A single polymorphism in HIV-1 subtype C SP1 is sufficient to confer natural resistance to the maturation inhibitor bevirimat

3-O-(3',3'-Dimethylsuccinyl) betulinic acid (DSB), also known as PA-457, bevirimat (BVM), or MPC-4326, is a novel HIV-1 maturation inhibitor. Unlike protease inhibitors, BVM blocks the cleavage of the Gag capsid precursor (CA-SP1) to mature capsid (CA) protein, resulting in the release of immature, noninfectious viral particles. Despite the novel mechanism of action and initial progress made in small-scale clinical trials, further development of bevirimat has encountered unexpected challenges, because patients whose viruses contain genetic polymorphisms in the Gag SP1 (positions 6 to 8) protein do not generally respond well to BVM treatment. To better define the role of amino acid residues in the HIV-1 Gag SP1 protein that are involved in natural polymorphisms to confer resistance to the HIV-1 maturation inhibitor BVM, a series of Gag SP1 chimeras involving BVM-sensitive (subtype B) and BVM-resistant (subtype C) viruses was generated and characterized for sensitivity to BVM. We show that SP1 residue 7 of the Gag protein is a primary determinant of SP1 polymorphism-associated drug resistance to BVM.     

Alkyl Amine Bevirimat Derivatives Are Potent and Broadly Active HIV-1 Maturation Inhibitors

Concomitant with the release of human immunodeficiency virus type 1 (HIV-1) particles from the infected cell, the viral protease cleaves the Gag polyprotein precursor at a number of sites to trigger virus maturation. We previously reported that a betulinic acid-derived compound, bevirimat (BVM), blocks HIV-1 maturation by disrupting a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. BVM was shown in multiple clinical trials to be safe and effective in reducing viral loads in HIV-1-infected patients. However, naturally occurring polymorphisms in the SP1 region of Gag (e.g., SP1-V7A) led to a variable response in some BVM-treated patients. The reduced susceptibility of SP1-polymorphic HIV-1 to BVM resulted in the discontinuation of its clinical development. To overcome the loss of BVM activity induced by polymorphisms in SP1, we carried out an extensive medicinal chemistry campaign to develop novel maturation inhibitors. In this study, we focused on alkyl amine derivatives modified at the C-28 position of the BVM scaffold. We identified a set of derivatives that are markedly more potent than BVM against an HIV-1 clade B clone (NL4-3) and show robust antiviral activity against a variant of NL4-3 containing the V7A polymorphism in SP1. One of the most potent of these compounds also strongly inhibited a multiclade panel of primary HIV-1 isolates. These data demonstrate that C-28 alkyl amine derivatives of BVM can, to a large extent, overcome the loss of susceptibility imposed by polymorphisms in SP1.     

New Small-Molecule Inhibitor Class Targeting Human Immunodeficiency Virus Type 1 Virion Maturation

A new small-molecule inhibitor class that targets virion maturation was identified from a human immunodeficiency virus type 1 (HIV-1) antiviral screen. PF-46396, a representative molecule, exhibits antiviral activity against HIV-1 laboratory strains and clinical isolates in T-cell lines and peripheral blood mononuclear cells (PBMCs). PF-46396 specifically inhibits the processing of capsid (CA)/spacer peptide 1 (SP1) (p25), resulting in the accumulation of CA/SP1 (p25) precursor proteins and blocked maturation of the viral core particle. Viral variants resistant to PF-46396 contain a single amino acid substitution in HIV-1 CA sequences (CAI201V), distal to the CA/SP1 cleavage site in the primary structure, which we demonstrate is sufficient to confer significant resistance to PF-46396 and 3-O-(3′,3′-dimethylsuccinyl) betulinic acid (DSB), a previously described maturation inhibitor. Conversely, a single amino substitution in SP1 (SP1A1V), which was previously associated with DSB in vitro resistance, was sufficient to confer resistance to DSB and PF-46396. Further, the CAI201V substitution restored CA/SP1 processing in HIV-1-infected cells treated with PF-46396 or DSB. Our results demonstrate that PF-46396 acts through a mechanism that is similar to DSB to inhibit the maturation of HIV-1 virions. To our knowledge, PF-46396 represents the first small-molecule HIV-1 maturation inhibitor that is distinct in chemical class from betulinic acid-derived maturation inhibitors (e.g., DSB), demonstrating that molecules of diverse chemical classes can inhibit this mechanism.Despite the wide-ranging success of highly active retroviral therapies against human immunodeficiency virus type 1 (HIV-1), there still remain several powerful drivers to discover and develop new classes of HIV inhibitors. First and foremost is the continued acquisition of HIV-1 resistance to currently administered antiretroviral drugs. The discovery of compounds that inhibit the replication of HIV-1 via new mechanisms offers the best hope of generating drugs that are active against all HIV-1 variants in the clinic. In principle, viral mutations conferring resistance to any existing drug classes would not confer cross-resistance to drugs targeting a new mechanism. Also, as the current standard of care requires lifelong therapy for HIV-infected individuals, it is imperative that highly active retroviral therapy-related adverse effects and toxicity are minimized with new drugs.Recently, two new classes of antiretroviral medications have been approved for use in patients for whom previous HIV treatment regimens have failed; the CCR5 [chemokine (C-C motif) receptor 5] inhibitor maraviroc (9, 11) and the HIV-1 integrase (IN) inhibitor raltegravir (22). Additional novel mechanism drug classes will add similar value to the treatment of HIV-1 by further expanding the possibilities for combination regimens and providing a wider range of options for treatment of experienced patients failing first- and second-line therapies.Virion maturation represents one new antiviral mechanism that is currently being evaluated in the clinic (20, 21). During maturation of the HIV-1 viral particle, the Gag polyprotein undergoes several cleavage events mediated by the viral protease that yield the individual structural proteins, matrix (MA), capsid (CA), nucleocapsid (NC), and the p6 protein. In addition, there are spacer peptides that must be excised from between CA and NC (spacer peptide 1 [SP1]) and from between NC and p6 (SP2). Although the functions of the spacer peptides have not been clearly delineated, recent evidence suggests that SP1 stabilizes CA hexamers (25). Gag cleavage events occur in a sequential fashion as dictated by the relative kinetics of proteolysis at each site. The last of these proteolytic events cleaves at the CA-SP1 junction to convert p25 Gag to the mature p24 CA protein. This is a critical step in the replication cycle of HIV-1, as only in its mature form is CA capable of forming the higher-order complexes that comprise the mature viral core and in the absence of this maturation, viral particles remain noninfectious. Currently, two HIV-1 inhibitors [3-O-(3′,3′-dimethylsuccinyl) betulinic acid (DSB/PA-457/bevirimat) and MPC-9055] targeting HIV-1 virion maturation are being evaluated in clinical trials (3, 4, 14, 16). The hallmark of this class of inhibitors is the accumulation of the CA/SP1 (p25) precursor in cells and HIV-1 viral particles in the presence of the compounds. However, to date, all of the Gag maturation inhibitors reported belong to the same general chemical class of betulinic acid derivatives.In this report, we describe a specific small-molecule inhibitor of HIV-1 Gag maturation, PF-46396 {1-[2-(4-tert-butylphenyl)-2-(2,3-dihydro-1H-inden-2-ylamino)ethyl]-3-(trifluoromethyl)pyridin-2(1H)-one}. This pyridone-based compound was first discovered as a hit from our HIV-1 high-throughput full-replication screen that incorporates all of the HIV-1 targets required for viral replication in cell culture (8). This approach allowed us to screen for multiple targets in the context of a full replication cycle and to identify compounds directed against new HIV-1 mechanisms. We demonstrate that PF-46396 is a late stage (postintegration) HIV-1 inhibitor by showing that the compound inhibits the production of infectious viral particles by HIV-1 producer cells and that the compound does not inhibit early events in HIV-1 replication. We present mechanism-of-action studies demonstrating that PF-46396 does not inhibit HIV protease but interferes specifically with the cleavage of the CA/SP1 (p25) Gag precursor to the mature CA (p24) protein and show a correlation between this mechanism and the antiretroviral activity of the compound. Consistent with these mechanistic data, we show that selection of HIV-1 isolates that are resistant to PF-46396 yields a mutation in the C-terminal region of HIV-1 CA. This single mutation is sufficient to cause significant loss of susceptibility to the compound and displays cross-resistance to the previously reported HIV-1 Gag maturation inhibitor, DSB. Conversely, we demonstrate that a mutation selected for resistance to DSB is cross-resistant to PF-46396.Our data show that PF-46396 is a specific Gag maturation inhibitor that is clearly differentiated from previously reported compounds that share this mechanism of action. The structure and, in particular, the low molecular weight of PF-46396 show that this mechanism of action can be prosecuted through a more diverse set of chemical structures than previously represented in the literature.     

HIV-1 proprotein processing as a target for gene therapy

The central role of endoconvertases and HIV-1 protease (HIV-1 PR) in the processing of HIV proproteins makes the design of specific inhibitors important in anti-HIV gene therapy. Accordingly, we tested native alpha(1) antitrypsin (alpha(1)AT) delivered by a recombinant simian virus-40-based vector, SV(AT), as an inhibitor of HIV-1 proprotein maturation. Cell lines and primary human lymphocytes were transduced with SV(AT) without selection and detectable toxicity. Expression of alpha(1)AT was confirmed by Northern blotting, immunoprecipitation and immunostaining. SV(AT)-transduced cells showed no evidence of HIV-1-related cytopathic effects when challenged with high doses of HIV-1(NL4-3). As measured by HIV-1 p24 assay, SV(AT)-transduced cells were protected from HIV-1(NL4-3) at challenge dose of 40 000 TCID(50) (MOI = 0.04). In addition, peripheral blood lymphocytes treated with SV(AT) were protected from HIV doses challenge up to 40 000 TCID(50) (MOI = 0.04). By Western blot analyses, the delivered alpha(1)AT inhibited cellular processing of gp160 to gp120 and decreased HIV-1 virion gp120. SV(AT) inhibited processing of p55(Gag) as well. Furthermore, high levels of uncleaved p55(Gag) protein were detected in HIV virus particles recovered from SV(AT)-transduced cells lines and primary lymphocytes. Thus, delivering alpha(1)AT using SV(AT) to human lymphocytes strongly inhibits replication of HIV-1, most likely by inhibiting the activities both of the cellular serine proteases involved in processing gp160 and of the aspartyl protease, HIV-1 PR, which cleaves p55(Gag). alpha(1)AT delivered by SV(AT) may represent a novel and effective strategy for gene therapy to interfere with HIV replication, by blocking a stage in the virus replicative cycle that has until now been inaccessible to gene therapeutic intervention.     

Comparison of human immunodeficiency virus type 1 Pr55(Gag) and Pr160(Gag-pol) processing intermediates that accumulate in primary and transformed cells treated with peptidic and nonpeptidic protease inhibitors

Human immunodeficiency virus type 1 (HIV-1) produces two polyproteins, Pr55(Gag) and Pr160(Gag-Pol), that are cleaved into mature functional subunits by the virally encoded protease. Drugs that inhibit this protease are an important part of anti-HIV therapy. We studied the ordered accumulation of Gag and Gag-Pol processing intermediates by variably blocking the protease with HIV-1 protease inhibitors (PIs). Variable protease inhibition caused accumulation of a complex pattern of processing intermediates, which was the same after incubating HIV-1-infected cells with increasing concentrations of either one of the peptidomimetic inhibitors indinavir, saquinavir (SQV), ritonavir (RTV), nelfinavir, and SC-52151 or one of the nonpeptidomimetic inhibitors DMP450, DMP323, PNU-140135, and PNU-109112 for 3 days. The patterns of Gag and Gag-Pol processing intermediate accumulation were nearly identical when the following were compared: cell- versus virion-associated proteins, HIV-1-infected transformed cell lines versus primary human peripheral blood mononuclear cells (PBMCs) and HIV-1(MN) versus HIV-1(IIIB) virus strains. RTV was a more potent inhibitor of p24 production in PBMCs than SQV by approximately 7-fold, whereas SQV was a more potent inhibitor in transformed cells than RTV by approximately 30-fold. Although the antiretroviral potency of HIV-1 PIs may change as a function of cell type, the polyprotein intermediates that accumulate with increasing drug concentrations are the same. These results support sequential processing of Gag and Gag-Pol polyproteins by the HIV-1 protease and may have important implications for understanding common cross-resistance pathways.     

In vitro anti-human immunodeficiency virus (HIV) activities of transition state mimetic HIV protease inhibitors containing allophenylnorstatine.

Transition state mimetic tripeptide human immunodeficiency virus (HIV) protease inhibitors containing allophenylnorstatine [(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] were synthesized and tested for activity against HIV in vitro. Two compounds, KNI-227 and KNI-272, which were highly potent against HIV protease with little inhibition of other aspartic proteases, showed the most potent activity against the infectivity and cytopathic effect of a wide spectrum of HIV strains. As tested in target CD4+ ATH8 cells, the 50% inhibitory concentrations of KNI-227 against HIV type 1 LAI (HIV-1LAI), HIV-1RF, HIV-1MN, and HIV-2ROD were 0.1, 0.02, 0.03, and 0.1 microM, respectively, while those of KNI-272 were 0.1, 0.02, 0.04, and 0.1 microM, respectively. Both agents completely blocked the replication of 3'-azido-2',3'-dideoxythymidine-sensitive and -insensitive clinical HIV-1 isolates at 0.08 microM as tested in target phytohemagglutinin-activated peripheral blood mononuclear cells. The ratios of 50% cytotoxic concentrations to 50% inhibitory concentrations for KNI-227 and KNI-272 were approximately 2,500 and > 4,000, respectively, as assessed in peripheral blood mononuclear cells. Both compounds blocked the posttranslational cleavage of the p55 precursor protein to generate the mature p24 Gag protein in stably HIV-1-infected cells. The n-octanol-water partition coefficients of KNI-227 and KNI-272 were high, with log Po/w values of 3.79 and 3.56, respectively. Degradation of KNI-227 and KNI-272 in the presence of pepsin (1 mg/ml, pH 2.2) at 37 degrees C for 24 h was negligible. Current data warrant further careful investigations toward possible clinical application of these two novel compounds.     

CGP 53437, an orally bioavailable inhibitor of human immunodeficiency virus type 1 protease with potent antiviral activity.

CGP 53437 is a peptidomimetic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease containing a hydroxyethylene isostere. The compound inhibited recombinant HIV-1 protease with a Ki of 0.2 nM. The inhibition constant versus human cathepsin D and human cathepsin E was 4 nM. Human pepsin and gastricsin were inhibited with Kis of 8 and 500 nM, respectively, and human renin was inhibited with a Ki of 190 microM. The replication of HIV-1/LAV, HIV-1/Z-84, and HIV-1/pLAI was inhibited with a 90% effective dose of 0.1 microM in acutely infected MT-2 cells. The 50% cytotoxic dose was 100 microM. Similar antiviral activity was observed when the compound was added up to 10 h after infection. At the effective concentration, processing of Gag precursor protein p55 was greatly reduced, confirming an action on the late stage of the virus life cycle, as expected. The efficacy of the inhibitor was also demonstrated by using primary human peripheral blood lymphocytes infected with the HIV-1/LAV strain, low-passage clinical isolates obtained from HIV-1-seropositive individuals (including a zidovudine-resistant strain), and HIV-2/ROD. In these cells, CGP 53437 delayed the onset of HIV replication in a dose-dependent fashion (substantial effects with concentrations of > or = 0.1 microM) as long as the inhibitor was maintained in the culture. CGP 53437 was orally bioavailable in mice. Concentrations in plasma 10-fold in excess of the in vitro antiviral 90% effective dose could be sustained for several hours after oral application of 120 mg/kg. Therefore, CGP 53437 has the potential to be a therapeutically useful anti-HIV agent for the treatment of AIDS.     

Role of human immunodeficiency virus (HIV) type 1 envelope in the anti-HIV activity of the betulinic acid derivative IC9564

The betulinic acid derivative IC9564 is a potent anti-human immunodeficiency virus (anti-HIV) compound that can inhibit both HIV primary isolates and laboratory-adapted strains. However, this compound did not affect the replication of simian immunodeficiency virus and respiratory syncytial virus. Results from a syncytium formation assay indicated that IC9564 blocked HIV type 1 (HIV-1) envelope-mediated membrane fusion. Analysis of a chimeric virus derived from exchanging envelope regions between IC9564-sensitive and IC9564-resistant viruses indicated that regions within gp120 and the N-terminal 25 amino acids (fusion domain) of gp41 are key determinants for the drug sensitivity. By developing a drug-resistant mutant from the NL4-3 virus, two mutations were found within the gp120 region and one was found within the gp41 region. The mutations are G237R and R252K in gp120 and R533A in the fusion domain of gp41. The mutations were reintroduced into the NL4-3 envelope and analyzed for their role in IC9564 resistance. Both of the gp120 mutations contributed to the drug sensitivity. On the contrary, the gp41 mutation (R533A) did not appear to affect the IC9564 sensitivity. These results suggest that HIV-1 gp120 plays a key role in the anti-HIV-1 activity of IC9564.     

The benzamide derivative N-[1-(7-tert-Butyl-1H-indol-3-ylmethyl)-2-(4-cyclopropanecarbonyl-3-methyl-piperazin-1-yl)-2-oxo-ethyl]-4-nitro-benzamide (SP-10) reduces HIV-1 infectivity in vitro by modifying actin dynamics

Current treatments for patients infected with HIV are suboptimal. There is a need for new HIV therapies that act through different mechanisms than current treatments. We investigated the in vitro efficacy, safety and mechanism of action of the benzamide derivative N-[1-(7-tert-Butyl-1H-indol-3-ylmethyl)-2-(4-cyclopropanecarbonyl-3-methyl-piperazin-1-yl)-2-oxo-ethyl]-4-nitro-benzamide (SP-10), a potential new HIV treatment. When HIV-1-responsive engineered HeLa cells were pre-incubated for 48 h with either SP-10 or zidovudine (AZT), SP-10 was able to inhibit viral replication at much lower concentrations (IC50 = 0.036 nM) than AZT (IC50 = 27.4 nM). In contrast to AZT, SP-10 also inhibited replication of the multidrug-resistant HIV-1 strain MDR-769 in the HeLa cell model. In co-incubation experiments, SP-10 also inhibited the CCR5-sensitive HIV-1 BaL virus replication in human peripheral blood mononuclear cells. SP-10 displayed very low toxicity compared with current antiviral treatments. Confocal laser scanning microscopy and immunoprecipitation studies showed that SP-10 reduced the expression of CD4 and CCR5 on the surface of the host cell. SP-10 also reduced the level of gp120 binding to the cell surface. Confocal laser scanning microscopy studies showed that SP-10 blocked the formation of actin filaments (F-actin) and altered actin accumulation near the cell surface. These promising results suggest that SP-10 has a novel mechanism of action that enables effective inhibition of HIV-1 binding and cell entry. Further development of SP-10 as a new HIV treatment appears warranted.     

Synthesis of dual function (5R,6R)- and (5S,6S)-5-bromo-6-methoxy-5,6-dihydro-AZT-5'-(para-bromophenyl methoxyalaninyl phosphate) as novel spermicidal and anti-HIV agents

We synthesized a novel compound, 5-bromo-6-methoxy-5,6-dihydro-AZT-5'- (p-bromophenyl methoxyalaninyl phosphate), which had an EC50 value of 5 microM in sperm motility assays. This is > 1 log10 better than that of the detergent spermicide nonoxynol-9 (EC50 81 microM). The compound also displayed a potent anti-human immunodeficiency virus (HIV) activity with an IC50 value of 0.005 microM in HIV replication assays, which was virtually identical to that of AZT (IC50 0.006 microM) and > 2 log10 more potent than that of nonoxynol-9 (IC50 2.2 microM). The promising results reported herein recommend the further development of the dual function 5-halo-6-alkoxyl-5,6-dihydro-AZT derivatives as a new class of vaginal contraceptives capable of preventing the sexual transmission of HIV while providing fertility control for women who are at high risk of acquiring HIV by heterosexual transmission. These dual function 5-halo-6-alkoxyl-5,6-dihydro-AZT derivatives may also have utility in curbing domestic and wildlife animal retroviral transmissions.     

(-)-2''-deoxy-3''-thiacytidine is a potent, highly selective inhibitor of human immunodeficiency virus type 1 and type 2 replication in vitro.

The (-)-enantiomer of 2'-deoxy-3'-thiacytidine (3TC) was found to be a potent and selective inhibitor of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) in vitro. We determined its antiviral activity against a number of laboratory strains of HIV-1 and HIV-2 in a range of CD4-bearing lymphocyte cell lines (mean 50% inhibitory concentration [IC50] range, 4 nM to 0.67 microM). 3TC was also active against a range of HIV-1 strains in peripheral blood lymphocytes (mean IC50 range, 2.5 to 90 nM). The IC50 for cytotoxicity in seven lymphocyte cell cultures, including human peripheral blood lymphocytes, ranged from 0.5 to 6 mM. 3TC had no detectable antiviral activity against a range of other viruses or in cells chronically infected with HIV-1 or HIV-2. The effects of time of addition of the compound and varying the multiplicity of infection on the antiviral activity of 3TC were determined. The results showed that 3TC is a potent and selective inhibitor of HIV-1 and HIV-2 replication in vitro.     

Efficacy of constant infusion of A-77003, an inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease, in limiting acute HIV-1 infection in vitro.

A-77003, a human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is effective for both acute and chronic infection in vitro and was evaluated clinically by continuous intravenous infusion administration. The minimum effective dose (the concentration required to completely inhibit viral replication) was determined in vitro in a population of uninfected (99%) and HIV-infected (1%) cells exposed to A-77003 by continuous infusion in hollow-fiber bioreactors. The production of infectious HIV and release of p24 antigen from infected cells were completely inhibited in cultures exposed to A-77003 at or above a concentration of 0.5 microM. Measurement of unintegrated HIV-1 DNA synthesis and flow cytometric analysis for cells expressing HIV p24 antigen demonstrated that the spread of HIV to uninfected cells was also blocked at 0.5 microM A-77003. Dose deescalation to 0.25 microM or removal of A-77003 resulted in the limited spread of the virus throughout the culture, the resumption of viral DNA synthesis, and release of p24. HIV produced after exposure to 0.5 microM A-77003 was noninfectious for a period of 72 h after the removal of the drug. Addition of 1 mg of alpha 1-acid glycoprotein per ml to this in vitro system completely ablated the anti-HIV effect of 0.5 microM A-77003. These data suggest that determination of the minimum effective dose under conditions which simulate human pharmacodynamic patterns may be useful in determining the initial dose and schedule for clinical trials. However, other factors, such as serum protein binding, may influence the selection of a therapeutic regimen.     

Crippling HIV one mutation at a time

Accumulating data suggest that not all human immunodeficiency virus (HIV)-1-specific immune responses are equally effective at controlling HIV-1 replication. A new study now demonstrates that multiple immune-driven sequence polymorphisms in the highly conserved HIV-1 Gag region of transmitted viruses are associated with reduced viral replication in newly infected humans. These data suggest that targeting these and other conserved viral regions may be the key to developing an effective HIV-1 vaccine.     

In vitro inhibition of human immunodeficiency virus (HIV) type 1 replication by C2 symmetry-based HIV protease inhibitors as single agents or in combinations.

C2 symmetry-based human immunodeficiency virus (HIV) protease inhibitors were examined in vitro as single agents or in combination with 3'-azido-2',3'-dideoxythymidine (AZT) or 2',3'-dideoxyinosine for activity against HIV type 1 (HIV-1). Ten C2 symmetry-based or pseudo-C2 symmetry-based HIV protease inhibitors were active against a laboratory strain (HIV-1IIIB) in the HIV-1 cytopathic effect inhibition assay. Three inhibitors, A75925, A76928, and A77003, selected to represent a range of aqueous solubility and antiviral activity, were active against four different HIV-1 strains tested. These three inhibitors exhibited a significant inhibition of the cytopathic effect of HIV-1 against the CD4+ ATH8 cell line, with 90% inhibitory concentrations ranging from 0.1 to 4 microM. Cellular toxicity was negligible at up to 20 microM. Furthermore, they completely inhibited the replication of monocytotropic strain HIV-1Ba-L in purified monocytes and macrophages at 0.75 to 2 microM. Potent inhibitory activity against a primary HIV-1 isolate and an AZT-resistant HIV-1 variant was also observed for all three inhibitors in phytohemagglutinin-activated peripheral blood mononuclear cells. When these three HIV protease inhibitors and AZT or 2',3'-dideoxyinosine were used in combinations against a primary HIV isolate in phytohemagglutinin-activated peripheral blood mononuclear cells and the results were analyzed with the COMBO program package, their antiviral activities were identified to be synergistic in some cases and additive in others. The present data warrant further investigations of these compounds as potential antiviral agents for the therapy of HIV infections.     

Effective transduction and stable transgene expression in human blood cells by a third-generation lentiviral vector

Difficulty in gene transduction of human blood cells, including hematopoietic stem cells, has hampered the development of gene therapy applications for hematological disorders, encouraging the development and use of new gene delivery systems. In this study, we used a third-generation self-inactivating (SIN) lentiviral vector system based on human immunodeficiency virus type 1 (HIV-1) to improve transduction efficiency and prevent vector-related toxicity. The transduction efficiency of the HIV-1-based vector was compared directly with the Moloney murine leukemia virus (MLV) SIN vector in human leukemia cell lines. Initial transduction efficiencies were almost 100% for the HIV and less than 50% for the MLV vectors. Similar results were observed in 11 types of primary cells obtained from leukemia or myeloma patients. Transgene expression persisted for 8 weeks in cells transduced with the HIV vector, but declined with the MLV vector. In addition, resting peripheral blood lymphocytes and CD34(+) hematopoietic cells were transduced successfully with the HIV vector, but not with the MLV vector. Finally, we confirmed vector gene integration in almost all colony-forming cells transduced with the HIV vector, but not with the MLV vector. In conclusion, this lentiviral vector is an excellent gene transduction system for human blood cells because of its high gene transduction and host chromosome integration efficiency.     

Prevalence and clinical implications of insertions in the HIV-1 p6Gag N-terminal region in drug-naive individuals initiating antiretroviral therapy

We assessed the prevalence and clinical impact of insertions within the HIV-1 p6Gag proline-rich (PTAP) region on initial antiretroviral therapy response in 461 HIV-infected, drug-naive individuals initiating therapy in British Columbia, Canada between June 1996 and August 1998. HIV p6Gag insertions were detected by nested RT-PCR of extracted patient plasma followed by direct DNA sequencing. Insertions were observed in 70 of 423 successfully genotyped samples (16.5%). HIV p6Gag insertions were significantly associated with a lower baseline CD4 cell count (P<0.05) and the presence of basic amino acids at key positions in the HIV envelope V3 loop linked to a syncytium-inducing phenotype (P<0.05). After adjusting for baseline factors, no effect of HIV p6Gag insertions was observed on time to virological success (pVL < or = 500 copies/ml), virological failure (subsequent confirmed pVL > or = 500 copies/ml) or immunological failure (confirmed CD4 count below baseline), as evaluated by Kaplan-Meier methods and Cox proportional hazard regression (P>0.1). The data suggest that HIV p6Gag insertions are not exclusively related to drug resistance and may not influence response to antiretroviral therapy, but may be linked to sequence variations in the HIV envelope.