Gadi Borkow

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.

The K65R Mutation Confers Increased DNA Polymerase Processivity to HIV-1 Reverse Transcriptase

The K65R mutation in HIV-1 reverse transcriptase (RT) is associated with viral cross-resistance to 2′,3′-dideoxyinosine, 2′,3′-dideoxycytidine, and 2′,3′-dideoxy-3′-thiacytidine. We have found that in vitro DNA synthesis by K65R RT is significantly more processive than that of wild type (wt) RT. Depending on the template/primer (T/P) used, the total incorporation of nucleotides under single processive cycle conditions was 20-50% higher with K65R RT than with wt RT. With heteropolymeric T/P, the total incorporation of dNMP by K65R and wt RT was similar under continuous DNA synthesis reaction conditions. However, under single processive cycle conditions, the rate of full-length polymerization product synthesis by K65R RT was about 2-fold higher than that by wt RT. We also found a decreased rate of T/P dissociation during K65R RT DNA synthesis, which is consistent with the increased processivity of the enzyme. We postulate that the increased processivity of the K65R RT may be a compensatory response to the decreased affinity of this mutant for certain dNTP substrates, allowing normal viral replication kinetics.

Effectiveness of 3TC in HIV clinical trials may be due in part to the M184V substitution in 3TC-resistant HIV-1 reverse transcriptase.

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 × 106/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 10‐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.

Attenuated Infectivity of HIV Type 1 from Epithelial Cells Pretreated with a Tight-Binding Nonnucleoside Reverse Transcriptase Inhibitor

Short exposure of uninfected lymphocytes to UC781, a tight-binding nonnucleoside reverse transcriptase inhibitor (NNRTI), renders these cells refractory to subsequent HIV infection in the absence of exogenous drug (Borkow et al: J Virol 1997;71:3023-3030). Since epithelial cells may play a role in the sexual transmission of HIV-1, we examined the ability of NNRTI pretreatment to protect ME180 cervical epithelial cells and I407 intestinal epithelial cells from subsequent HIV-1 infection. Epithelial cells were pretreated with NNRTI, then exposed to HIV-1 chronically infected H9(+) cells for a short time following removal of the exogenous drug. The epithelial cells were productively infected by HIV-1, as shown by the presence of integrated HIV-1 proviral DNA, the presence of intracellular p24 antigen, and the production of nascent HIV-1 virions (cell-free p24) at various times postinfection. UC781 pretreatment of the epithelial cells did not prevent HIV-1 infection, since the cells had integrated proviral DNA, but the infectivity of virus subsequently produced from the UC781-treated cells was attenuated in a dose-dependent manner and virtually abolished at UC781 concentrations readily attainable in vivo. In contrast, nevirapine was ineffective in this respect, suggesting that not all NNRTI have microbicidal potential. The abrogation of infectivity of virus produced from UC781-pretreated epithelial cells suggests that this NNRTI may be useful in vaginal microbicide formulations targeted to inhibit HIV-1 in the vaginal/cervical or rectal milieus of a newly exposed individual.

The helminth HIV connection: time to act.

In 1995, we hypothesized that helminthic infections have a major impact on the pathogenesis of AIDS in Africa by increasing the susceptibility to HIVand undermining the host capacity to generate a potent immune response against the viral infection [1]. These ideas were driven by the profound immunological changes we found in the Ethiopian immigrants (ETH) to Israel [2,3]. ETH were seemingly healthy, only 1–2% of them were infected with HIV or tuberculosis, but they were heavily infected with helminthic parasites, mainly Ascaris lumbricoides, Schistosoma mansoni, and Necator americanus [3,4]. They had a dominant TH2 immune profile with extreme elevation of eosinophils and serum IgE, elevated secretion of IL-4, IL5, and IL-10, decreased secretion of IL-2 and IFN-g, and marked degree of immune activation, manifested by elevated levels of immune activated CD4(þ) and CD8(þ) cells, with spontaneous apoptosis and lowered levels of CD4(þ) cells [2,3]. These impressive immunological impairments were normalized in ETH living in Israel for over 5 years and in whom the helminthes were eradicated [3,5,6], supporting our notion that the helminths caused the immune impairments. Being an immunologist, I thought that the profound TH2 dominance and the chronic immune activation had to have an impact on the pathogenesis of HIV infection. We hypothesized that because people infected with helminths are immune activated, they will be more prone to HIV infection, have enhanced HIV-1 replication (and thus higher viral load), transmit the virus more easily and the infection will progress faster. Due to their dominant TH2 response, they will not be able to develop potent HIV-1-specific cellular immune responses, being detrimental to the disease progression and for responding to vaccination against HIV-1 [1,7–10]. The preexisting immune activation of the host, like that found in the helminth-

Analysis of the Effect of Wearing Copper Oxide Impregnated Socks on Tinea Pedis Based on "Before and After" Pictures - A Statistical Follow-up Tool

The assessment of skin conditions by digital images as part of the evaluation of treatment efficacy is not widely used in podiatry. The main objective of our study was to evaluate quantitative measuring of tinea pedis (athletes foot) related medical endpoints via digital images of the affected feet areas before and after treatment as a supporting tool for podiatrists. In order to do so, we analyzed photographs taken of patients who had participated in a previous clinical study. During this already published study, the patients fungal feet infections were treated only by wearing of antifungal socks containing copper oxide. The efficacy of the treatment was then determined solely by clinical observations of the podiatrist. In the current study, we randomly and blindly analyzed 282 digital images of patients feet taken before and after using the socks in the pilot study. The affected feet areas, in which the tinea pedis infection was manifested by fissuring, scaling, erythema and/or vesicular eruptions, were determined before and after treatment by using ImageTool software. Statistical analysis of these determinations demonstrated a significant reduction in the severity of all 4 endpoints analyzed (p<0.05). This is in accordance with the results described in the published study, further establishing that using socks containing copper oxide is efficacious in treating tinea pedis. The present study thus demonstrates that statistical analysis of quantitative data obtained from digital images taken during treatment of tinea pedis is feasible and may serve as a tool for podiatrists in monitoring treatment.

Inhibitory potency of R-region specific antisense oligonucleotides against in vitro DNA polymerization and template-switching reactions catalysed by HIV-1 reverse transcriptase

Antisense oligonucleotides (AONs) targeted to the R-region near the 5-LTR of HIV-1 genomic RNA inhibited both the synthesis of (-) strong stop DNA and the first template-switch reaction catalysed by HIV-1 reverse transcriptase (RT) in vitro. The 18 nucleotide (nt) AONs used were identical in sequence but differed in the sugar component of the 3-terminal nucleotide, with either 2-deoxy-D-ribose (DNA), 2-deoxy-L-ribose (L), or arabinose (ARA) in this position. All three AONs hybridized to complementary 18 nt RNA (T(m) approximately 70 degrees C) and specifically interacted with the target RNA HIV-1 sequence at 37 degrees C. L was unable to serve as primer for RT-catalysed DNA polymerization, whereas priming from ARA was about 30% that noted with DNA. Each of the three AONs resulted in similar 85-95% decreases in the amount of full length (-) strong stop DNA and up to 75% decreases in the first template-switch reaction products formed by RT, implying that elongation of the AONs did not enhance the inhibitory activity in vitro. A concomitant increase in a truncated DNA product corresponding to polymerization termination at the 5-end of the AON was noted, indicating that RT was unable to displace the AON. Interestingly, near maximal inhibition in vitro an AON:target RNA template ratio of 1:1 was noted. Our results confirm the validity of our in vitro system for the analysis of potential antisense oligonucleotide inhibitors, and suggest that antisense oligonucleotides directed to the R-region of HIV-1 RNA may be effective inhibitors of the initial stages of HIV-1 proviral DNA synthesis.