Retroviral interference on STAT activation in individuals coinfected with human T cell leukemia virus type 2 and HIV-1

Human T cell leukemia virus (HTLV) type-2 is a human retrovirus whose infection has not been tightly linked to human diseases. However, the fairly high prevalence of this infection among HIV-1-positive individuals indicates the importance of better understanding the potential interference of HTLV-2 infection on HIV-1 infection and AIDS. We previously demonstrated that one signature of PBMC freshly derived from HIV-1-infected individuals is the constitutive activation of a C-terminal truncated STAT5 (STAT5Delta). Therefore, we analyzed the potential activation of STATs in HTLV-2 monoinfected and HTLV-2/HIV-1 dually infected individuals. We observed that PBMC of HTLV-2-infected individuals do not show STAT activation unless they are cultivated ex vivo, in the absence of any mitogenic stimuli, for at least 8 h. The emergence of STAT activation, namely of STAT1, in culture was mostly related to the secretion of IFN-gamma. Of note, this phenomenon is not only a characteristic feature of HTLV-2-infected individuals but also occurred with PBMC of HIV-1(+) individuals. Surprisingly, HTLV-2/HIV-1 coinfection resulted in low/absent STAT activation in vivo that paralleled a diminished secretion of IFN-gamma after ex vivo cultivation. Our findings indicate that both HTLV-2 and HIV-1 infection prime T lymphocytes for STAT1 activation, but they also highlight an interference exerted by HTLV-2 on HIV-1-induced STAT1 activation. Although the nature of such a phenomenon is unclear at the present, these findings support the hypothesis that HTLV-2 may interfere with HIV-1 infection at multiple levels.     

Diagnosis of infection with human T-lymphotropic virus type II (HTLV-II) in Norwegian HIV-infected individuals

Sera from 298 HIV-infected individuals from Southern Norway were examined for antibodies against HTLV. 30 sera (10.1%) were HTLV-II positive and 1(0.3%) HTLV-I positive. 25 of the HTLV-II infected subjects were intravenous drug abusers (IVDAs), giving a prevalence of HTLV-II infection of 24.5% in this group. Examination of blood samples by polymerase chain reaction followed by restriction enzyme analysis or sequencing confirmed the serological diagnosis. To evaluate current screening and verification HTLV tests, 44 sera were examined using a gelatin particle agglutination test, 5 different enzyme-linked immunoassays (ELISA) and 4 Western blots (WB). While earlier ELISAs and WBs were inadequate, a recent ELISA and WB including recombinant envelope glycoproteins from both viruses permitted serological diagnosis and distinction between HTLV-I and HTLV-II. Thus, HTLV-II now spreads among IVDAs in a North-European country. Health authorities in other countries should estimate the magnitude of the problem to decide upon measures to avoid transmission through blood transfusion.     

Human immunodeficiency virus type 1 (HIV-1) antigen secretion by latently infected resting CD4+ T lymphocytes from HIV-1-infected individuals

In resting CD4(+) T lymphocytes harboring human immunodeficiency virus type 1 (HIV-1), replication-competent virus persists in patients responding to highly active antiretroviral therapy (HAART). This small latent reservoir represents between 10(3) and 10(7) cells per patient. However, the efficiency of HIV-1 DNA-positive resting CD4(+) T cells in converting to HIV-1-antigen-secreting cells (HIV-1-Ag-SCs) after in vitro CD4(+)-T-cell polyclonal stimulation has not been satisfactorily evaluated. By using an HIV-1-antigen enzyme-linked immunospot assay, 8 HIV-1-Ag-SCs per 10(6) CD4(+) resting T cells were quantified in 25 patients with a plasma viral load of <20 copies/ml, whereas 379 were enumerated in 10 viremic patients. In parallel, 369 and 1,238 copies of HIV-1 DNA per 10(6) CD4(+) T cells were enumerated in the two groups of patients, respectively. Only a minority of latently HIV-1 DNA-infected CD4(+) T cells could be stimulated in vitro to become HIV-1-Ag-SCs, particularly in aviremic patients. The difference between the number of HIV-1 immunospots in viremic versus aviremic patients could be explained by HIV-1 unintegrated viral DNA that gave additional HIV-1-Ag-SCs after in vitro CD4(+)-T-cell polyclonal stimulation. The ELISPOT approach to targeting the HIV-1-Ag-SCs could be a useful method for identifying latently HIV-1-infected CD4(+) T cells carrying replication-competent HIV-1 in patients responding to HAART.     

Superinfection of human immunodeficiency virus type 1 (HIV-1) to cell clone persistently infected with defective virus induces production of highly cytopathogenic HIV-1

Superinfection with human immunodeficiency virus type 1 (HIV-1) in human subjects, defined as reinfection with a heterologous strain of HIV-1, has become a topic of great interest. To illustrate the significance of this occurrence, we performed HIV-1 superinfection of L-2 cells, which were isolated from MT-4 cells persistently infected with subtype B HIV-1 as a cell clone continuously producing defective HIV-1 particles. L-2 cells carrying provirus with a one-base insertion in the pol protease were superinfected with HIV-1 derived from primary isolates of subtype B or CRF01_AE. The kinetics of the superinfection in L-2 were very slow compared with those of primary infections in MT-4. Interestingly, L-2 shifted after superinfection to become a producer of highly cytopathogenic HIV-1. Molecular characterization revealed that superinfection occurred in only about 10% of the CRF01_AE-superinfected L-2, which carried provirus of both subtypes and produced viral particles containing genomic RNA of both subtypes. Surprisingly, such cytopathogenic HIV-1 showed predominantly the original subtype B phenotype. Thus, the mechanism of the production of cytopathic HIV-1 seemed to be mediated by trans complementation with pol products of superinfected CRF01_AE. These findings suggest the significance of long-lived infected cells as recipients for superinfection that could result in the generation of new HIV-1 variants with high virulence in patients who are off therapy or do not adhere to treatment, and may indicate the need for precautions against such superinfection.     

Expansion and diversification of virus-specific T cells following immunization of human immunodeficiency virus type 1 (HIV-1)-infected individuals with a recombinant modified vaccinia virus Ankara/HIV-1 Gag vaccine

Affordable therapeutic strategies that induce sustained control of human immunodeficiency virus type 1 (HIV-1) replication and are tailored to the developing world are urgently needed. Since CD8(+) and CD4(+) T cells are crucial to HIV-1 control, stimulation of potent cellular responses by therapeutic vaccination might be exploited to reduce antiretroviral drug exposure. However, therapeutic vaccines tested to date have shown modest immunogenicity. In this study, we performed a comprehensive analysis of the changes in virus-specific CD8(+) and CD4(+) T-cell responses occurring after vaccination of 16 HIV-1-infected individuals with a recombinant modified vaccinia virus Ankara-vectored vaccine expressing the consensus HIV-1 clade A Gag p24/p17 sequences and multiple CD8(+) T-cell epitopes during highly active antiretroviral therapy. We observed significant amplification and broadening of CD8(+) and CD4(+) gamma interferon responses to vaccine-derived epitopes in the vaccinees, without rebound viremia, but not in two unvaccinated controls followed simultaneously. Vaccine-driven CD8(+) T-cell expansions were also detected by tetramer reactivity, predominantly in the CD45RA(-) CCR7(+) or CD45RA(-) CCR7(-) compartments, and persisted for at least 1 year. Expansion was associated with a marked but transient up-regulation of CD38 and perforin within days of vaccination. Gag-specific CD8(+) and CD4(+) T-cell proliferation also increased postvaccination. These data suggest that immunization with MVA.HIVA is a feasible strategy to enhance potentially protective T-cell responses in individuals with chronic HIV-1 infection.     

Complete nucleotide sequence of an Amerindian human T-cell lymphotropic virus type II (HTLV-II) isolate: identification of a variant HTLV-II subtype b from a Guaymi Indian.

The complete nucleotide sequence of a human T-cell lymphotropic virus type II (HTLV-II) isolate from a Panamanian Guaymi Indian was determined and analyzed. When this new viral isolate (HTLV-IIG12) was compared with prototypic HTLV-IIMoT, the overall nucleotide sequence similarity was 95.4%, while the predicted amino acid sequence similarity was 97.5%. Although the overall percentage of nucleotide and amino acid identity with prototypic HTLV-IIMoT (subtype a) was high, HTLV-IIG12 displayed several distinctive features that defined it as an HTLV-II subtype b. However, there were several characteristics unique to this isolate, which included a cluster of nucleotide substitutions in the pre-gag region and changes in restriction enzyme sites within the pre-gag region and the gag, pol, env, and pX genes. In addition, two nucleotide changes in the C terminus of the Tax protein coding sequence inserted an Arg residue for a stop codon and appeared to result in a larger tax gene product in HTLV-IIG12. Although the HTLV-IIG12 isolate appears to be a variant of the prototypic HTLV-IIb, this information represents the first complete nucleotide sequence of any HTLV-II subtype b. These data will allow further studies on the evolutionary relationships between the HTLV-II subtypes and between HTLV-I and HTLV-II.     

Patterns of cytokine production in human immunodeficiency virus type 1 (HIV-1)-specific human CD8+ T cells after stimulation with HIV-1-infected CD4+ T cells

Although human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells can produce various cytokines that suppress HIV-1 replication or modulate anti-HIV-1 immunity, the extent to which HIV-1-specific CD8+ T cells produce cytokines when they recognize HIV-1-infected CD4+ T cells in vivo still remains unclear. We first analyzed the abilities of 10 cytotoxic T-lymphocyte (CTL) clones specific for three HIV-1 epitopes to produce gamma interferon, macrophage inflammatory protein 1beta, and tumor necrosis factor alpha after stimulation with epitope peptide-pulsed cells. These CTL clones produced these cytokines in various combinations within the same specificity and among the different specificities, suggesting a functional heterogeneity of HIV-1-specific effector CD8+ T cells in cytokine production. In contrast, the HIV-1-specific CTL clones for the most part produced a single cytokine, without heterogeneity of cytokine production among the clones, after stimulation with HIV-1-infected CD4+ T cells. The loss of heterogeneity in cytokine production may be explained by low surface expression of HLA class I-epitope peptide complexes. Freshly isolated HIV-1-specific CD8+ T cells with an effector/memory or memory phenotype produced much more of the cytokines than the same epitope-specific CTL clones when stimulated with HIV-1-infected CD4+ T cells. Cytokine production from HIV-1-specific memory/effector and memory CD8+ T cells might be a critical event in the eradication of HIV-1 in HIV-1-infected individuals.     

Human immunodeficiency virus type 1 (HIV-1)-induced GRO-alpha production stimulates HIV-1 replication in macrophages and T lymphocytes

We examined the early effects of infection by CCR5-using (R5 human immunodeficiency virus [HIV]) and CXCR4-using (X4 HIV) strains of HIV type 1 (HIV-1) on chemokine production by primary human monocyte-derived macrophages (MDM). While R5 HIV, but not X4 HIV, replicated in MDM, we found that the production of the C-X-C chemokine growth-regulated oncogene alpha (GRO-alpha) was markedly stimulated by X4 HIV and, to a much lesser extent, by R5 HIV. HIV-1 gp120 engagement of CXCR4 initiated the stimulation of GRO-alpha production, an effect blocked by antibodies to CXCR4. GRO-alpha then fed back and stimulated HIV-1 replication in both MDM and lymphocytes, and antibodies that neutralize GRO-alpha or CXCR2 (the receptor for GRO-alpha) markedly reduced viral replication in MDM and peripheral blood mononuclear cells. Therefore, activation of MDM by HIV-1 gp120 engagement of CXCR4 initiates an autocrine-paracrine loop that may be important in disease progression after the emergence of X4 HIV.     

Serum leptin and adiponectin concentrations in patients infected with human immunodeficiency virus type 1 (HIV-1) on antiretroviral therapy

The lipodystrophy syndrome with dyslipidaemia and insulin resistance is side-effect of combined antiretroviral therapy (CART). Aim of the study: to describe the influence of CART on leptin and adiponectin concentration in connection with lipids levels in HIV-infected patients on antiretroviral therapy. BMI, serum leptin, adiponectin, triglycerides, total cholesterol, HDL- and LDL-cholesterol concentrations were measured in 56 HIV(+) patients before and on CART; average of treatment duration 38.4 +/-13.2 months. Significant increase of BMI (p=0.0268) of (22.6 +/- 3.3 before and 23.5 +/- 3.4 kg/m2 on therapy, respectively) and all analyzed lipids were found. Mean adiponectin concentration in treated patients was significantly (7.256 +/- 3.551 microg/ml) lower than mean value before treatment (8.395 +/- 3.568 microg/ml; p=0.0011). Mean values of leptin concentrations did not differ significantly (before therapy 3.721 +/- 0.347 log10; on therapy 3.1737 +/- 0.353 log10). Significant positive correlation between BMI and leptin concentrations was found before, as well as during CART (r=0.5333; p<0.0001), but negative correlation between adiponectin and leptin concentrations (r=-0.2677; p=0.042). Leptin and adiponectin concentrations did not revealed significant correlation with lipids levels before therapy. The decrease of adiponectin concentration on CART correlated negatively with total (r=-0.2912; p=0.0310) and LDL-cholesterol (r=-0.310; p=0.0225). CART lasting longer than 2 years resulted in the decrease of adiponectin concentration, with lack of influence on leptin concentration in analyzed group. The increase of total cholesterol and LDL-cholesterol in correlation with the decrease of adiponectin concentration confirms that CART induces metabolic disturbances related to higher risk of atherosclerosis and its sequel.     

Robust growth of human immunodeficiency virus type 1 (HIV-1)

The persistence of human immunodeficiency virus type-1 (HIV-1) has long been attributed to its high mutation rate and the capacity of its resulting heterogeneous virus populations to evade host immune responses and antiviral drugs. However, this view is incomplete because it does not explain how the virus persists in light of the adverse effects mutations in the viral genome and variations in host functions can potentially have on viral functions and growth. Here we show that the resilience of HIV-1 can be credited, at least in part, to a robust response to perturbations that emerges as an intrinsic property of its intracellular development. Specifically, robustness in HIV-1 arises through the coupling of two feedback loops: a Rev-mediated negative feedback and a Tat-mediated positive feedback. By employing a mechanistic kinetic model for its growth we found that HIV-1 buffers the effects of many potentially detrimental variations in essential viral and cellular functions, including the binding of Rev to mRNA; the level of rev mRNA in the pool of fully spliced mRNA; the splicing of mRNA; the Rev-mediated nuclear export of incompletely-spliced mRNAs; and the nuclear import of Tat and Rev. The virus did not, however, perform robustly to perturbations in all functions. Notably, HIV-1 tended to amplify rather than buffer adverse effects of variations in the interaction of Tat with viral mRNA. This result shows how targeting therapeutics against molecular components of the viral positive-feedback loop open new possibilities and potential in the effective treatment of HIV-1.     

Anti-CD95 (APO-1/Fas) autoantibodies and T cell depletion in human immunodeficiency virus type 1 (HIV-1)-infected children

Advanced stages of HIV-1-infection are characterized by progressive CD4+ T cell depletion. Peripheral T cells from HIV-1+ donors show accelerated apoptosis in vitro. The CD95 (APO-1/Fas) receptor/ligand system is involved in this process. To further study deregulation of the CD95 system in peripheral T cells during HIV-1-infection, we measured CD95-expression on CD4+ and CD8+ T cells together with serum levels of soluble CD95 (sCD95) and anti-CD95 autoantibodies in HIV-1+ children and healthy controls. Anti-CD95 levels in HIV-1+ children were significantly elevated when compared to uninfected controls, whereas serum levels of sCD95 were not different. In HIV-1+ children, CD95-expression on CD4+ and CD8+ T cells increased with age. A strong correlation between depletion of CD4+ cells in vivo and increase in CD95-expression on CD4+ T cells was observed. In contrast, such a correlation was not found for CD8+ T cells. A negative correlation between anti-CD95 autoantibody levels and CD4+ T cell counts, that was predicted by multiple linear regression analysis of pooled data, was found in individual patients observed longitudinally by repeated measurements. Since anti-CD95 autoantibodies isolated from HIV-infected adults have previously been shown to induce apoptosis of sensitive target cells in vitro, we speculate that the interaction of these antibodies with CD95-positive and CD95-sensitive T cells in vivo might be involved in progressive T cell loss during HIV-1-infection.     

Diversity of the human immunodeficiency virus type 1 (HIV-1) env sequence after vertical transmission in mother-child pairs infected with HIV-1 subtype A

Although several virologic and immunologic factors associated with an increased risk of perinatal human immunodeficiency virus type 1 (HIV-1) transmission have been described, the mechanism of mother-to-child transmission is still unclear. More specifically, the question of whether selective pressures influence the transmission remains unanswered. The aim of this study was to assess the genetic diversity of the transmitted virus after in utero transmission and after peripartum transmission and to compare the viral heterogeneity in the child with the viral heterogeneity in the mother. To allow a very accurate characterization of the viral heterogeneity in a single sample, limiting-dilution sequencing of a 1016-bp fragment of the env gene was performed. Thirteen children were tested, including 6 with in utero infections and 7 with peripartum infections. Samples were taken the day after birth and at the ages of 6 and 14 weeks. A homogeneous virus population was seen in six (46.2%) infants, of whom two were infected in utero and four were infected peripartum. A more heterogeneous virus population was detected in seven infants (53.8%), four infected in utero and three infected peripartum. The phylogenetic trees of the mother-child pairs presented a whole range of different tree topologies and showed infection of the child by one or more maternal variants. In conclusion, after HIV-1 transmission from mother to child a heterogeneous virus population was detected in approximately one-half of the children examined. Heterogeneous virus populations were found after peripartum infection as well as after in utero infection. Phylogenetic tree topologies argue against selection processes as the major mechanism driving mother-to-child transmission but support the hypothesis that virus variability is mainly driven by the inoculum level and/or exposure time.     

Random T-cell receptor recruitment in human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells from genetically identical twins infected with the same HIV-1 strain

Human immunodeficiency virus type 1 (HIV-1) cytotoxic T-lymphocyte escape mutations represent both a major reason for loss of HIV immune control and a considerable challenge for HIV-1 vaccine design. Previous data suggest that initial HIV-1-specific CD8⁺ T-cell responses are determined largely by viral and host genetics, but the mechanisms influencing the subsequent viral evolution are unclear. Here, we show a random recruitment of T-cell receptor (TCR) alpha and beta clonotypes of the initial HIV-1-specific CD8⁺ T cells during primary infection in two genetically identical twins infected simultaneously with the same virus, suggesting that stochastic TCR recruitment of HIV-1-specific CD8⁺ T cells contributes to the diverse and unpredictable HIV-1 sequence evolution.