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Objectives Dolutegravir (DTG) has achieved better long-term suppression of HIV-1 replication than other integrase strand transfer inhibitors (INSTIs), such as raltegravir (RAL) and elvitegravir (EVG). In in-vitro drug washout experiments, we previously showed that removal of DTG from pretreated MT-2 cells infected with wild-type HIV-1 showed slower rebound in viral replication as compared to removal of RAL. Now, we performed DTG, EVG and RAL washout experiments to compare the recovery of viral integration and production of 2-long terminal repeat (LTR) circles using wild-type HIV-1 clones, R263K viruses with low-level resistance to DTG and viruses with G140S/Q148H mutations showing cross-resistance against all currently approved INSTIs. Design and methods MT-2 cells infected with wild-type, R263K or G140S/Q148H HIV-1 clones were treated with DTG, RAL or EVG for 3 days. Viral rebound following drug washout was assessed, monitoring viral integration and 2-LTR circle production by qPCR. Results Viral integration did not resume for up to 8 days after DTG washout from the wild-type or R263K infections but increased soon after washout of either RAL or EVG. With the G140S/Q148H virus, levels of integration were not significantly affected by the presence of either RAL or EVG. With DTG, integration was much lower at 3 days after infection than for the no-drug control. At 8 days after DTG washout, viral integration resumed but remained relatively low. Conclusion DTG antiretroviral activity in tissue culture is more durable than that of either RAL or EVG after drug washout and this is true for both wild-type and drug-resistant viruses.

Objective Recommended treatments for newly diagnosed HIV-positive individuals now focus on the integrase strand transfer inhibitors, raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG). In treatment-naive individuals, cases of RAL-based and EVG-based virological failure, although rare, are associated with the occurrence of resistance mutations in integrase and/or reverse transcriptase coding sequences. In such cases, common resistance substitutions in reverse transcriptase that were associated with nucleos(t)ide reverse transcriptase inhibitors included M184I/V and K65R and these occurred together with various mutations in integrase. In some instances, these mutations in reverse transcriptase preceded the emergence of mutations in integrase. In contrast, no resistance substitutions in either integrase or reverse transcriptase have been observed to date in viruses isolated from treatment-naive individuals who experienced treatment failure with DTG-based regimens. Design The objective of this study was to determine the effects of the M184I/V and K65R substitutions in reverse transcriptase on the ability of HIV-1 to become resistant against RAL, EVG or DTG. Methods We performed tissue culture selection experiments using reverse transcriptase inhibitor-resistant viruses containing resistance substitutions at positions K65R, M184I or M184V in the presence of increasing concentrations of RAL, EVG or DTG and monitored changes in integrase sequences by genotyping. Results Selections using EVG and RAL led to the emergence of resistance mutations in integrase. In contrast, only the wild-type virus was able to acquire resistance mutations for DTG. Conclusion Resistance mutations against nucleos(t)ide reverse transcriptase inhibitors antagonized the development of HIV-1 resistance against DTG but not RAL or EVG.

Objective Although the integrase inhibitor dolutegravir (DTG) has demonstrated greater resilience than other antiretroviral drugs at withstanding the emergence of HIV-1 resistance mutations, such substitutions can develop, albeit rarely, in treatment-experienced integrase inhibitor-naive individuals. The most common substitution in integrase under those circumstances is R263K whereas another substitution that was selected against DTG in tissue culture was G118R. The objective of this study was to determine the effects of these DTG-specific resistance substitutions on the ability of HIV-1 to become resistant against either of two other integrase inhibitors, raltegravir (RAL) and elvitegravir (EVG). Design and methods We performed tissue culture selection experiments using DTG-resistant viruses containing integrase substitutions at positions R263K, H51Y/R263K, E138K/R263K, G118R and H51Y/G118R in the presence of increasing concentrations of either RAL or EVG. Changes in integrase sequences were monitored by genotyping. Results The presence of the R263K substitution delayed the emergence of resistance against RAL whereas the simultaneous presence of either the H51Y or E138K secondary substitutions in combination with R263K somewhat mitigated this inhibitory effect. In contrast, resistance against EVG appeared earlier than in wild-type virus in viruses containing the R263K and E138K/R263K DTG-associated resistance substitutions. Conclusion The DTG-resistant R263K substitution antagonized the development of HIV-1 resistance against RAL while partially facilitating the occurrence of resistance against EVG.

OBJECTIVE In treatment-naive HIV-positive individuals, the integrase strand-transfer inhibitor dolutegravir (DTG) has not been associated with emergent drug-resistance mutations, neither against this drug nor against other antiretroviral drugs that were used in combination with it. This is in contrast to all other antiretroviral drugs tested so far, including the integrase strand-transfer inhibitors raltegravir (RAL) and elvitegravir that can lead to treatment failure with the emergence of drug-resistance mutations. DESIGN These observations suggest that DTG may provide an additional protection against resistance compared to other drugs by decreasing HIV-1 genetic evolution. METHODS Here, we tested this hypothesis by measuring the genetic and amino-acid diversity of Env/gp160 from two HIV-1 primary isolates that were grown in the presence of increasing concentrations of DTG or RAL over the course of 38-55 weeks. RESULTS The results show that treatment with DTG led to less HIV-1 genetic and amino-acid diversification over time, as compared to treatment with RAL or the absence of drug. CONCLUSION These results may help to explain the absence of emergent resistance mutations in treatment-naive individuals treated with DTG.

Objective Among 1222 antiretroviral-naive patients who received dolutegravir (DTG) as part of first-line therapy, none has developed resistance against this compound after 48-96 weeks of follow-up. Moreover, only four occurrences of virological failure with resistance mutations have been documented in previously drug-experienced patients who received DTG as a first time integrase inhibitor as a component of a second-line regimen. The R263K integrase resistance mutation was observed in two of these individuals who received suboptimal background regimens. We have previously selected mutations at position R263K, G118R, H51Y, and E138K as being associated with low-level resistance to DTG. Now, we sought to investigate the facility with which resistance on the part of R263K-containing viruses might develop. Design and methods We tested the ability of DTG-resistant viruses containing either the R263K or G118R and/or H51Y mutations to develop further resistance against several reverse transcriptase inhibitors during in-vitro selection experiments. Results Our results show that DTG-resistant viruses are impaired in their ability to acquire further resistance to each of nevirapine and lamivudine as a consequence of their relative inability to develop resistance mutations associated with these two compounds. Conclusion Our findings provide an explanation for the fact that no individual has yet progressed to virological failure with resistance mutations associated with dolutegravir in clinical trials in which patients received dolutegravir together with an optimized background regimen.

Objective The M184V mutation in the HIV-1 reverse transcriptase gene is frequent (>50%) in patients, both in resource-rich and resource-limited countries, conferring high-level resistance (>100-fold) to the cytosine analog reverse transcriptase inhibitors lamivudine and emtricitabine. The reverse transcriptase enzyme of M184V HIV-1 mutants has reduced processivity, resulting in reduced viral replication, particularly at low deoxynucleotide (dNTP) levels. We hypothesized that lowering intracellular dNTPs with resveratrol, a dietary supplement, could interfere with replication of M184V HIV-1 mutants. Design and methods Evaluation of the activity of resveratrol on infection of primary peripheral blood lymphocytes by wild-type and M184V mutant HIV-1. We assayed both molecular clones and primary isolates of HIV-1, containing M184V alone and in combination with other reverse transcriptase mutations. Viral infection was quantified by p24 ELISA and by quantitative real-time PCR analysis. Cell viability was measured by colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. Results In virus-infectivity assays, resveratrol did not inhibit replication of wild-type NL4-3 (resveratrol EC50 > 10 μmol/l), but it inhibited NL4-3 184V mutant (resveratrol EC50 = 5.8 μmol/l). These results were confirmed by real-time PCR analysis of early and late products of reverse transcription. Resveratrol inhibited molecular clones and primary isolates carrying M184V, alone or in combination with other reverse transcriptase mutations (resveratrol EC50 values ranging from 2.5 to 7.7 μmol/l). Conclusions Resveratrol inhibits HIV-1 strains carrying the M184V mutation in reverse transcriptase. We propose resveratrol as a potential adjuvant in HIV-1 therapy, particularly in resource-limited settings, to help control emtricitabine-resistant M184V HIV-1 mutants.

Objectives: To determine the underlying biochemical mechanisms responsible for the diminished viral replicative capacity associated with K65R/M184V-containing viruses. Methods: We studied the efficiency of (-)ssDNA synthesis by recombinant wild-type and mutated HIV-1 reverse transcriptases in cell-free assays. In addition, we determined susceptibility levels to nucleoside analog reverse transcriptase inhibitors (NRTIs) both in cell-free and cell culture assays. Results: We observed that the K65R/M184V mutations in reverse transcriptase caused reductions in the efficiency of initiation of (-)ssDNA synthesis by increasing pausing at positions +3 and +5 as well as diminished RNA usage. These findings were confirmed in cell culture data using MT-4 cells and cord blood mononuclear cells. Conclusions: The simultaneous presence of K65R and M184V in reverse transcriptase has a negative impact with regard to the efficiency of initiation of (-)ssDNA synthesis and RNA usage, that exceeds the effect of either mutation on its own. These mechanisms, among others, are responsible for the diminished viral replicative capacity observed in tissue culture when K65R/M184V-containing viruses are studied.

Genetic diversity is a hallmark of HIV-1 infection with regard to the expansion of distinct viral subtypes (clades A, B, C, D, E, F, G, K, and O) in different geographical regions. Here, we discuss the issues of HIV-1 sensitivity to antiretroviral drugs and drug resistance in the context of HIV-1 subtype diversity. Virtually all available evidence suggests that all subtypes of HIV display similar sensitivity to antiviral drugs, but viruses from some subtypes or geographical regions may occasionally have a greater propensity to develop resistance against certain drugs than other viral variants. In some situations, the types of mutations associated with resistance may vary, as a result of subtle differences among subtypes with regard to the genetic code. This consideration notwithstanding, drug resistance is unlikely to become a more serious issue in developing than developed countries, and there is an urgency to make anti-HIV drugs available to all who are in need.

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 (Iamivudine), 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.

Objective: To assess the potential discrepancies in reported changes in plasma viral load (PVL) depending on how values below the detection limit of the assay are handled in the data analysis phase of a randomized controlled clinical trial. Design: Data from a recently completed clinical trial comparing combinations of zidovudine, didanosine and nevirapine were analysed. In this trial, PVL was measured using an assay with a lower quantification limit of 400 HIV-1 RNA copies/ml initially. All PVL values less than 500 copies/ml were retested with a more sensitive assay with a lower quantification limit of 20 copies/ml. Methods: Several summary measures for assessing change in PVL were calculated using three different methods to adjust for PVL values less than the quantification limit of the assay. The differences between these measures were evaluated. Results: We found that the magnitude of the discrepancy between summary measures used to report changes in PVL depended on the proportion of subjects with PVL less than the quantification limit of the assay, how those observations were handled in the data analysis, and the relative difference between the quantification limits of the conventional and more sensitive assay. Conclusion: The lack of consensus in reporting of PVL data in the literature makes the interpretation of published trial results difficult. In the absence of agreement on the most appropriate summary measure of PVL data, we recommend that all summaries include information on the quantification limit of the assay used, the proportion of observations at or below the quantification limit and how these observations were handled in the data analysis.

New combination therapies for HIV/AIDS demand up-to-date knowledge of rapidly developing complex therapeutic regimes. These often arduous regimens require careful adherence by patients regular monitoring and rapid response to adverse reactions. The risk of failure is high since days or weeks of missed or incomplete medication can render antiviral medications ineffective for the individual patient through the rapid development of drug resistance. Although little is yet known about how transmissible such resistant strains really are there has been considerable public discussion of whether physicians should refuse to prescribe combination therapy to patients whose life circumstances make it difficult to follow prescribed regimens and thus make them likely incubators of resistant strains. The ethical issue of denial of treatment is usually posited as a conflict between public health and the individual. From this perspective therapy may potentially benefit individuals but prescribing to non-adherent individuals may pose a risk to public health. Physicians obliged to attend to the interests of both the individual and public health are forced to mediate between these interests. (excerpt)

Objective: To determine whether disruption of the cleavage site between protease and reverse transcriptase (RT) or the HIV-1 frameshift site could yield trans-dominant negative HIV-1 variants that interfere with wild-type viral replication. Design: Residues at the cleavage site between the HIV-1 protease and RT coding regions were mutagenized to produce a protease-RT (PR-RT) fusion protein that was expressed in the context of a full-length provirus. The PR-RT cleavage site mutation was also combined with a read-through mutation at the frameshift site in order to overexpress the mutant Gag-Pol polyproteins. Methods: COS-7 cells were transiently transfected with the mutant constructs to produce viruses harbouring the PR-RT fusion protein. In addition, we performed cotransfection studies in various cell types to analyze the inhibition of wild-type replication by the mutant constructs. Results: Immunoblot analysis revealed that this novel mutation prevented cleavage between the two proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. While both the protease and RT components of this fusion protein remained functionally active, viruses containing the cleavage site mutation were less infectious in tissue culture than wild-type viruses produced by COS-7 cells. This defect was further pronounced when the cleavage site mutation between protease and RT was overexpressed as a consequence of an additional mutation that interfered with frameshifting. Cotransfection of COS-7 cells with the mutant constructs and wild-type HIV-1 interfered with the replication of the latter and reduced the infectiousness of the virus particles produced. Conclusions: HIV-1 constructs harbouring a cleavage site mutation between protease and RT can probably act as trans-dominant negative mutants to interfere with wild-type viral replication.

Objective: To measure the extent of HIV resistance to (-)-2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine) within the context of monotherapy and to assess the presence of the M184V substitution in the case of 3TC-resistant viruses. Whether the success of 3TC in clinical trials could be due, in part, to an increase in the fidelity of HIV reverse transcriptase conferred by the M184V substitution was also considered. Methods: Two separate monotherapy studies were evaluated, one involving adults with CD4 counts > 300 x 10 6 /l, and the second involving children, some of whom had received antiretroviral treatment previously, while others were drug naive. Peripheral blood and plasma samples were collected regularly, and HIV isolation and determinations of drug median inhibitory concentration values were performed using umbilical cord mononuclear cells as targets. Amplification of the 184 mutation was performed by the polymerase chain reaction, using specific primer pairs. Fidelity determinations using purified, recombinant HIV reverse transcriptase derived from either wild-type virus or viruses that contained the 184V substitution were performed. Results: Phenotypic resistance was detected in almost all subjects at times ranging from 8-20 weeks after initiation of therapy. The 184V substitution was usually detected prior to the occurrence of phenotypic resistance to 3TC. Fidelity determinations revealed that the 184V substitution conferred an approximately 5- to 1 0-fold increase in HIV reverse transcriptase fidelity. In addition, titres of patient sera tested for their ability to neutralize autologous sequential viral isolates were stabilized in patients receiving 3TC therapy as opposed to other drugs. Conclusions: Resistance to 3TC developed in virtually all subjects treated with this drug, and was associated with the appearance of an M184V mutation in HIV reverse transcriptase. The clinical benefit of 3TC therapy may be attributable in part to selection of viruses that are less able to replicate and mutate than the wild types.

As expected, the productive infection of several monocytic cell lines by HIV-1 led to a diminution of cell-surface CD4 antigen. However, unlike findings reported for HIV-1-infected T cells, this decrease was not accompanied by a similar reduction in levels of CD4 transcripts. OKT4 monoclonal antibodies (MAbs) to CD4 were used in immunoprecipitation experiments to show that intracellular CD4 levels were diminished in U-937 monocytic cells that had been infected by HIV-1. These MAbs also coprecipitated viral gp120, indicating that CD4-gp120 complexes are present in infected monocytes. Our results therefore demonstrate that cell-surface down-modulation of CD4 is exclusively a post-transcriptional event in HIV-1 infected monocytic cells. These data suggest that HIV-1-mediated depletion of cell-surface CD4 in monocytes does not involve transcript down-modulation as has been reported in T lymphocytes.

HIV-infected patients who fail standard therapeutic regimens are in need of new drugs that will remain active against drug-resistant viral variants. There is a constant need to develop new compounds that will not be compromised by problems of cross-resistance, as so often occurs among members of the same family of drugs, for example, nucleoside reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors. Indeed, successful second-line and salvage regimens will commonly include members of drug classes to which a patient has not previously been exposed, for example, a protease inhibitor and an integrase strand transfer inhibitor (INSTI) in the case of an individual who began therapy with two NRTIs and a NNRTI, as well as the use of fusion inhibitors, for example, enfuvirtide, and CCR5 antagonists, for example, maraviroc.

Journalists more commonly than other professionals may be able to transmit complex scientific information about HIV/AIDS in a language that is understood by the general public. This premise led to establishment of a journalist-to-journalist (J2J) HIV/AIDS training program as a component of the International AIDS Conference in Barcelona in 2002. We were asked to evaluate the J2J program in order to assess the suitability of the curriculum content and didactic quality (process evaluation) and explore the effects of the program on journalists reporting of HIV/ AIDS (outcome evaluation). We also wished to assess journalists perceptions as to how their training impacted coverage of HIV/AIDS and determine whether the program had helped to better inform communities about truths (and nontruths) regarding HIV/AIDS. (excerpt)

We determined whether drug-resistant variants of HIV-1 could be isolated from the peripheral blood mononuclear cells of 20 individuals with HIV infection (Centers for Disease Control groups II and III) on long-term zidovudine (AZT) therapy. Toward this end, zidovudine (10 microM) has been included in the tissue culture medium used to isolate HIV-1. Under these circumstances, virus with a zidovudine-resistant phenotype was successfully obtained in five out of 20 cases. This property of drug resistance appeared to be stable, and did not disappear upon extended replication of such virus in the absence of drug pressure. Drug-resistant virus could also be isolated from these subjects on subsequent occasions, but was not present in samples obtained prior to therapy. Replication of these zidovudine-resistant isolates in tissue culture was inhibited by each of four other nucleoside analogues. Thus, other drugs may be useful in controlling selective zidovudine-resistant variants of HIV-1.