Human immunodeficiency virus type 1 proteinase resistance to symmetric cyclic urea inhibitor analogs

Resistant virus was isolated from virus propagated in cell culture in the presence of the human immunodeficiency virus type 1 (HIV-1) proteinase inhibitor DMP 323, Ro 31-8959, or A-75925. The proteinase gene of resistant virus was sequenced, and key mutations (G48V, V82A, I84V, L90M, and G48V/L90M) were introduced into clones used for the expression, purification, and further characterization of the enzyme. The mutant enzymes were all less active than the wild-type enzyme, as judged by k(cat) and k(cat)/Km values. L90M had a lower Km than the wild type, whereas the G48V/L90M double mutant had an increased Km compared with that of the wild type, contributing to a 10-fold reduction in the k(cat)/Km. Vitality values were used to show that the enzyme of the I84V mutant is the enzyme most resistant to the two cyclic urea inhibitors DMP 323 and AHA 008. Virus with the same mutation is also resistant, although the double mutation L10F/I84V confers even greater resistance. All of these mutants are more resistant to DMP 323 than to AHA 008. The resistance of the I84V mutant may be attributed to a loss of van der Waals interactions with the inhibitor, since the larger amino acid side chain involved in the interaction is replaced by a smaller side chain. This is supported by the lower level of resistance to AHA 008 that was observed. The phenyl groups of AHA 008 should protrude deeper into the S1 and S1' subsites than those of the smaller compound DMP 323, reducing the loss of interaction energy. These results reveal that small structural modifications of inhibitors that do not affect the inhibitory effect on wild-type virus can influence the inhibition of resistant strains. This is of importance for optimizing drugs with respect to their potency and resistance.     

Characterization of human immunodeficiency virus type 1 mutants with decreased sensitivity to proteinase inhibitor Ro 31-8959

A human immunodeficiency virus type 1 (HIV-1) variant with highly reduced susceptibility to Ro 31-8959, an inhibitor of the viral proteinase, has been selected by repeated passage of wild-type virus in CEM cells in the presence of increasing concentrations of the inhibitor. Peptide sequences of the proteinase of selected virus were obtained from proviral DNA. Sequence comparison to wild-type (wt) proteinase demonstrated two amino acid substitutions in the resistant virus, a Gly to Val exchange at position 48 and a Leu to Met exchange at position 90. Furthermore, sequences of intermediate passage virus suggest contributions from positions 12, 36, 57, and 63 in early steps of resistance development. The selected virus showed a ca. 40-fold increase in 50% inhibitory concentration of Ro 31-8959. Growth kinetics of resistant virus were comparable to wild-type virus and the resistant genotype proved to be stable in the absence of inhibitor. Directed mutagenesis of the HIV-1 HXB2 proteinase at positions 48 and 90 suggested that each mutation alone led to a moderate decrease in sensitivity of the recombinant virus to proteinase inhibitor. However, a recombinant virus carrying both mutations in the proteinase gene showed a significant reduction in its sensitivity to Ro 31-8959 thus proving the importance of these exchanges for the resistance phenotype.     

A novel, picomolar inhibitor of human immunodeficiency virus type 1 protease

The design, synthesis, and molecular modeling studies of a novel series of azacyclic ureas, which are inhibitors of human immunodeficiency virus type 1 (HIV-1) protease that incorporate different ligands for the S1', S2, and S2' substrate-binding sites of HIV-1 protease are described. The synthesis of this series is highly flexible in the sense that the P1', P2, and P2' residues of the inhibitors can be changed independently. Molecular modeling studies on the phenyl ring of the P2 and P2' ligand suggested incorporation of hydrogen-bonding donor/acceptor groups at the 3' and 4-positions of the phenyl ring should increase binding potency. This led to the discovery of compound 7f (A-98881), which possesses high potency in the HIV-1 protease inhibition assay and the in vitro MT-4 cell culture assay (Ki = approximately 5 pM and EC50 = 0.002 microM). This compares well with the symmetrical cyclic urea 1 pioneered at DuPont Merck.     

Inhibition of human immunodeficiency virus type 1 replication by a Tat-activated, transduced interferon gene: targeted expression to human immunodeficiency virus type 1-infected cells

We have examined the feasibility of using interferon (IFN) gene transfer as a novel approach to anti-human immunodeficiency virus type 1 (HIV-1) therapy in this study. To limit expression of a transduced HIV-1 long terminal repeat (LTR)-IFNA2 (the new approved nomenclature for IFN genes is used throughout this article) hybrid gene to the HIV-1-infected cells, HIV-1 LTR was modified. Deletion of the NF-kappa B elements of the HIV-1 LTR significantly inhibited Tat-mediated transactivation in T-cell lines, as well as in a monocyte line, U937. Replacement of the NF-kappa B elements in the HIV-1 LTR by a DNA fragment derived from the 5'-flanking region of IFN-stimulated gene 15 (ISG15), containing the IFN-stimulated response element, partially restored Tat-mediated activation of LTR in T cells as well as in monocytes. Insertion of this chimeric promoter (ISG15 LTR) upstream of the human IFNA2 gene directed high levels of IFN synthesis in Tat-expressing cells, while this promoter was not responsive to tumor necrosis factor alpha-mediated activation. ISG15-LTR-IFN hybrid gene inserted into the retrovirus vector was transduced into Jurkat and U937 cells. Selected transfected clones produced low levels of IFN A (IFNA) constitutively, and their abilities to express interleukin-2 and interleukin-2 receptor upon stimulation with phytohemagglutinin and phorbol myristate acetate were retained. Enhancement of IFNA synthesis observed upon HIV-1 infection resulted in significant inhibition of HIV-1 replication for a period of at least 30 days. Virus isolated from IFNA-producing cells was able to replicate in the U937 cells but did not replicate efficiently in U937 cells transduced with the IFNA gene. These results suggest that targeting IFN synthesis to HIV-1-infected cells is an attainable goal and that autocrine IFN synthesis results in a long-lasting and permanent suppression of HIV-1 replication.     

Common themes of antibody maturation to simian immunodeficiency virus, simian-human immunodeficiency virus, and human immunodeficiency virus type 1 infections

Characterization of virus-specific immune responses to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) is important to understanding the early virus-host interactions that may determine the course of virus infection and disease. Using a comprehensive panel of serological assays, we have previously demonstrated a complex and lengthy maturation of virus-specific antibody responses elicited by attenuated strains of SIV that was closely associated with the development of protective immunity. In the present study, we expand these analyses to address several questions regarding the nature of the virus-specific antibody responses to pathogenic SIV, SIV/HIV-1 (SHIV), and HIV-1 infections. The results demonstrate for the first time a common theme of antibody maturation to SIV, SHIV, and HIV-1 infections that is characterized by ongoing changes in antibody titer, conformational dependence, and antibody avidity during the first 6 to 10 months following virus infection. We demonstrate that this gradual evolution of virus-specific antibody responses is independent of the levels of virus replication and the pathogenicity of the infection viral strain. While the serological assays used in these studies were useful in discriminating between protective and nonprotective antibody responses during evaluation of vaccine efficacy with attenuated SIV, these same assays do not distinguish the clinical outcome of infection in pathogenic SIV, SHIV, or HIV-1 infections. These results likely reflect differences in the immune mechanisms involved in mediating protection from virus challenge compared to those that control an established viral infection, and they suggest that additional characteristics of both humoral and cellular responses evolve during this early immune maturation.     

In vitro combination of PNU-140690, a human immunodeficiency virus type 1 protease inhibitor, with ritonavir against ritonavir-sensitive and -resistant clinical isolates

PNU-140690 (sulfonamide-containing 5,6-dihydro-4-hydroxy-2-pyrone) is a potent, nonpeptidic inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease currently under clinical evaluation. PNU-140690 and ritonavir were studied in two-drug combinations against the replication of HIV-1 clinical isolates in peripheral blood mononuclear cells. A ritonavir-sensitive (301-1x) and -resistant (301-6x) isolate pair derived from an individual before and after monotherapy with ritonavir were used. These isolates showed no significant difference in sensitivity to PNU-140690, but isolate 301-6x was more than 50-fold less sensitive to ritonavir than isolate 301-1x. Mathematical analysis showed that the combination of various concentrations of PNU-140690 with ritonavir yielded additive to moderately synergistic antiviral effects against the ritonavir-sensitive isolate and stronger synergy against the ritonavir-resistant isolate. The mechanism of synergy was not investigated, but the results suggested that both the virological and the observed in vitro pharmacological effects may have contributed to the observed synergy. Importantly, no significant antagonism was observed with the drug combinations studied. These data suggest that PNU-140690 may be useful in combination regimens with a structurally unrelated protease inhibitor such as ritonavir.     

Impaired fitness of human immunodeficiency virus type 1 variants with high-level resistance to protease inhibitors

One hope to maintain the benefits of antiviral therapy against the human immunodeficiency virus type 1 (HIV-1), despite the development of resistance, is the possibility that resistant variants will show decreased viral fitness. To study this possibility, HIV-1 variants showing high-level resistance (up to 1,500-fold) to the substrate analog protease inhibitors BILA 1906 BS and BILA 2185 BS have been characterized. Active-site mutations V32I and I84V/A were consistently observed in the protease of highly resistant viruses, along with up to six other mutations. In vitro studies with recombinant mutant proteases demonstrated that these mutations resulted in up to 10(4)-fold increases in the Ki values toward BILA 1906 BS and BILA 2185 BS and a concomitant 2,200-fold decrease in catalytic efficiency of the enzymes toward a synthetic substrate. When introduced into viral molecular clones, the protease mutations impaired polyprotein processing, consistent with a decrease in enzyme activity in virions. Despite these observations, however, most mutations had little effect on viral replication except when the active-site mutations V32I and I84V/A were coexpressed in the protease. The latter combinations not only conferred a significant growth reduction of viral clones on peripheral blood mononuclear cells but also caused the complete disappearance of mutated clones when cocultured with wild-type virus on T-cell lines. Furthermore, the double nucleotide mutation I84A rapidly reverted to I84V upon drug removal, confirming its impact on viral fitness. Therefore, high-level resistance to protease inhibitors can be associated with impaired viral fitness, suggesting that antiviral therapies with such inhibitors may maintain some clinical benefits.     

Oligonucleotide ligation assay for detecting mutations in the human immunodeficiency virus type 1 pol gene that are associated with resistance to zidovudine, didanosine, and lamivudine

Nonpregnant adults with group B streptococcus bacteremia were identified by active surveillance in three hospitals. Serum samples collected within 2 days of the time of blood culture were assayed for IgG antibodies to the capsular polysaccharide of the infecting strain: serotype Ia (3 isolates), III (5 isolates), or V (4 isolates). In 7 of 12 bacteremia episodes, the serum level of IgG to the infecting isolate was > or = 3.5 microg/mL, higher than the 1-2 microg/mL level thought to be protective in neonates. Among selected acute-phase sera, 4 of 5 that contained > or = 3.5 microg/mL specific IgG mediated efficient opsonophagocytic killing of the corresponding group B streptococcus isolate in vitro. High levels of specific antibodies during the acute phase of invasive group B streptococcus infection in nonpregnant adults may reflect a rapid antibody response to infection or, in some cases, may indicate that susceptibility is due to defects in other immune effectors.     

Kinetic properties of saquinavir-resistant mutants of human immunodeficiency virus type 1 protease and their implications in drug resistance in vivo

In order to study the basis of resistance of human immunodeficiency virus, type 1 (HIV-1), to HIV-1 protease inhibitor saquinavir, the catalytic and inhibition properties of the wild-type HIV-1 protease and three saquinavir resistant mutants, G48V, L90M, and G48V/L90M, were compared. The kinetic parameter kcat/Km was determined for these proteases using eight peptide substrates whose sequences were derived from the natural processing site sequences of HIV-1. The kcat/Km values were determined using conventional steady-state kinetics as well as initial velocities of mixed substrate cleavages under the condition where the substrate concentrations [S]o < Km. The independently determined kcat and Km values for some of the substrates confirmed the accuracy of the mixed-substrate method and also permitted the calculation in all cases of true rather than relative kcat/Km values. The Ki values were also determined. Using a previously described kinetic model [Tang, J., & Hartsuck, J. A. (1995) FEBS Lett. 367, 112-116], the relative processing activities of HIV-1 protease variants were estimated in the saquinavir concentration range of 0-10(-7) M. Although the protease activity of G48V, L90M, and G48V/L90M are only about 10, 7, and 3% of that of the wild-type HIV-1 protease in the absence of inhibitor, the resistance tendencies of the three mutants are clearly manifest by relatively less activity loss as inhibitor concentration becomes higher. Also, the ratios of the activities of the four protease species at certain saquinavir concentrations appear to correlate with the population ratios of the four protease species at different time points of clinical trials. This correlation suggests that the population ratio of the protease species is driven by in vivo saquinavir concentration, which appears to be in the range 10(-10)-10(-9) M during the clinical trials.     

Vasectomy and human immunodeficiency virus type 1 in semen

PURPOSE: Human immunodeficiency virus type 1 (HIV) is cultured more often from seminal cells than seminal plasma. Because vasectomy causes dramatic reductions in seminal cells and also eliminates secretions from proximal sites in the male reproductive tract, vasectomy may change the potential infectiousness of semen. MATERIALS AND METHODS: We used polymerase chain reaction (PCR) assays to measure HIV ribonucleic acid (RNA) in seminal plasma and HIV deoxyribonucleic acid (DNA) in seminal cells from 46 asymptomatic, seropositive men before and after vasectomy. RESULTS: HIV RNA levels in semen correlated only weakly with blood levels (r = 0.22, p = 0.03). Of 183 semen specimens assayed for cell-free HIV RNA and proviral DNA 37 (20%) were positive for HIV RNA only, 41 (22%) were positive for HIV DNA only, and 18 (10%) were positive for RNA and DNA. Thus, detection of HIV RNA in seminal plasma was not associated with detection of HIV DNA in seminal cells. HIV RNA was present in 23 of 82 specimens (28%) (mean 2.87 log copies/ml.) before vasectomy and in 38 of 121 specimens (31%) after vasectomy (mean 2.81 log copies/ml.). CONCLUSIONS: These findings suggest that direct measurement of HIV levels in semen is necessary to assess the potential for sexual transmission, most cell-free HIV in seminal plasma arises distal to the vas deferens, and vasectomy may have minimal impact on the infectiousness of HIV seropositive men on sexual partners.     

In vivo sequence diversity of the protease of human immunodeficiency virus type 1: presence of protease inhibitor-resistant variants in untreated subjects

We have evaluated the sequence diversity of the protease human immunodeficiency virus type 1 in vivo. Our analysis of 246 protease coding domain sequences obtained from 12 subjects indicates that amino acid substitutions predicted to give rise to protease inhibitor resistance may be present in patients who have not received protease inhibitors. In addition, we demonstrated that amino acid residues directly involved in enzyme-substrate interactions may be varied in infected individuals. Several of these substitutions occurred in combination either more or less frequently than would be expected if their appearance was independent, suggesting that one substitution may compensate for the effects of another. Taken together, our analysis indicates that the human immunodeficiency virus type 1 protease has flexibility sufficient to vary critical subsites in vivo, thereby retaining enzyme function and viral pathogenicity.     

Rapid, transient changes at the env locus of plasma human immunodeficiency virus type 1 populations during the emergence of protease inhibitor resistance

Plasma human immunodeficiency virus type 1 (HIV-1) populations were genetically analyzed at their most variable locus, the envelope gene, during the rapid emergence of resistance to protease inhibitor monotherapy. Plasma virus populations remained genetically constant prior to drug treatment and during the 1 to 2 weeks following initiation of therapy, while viremia fell 10- to 100-fold. Concomitant with rapid plasma viremia rebounds associated with the emergence of drug-resistant virus, marked alterations were then detected at the env locus. Plasma population changes lasted only a few weeks before the reappearance of the pretreatment envelope variants. The emergence of resistance to single protease inhibitors was therefore associated with major but transient changes at a nonselected locus. Selection for resistance to single protease inhibitors thus appears to be more complex than the continued replication of a large, random, and therefore genetically representative sampling of the pretreatment plasma population. The possibility that drug-privileged anatomical sites containing distinct envelope variants and/or a small effective HIV-1 population size account for these results is discussed.     

Intrinsic human immunodeficiency virus type 1 resistance of hematopoietic stem cells despite coreceptor expression

Interactions of human immunodeficiency virus type 1 (HIV-1) with hematopoietic stem cells may define restrictions on immune reconstitution following effective antiretroviral therapy and affect stem cell gene therapy strategies for AIDS. In the present study, we demonstrated mRNA and cell surface expression of HIV-1 receptors CD4 and the chemokine receptors CCR-5 and CXCR-4 in fractionated cells representing multiple stages of hematopoietic development. Chemokine receptor function was documented in subsets of cells by calcium flux in response to a cognate ligand. Productive infection by HIV-1 via these receptors was observed with the notable exception of stem cells, in which case the presence of CD4, CXCR-4, and CCR-5, as documented by single-cell analysis for expression and function, was insufficient for infection. Neither productive infection, transgene expression, nor virus entry was detectable following exposure of stem cells to either wild-type HIV-1 or lentivirus constructs pseudotyped in HIV-1 envelopes of macrophage-tropic, T-cell-tropic, or dualtropic specificity. Successful entry into stem cells of a vesicular stomatitis virus G protein-pseudotyped HIV-1 construct demonstrated that the resistance to HIV-1 was mediated at the level of virus-cell membrane fusion and entry. These data define the hematopoietic stem cell as a sanctuary cell which is resistant to HIV-1 infection by a mechanism independent of receptor and coreceptor expression that suggests a novel means of cellular protection from HIV-1.