBV-coinfected individuals is discussed.

Examination of real-time PCR for HIV-1 RNA and DNA quantitation in patients infected with HIV-1 BF intersubtype recombinant variants.

The impact of HIV-1 genetic diversity on the performance of laboratory testing is an issue that has to be monitored continuously. An "in-house" real-time PCR assay was developed by the Agence Nationale de Recherche sur le SIDA (ANRS) in France for viral load (VL) quantitation based on the amplification of the HIV-1 long terminal repeat (LTR) region. This technology has not been used in Argentina yet and considering the HIV-1 diversity in the country, a comparative analysis of this assay was undertaken versus the Versant HIV-1 RNA 3.0 Assay (b-DNA). The performance was assessed on 30 drug-naïve HIV-1 infected patients who were characterized previously by phylogenetic analysis of the pol and vpu gene. The results showed that there is a significant linear correlation between values of transformed viral load logarithms measured by both, bDNA and real-time PCR assay and that this assay can be used to quantify viral load in samples from BF-infected patients with the same accuracy and reliability as for B subtype samples. The use of "in-house" real-time PCR to measure DNA in PBMCs correlated strongly with the HIV-1 RNA levels in all specimens.

HCV genotype distribution among HIV co-infected individuals in Argentina: relationship with host and viral factors.

Coinfection with hepatitis C virus (HCV) in individuals infected with HIV is associated with a higher incidence of liver injury hepatic decompensation, and decreased survival than that observed in an HIV-monoinfected population. While prevalence studies on HIV/HCV coinfection have been performed in the U.S. and in some European countries, little is known about HCV genotype distribution in Latin America. The main objective was to evaluate the HCV prevalence and genotypes among HIV co-infected patients, and their relationship with HCV viral load, serum ALT level and T lymphocyte CD4+ cell count. These data pursue to increase the knowledge from South America about a pressing problem from HIV-infected patients. Retrospectively collected specimens from 593 HIV-positive individuals in Argentina were tested for anti-HCV These were analyzed for HCV-RNA qualitatively and quantitatively. The HCV genotype was determined by the RFLP method. One hundred and twenty-nine (21.7%) HIV-infected individuals were anti-HCV positive; 65.9% of them exhibited detectable HCV-RNA. Genotype 1 (43, la/c; 9, 1b; and 5, 1a/c+1b) was present in 57, while 1, 14 and 13 were infected with genotype 2, 3 or a mix, respectively. Co-infected individuals were more likely to be male, without significant differences in age and CD4+ cell counts than HIV-monoinfected individuals. HCV infection prevalence in patients co-infected with HIV highlights the impending public health impact of this problem. Considering the increasing rate of HCV genotypes with lower response rates to treatment among HIV co-infected patients, antiretroviral therapy success might be jeopardized by HCV coinfection.

HIV-1 infection downregulates nuclear telomerase activity on lymphoblastoic cells without affecting the enzymatic components at the transcriptional level.

Both increased lymphocyte renewal with subsequent exhaustion of the immune system and impaired T cell renewal have been put into view to account for CD4+ T cell depletion and development of AIDS in HIV-1- infected humans. Telomerase is an enzyme that is involved in mechanisms that control cell life span and replicative potential. The effect of HIV-1 on telomerase activity, certain regulators, and telomeric terminal restriction fragment length on lymphoid Jurkat cells was used in measuring the proliferative activity of T lymphoid cells before and after being infected. At the cellular level, the enzymatic activity remains almost stable but further analyses of fractionated cells revealed that telomerase activity in the nuclear compartment was diminished whereas in the cytoplasmic compartment it was relatively increased on HIV-1 infection. Two key components of telomerase regulation were further considered at the transcriptional level, that is, the mRNA levels of both human telomerase reverse transcriptase (hTERT)--including the relative amount of its alternative splicing variants--and hTR. They were unaffected on HIV-1 infection. Telomeric length was also conserved in infected cells. Overall, these findings demonstrate that HIV-1 infection of Jurkat cells down modulate telomerase activity in the nuclear compartment by affecting its cellular localization.

Lack of association between cancer incidence and residence near petrochemical industry in the San Francisco Bay area.

Estimated age-adjusted incidence rates for cancer during 1971--77 among Kaiser Foundation Health Plan (KFHP) members living in a portion of the San Francisco Bay area (SFBA) characterized by a heavy concentration of petroleum and chemical industries were compared to estimated rates among KFHP members in the remainder of the SFBA. One hundred fifty-four comparisons were done for 41 selected cancer sites. The number of significant differences did not appear inconsistent with what might be expected by chance alone; furthermore, in most of these instances the so-called exposed area showed the lower rate. These findings provided some assurance that place of residence near petrochemical industries is not associated with increased cancer risk.

Development of HIV-1 resistance to (-)2'-deoxy-3'-thiacytidine in patients with AIDS or advanced AIDS-related complex.

OBJECTIVE: To determine the rate of development of in vitro HIV resistance to (-)2'-deoxy-3'-thiacytidine (3TC) and relate the effect of dose to emergence of resistance. METHODS: HIV-infected men and non-pregnant women, aged > or = 18 years, with a CD4 count < or = 300 x 10(6)/l cells were followed in a Phase I/II study, in which they were evaluated for tolerance to 3TC and effect of this agent with regard to viral susceptibility. Peripheral blood and plasma samples were collected at regular intervals for analysis. HIV was isolated using umbilical cord blood mononuclear cells as targets. These cells were also used in determinations of median inhibitory drug concentration. Specific amplification of the 184 mutation site, associated with HIV resistance to 3TC, was performed by polymerase chain reaction, using specific primer pairs, on DNA harvested from infected peripheral blood mononuclear cells (PBMC) of donors or, alternatively, on DNA that had been reverse transcribed from plasma-associated HIV RNA. RESULTS: Phenotypic resistance was detected in approximately one-third of individuals studied, who were followed between 8 and 56 weeks. Development of 3TC resistance occurred independently of dose, although time of first appearance of resistant HIV-1 variants appeared reduced at high 3TC doses. Amino-acid changes at codon 184 in HIV-1 reverse transcriptase were associated with, and preceded, the development of phenotypic 3TC resistance. Most commonly, a Met to Ile substitution appeared transiently before being superceded by a Val substitution at codon 184. CONCLUSIONS: In vitro resistance to 3TC developed in a high proportion of subjects who received prolonged monotherapy with this drug. The development of resistance to 3TC was associated with appearance of mutated viral forms and the disappearance of wild-type virus, with regard to codon 184, in both patient plasma and PBMC.

Clinical correlates of in vitro HIV-1 resistance ot zidovudine. Results of the Multicentre Canadian AZT Trial.

OBJECTIVE: To describe the rate of development of in vitro HIV resistance to zidovudine (ZDV) and its prognostic implications within the Multicentre Canadian AZT Trial (MCAT). METHODS: HIV-infected subjects in Centers for Disease Control (CDC) stages IIB, III and IVC-2 with CD4 cell counts > 270 x 10(6)/l were treated with ZDV as part of a dose-range study. Participating volunteers underwent prospective clinical and laboratory evaluations at regular intervals. Viral cultures and sensitivity testing were performed every 12 weeks in a predefined subset of 50 volunteers. An isolate was designated ZDV-resistant if it had a median inhibitory concentration (IC50) for ZDV at least 50-fold higher than that of virus isolated from the same subject before initiation of antiviral chemotherapy. The relationship between resistance and subsequent disease progression was studied using the Mantel and Byar method, for which, at each instance of disease progression, 2 x 2 tables classifying progression versus resistance status were constructed. The observed number of progressions was compared with that expected under the null hypothesis using Mantel-Haenszel methods adjusted for baseline CD4:CD8 ratio. RESULTS: The Kaplan-Meier estimate for the cumulative development of in vitro resistance was 64% [95% confidence interval (CI), 41-78] at 180 weeks. Baseline CD4:CD8 ratio was negatively associated (P = 0.10) with the subsequent development of resistance (proportional hazard, 0.44; 95% CI, 0.17-1.10). After adjusting for baseline CD4:CD8 ratio, the numbers of observed and expected progressions following the development of resistance were 15 and 7.6, respectively (P = 0.008). A similar relative risk of progression between resistant and non-resistant states was found in the two CD4:CD8 strata; observed and expected progressions were 4 and 2.3 and 11 and 5.2 in the high and low CD4:CD8 strata, respectively. CONCLUSIONS: In vitro resistance to ZDV developed in 64% of subjects after 180 weeks of ZDV therapy. Lower CD4:CD8 ratio at baseline was associated with faster development of resistance. In addition, the development of resistance was found to be a marker of subsequent disease progression. This association persisted after adjustment for baseline CD4:CD8 ratio. Whether in vitro resistance to ZDV is merely a surrogate marker or a determinant of disease progression remains to be established.

Diverse BF recombinants have spread widely since the introduction of HIV-1 into South America.

OBJECTIVE: To describe the genetic diversity of HIV-1 in South America by full genome sequencing and analysis. METHODS: Purified peripheral blood mononuclear cell DNA from HIV-infected individuals in Argentina, Uruguay and Bolivia was used to amplify full HIV-1 genomes. These were sequenced using the ABI 3100 automated sequencer and phylogenetically analysed. RESULTS: Twenty-one HIV-1 strains from three South American countries, 17 of which were pre-screened by envelope heteroduplex mobility assay (HMA), were studied. Ten out of 10 HMA subtype F and four out of seven HMA subtype B strains were actually BF recombinants upon full genome analysis. Two BF recombinants from Argentina and two from Uruguay had the same structure, representing a new circulating recombinant form termed CRF12_BF(ARMA159). Twelve other BF recombinants had structures related to CRF12 but with additional segments of subtype B; each was unique. BF recombinants were temporally and geographically widespread, found as early as 1986-1987 in vertically infected Argentinian children and in Argentina, Uruguay, and Bolivia.

Prevalence of HIV-1 resistant to antiretroviral drugs in 81 individuals newly infected by sexual contact or injecting drug use. Investigators of the Quebec Primary Infection Study.

OBJECTIVE: Prolonged treatment with antiretroviral drugs results in the selection of HIV-1 variants with mutations conferring resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTI and NNRTI) or to protease inhibitors (PI). There is serious concern about transmission of resistant viruses to newly infected persons. This study monitored the prevalence of resistant viruses in individuals undergoing primary HIV infection. DESIGN: Resistance testing was performed on 81 individuals infected between 1997 and 1999 by injecting drug use (n =21), sexual (n = 56), or unknown (n = 4) transmission. METHODS: Automated sequencing was used to genotype the reverse transcriptase (RT) and protease regions of virus isolated from patients' plasma. The phenotypic susceptibility of stimulated peripheral blood mononuclear cells to antiretroviral drugs was assayed. Line probe assays detected quasispecies variations in wild-type and mutated RT codons. RESULTS: A high prevalence of PI and RT genotypic variants, associated with high-level resistance to antiretroviral drugs, was observed in individuals newly infected by injecting drug use (PI = 24%, RT = 24%) or sexual transmission (PI = 12%, RT = 22%). The PI mutations, L101, V82A, and L90M, were found in 10.5, 3 and 4% of cases, respectively; whereas for RT, primary mutations at positions T215Y (zidovudine), M184V (lamivudine), T69D/A (zalcitabine), and K103N (multi-NNRTI) were present in 8, 5, 4, and 4% of subjects, respectively. Resistance to NRTI was demonstrated by phenotypic, genotypic, and line probe analyses. Transmission of multidrug (NRTI/NNRTI/PI) resistance in eight subjects (9.9%) was confirmed by showing that source partners possessed viruses of similar genotype. CONCLUSIONS: The transmission of drug-resistant HIV is a serious problem that merits further attention by public health officials as well as virologists and clinicians.

Antiviral activity and protection of cells against human immunodeficiency virus type-1 using an antisense oligodeoxyribonucleotide phosphorothioate complementary to the 5'-LTR region of the viral genome.

A COS-like monkey kidney cell line stably transfected with the plasmids pCMVgagpol-rre-r with the gag and pol genes, and pCMV rev with the rev gene of HIV-1 derived from the cDNA clone BH10, was used as a model for assessing the effectiveness of antisense (AS) constructs, A 20-mer oligodeoxyribonucleotide (oligo) phosphorothioate sequence (5'-CCG CCC CTC GCC TCT TGC CG) complementary to a portion of the 5'-long terminal repeat (5'-LTR) of the HIV-1 genome was tested for its inhibitory effects on the biologically important processes of HIV-1 replication and proliferation. We observed a concentration-dependent inhibition of HIV protein synthesis. Desitometric analysis of data from Western blot analysis showed sequence-specific and concentration-dependent oligo inhibition of p24 viral core antigen formation in the low-microM range. When lipofectin was used as a delivery vehicle, a markedly increased potentiation of the AS activity of the sequence was observed at a lower concentration (0.1 microM), following a 24-h preincubation. The AS construct specifically inhibited intracellular p24 production in chronically HIV-1-infected cells of lymphoid origin (H9/IIIB cells) by 95%, resulting in a 15-fold inhibitory effect relative to a similar sequence thiolated at only seven single-base positions. A concentration-dependent attenuation in the reverse transcriptase activity and a reduction in viral p24 level was observed in the culture supernatant of AS-pretreated HIV-1-infected phytohemagglutinin A-stimulated human cord blood mononuclear cells. Incubation of a HIV-1-infected lymphoid cell line with AS sequence resulted in a marked reduction in syncytium formation, and therefore protected cells from the cytopathic effects of the virus. Furthermore, the AS oligo did not appear to be cytotoxic in cell growth rate and colony-forming ability assays. The AS oligo described in this report is a useful new tool for the molecular analysis of HIV-1 gene expression and proliferation, and may have potential as a therapeutic agent.

Treatment of primary HIV infection: a pilot study of stavudine and didanosine plus nevirapine with or without hydroxyurea.

Treatment of primary human immunodeficiency virus (HIV) infection (PHI) may provide an opportunity to achieve a long lasting suppression of viral replication. Although there is growing evidence of the benefit of treating PHI, clinical data are still very limited. Special therapeutic considerations in this clinical setting include the prevalence of resistant viruses in the community, complexity of regimens and their long-term toxicity. In addition, adjunctive therapies aimed at exploring the role of immune modulation and intensification of antiretroviral therapy are becoming areas of great interest. In this regard, the role of hydroxyurea, a cytostatic agent that potentiates the antiviral effect of didanosine, and possibly of stavudine is being investigated. A pilot study to assess the antiviral effect of a combination of didanosine plus stavudine plus nevirapine with or without hydroxyurea in the treatment of PHI is currently under way. Preliminary results on 22 patients who completed at least 36 weeks of therapy suggest that the combination is safe, well tolerated and effective for the treatment of PHI.

Diminished HIV-1 sensitivity to stavudine in patients on prolonged therapy occurs only at low levels and cannot be attributed to any single amino acid substitution in reverse transcriptase.

To study the extent to which phenotypic resistance to stavudine occurs under therapy, we studied 18 pairs of human immunodeficiency virus type 1 (HIV-1) isolates from patients both prior to and following 24-48 weeks of treatment with stavudine monotherapy or stavudine in combination with either didanosine or lamivudine. We also used a nested polymerase chain reaction (PCR) assay to probe for the presence of specific mutations associated in culture with stavudine resistance. The results showed that resistance to stavudine (approximately 3-10 fold) was observed in nine of ten cases of monotherapy, in three of four cases of therapy involving both stavudine and didanosine, and in two of four cases involving stavudine and lamivudine. Viruses from the four patients receiving stavudine plus didanosine became resistant to didanosine in only one instance while the use of lamivudine plus stavudine yielded resistance to lamivudine each time. Whereas changes in the reverse transcriptase (RT) genes of resistant isolates were frequently observed, two mutations, previously identified with stavudine resistance in tissue culture (i.e., V75T and I50T), could not be identified in the clinical samples by either direct sequencing of the RT gene or by PCR amplification. Thus, resistance to stavudine can occur, albeit at low levels, in the context of prolonged therapy with this drug but is not associated with specific mutations in HIV RT at either codons 75 or 50 in clinical samples.

Resistance profiles to antiretroviral drugs in HIV-1 drug-naive patients in Argentina.

The drug resistance profile of treatment-naive HIV-infected individuals living in Buenos Aires, Argentina, was studied. Samples taken from 94 drug-naive individuals with established HIV infection and 13 patients with primary HIV infection were assessed by nucleotide sequencing and LIPA. The prevalence of drug-associated primary mutations in individuals with established infection was very low. In the viral protease region, 1/86 (1.2%) individuals carried the D30N mutation, whereas 1/85 (1.2%) had the M41L mutation in the reverse transcriptase (RT) region. Secondary mutations in both the protease and RT regions were found in almost 90% of the individuals. In individuals with primary infection, primary mutations were detected in 2/13 (15.4%) patients, one of them carrying M461 mutation in the protease while the other patient had a mutation at codon 184 of the RT. In accordance with current drug resistance testing guidelines, the results of this study suggest that susceptibility tests need not be performed at this time prior to initiation of antiretroviral therapy in HIV-1-infected people in Argentina. However, the public health implications of this subject warrant follow-up studies that will examine a larger number of drug-naive patients, not only in Buenos Aires but also in other major Argentinian cities and in rural areas.

Resistance to (-)-2',3'-dideoxy-3'-thiacytidine (3TC) in HIV-1 isolated from paediatric patients.

We conducted detailed virological evaluations of 16 HIV-1-infected paediatric patients treated with 3TC (lamivudine) monotherapy. High-level phenotypic resistance against this compound (up to 2,500-fold) was seen in virtually all cases, usually within 8-12 weeks of initiation of therapy. This was concomitant with the appearance of the M184V mutation in viral reverse transcriptase, previously shown to be responsiblefor such resistance. Viral burden fell in virtually all cases after commencement of therapy, and remained below baseline in each instance studied, despite a rebound effect and the appearance of drug resistance. Viral isolates from some patients underwent a switch from a non-syncytium-inducing (NSI) to a syncytium-inducing (SI) phenotype during the course of the study, although no relationship was apparent between dose of drug employed, time to development of drug resistance or time of appearance of SI phenotype.

Selection and characterization of HIV-1 variants resistant to the (+) and (-) enantiomers of 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC).

Human immunodeficiency virus type 1 (HIV-1) variants were selected for resistance against the (+) and (-) enantiomers of a novel nucleoside analogue, 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC), using the infectious molecular clone HXB2D grown in the MT-4 line of human T cells. The variants selected with (+) dOTC were approximately 6-7-fold less sensitive than wild-type virus to this drug. Cloning and sequencing of the complete reverse transcriptase (RT) coding region of these variants identified the M1841 mutation and further selection with virus containing the M1841 substitution led to the appearance of an M184V mutation. In contrast, selection experiments performed with (-) dOTC yielded variants capable of growing in drug concentrations as high as 100 microM, but phenotypic analysis of these viruses revealed near wild-type 50% inhibitory concentration (IC50) values for this compound. Site-directed mutagenesis experiments in which the M1841 and M184V mutations were introduced into HXB2D confirmed the importance of these mutations when viruses were grown in MT4 cells. However, wild-type IC50 values in regard to both (-) and (+) dOTC were obtained when these recombinant viruses were grown in cord blood mononuclear cells (CBMC). Clinical isolates of HIV-1 resistant to lamivudine and containing the M184V substitution also displayed low-level resistance to both (-) and (+) dOTC when grown in CBMC. Finally, cell-free RT assays were performed in the presence of either (-) dOTC triphosphate, (+) dOTC triphosphate, or the triphosphate of a racemic mixture of (+) and (-) dOTC with wild-type and mutated M184V-containing recombinant RT. The data demonstrate chain termination effects of these compounds with regard to both wild-type and mutated enzyme and that the latter was approximately twofold less sensitive than the former to these drugs.

In vitro selection and characterization of HIV-1 with reduced susceptibility to PMPA.

9-(2-phosphonomethoxypropyl)adenine (PMPA) has demonstrated remarkable anti-simian immunodeficiency virus (SIV) activity in macaque models of SIV infection and transmission prevention. Recently, PMPA and its oral prodrug, bis-POC PMPA, have also shown potent anti-human immunodeficiency virus type 1 (HIV-1) activity in Phase I clinical studies. In vitro experiments were performed to address the resistance properties of PMPA. After eight passages in increasing concentrations of PMPA, HIV-1IIIB was able to grow in the presence of 2 microM PMPA, fivefold above the IC50 of PMPA for wild-type parental virus. Sequence analysis of the reverse transcriptase (RT) genes from four of 15 RT clones demonstrated the presence of a K65R substitution in RT and recombinant HIV expressing the K65R RT mutation showed a threefold to fourfold increase in IC50 value for PMPA as compared to wild-type. Additional experiments demonstrated that viruses expressing other nucleoside-associated RT resistance mutations all showed wild-type or < threefold reduced susceptibility to PMPA in vitro. Interestingly, lamivudine-resistant viruses expressing the M184V RT mutation showed wild-type to slightly increased susceptibility to PMPA in vitro and addition of the M184V mutation to HIV with the K65R mutation resulted in reversion to wild-type susceptibility for PMPA. In agreement with the cell culture findings, Escherichia coli-expressed K65R RT showed fivefold reduced susceptibility to PMPA diphosphate, the active moiety of PMPA. Furthermore, in combination experiments, PMPA with hydroxyurea showed synergistic inhibition of HIV replication in vitro. The potent antiretroviral activity and favourable resistance profile of PMPA and bis-POC PMPA are being further investigated in ongoing clinical trials.