HIV-1-specific CTLs effectively suppress replication of HIV-1 in HIV-1-infected macrophages

Both CD4+ T cells and macrophages are major reservoirs of HIV-1. Previous study showed that HIV-1-specific cytolytic T lymphocytes (CTLs) hardly recognize HIV-1-infected CD4+ T cells because of Nef-mediated HLA class I down-regulation, suggesting that HIV-1 escapes from HIV-1-specific CTLs and continues to replicate in HIV-1-infected donors. On the other hand, the CTL recognition of HIV-1-infected macrophages and the effect of Nef-mediated HLA class I down-regulation on this recognition still remain unclear. We show a strong HIV-1 antigen presentation by HIV-1-infected macrophages. HIV-1-specific CTLs had strong abilities to suppress HIV-1R5 virus replication in HIV-1-infected macrophages and to kill HIV-1R5-infected macrophages. Nef-mediated HLA class I down-regulation minimally influenced the recognition of HIV-1-infected macrophages by HIV-1-specific CTLs. In addition, HIV-1-infected macrophages had a stronger ability to stimulate the proliferation of HIV-1-specific CTLs than HIV-1-infected CD4+ T cells. Thus, the effect of Nef-mediated HLA class I down-regulation was less critical with respect to the recognition by HIV-1-specific CTLs of HIV-infected macrophages than that of HIV-1-infected CD4+ T cells. These findings support the idea that the strong HIV-1 antigen presentation by HIV-1-infected macrophages is one of the mechanisms mediating effective induction of HIV-1-specific CTLs in the acute and early chronic phases of HIV-1 infection.     

Dynamics of HIV-1 viral load rebound among patients with previous suppression of viral replication

OBJECTIVE: To model the dynamics of HIV-1 rebound in patients receiving suboptimal therapy after suppression of plasma viremia to < 200 copies/ml by triple combination therapy. DESIGN: Mathematical modeling of data from 23 patients switched to indinavir maintenance therapy after viral replication was suppressed with a combination of indinavir, zidovudine and lamivudine. Modeling of HIV-1 rebound among 24 patients on zidovudine/lamivudine maintenance was also performed for comparison. METHODS: Evaluation of slopes of rebound and of their heterogeneity; calculation of the basic reproductive number (Ro, the number of newly infected cells arising from each productively infected cell); regression analyses for predictors of the slope of rebound. RESULTS: Rebound of plasma HIV RNA followed a sigmoid curve with an initial exponential phase. There was significant heterogeneity in the slopes of rebound for individual patients (P < 0.001). In the indinavir maintenance rebounds, the average initial slope was estimated to be 0.587/day (doubling time 1.2 days). The slopes of rebound in patients on zidovudine/lamivudine maintenance tended to be less steep on average (P = 0.025). Among patients taking indinavir maintenance, the average Ro for the initial rebound of viremia was 4.3; in multivariate regressions, the slope of rebound was steeper during early rebound and in patients with higher viral load at the start of triple therapy or a higher CD4 cell count when indinavir monotherapy was initiated. The slope was less steep in patients with a greater increase in the number of CD4 cells during triple therapy. CONCLUSIONS: The rates of viral load increase among patients with viral rebound while receiving less than triple therapy are similar to those reported in patients interrupting therapy. Variability among patients may depend on viral fitness, target cell availability and extent of immune reconstitution.     

Inhibition of HIV-1 replication by RNA-based strategies

The major etiologic agent of the acquired immunodeficiency syndrome (AIDS) is the human immunodeficiency virus type 1 (HIV-1), which belongs to the family of human retroviruses. This pandemic infection affects millions of people worldwide. The most efficient current treatment regimen for HIV-infected individuals combines two or more drugs targeting different HIV-specific enzymes. However, the emergence of multiple drug-resistant HIV-1 strains and the side effects of drug-based therapies make alternative approaches for the treatment of HIV infection and AIDS necessary. RNA-based antiviral approaches are among the most promising for developing long-term anti-HIV therapies. Anti-HIV-1 RNA-based strategies include ribozymes, antisense RNAs, RNA aptamers, RNA decoys, external guide sequences (EGS) for site-specific cleavage of RNA molecules with human ribonuclease P (RNase P), modified small nuclear RNA (RNAu) and small interfering RNAs (siRNAs). This review describes the main features and functions of viral and cellular targets as well as the different classes of RNA molecules that have been explored in developing therapeutic strategies against HIV infection. Many RNA-based strategies are already being tested in human clinical trials or are currently being developed for future trials.     

HIV-1 replication in the plasma and cerebrospinal fluid

Summary The HIV-1 RNA in plasma and CSF samples from 40 HIV-1 infected patients was measured by a polymerase chain reaction (PCR) technique. The possible implication of cytokines in HIV-1 replication was investigated by measuring the concentrations of tumor necrosis factor (TNF-), macrophage colony stimulation factor (M-CSF) and interleukin-6 (IL-6) in these fluids. HIV-1 RNA was quantified in all plasma samples and in 87.5% of the CSF samples. CSF HIV-1 RNA titers did not correlate with the stage of disease or the CD4⁺ T cell counts, unlike the plasma HIV-1 RNA titers. These results were confirmed when patients with a blood brain barrier damage, as assessed by the CSF/plasma albumin ratio, were excluded from the analysis. TNF- levels were statistically correlated with the HIV-1 RNA in plasma and CSF. These data demonstrate that HIV-1 replication in the CSF at each clinical stage can be accurately measured with PCR and, although the titers of HIV-1 RNA copies in the CSF are correlated with those in the plasma, the magnitude of HIV-1 replication in CSF is not directly linked to the stage of disease, or to the CD4⁺ T cell count. The significance of early high levels of HIV-1 RNA in CSF is now being studied prospectively.

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HIV-1-Replikation im Plasma und Liquor cerebrospinalis

Zusammenfassung HIV-1-RNA wurde mittels Polymerasekettenreaktion (PCR) in Plasma- und Liquorproben von 40 HIV-1-infizierten Patienten quantifiziert. Um mögliche Einflüsse durch Zytokine auf die HIV-1-Replikation zu erfassen, wurden Tumornekrosefaktor- (TNF-), Makrophagen-Kolonie-stimulierender Faktor (M-CSF) und Interleukin-6 (IL-6) in diesen Flüssigkeiten ebenfalls bestimmt. Eine Quantifizierung von HIV-1 RNA war in allen Plasmaproben und in 87,5% der Liquorproben möglich. Im Gegensatz zu den HIV-1-RNA-Titern im Plasma fand sich zwischen HIV-1-RNA-Titern im Liquor und dem Krankheitsstadium oder den CD4⁺-T-Zell-Zahlen keine Korrelation. Diese Ergebnisse bestätigten sich auch bei Patienten, bei denen gemessen am Liquor-Ausschluß von Plasma-Albumin-Quotienten eine Blut-Liquor-Schrankenstörung bestand. Die HIV-1-Replikation kann in allen klinischen Stadien mittels PCR exakt quantifiziert werden. Obwohl die Liquor-Titer an HIV-1-RNA-Kopien mit den Plasmatitern korrelieren, besteht dennoch keine direkte Beziehung zum Krankheitsstadium oder zur CD4⁺-Zellzahl. In einer prospektiven Studie wird derzeit die Bedeutung frühzeitig auftretender HIV-1-RNA-Spiegel im Liquor untersucht.

     

HIV-1 replication and pathogenesis in the human thymus

How HIV replicates and causes destruction of the thymus, and how to restore thymic function, are among the most important questions of HIV-1 pathogenesis and therapy in adult as well as pediatric patients. The thymus appears to function, albeit at reduced levels, throughout the life of adults, to respond to T cell depletion induced by HIV and to be suppressed by HIV. In this review, we summarize recent findings concerning HIV replication and pathogenesis in the human thymus, focusing on mechanistic insights gleaned from studies in the SCID-hu Thy/Liv mouse and human fetal-thymus organ culture (HF-TOC) models. First, we discuss HIV viral determinants and host factors involved in the replication of HIV in the thymus. Second, we consider evidence that both viral factors and host factors contribute to HIV-induced thymocyte depletion. We thus propose that multiple mechanisms, including depletion and suppression of progenitor cells, paracrine and direct lytic depletion of thymocytes, and altered thymocyte selection are involved in HIV-induced pathology in the thymus. With the SCID-hu Thy/Liv mouse and HF-TOC models, it will be important in the coming years to further clarify the virological, cell biological, and immunological mechanisms of HIV replication and pathogenesis in human thymus, and to correlate their significance in HIV disease progression.     

HIV-1 pol replication capacity predicts disease progression

OBJECTIVE: To determine the influence of pol replication capacity on the natural history of HIV-1 infection. DESIGN: Pol replication capacity was measured using a recombinant virus single cycle assay on baseline plasma specimens from subjects enrolled in the Hemophilia Growth and Development Study. SETTING: Children and adolescents with hemophilia and HIV-1 infection were enrolled at multiple sites across the USA into a natural history study. PARTICIPANTS: The Hemophilia Growth and Development Study enrolled 207 HIV-1-infected hemophiliacs between 6 and 19 years of age in 1989 and 1990. Subjects were followed every 6 months through 1997 with pol replication capacity measurements available from 128 of the subjects. MAIN OUTCOME MEASURES: A univariate model defined the relationship between pol replication capacity and HIV-1 RNA and CD4 T-cell number. A random effects model assessed the ability of this measure to predict CD4 T-cell decline over time and a Cox proportional hazards model and Kaplan-Meier analyses defined how it predicts clinical progression. RESULTS: Pol replication capacity measures correlated with baseline HIV-1 RNA, R = 0.189 (P = 0.03) and CD4 T-cell number, -0.197 (P = 0.03). It also independently predicted CD4 T-cell decline over time and progression to AIDS. CONCLUSIONS: This study demonstrates that pol replication capacity independently influences the natural history of HIV-1 infection.     

Inhibitory effect of novel 1-deoxynojirimycin derivatives on HIV-1 replication

The effect of 11 derivatives of 1-deoxynojirimycin (DNM) on the replication of HIV-1 was studied. Compared with DNM, seven of them showed remarkable inhibition of HIV-1-induced syncytium formation at significantly low concentrations which were not cytotoxic. Two derivatives were found to markedly reduce the infectious virus yields from cell lines chronically infected with HIV. Analysis of HIV-1 envelope glycoproteins showed that the derivatives induced modification of the processing of not only gp120/160 but also the transmembrane glycoprotein gp41. The modification of the processing of the transmembrane glycoprotein gp41 might play an important role in the inhibition of virus replication at a step after the binding of gp120 to CD4. The enhanced anti-HIV activity of DNM derivatives reported here could increase the possibility of non-toxic therapeutic intervention in HIV infections.     

A conditionally replicating HIV-1 vector interferes with wild-type HIV-1 replication and spread.

Defective-interfering viruses are known to modulate virus pathogenicity. We describe conditionally replicating HIV-1 (crHIV) vectors that interfere with wild-type HIV-1 (wt-HIV) replication and spread. crHIV vectors are defective-interfering HIV genomes that do not encode viral proteins and replicate only in the presence of wt-HIV helper virus. In cells that contain both wt-HIV and crHIV genomes, the latter are shown to have a selective advantage for packaging into progeny virions because they contain ribozymes that cleave wt-HIV RNA but not crHIV RNA. A crHIV vector containing a triple anti-U5 ribozyme significantly interferes with wt-HIV replication and spread. crHIV vectors are also shown to undergo the full viral replicative cycle after complementation with wt-HIV helper-virus. The application of defective interfering crHIV vectors may result in competition with wt-HIVs and decrease pathogenic viral loads in vivo.     

Decay of cell-associated HIV-1 DNA correlates with residual replication in patients treated during acute HIV-1 infection

OBJECTIVES: To evaluate the decay rate of cell-associated HIV-1 RNA and DNA and to identify factors associated with residual viral load in patients treated at the time of primary HIV-1 infection. PATIENTS: A group of 15 patients adherent to highly active antiretroviral therapy (HAART) with sustained undetectable HIV-1 viremia for at least 24 months. METHODS: Viremia, cell-associated HIV-1 RNA and DNA in blood and lymph node mononuclear cells were measured using ultrasensitive assays. RESULTS: Viremia decreased rapidly in all patients; HIV RNA remained < 3 copies/ml in nine patients and fluctuated between 3 and 50 copies/ml in five patients and between 50 and 200 copies/ml in one patient. Decay rates of cell-associated RNA and DNA presented an inflexion point at 1 and 3 months, respectively: first-phase mean half-lives were 0.15 and 0.84 months, respectively, and second-phase mean half-lives were 13.7 and 6.6 months, respectively (95% confidence interval 4.4-13.8). The second phase decay rates were markedly slower, with a DNA decay rate that was highly associated with the mean levels of cell-associated RNA measured in blood from 6 to 33 months (P= 0.001) and in lymph nodes collected at 14 months (P= 0.02). CONCLUSIONS: The clearance of HIV-1 infected cells is correlated with the extent of viral replication as measured by cell-associated RNA levels in both blood and lymph nodes. Quantification of cell-associated RNA and DNA further defines treatment efficacy in 'aviremic' patients.     

HIV-1 replication and potential targets for intervention.

Intense research into fundamental processes of human immunodeficiency syndrome type 1 (HIV-1) replication has yielded knowledge that in many aspects equals or exceeds that of the oncogenic retroviruses. The availability of sensitive virus detection methods has allowed a more thorough characterization of the biology of virus persistence and latency in vivo and removed the dependence on in vitro models. As a clearer picture of the pattern of HIV-1 replication in vivo evolves, it becomes apparent that HIV-1 biology is distinct from that of the prototypic oncogenic retroviruses in several key aspects, particularly with regard to host cell range and determinants of viral permissiveness. In this respect it may be appropriate to examine the lentivirus, rather than the oncovirus model system to better understand the biology and pathogenesis of HIV-1 infection. This synopsis of recent and ongoing research developments in HIV-1 replication and pathogenesis emphasizes the determinants of host cell permissiveness, early events in virus replication, and underlying features in HIV-1 cytopathogenesis. In addition, basic viral replication processes which can be exploited for therapeutic intervention are discussed.     

The antimicrobial peptide LL-37 inhibits HIV-1 replication

The antimicrobial peptide LL-37 is the only cathelicidin that has been described in humans. LL-37 exerts chemotactic, immunomodulatory and angiogenic effects; activities that are mediated through binding to the formyl peptide receptor like (FPRL)-1 receptor. Agonistic ligation of FPRL-1 can also induce down-regulation of HIV-1 chemokine receptors and reduce susceptibility to HIV-1 infection in vitro. Therefore, we have evaluated the capacity of LL-37 to inhibit HIV-1 infection in vitro. Here we demonstrate that LL-37 inhibits HIV-1 replication in PBMC, including primary CD4(+) T cells. This inhibition was readily reproduced using various HIV-1 isolates without detectable changes in the target cell expression of HIV-1 chemokine receptors. Accordingly, the HIV-1 inhibitory effect was shown to be independent of FPRL-1 signalling. Given the epithelial expression of LL-37, it may contribute to the local protection against HIV-1 infection.     

Dynamics of HIV-1 recombination in its natural target cells

Genetic recombination is believed to assist HIV-1 diversification and escape from host immunity and antiviral therapies, yet this process remains largely unexamined within the natural target-cell populations. We developed a method for measuring HIV-1 recombination directly that employs reporter viruses bearing functional enhanced yellow fluorescent protein (YFP) and enhanced cyan fluorescent protein (CFP) genes in which recombination produces a modified GFP gene and GFP fluorescence in the infected cells. These reporter viruses allow simultaneous quantification of the dynamics of HIV-1 infection, coinfection, and recombination in cell culture and in animal models by flow-cytometric analysis. Multiround infection assays revealed that productive cellular coinfection was subject to little functional inhibition. As a result, generation of recombinants proceeded according to the square of the infection rate during HIV-1 replication in T lymphocytes and within human thymic grafts in severe combined immunodeficient (SCID)-hu (Thy/Liv) mice. These results suggest that increases in viral load may confer a compounding risk of virus escape by means of recombinational diversification. A single round of replication in T lymphocytes in culture generated an average of nine recombination events per virus, and infection of macrophages led to approximately 30 crossover events, making HIV-1 up to an order of magnitude more recombinogenic than recognized previously and demonstrating that the infected cell exerts a profound influence on the frequency of recombination.     

Inhibitors of HIV-1 replication that inhibit HIV integrase.

HIV-1 replication depends on the viral enzyme integrase that mediates integration of a DNA copy of the virus into the host cell genome. This enzyme represents a novel target to which antiviral agents might be directed. Three compounds, 3,5-dicaffeoylquinic acid, 1-methoxyoxalyl-3,5-dicaffeoylquinic acid, and L-chicoric acid, inhibit HIV-1 integrase in biochemical assays at concentrations ranging from 0.06-0.66 microgram/ml; furthermore, these compounds inhibit HIV-1 replication in tissue culture at 1-4 microgram/ml. The toxic concentrations of these compounds are fully 100-fold greater than their antiviral concentrations. These compounds represent a potentially important new class of antiviral agents that may contribute to our understanding of the molecular mechanisms of viral integration. Thus, the dicaffeoylquinic acids are promising leads to new anti-HIV therapeutics and offer a significant advance in the search for new HIV enzyme targets as they are both specific for HIV-1 integrase and active against HIV-1 in tissue culture.     

Inhibition of HIV-1 replication by an antiviral xanthate compound in vitro

The antiviral xanthate compound tricyclodecan-9-yl-xanthogenate (code name D609) is capable of inhibiting DNA and RNA viruses in vitro. It can also inhibit the shedding of infectious HIV into the tissue culture medium from chronically infected lymphoma cells (KE37-III) as shown by infectivity assays and Western blots of the supernatant. HIV-specific proteins, however, were accumulated intracellularly. The initiation of a de novo HIV replication after infection of permissive KE37-1 cells was completely inhibited at concentrations of D609 which still permitted mitotic divisions of the cells. Furthermore, the selective antiviral activity of the xanthate compound was evidenced by the absence of HIV replicative intermediate DNA. The expression of cellular genes, such as c-myc, remained unimpaired within these cells.