Infrequent recovery of HIV from but robust exogenous infection of activated CD4(+) T cells in HIV elite controllers.

BACKGROUND. Human immunodeficiency virus (HIV) elite controllers are able to control infection with HIV-1 spontaneously to undetectable levels in the absence of antiretroviral therapy, but the mechanisms leading to this phenotype are poorly understood. Although low frequencies of HIV-infected peripheral CD4(+) T cells have been reported in this group, it remains unclear to what extent these are due to viral attenuation, active immune containment, or intracellular host factors that restrict virus replication. METHODS. We assessed proviral DNA levels, autologous viral growth from and infectability of in vitro activated, CD8(+) T cell-depleted CD4(+) T cells from HIV elite controllers (mean viral load, <50 copies/mL), viremic controllers (mean viral load, <2000 copies/mL), chronic progressors, and individuals receiving highly active antiretroviral therapy. RESULTS. Although we successfully detected autologous virus production in ex vivo activated CD4(+) T cells from all chronic progressors and from most of the viremic controllers, we were able to measure robust autologous viral replication in only 2 of 14 elite controllers subjected to the same protocol. In vitro activated autologous CD4(+) T cells from elite controllers, however, supported infection with both X4 and R5 tropic HIV strains at comparable levels to those in CD4(+) T cells from HIV-uninfected subjects. Proviral DNA levels were the lowest in elite controllers, suggesting that extremely low frequencies of infected cells contribute to difficulty in isolation of virus. CONCLUSIONS. These data indicate that elite control is not due to inability of activated CD4(+) T cells to support HIV infection, but the relative contributions of host and viral factors that account for maintenance of low-level infection remain to be determined.

Structure-based design, synthesis, and biological evaluation of indomethacin derivatives as cyclooxygenase-2 inhibiting nitric oxide donors.

Indomethacin, a nonselective cyclooxygenase (COX) inhibitor, was modified in three distinct regions in an attempt both to increase cyclooxygenase-2 (COX-2) selectivity and to enhance drug safety by covalent attachment of an organic nitrate moiety as a nitric oxide donor. A human whole-blood COX assay shows the modifications on the 3-acetic acid part of the indomethacin yielding an amide-nitrate derivative 32 and a sulfonamide-nitrate derivative 61 conferred COX-2 selectivity. Along with their respective des-nitrate analogs, for example, 31 and 62, the nitrates 32 and 61 were effective antiinflammatory agents in the rat air-pouch model. After oral dosing, though, only 32 increased nitrate and nitrite levels in rat plasma, indicating that its nitrate tether served as a nitric oxide donor in vivo. In a rat gastric injury model, examples 31 and 32 both show a 98% reduction in gastric lesion score compared to that of indomethacin. In addition, the nitrated derivative 32 inducing 85% fewer gastric lesions when coadministered with aspirin as compared to the combination of aspirin and valdecoxib.

Synthesis and anti-inflammatory activity of a series of N-substituted naproxen glycolamides: nitric oxide-donor naproxen prodrugs.

A series of glycolamide naproxen prodrugs containing a nitrate group as a nitric oxide (NO) donor moiety has been synthesized. These compounds were evaluated for their anti-inflammatory activity, naproxen release, and gastric tolerance. Compounds 4a, 4b, 5a, 5b, 7b, and 7c exhibited anti-inflammatory activity equivalent to that of the parent NSAID, naproxen-Na, in the rat carrageenan paw edema model. At equimolar doses relative to naproxen-Na, the NO-donor glycolamide derivatives 4a, 4b, 5a, 5b, 7b, and 7c were gastro-sparing in the rat. Naproxen formation from these NO-donor glycolamides varied among the structures examined, with the N-substituent on the amide group having a particular influence, and demonstrated their prodrug nature. Compound 7b was selected for exemplary demonstration that the glycolamide nitrates can be bioactivated to release NO. These data open the possibility that naproxen glycolamide nitrates may represent a safer alternative to naproxen as anti-inflammatory medicines.

NMI-1182, a gastro-protective cyclo-oxygenase-inhibiting nitric oxide donor.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammation and to provide pain relief but suffer from a major liability concerning their propensity to cause gastric damage. As nitric oxide (NO) is known to be gastro-protective we have synthesized a NO-donating prodrug of naproxen named NMI-1182. We evaluated two cyclo-oxygenase (COX)-inhibiting nitric oxide donors (CINODs), NMI-1182 and AZD3582, for their ability to be gastro-protective compared to naproxen and for their anti-inflammatory activity. NMI-1182 and AZD3582 were found to produce similar inhibition of COX activity to that produced by naproxen. Both NMI-1182 and AZD3582 produced significantly less gastric lesions after oral administration than naproxen. All three compounds effectively inhibited paw swelling in the rat carrageenan paw edema model. In the carrageenan air pouch model all three compounds significantly reduced PGE2 levels in the pouch exudate but only NMI-1182 and naproxen inhibited leukocyte influx. These data demonstrate that NMI-1182 has comparable anti-inflammatory activity to naproxen but with a much reduced likelihood to cause gastric damage.

Synthesis and selective cyclooxygenase-2 inhibitory activity of a series of novel, nitric oxide donor-containing pyrazoles.

The synthesis of a series of novel pyrazoles containing a nitrate (ONO(2)) moiety as a nitric oxide (NO)-donor functionality is reported. Their COX-1 and COX-2 inhibitory activities in human whole blood are profiled. Our data demonstrate that pyrazole ring substituents play an important role in COX-2 selective inhibition, such that a cycloalkyl pyrazole (6b) was found to be a potent and selective COX-2 inhibitor. Other modifications at the 3 position of the central pyrazole ring (17b, 23b, 26b-I) enhanced COX-2 inhibitory potency. Among the pyrazoles synthesized, the oxime (23b) was identified as the most potent COX-2 selective inhibitor. Accordingly, 23b was profiled pharmacologically in the rat after oral administration and shown to possess potent antiinflammatory activity in the carrageenan-induced air-pouch model and less gastric toxicity than a standard COX-2 inhibitor when administered with background aspirin treatment. We suggest that the enhanced gastric tolerance of an NO-donor COX-2 selective inhibitor has the potential to augment the clinical profile of this drug class.

Combination of paclitaxel and nitric oxide as a novel treatment for the reduction of restenosis.

The combination of a nitric oxide (NO) donor and a paclitaxel-NO donor conjugate coated on a vascular stent was tested in a rabbit iliac artery model of stenosis as a potential therapy for restenosis. Paclitaxel was conjugated with a NO donor at the 7-position to give compound 7. An adamantane-based NO donor 14 was synthesized and combined with 7 to provide a burst of NO in the first few critical hours following injury to the vessel wall. Both 7 and 14 demonstrated antiproliferative activity (IC(50) = 20 nM and 15 microM, respectively) and antiplatelet activity (IC(50) = 10 and 1 microM, respectively). Stents were coated with a layer of a polymer containing test compounds. The total amount of NO eluted from the stents after a 6 h implantation in the rabbit iliac artery was 35%, 95%, and 69% of the original content for the stents coated with 7, 14, and the combination of 7 and 14, respectively. The antistenotic activity of 7 and 14 was determined in a 28-day rabbit model with two control groups (uncoated stents and polymer-coated stents) and two study groups (paclitaxel-coated stents and stents coated with the combination of 7 and 14). Polymer-coated stents caused inflammation and increased stenosis by 39% when compared to the uncoated stents. The stents coated with 7 plus 14 were as good as the uncoated stents, 41% better than the polymer-coated stents and 34% better than the paclitaxel-coated stents. These data indicate a beneficial effect of adding NO to an antiproliferative agent (paclitaxel) and suggest a potential therapeutic combination for the treatment of stenotic vessel disease.

Consistent cytotoxic-T-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities.

Although there is increasing evidence that virus-specific cytotoxic-T-lymphocyte (CTL) responses play an important role in the control of human immunodeficiency virus (HIV) replication in vivo, only scarce CTL data are available for the ethnic populations currently most affected by the epidemic. In this study, we examined the CD8(+)-T-cell responses in African-American, Caucasian, Hispanic, and Caribbean populations in which clade B virus dominates and analyzed the potential factors influencing immune recognition. Total HIV-specific CD8(+)-T-cell responses were determined by enzyme-linked immunospot assays in 150 HIV-infected individuals by using a clade B consensus sequence peptide set spanning all HIV proteins. A total of 88% of the 410 tested peptides were recognized, and Nef- and Gag-specific responses dominated the total response for each ethnicity in terms of both breadth and magnitude. Three dominantly targeted regions within these proteins that were recognized by >90% of individuals in each ethnicity were identified. Overall, the total breadth and magnitude of CD8(+)-T-cell responses correlated with individuals' CD4 counts but not with viral loads. The frequency of recognition for each peptide was highly correlated with the relative conservation of the peptide sequence, the presence of predicted immunoproteasomal cleavage sites within the C-terminal half of the peptide, and a reduced frequency of amino acids that impair binding of optimal epitopes to the restricting class I molecules. The present study thus identifies factors that contribute to the immunogenicity of these highly targeted and relatively conserved sequences in HIV that may represent promising vaccine candidates for ethnically heterogeneous populations.

Evolution and transmission of stable CTL escape mutations in HIV infection.

Increasing evidence indicates that potent anti-HIV-1 activity is mediated by cytotoxic T lymphocytes (CTLs); however, the effects of this immune pressure on viral transmission and evolution have not been determined. Here we investigate mother-child transmission in the setting of human leukocyte antigen (HLA)-B27 expression, selected for analysis because it is associated with prolonged immune containment in adult infection. In adults, mutations in a dominant and highly conserved B27-restricted Gag CTL epitope lead to loss of recognition and disease progression. In mothers expressing HLA-B27 who transmit HIV-1 perinatally, we document transmission of viruses encoding CTL escape variants in this dominant Gag epitope that no longer bind to B27. Their infected infants target an otherwise subdominant B27-restricted epitope and fail to contain HIV replication. These CTL escape variants remain stable without reversion in the absence of the evolutionary pressure that originally selected the mutation. These data suggest that CTL escape mutations in epitopes associated with suppression of viraemia will accumulate as the epidemic progresses, and therefore have important implications for vaccine design.

Persistence of human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte clones in a subject with rapid disease progression.

We longitudinally measured T-cell receptor transcript frequencies of human immunodeficiency virus type 1 (HIV-1) specific cytotoxic T lymphocytes (CTL) in an individual with rapidly progressive disease and high levels of viremia. CTL clones elicited during acute HIV-1 infection were present at the time of death, despite absent functional CTL responses, arguing against clonal deletion as a mechanism for the decline of CTL responses observed during HIV-1 infection.

Functionally inert HIV-specific cytotoxic T lymphocytes do not play a major role in chronically infected adults and children.

The highly sensitive quantitation of virus-specific CD8(+) T cells using major histocompatibility complex-peptide tetramer assays has revealed higher levels of cytotoxic T lymphocytes (CTLs) in acute and chronic virus infections than were recognized previously. However, studies in lymphocytic choriomeningitis virus infection have shown that tetramer assays may include measurement of a substantial number of tetramer-binding cells that are functionally inert. Such phenotypically silent CTLs, which lack cytolytic function and do not produce interferon (IFN)-gamma, have been hypothesized to explain the persistence of virus in the face of a quantitatively large immune response, particularly when CD4 help is impaired. In this study, we examined the role of functionally inert CTLs in chronic HIV infection. Subjects studied included children and adults (n = 42) whose viral loads ranged from <50 to >100,000 RNA copies/ml plasma. Tetramer assays were compared with three functional assays: enzyme-linked immunospot (Elispot), intracellular cytokine staining, and precursor frequency (limiting dilution assay [LDA]) cytotoxicity assays. Strong positive associations were observed between cell numbers derived by the Elispot and the tetramer assay (r = 0.90). An even stronger association between tetramer-derived numbers and intracellular cytokine staining for IFN-gamma was present (r = 0.97). The majority (median 76%) of tetramer-binding cells were consistently detectable via intracellular IFN-gamma cytokine staining. Furthermore, modifications to the LDA, using a low input cell number into each well, enabled LDAs to reach equivalence with the other methods of CTL enumeration. These data together show that functionally inert CTLs do not play a significant role in chronic pediatric or adult HIV infection.

Distinct trafficking pathways mediate Nef-induced and clathrin-dependent major histocompatibility complex class I down-regulation.

The human immunodeficiency virus type 1 Nef protein alters the post-Golgi stages of major histocompatibility complex class I (MHC-I) biogenesis. Presumed mechanisms involve the disclosure of a cryptic tyrosine-based sorting signal (YSQA) located in the cytoplasmic tail of HLA-A and -B heavy chains. We changed this signal for a prototypic sorting motif (YSQI or YSQL). Modified HLA-A2 molecules, termed A2-endo, displayed constitutively low surface levels and accumulated in a region close to or within the Golgi apparatus, a behavior reminiscent of wild-type HLA-A2 in Nef-expressing cells. However, several lines of evidence indicate that the action of prototypic signals on MHC-I trafficking differs from that of Nef. Internalization of surface A2-endo was more rapid and was associated with efficient recycling to the surface. A transdominant-negative mutant of dynamin-1 inhibited A2-endo constitutive internalization and Nef-induced CD4 down-regulation, whereas it did not affect the activity of Nef on MHC-I. Moreover, trafficking of A2-endo was still affected by the viral protein, indicating additive effects of prototypic signals and Nef. Therefore, distinct trafficking pathways regulate clathrin-dependent and Nef-induced MHC-I modulation.

Identification of dominant optimal HLA-B60- and HLA-B61-restricted cytotoxic T-lymphocyte (CTL) epitopes: rapid characterization of CTL responses by enzyme-linked immunospot assay.

Human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte (CTL) responses play a major role in the antiviral immune response, but the relative contribution of CTL responses restricted by different HLA class I molecules is less well defined. HLA-B60 or the related allele B61 is expressed in 10 to 20% of Caucasoid populations and is even more highly prevalent in Asian populations, but yet no CTL epitopes restricted by these alleles have been defined. Here we report the definition of five novel HLA-B60-restricted HIV-1-specific CTL epitopes, using peripheral blood mononuclear cells in enzyme-linked immunospot (Elispot) assays and using CTL clones and lines in cytolytic assays. The dominant HLA-B60-restricted epitope, Nef peptide KEKGGLEGL, was targeted by all eight subjects with B60 and also by both subjects with B61 studied. This study additionally establishes the utility of the Elispot assay as a more rapid and efficient method of defining novel CTL epitopes. This approach will help to define new CTL epitopes that may play an important role in the immune control of HIV-1.

Efficient processing of the immunodominant, HLA-A*0201-restricted human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitope despite multiple variations in the epitope flanking sequences.

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.

Effects of intensified antihypertensive treatment in diabetic nephropathy: mortality and morbidity results of a prospective controlled 10-year study.

OBJECTIVE: The aim of this study was to describe the effect of intensified antihypertensive therapy based on a structured teaching and treatment programme on the prognosis of hypertensive type 1 (insulin-dependent) diabetic patients with kidney disease. DESIGN: The study was a controlled, prospective, parallel, 10-year follow-up trial. PATIENTS AND INTERVENTIONS: A sequential sample of 91 hypertensive type 1 diabetic patients with overt diabetic nephropathy was prospectively followed for 10 years. Forty-five patients (intensified antihypertensive therapy; IT group) participated in an intensified antihypertensive therapy programme and 46 patients received routine antihypertensive treatment as provided by family physicians, consultants and local hospitals (routine antihypertensive therapy; RT group). OUTCOME MEASURES: The main endpoint was death; secondary endpoints were renal replacement therapy, blindness and amputation. RESULTS: Blood pressure was reduced in the IT group and increased in the RT group. During the follow-up period, 29 patients died, seven in the IT group and 22 in the RT group. The survival curves were significantly different (P = 0.0008). The main causes of death were cardiac. In a multiple Cox proportional hazards model, allocation to the IT group reduced the mortality risk [relative risk (RR) = 0.213; 95% confidence interval 0.089-0.509, P = 0.00051, while age (P = 0.0039) and mean blood pressure (P= 0.0113) increased this risk. In multiple Cox or multiple logistic regression models, the risks of dialysis (RR = 0.269, 95% confidence interval 0.110-0.656, P = 0.0039), blindness (odds ratio = 0.158, 95% confidence interval 0.037-0.684, P= 0.0136), and amputation (RR = 0.181, 95% confidence interval 0.047-0.703, P= 0.0135) were significantly lower in the IT group compared with the RT group (log rank P = 0.0008). CONCLUSION: We conclude that intensified antihypertensive treatment, based on a hypertension teaching and treatment programme, reduces long-term morbidity and mortality in patients with diabetic nephropathy.

Association between virus-specific cytotoxic T-lymphocyte and helper responses in human immunodeficiency virus type 1 infection.

Cellular immune responses are thought to be an important antiviral host defense, but the relationship between virus-specific T-helper and cytotoxic-T-lymphocyte (CTL) responses has not been defined. To investigate a potential link between these responses, we examined functional human immunodeficiency virus type 1 (HIV-1)-specific memory CTL precursor frequencies and p24-specific proliferative responses in a cohort of infected untreated persons with a wide range of viral loads and CD4 cell counts. Levels of p24-specific proliferative responses positively correlated with levels of Gag-specific CTL precursors and negatively correlated with levels of plasma HIV-1 RNA. These data linking the levels of HIV-specific CTL with virus-specific helper cell function during chronic viral infection provide cellular immunologic parameters to guide therapeutic and prophylactic vaccine development.

Levels of human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte effector and memory responses decline after suppression of viremia with highly active antiretroviral therapy.

Therapeutic suppression of human immunodeficiency virus type 1 (HIV-1) replication may help elucidate interactions between the host cellular immune responses and HIV-1 infection. We performed a detailed longitudinal evaluation of two subjects before and after the start of highly active antiretroviral therapy (HAART). Both subjects had evidence of in vivo-activated and memory cytotoxic T-lymphocyte precursor (CTLp) activity against multiple HIV-1 gene products. After the start of therapy, both subjects had declines in the levels of in vivo-activated HIV-1-specific CTLs and had immediate increases in circulating HIV-1-specific CTL memory cells. With continued therapy, and continued suppression of viral load, levels of memory CTLps declined. HLA A*0201 peptide tetramer staining demonstrated that declining levels of in vivo-activated CTL activity were associated with a decrease in the expression of the CD38(+) activation marker. Transient increases in viral load during continued therapy were associated with increases in the levels of virus-specific CTLps in both individuals. The results were confirmed by measuring CTL responses to discrete optimal epitopes. These studies illustrate the dynamic equilibrium between the host immune response and levels of viral antigen burden and suggest that efforts to augment HIV-1-specific immune responses in subjects on HAART may decrease the incidence of virologic relapse.

Processing of HIV-1 envelope glycoprotein for class I-restricted recognition: dependence on TAP1/2 and mechanisms for cytosolic localization.

Processing of viral proteins for recognition by CTL involves degradation of the proteins in the cytosol of an infected cell followed by transport of the resulting peptides into the endoplasmic reticulum (ER) by the TAP1/2 complex. Uncertainty exists over the site of processing of viral envelope (env) proteins since the extracellular domains of env proteins are not present in the cytosol where the class I Ag-processing pathway begins. Rather, the ectodomains of env proteins are cotranslationally translocated into the ER during biosynthesis. To analyze env protein processing, we used the herpes simplex virus protein ICP47 to block peptide transport by TAP1/2 and examined the effects of TAP blockade on the processing of the HIV-1 env protein. For the majority of env-specific CD8+ CTL, the processing pathway required TAP1/2-mediated transport of cytosolic peptides into the ER. To determine how env peptides are generated in the cytosol, we analyzed the processing of two TAP1/2-dependent epitopes containing N-linked glycosylation sites. In each case, processing involved glycosylation-dependent posttranslational modification of asparagine residues to aspartic acid. These results are consistent with cotranslational translocation of env into the ER, where glycosylation occurs. This is followed by export of a fraction of the newly synthesized protein into the cytosol, where it is deglycosylated, with conversion of the asparagines to aspartic acid residues. Following cytoplasmic proteolysis, env peptides are retransported by TAP1/2 into the ER, where association with class I occurs. Thus, the env protein can enter the class I pathway through multiple distinct processing mechanisms.

Detection of HIV-1-specific CTLs in the semen of HIV-infected individuals.

CTLs play an important role in controlling cell-associated HIV. Since the majority of HIV infections are acquired through sexual transmission, we investigated whether antiviral CTLs were present in the male urogenital tract using semen as a source of T cells. We were able to establish anti-HIV cytolytic lines in five of five HIV-infected men with CD4 counts of >500/microl, although cloning efficiencies were lower than with peripheral blood-derived T cells. CTLs generated from the semen of three men were analyzed in detail and showed a broadly active response, recognizing gag, env, and pol proteins. Detailed analysis of two gag-specific clones from one of the individuals demonstrated HLA class I restriction and recognition of the same p24 epitope (EQASQEVKNWMT). In summary, our results demonstrate the presence of a broad CTL response to HIV in the urogenital tract and provide a rationale for further studies of local enhancement of genital mucosal responses by anti-HIV immunization.

Immune modulation of human B lymphocytes by gene transfer with recombinant Epstein-Barr virus amplicons.

We described previously a novel mode of gene transfer by infection of human B lymphocytes with recombinant Epstein-Barr virus (EBV) amplicons. This system was explored for its potential use in expressing various recombinant genes, including the cytokine IL-4, the HIV envelope glycoprotein (gp120) and a suicide and gag gene. Recombinant genes were present as multiple copy episomes and stable, high level recombinant gene expression could be detected by antigenic and functional assays. Amplicon-infected B cells secreted high levels of recombinant cytokine and efficiently presented recombinant antigens through classes I and II MHC-restricted antigen processing pathways. Thus, recombinant EBV amplicons can be used to express components of the immune system or heterologous genes for immune recognition in human B cells. Combining gene transfer with EBV infection may provide unique advantages for in vitro and in vivo gene transfer.

Lack of strong immune selection pressure by the immunodominant, HLA-A*0201-restricted cytotoxic T lymphocyte response in chronic human immunodeficiency virus-1 infection.

Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.