HIV P24 Antigen Levels Research Articles

Characteristics of recent HIV infection among individuals newly diagnosed as HIV-positive in South Korea (2008–2015)

Most HIV-positive individuals diagnosed in Korea are infected through sexual contact, with the time of HIV infection therefore often being unknown. The aim of this study was to investigate the characteristics of diagnosed patients near the time of HIV seroconversion to establish useful HIV intervention strategies. Cross-sectional study. Newly diagnosed HIV cases based on the national HIV surveillance system in South Korea, 2008–2015. To distinguish recent from long-standing HIV infection among 5898 nationwide HIV-positive specimens, limiting antigen avidity assays (Sedia HIV-1 LAg-Avidity EIA, cut-off = 1.5) were performed. Data on CD4+ T cell count at HIV diagnosis and death from AIDS within one year after first HIV diagnosis were used to distinguish recent HIV infections. Acute HIV infection is characterized by detectable HIV RNA or HIV p24 antigen levels, along with a negative or indeterminate antibody western blot result, but with the subject subsequently testing positive after a predetermined period. We analyzed the characteristics of recent and acute HIV infections by sex, age, nationality, HIV testing site, region, and reason for HIV testing. Recent and acute HIV infections were described as the proportion of recent and acute HIV infections among newly-diagnosed cases in a given year. Recent and acute HIV infections over the 8-year study period were 20.5% (1210/5898) and 9.5% (562/5898), respectively. Both infections were generally higher in the following groups: males, younger age, Koreans, individuals who were tested due to disease, residents of smaller city or rural area, and individuals diagnosed in recent calendar years. Acute infections were significantly higher among individuals tested in hospitals and due to suspected HIV infection. The recent and acute HIV infection in younger age groups (< 30 years) has been increasing in a given year. There was an increase in the proportion of young individuals with recent HIV infection, and we identified risk groups more at risk of HIV infection recently in Korea. Therefore, our results could prove useful for the development and assessment of national HIV prevention strategies in Korea and provide valuable data for further HIV research.

The Use of Droplet Digital PCR to Quantify HIV-1 Replication in the Colorectal Explant Model

Ex vivo explant models are used to characterize in vitro efficacy of preexposure prophylaxis (PrEP) agents. Tissue is challenged with virus in culture and HIV-1 p24 levels are quantified with enzyme-linked immunosorbent assay (ELISA) on supernatants collected throughout a 14-21-day incubation. Due to the narrow dynamic range of HIV-1 p24 kits, we evaluated whether droplet digital PCR (ddPCR) provides an alternative method to quantify HIV-1 replication in supernatant samples. We used samples from the MWRI-01 study, which evaluated the pharmacokinetic/pharmacodynamic profile of long-acting rilpivirine using the explant model (McGowan et al. Lancet HIV 2016). HIV-1 pol RNA was measured with ddPCR, either directly with a one-step method or reverse transcribed to cDNA before ddPCR (two-step method) on supernatants from the MWRI-01 study. Previously analyzed HIV-1 p24 antigen levels (Alliance; Perkin-Elmer) were available for comparison purposes. Both ddPCR methods strongly correlated with HIV-1 p24 and displayed similar patterns of HIV-1 suppression before and after rilpivirine. Compared to the p24 ELISA, two-step and one-step ddPCR reduced the amount of hands-on time by approximately one-half and two-thirds, respectively. ddPCR also required less sample and based on p24 versus ddPCR correlation, could potentially reduce the explant culture time from 14 to 10 days (r2 = 0.78, p < .001) due to the increased sensitivity of ddPCR. We demonstrate that ddPCR is a suitable alternative to HIV-1 p24 ELISA to quantify HIV-1 infection in the explant model and has the potential to decrease explant culture time.

Development and characterization of a human microglia cell model of HIV-1 infection.

Microglia cells are the major reservoir of HIV-1 (HIV) within the CNS. However, current models using transformed cell lines are not representative of primary microglia and fetal brain samples for isolation of primary human microglia (HMG) are increasingly difficult to obtain. Here, we describe a monocyte-derived microglia (MMG) cell model of HIV infection that recapitulates infection of primary HMG. CD14+ cells isolated from healthy donors were cultured with M-CSF, beta-nerve growth factor, GM-CSF, and CCL2, and compared to HMG. MMG and HMG cells were infected with HIV and viral replication was detected by p24 antigen. Both MMG and HMG cells were found to acquire spindle shape with few branched or unbranched processes at their ends during the second week in culture and both were found to be CD11b+/ CD11c+/ CD14+/ CD45+/ CD195+/ HLADRlow/ CD86low/ CD80+. Whereas hT-Hμglia and HMC3 transformed cell lines are deficient in human microglia signature genes (C1Q, GAS6, GPR34, MERTK, PROS1, and P2RY12), MMG cells expressed all of these genes. Additionally, MMG expressed all the microglia signature miRNA (miR-99a, miR125b-5p, and miR-342-3p). Both MMG and HMG produced ROS and phagocytosed labeled zymosan particles upon PMA stimulation. MMG and HMG infected with HIV produced equivalent levels of HIV p24 antigen in culture supernatants for 30days post-infection. Thus, we have developed and characterized a microglia cell model of HIV infection derived from primary monocytes that recapitulates the phenotypic and molecular properties of HMG, is superior to transformed cell lines, and has similar HIV replication kinetics to HMG.

In vitro anti-HIV-1 activity of fucoidan from Sargassum swartzii

Sargassum swartzii, a marine brown algae with wide range of biological properties belongs to the family Sargassaceae. Bioactive fucoidan fractions (CFF, FF1 and FF2) were isolated from S. swartzii and characterized by linear gradient anion-exchange chromatography and FT-IR. The characterized fucoidan fractions contained mainly sugars, sulfate and uronic acid. In the present study, anti-HIV-1 property of the fucoidan fractions was investigated. Fraction FF2 was found to exhibit significant anti-HIV-1 activity at concentrations of 1.56 and 6.25μg/ml as observed by >50% reduction in HIV-1 p24 antigen levels and reverse transcriptase activity. Fucoidan fractions have no cytotoxic effects on PBMCs at the concentration range of 1.56–1000μg/ml. These results suggest that fucoidan fractions could have inhibitory activity against HIV and has potential as an anti-HIV-1 agent.

Recombinant Tax1 and Tax2 proteins inhibit HIV-1 replication in peripheral blood mononuclear cells

Patients with HIV-1 and HTLV-2 coinfections often exhibit a clinical course similar to HIV-1 infected long-term nonprogressors. This observation has been attributed in part to the ability of the HTLV Tax2 protein to activate production of antiviral chemokines and to downregulate the CCR5 co receptor on lymphocytes. Therefore we investigated the possibility that a recombinant Tax2 protein could suppress HIV-1 viral replication in vitro. R5-tropic HIV-1 (NLAD8)-infected-PBMCs were treated daily with recombinant Tax1 and Tax2 proteins (dosage range 1-100 pM). Culture supernatants were collected at intervals for 22 days post-infection and assayed for levels of HIV-1 p24 antigen. Treatment of PBMCs with Tax2 protein resulted in significant reduction in HIV-1 p24 antigen levels (p<0.05) at days 10, 14, and 18 post-infection compared to HIV-1-infected or mock-treated PBMCs. This was preceded by the detection of increased levels of CC-chemokines MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5, on days 1-7 of infection (p<0.05). Similar, but less robust inhibition was determined in Tax1 treated PBMCs. Addition of Tax2 proteins starting 48 hours previous to infection resulted in a significant inhibition of HIV-1 p24 levels (p<0.05 at days 7 to 14 compared to the untreated, R5 infected PBMCs. In contrast, when addition of Tax2 began two days after R5 infection, significant HIV-1 p24 levels were only determined at days 7 and 10 for 1 pM and at day 7 for 10 pM (p<0.05). These results support the contention that Tax1 and Tax2 play a role in generating antiviral responses against HIV-1 in vivo and in vitro.

Inhibition of HIV Type 1 Replication by Human T Lymphotropic Virus Types 1 and 2 Tax Proteinsin Vitro

Patients with HIV-1 and human T-lymphotropic virus type 2 (HTLV-2) coinfections often exhibit a clinical course similar to that seen in HIV-1-infected individuals who are long-term nonprogressors. These findings have been attributed in part to the ability of HTLV-2 to activate production of antiviral chemokines and to downregulate the CCR5 coreceptor on lymphocytes. To further investigate these observations, we tested the ability of recombinant Tax1 and Tax2 proteins to suppress HIV-1 viral replication in vitro. R5-tropic HIV-1 (NLAD8)-infected peripheral blood mononuclear cells (PBMCs) were treated daily with recombinant Tax1 and Tax2 proteins (dosage range 1-100 pM). Culture supernatants were collected at intervals from days 1 to 22 postinfection and assayed for levels of HIV-1 p24 antigen by ELISA. Treatment of PBMCs with Tax2 protein resulted in a significant reduction in HIV-1 p24 antigen levels (p<0.05) at days 10, 14, and 18 postinfection compared to HIV-1-infected or mock-treated PBMCs. This was preceded by the detection of increased levels of CC-chemokines MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5 on days 1-7 of infection. Similar, but less robust inhibition was observed in Tax1-treated PBMCs. These results support the contention that Tax1 and Tax2 play a role in generating antiviral responses against HIV-1 in vivo and in vitro.

Fetal cord blood mononuclear cells that are collected at term from HIV-1 infected women harbor transcriptionally active integrated proviral DNA

The purpose of this study was to determine levels of intrauterine infection and transcriptional activity in cord blood mononuclear cells that were collected at term from fetuses who were born to women who were infected with human immunodeficiency virus (HIV) and who received highly active antiretroviral therapy (HAART). RNA and DNA were isolated from maternal placental tissues and fetal cord blood specimens that were obtained at term from pregnant women who were infected with HIV and who received HAART. Levels of integrated HIV provirus and messenger RNA transcripts were determined by real-time polymerase chain reaction. Detectable levels of transcriptionally active integrated provirus were present in approximately 27% of cord blood samples (n = 22) that were collected from fetuses who born to HIV-positive mothers who received HAART. Levels of HIV-p24 antigen in cultures that were detected in randomly selected cord blood samples confirmed the presence of inducible infectious virus. These findings suggest that some fetuses from HIV-infected mothers who receive HAART and who may be HIV-negative infants after delivery can harbor circulating leukocytes that are infected productively by intrauterine transmission of HIV.

Alcohol Abuse Enhances Neuroinflammation and Impairs Immune Responses in an Animal Model of Human Immunodeficiency Virus-1 Encephalitis

Neuroinflammatory disorders (including human immunodeficiency virus-1 encephalitis, HIVE) are associated with oxidative stress and inflammatory brain injury, and excessive alcohol use can exacerbate tissue damage. Using a murine model of HIVE, we investigated the effects of alcohol abuse on the clearance of virus-infected macrophages and neuroinflammation. Severe combined immunodeficient mice were reconstituted with human lymphocytes, and encephalitis was induced by intracranial injection of HIV-1-infected monocyte-derived macrophages (HIV-1(+) MDM). Animals were fed an ethanol-containing diet beginning 2 weeks before lymphocyte engraftment and for the entire duration of the experiment. Lymphocyte engraftment was not altered by ethanol exposure. Alcohol-mediated immunosuppression in ethanol-fed mice was manifested by a significant decrease in CD8(+)/interferon-gamma(+) T lymphocytes, a fivefold increase in viremia, and diminished expression of immunoproteasomes in the spleen. Although both groups showed similar amounts of CD8(+) T-lymphocyte infiltration in brain areas containing HIV-1(+) MDMs, ethanol-fed mice featured double the amounts of HIV-1(+) MDMs in the brain compared to controls. Ethanol-exposed mice demonstrated higher microglial reaction and enhanced oxidative stress. Alcohol exposure impaired immune responses (increased viremia, decreased immunoproteasome levels, and prevented efficient elimination of HIV-1(+) MDMs) and enhanced neuroinflammation in HIVE mice. Thus, alcohol abuse could be a co-factor in progression of HIV-1 infection of the brain.

Inhibition of human immunodeficiency virus type 1 replication in latently infected cells by a novel IkappaB kinase inhibitor.

In human immunodeficiency virus type 1 (HIV-1) latently infected cells, NF-kappaB plays a major role in the transcriptional induction of HIV-1 replication. Hence, downregulation of NF-kappaB activation has long been sought for effective anti-HIV therapy. Tumor necrosis factor alpha (TNF-alpha) stimulates IkappaB kinase (IKK) complex, a critical regulator in the NF-kappaB signaling pathway. A novel IKK inhibitor, ACHP {2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl-nicotinonitrile}, was developed and evaluated as a potent and specific inhibitor for IKK-alpha and IKK-beta. In this study, we examined the ability of this compound to inhibit HIV-1 replication in OM10.1 cells latently infected with HIV. When these cells were pretreated with ACHP, TNF-alpha-induced HIV-1 replication was dramatically inhibited, as measured by the HIV p24 antigen levels in the culture supernatants. Its 50% effective concentration was approximately 0.56 microM, whereas its 50% cytotoxic concentration was about 15 microM. Western blot analysis revealed inhibition of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 nuclear translocation, and p65 phosphorylation. ACHP was also found to suppress HIV-1 long terminal repeat (LTR)-driven gene expression through the inhibition of NF-kappaB activation. Furthermore, ACHP inhibited TNF-alpha-induced NF-kappaB (p65) recruitment to the HIV-1 LTR, as assessed by chromatin immunoprecipitation assay. These findings suggest that ACHP acts as a potent suppressor of TNF-alpha-induced HIV replication in latently infected cells and that this inhibition is mediated through suppression of IKK activity.

Co-receptor usage was more predictive than NSI/SI phenotype for HIV replication in macrophages: is NSI/SI phenotyping sufficient?

A monocyte-derived macrophage (MDM) culture assay was used to define the replication kinetics of HIV isolates. Ten-day-old MDMs were infected with HIV. Supernatants were collected and assayed for HIV p24 on days 3, 7, 10, and 14 post-infection (PI). In this assay, SF162 (macrophage tropic, NSI) produced increasing amounts of HIV p24 antigen with increasing time in culture. BRU (nonmacrophage tropic, SI) infection resulted in low levels of HIV p24 antigen with no increase in production during the culture period. A panel of 12 clinical isolates was evaluated. All isolates produced detectable levels of HIV p24 antigen in MDMs. However, the NSI viruses had significantly higher log10 HIV p24 antigen values at all times PI (P < 0.01). Co-receptor usage was determined for all 12 isolates (8 NSI and 4 SI). All SI isolates used CXCR4 for entry; two used CXCR4 only, one used CXCR4, CCR5, and CCR3, and one was a mixture of two isolates using CXCR4 and CCR5. None of the NSI viruses used CXCR4 for entry. All used CCR5 as their predominant co-receptor. Of the eight NSI isolates, three used CCR5 only, two used CCR5 and CCR2b, one used CCR5 and CCR3, and one used CCR5, CCR3, and CCR2b. Log10 HIV p24 antigen production on day 14 PI for viruses that used CCR5+CCR3 (3.79 + 1.40) was greater than for viruses that used CCR5+CCR2b (3.22 + 1.55) or CCR5 (3.32 + 1.49), and all were greater than those that used CXCR4 only (1.69 + 0.28), regardless of SI phenotype (P < 0.05). Thus, in these primary isolates, macrophage tropism and replication kinetics were closely linked to CCR5 utilization, whereas SI capacity was closely linked to CXCR4 utilization. Furthermore, viruses, which could use CCR5 and CCR3 for entry, had a replication advantage in macrophages, regardless of SI phenotype.

CCR5 expression correlates with susceptibility of maturing monocytes to human immunodeficiency virus type 1 infection.

The chemokine receptor CCR5 and to a lesser extent CCR3 and CCR2b have been shown to serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry into blood- or tissue-derived macrophages. Therefore, we examined the expression of the chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 as RNAs or as membrane-expressed antigens in monocytes maturing into macrophages and correlated these results with the susceptibility of macrophages to HIV-1 infection, as measured by their concentrations of extracellular p24 antigen and levels of intracellular HIV DNA by quantitative PCR. There was little change in levels of CCR1, CCR2b, and CCR5 RNAs. CCR3 RNA and surface antigen were undetectable throughout maturation of adherent monocytes over 10 days. CXCR4 RNA and membrane antigen were strongly expressed in newly adherent monocytes, but their levels declined at day 7. The amounts of CCR5 RNA remained stable, but the amounts of CCR5 antigen increased from undetectable to peak levels at day 7 and then declined slightly at day 10. Levels of susceptibility to laboratory (HIV-1BaL) and clinical strains of HIV-1 showed parallel kinetics, peaking at day 7 and then decreasing at days 10 to 14. The concordance of levels of HIV DNA and p24 antigen suggested that the changes in susceptibility with monocyte maturation were at or immediately after entry and correlated well with CCR5 expression and inversely with CXCR4 expression.

Elevated serum macrophage inhibitory factor-related protein (MRP) 8/14 levels in advanced HIV infection and during disease exacerbation.

To assess the value of MRP 8, MRP 14, and MRP 8/14 serum concentrations as markers of disease progression in HIV infection and as markers of intercurrent infections. We measured MRP 8, MRP 14, and MRP 8/14 serum concentrations in 184 HIV-infected patients in various stages of disease with or without disease exacerbation and in 50 healthy control subjects. In clinically stable HIV-infection correlations of MRP levels with stage of HIV disease, CD4 counts, p24 antigen, and beta-2 microglobulin levels were studied. In patients with intercurrent illnesses, correlations of MRP levels with type of disease exacerbation and with CRP were calculated and compared with those found in stable HIV infection. MRP 8/14 levels were significantly elevated and MRP 8 levels slightly decreased in stable HIV infection compared with HIV-negative controls. The CD4 cell count and MRP 8/14 levels correlated significantly in patients with AIDS. Despite higher values of MRP 8/14 during advanced disease, these were not significant predictors of progression to death. In patients with acute infections, MRP 8/14 levels were significantly elevated, compared with patients with illnesses of noninfectious origin. Levels of MRP 8/14 associated with acute infections were significantly higher in patients with AIDS than in patients during earlier stages of HIV infection. Both stable HIV infection and advanced immunedeficiency are associated with an elevation of the MRP 8/14 complex and probably with a decline of MRP 8 serum levels. MRP 8/14 is preserved as a marker of acute infection in immunecompromised patients.

Increased frequency of CCR-5 delta 32 heterozygotes among long-term non-progressors with HIV-1 infection. The Australian Long-Term Non-Progressor Study Group.

The beta-chemokine receptor CCR-5 is used as a coreceptor by macrophage-tropic strains of HIV-1 to gain entry into CD4+ cells. To determine the effect of a common 32 base-pair deletion mutation in the CCR-5 gene (CCR-5 delta 32) on progression of HIV infection to AIDS, and to assess the level of heterozygosity for this mutation in a well-defined group of long-term non-progressors (LTNP). Sixty-four HIV-1-infected LTNP (CD4+ T lymphocyte count > 500 x 10(6)/l after 8 years) were compared with 95 individuals infected within a similar period (1983-1986) but who had rapidly progressed to AIDS and death, and with a further 120 HIV-positive individuals with CD4+ counts < 500 x 10(6)/l. The presence of the CCR-5 delta 32 mutation was assessed using polymerase chain reaction with primers spanning the 32 base-pair deletion. CD4+ and CD8+ counts, plasma HIV-1 RNA, p24 antigen and beta 2-microglobulin levels in LTNP carrying the CCR-5 delta 32 mutation were compared with LTNP lacking the mutation. A marked increase in the frequency of CCR-5 delta 32 heterozygosity was found among LTNP (35.9%) compared with rapid progressors (12.6%; P = 0.0005) and patients selected on the basis of a CD4+ T-cell count < 500 x 10(6)/l (12.5%; P = 0.0004). LTNP heterozygous for CCR-5 delta 32 had a significantly higher CD8+ T-cell count than those without the mutation (1218 versus 972 x 10(6)/l; P = 0.044). No significant correlation was observed between heterozygosity and CD4 count, viral load, p24 antigen or beta 2-microglobulin within the LTNP group. This study provides the strongest evidence to date for the importance of a single copy of the CCR-5 delta 32 mutation in long-term non-progression of HIV infection, which may involve, in part, CD8+ T lymphocytes.

Protection of primary human T cells from HIV infection by Trev: a transdominant fusion gene.

Gene therapy is one of several approaches that are being tested in the search for an effective anti-human immunodeficiency virus (HIV) treatment. In this strategy, a "protective" gene would be introduced into target cells, rendering them relatively resistance to the virus-induced cytopathicity. Tat and Rev are viral proteins essential for HIV gene expression. Tat increases viral gene transcription and Rev is responsible for the nuclear export of mRNA encoding structural viral proteins. A fusion protein (Trev) was constructed, joining Tat and Rev transdominant mutant gene sequences. Previously, we showed that Trev inhibits both Tat and Rev activities in Jurkat T cells. To determine whether Trev could inhibit HIV replication in primary cells, we transferred the trev gene to peripheral blood lymphocytes and challenged them with different HIV strains. Levels of HIV p24 antigen (Ag) were reduced 4- to 15-fold in cultures of Trev-CD4+ T cells infected with two HIV primary clinical isolates and were not detectable in cultures infected with HIV strains NL4-3 and SF2. In contrast, cultures of nontransduced CD4+ T cells infected with the same viruses had levels of HIV p24 Ag up to 10 ng/ml. Trev-transduced CD4+ T cells demonstrated increased survival following HIV challenge for the length of the experiments (30 days). We did not observe rapid emergence of Trev-resistant HIV in our cultures. Following HIV challenge, cell-associated Trev protein was increased, supporting the hypothesis that cells surviving Trev expression provided a cell survival advantage. This work showed that Trev was able to inhibit HIV replication in primary CD4+ T cells, and, therefore the trev gene could be a candidate for gene therapy against HIV.

In VivoEffects of Interleukin 3 in HIV Type 1-Infected Patients with Cytopenia

To determine the safety, tolerance, and hematological and virological effects of the recombinant hematopoietic growth factor interleukin 3 (IL-3) in HIV-1-infected individuals with cytopenia. A phase I single-center trial was conducted with patients in cohorts of three receiving one of four dose levels of self-administered, subcutaneously injected IL-3 (0.5, 1.0, 2.5, or 5.0 micrograms/kg/day). Toxicities, hematological effects, and virological effects were recorded. Viral studies included serum HIV p24 antigen levels, quantitative plasma and peripheral blood mononuclear cell cultures, and quantitative, competitive polymerase chain reaction of patient plasma. Increases in white blood cell counts (WBC) and absolute neutrophil counts (ANC) were noted at the higher dose levels while absolute eosinophil counts (AEC) increased in all patients. The percent changes in WBC from baseline ranged from 52 to 309 and in ANC from 20 to 262 in the 2.5- and 5.0-micrograms/kg/day groups. The mean AEC change was 17-fold (range, 2- to 59-fold). Hemoglobin, hematocrit, platelets, and CD4 and CD8 counts were generally unaffected although individual patients demonstrated increases in hemoglobin and platelet levels. Toxicities were generally mild, but one patient developed a transient local erythematous rash at the sites of IL-3 injection which pathologically demonstrated hypersensitivity vasculitis. Of note, viral studies did not demonstrate any consistent changes in HIV-1 activity. These data demonstrate limited hematological effects of IL-3 monotherapy in HIV-1-infected patients with cytopenia. However, should IL-3 be incorporated into combination cytokine therapies for HIV disease, these data suggest that IL-3 does not enhance in vivo HIV-1 activity.

Distinct Changes in HIV Type 1 RNA versus p24 Antigen Levels in Serum during Short-Term Zidovudine Therapy in Asymptomatic Individuals with and without Progression to AIDS

Serum HIV-1 RNA and p24 antigen levels were examined in 28 seropositive asymptomatic individuals participating in a trial on the efficacy of zidovudine. Sixteen individuals remained asymptomatic until 4 years after the onset of the trial, whereas 12 individuals were diagnosed with an AIDS-defining event. The serum HIV-1 RNA load and p24 antigen levels were determined before the onset of therapy and during the first 8 weeks of therapy to establish whether the patterns of change were predictive of clinical outcome. Among the 28 participants 43% had measurable pretreatment concentrations of p24 antigen. Initiation of zidovudine therapy was followed by a similar decline of p24 antigen levels in nonprogressors as well as progressors and, therefore, these groups could not be distinguished on the basis of this parameter. HIV-1 RNA was detected in the pretreatment samples of 82% of the individuals and could be detected in p24 antigen-positive as well as p24 antigen-negative individuals. Similar changes in HIV-1 RNA load during zidovudine therapy were observed in p24 antigen-positive and -negative individuals. Analysis of the HIV-1 RNA response according to clinical outcome demonstrated that HIV-1 RNA copy numbers had declined significantly after 4 weeks of therapy in both nonprogressors and progressors, but the decline in RNA load was much stronger in the nonprogressors. Our data show that the HIV-1 RNA load in serum can be used to monitor the response to antiviral therapy in p24 antigen-positive as well as -negative individuals. Posttreatment changes in p24 antigen levels are not indicative for clinical outcome, whereas RNA copy numbers are.(ABSTRACT TRUNCATED AT 250 WORDS)

Zidovudine and Interferon- Combination Therapy versus Zidovudine Monotherapy in Subjects with Symptomatic Human Immunodeficiency Virus Type 1 Infection

Forty-five subjects with symptomatic human immunodeficiency virus type 1 (HIV-1) infection, CD4+ lymphocyte counts of > or = 150 x 10(6)/L, and Karnofsky scores > or = 60 were enrolled in a multicenter, randomized, controlled trial that compared zidovudine monotherapy and combination therapy for 48 weeks with zidovudine and interferon-alpha (IFN-alpha). Zidovudine with IFN-alpha (n = 25) had a favorable effect on CD4+ cell counts compared with zidovudine alone (n = 20). At all time points analyzed, the mean change from baseline was higher, reaching significance at week 24 (+10% versus -21%; P = .029). At week 48 the difference was -12% versus -45% (P = .07). Anti-CD3 monoclonal antibody-induced T cell reactivity improved temporarily in both groups. Serum HIV p24 antigen levels decreased maximally during the first 12 weeks of treatment. At weeks 0 and 48, polymerase chain reaction analysis for mutations at codons 67 and 215 of the HIV-1 reverse transcriptase gene conferring zidovudine resistance was conducted in 10 subjects receiving zidovudine and in 8 subjects receiving combination therapy. At week 48, 1 of 8 and 4 of 6 samples from the groups receiving zidovudine only or combination therapy, respectively, contained wild type virus at codon 215. Grade 3 or 4 toxicity was uncommon. Drug-related malaise and anorexia were observed more frequently in patients receiving both zidovudine and IFN-alpha.

Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy.

Drug susceptibility and mutations in the reverse transcriptase (RT) gene were analyzed with 167 virus isolates from 38 patients treated with nevirapine, a potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) RT. Resistant isolates emerged quickly and uniformly in all patients administered nevirapine either as monotherapy or in combination with zidovudine (AZT). Resistance developed as early as 1 week, indicating rapid turnover of the virus population. The development of resistance was associated with the loss of antiviral drug activity as measured by CD4 lymphocyte counts and levels of HIV p24 antigen and RNA in serum. In addition to mutations at amino acid residues 103, 106, and 181 that had been identified by selection in cell culture, mutations at residues 108, 188, and 190 were also found in the patient isolates. Sequences from patient clones documented cocirculating mixtures of populations of different mutants. The most common mutation with monotherapy, tyrosine to cysteine at residue 181, was prevented from emerging by coadministration of AZT, which resulted in the selection of alternative mutations. The observations documented that, under selective drug pressure, the circulating virus population can change rapidly, and many alternative mutants can emerge, often in complex mixtures. The addition of a second RT inhibitor, AZT, significantly altered the pattern of mutations in the circulating population of HIV.

Morphine amplifies HIV-1 expression in chronically infected promonocytes cocultured with human brain cells.

Previous studies have shown that morphine promotes the replication of human immunodeficiency virus (HIV)-1 in peripheral blood mononuclear cell cocultures. In the present study, we tested the hypothesis that morphine would amplify HIV-1 expression in the chronically infected promonocytic clone U1 when cocultured with lipopolysaccharide-stimulated human fetal brain cells. Marked upregulation of HIV-1 expression was observed in these cocultures (quantified by measurement of HIV-1 p24 antigen levels in supernatants), and treatment of brain cells with morphine resulted in a bell-shaped dose-dependent enhancement of viral expression. The mechanism of morphine's amplifying effect appears to be opioid receptor-mediated and to involve enhanced production of tumor necrosis factor-alpha by microglial cells.

Physiological effects of HIV infection on human intestinal epithelial cells: an in vitro model for HIV enteropathy.

To determine the role of direct infection of intestinal cells with HIV-1 in the pathogenesis of HIV-related enteropathy. We infected HT-29-18-C1 intestinal cells with the IIIB strain of HIV and examined the physiologic effects of enterocyte function. Dipeptidase-IV, aminopeptidase-N, gamma glutamic transferase, and alkaline phosphatase were measured in HIV-infected and control cultures. The cellular second messengers intracellular calcium and cyclic adenosine monophosphate were also measured in infected and control cultures. A persistent infection was established for > 95 days with peak supernatant reverse transcriptase and HIV p24 antigen levels of 5.17 log10 c.p.m./ml and 45 ng/ml, respectively. Brush-border enzyme activity (nmol of product/min/mg protein) tended to be lower in infected cell cultures compared with controls early in infection (P < 0.02). Baseline second messenger concentrations were similar but infected cultures responded to stimulation with a calcium ionophore with an exaggerated increase in intracellular calcium (P = 0.03). These results suggest that absorptive and secretory function of enterocytes may be altered by direct HIV infection and that additional physiologic experiments with this in vitro model may lead to a better understanding of the clinical syndrome of HIV enteropathy.