Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease.

Highly active antiretroviral therapy (HAART) increases CD4(+) cell numbers, but its ability to correct the human immunodeficiency virus (HIV)-induced immune deficiency remains unknown. A three-phase T cell reconstitution was demonstrated after HAART, with: (i) an early rise of memory CD4(+) cells, (ii) a reduction in T cell activation correlated to the decreasing retroviral activity together with an improved CD4(+) T cell reactivity to recall antigens, and (iii) a late rise of "naïve" CD4(+) lymphocytes while CD8(+) T cells declined, however, without complete normalization of these parameters. Thus, decreasing the HIV load can reverse HIV-driven activation and CD4(+) T cell defects in advanced HIV-infected patients.

T cell changes after combined nucleoside analogue therapy in HIV primary infection.

OBJECTIVE: To characterize the immune changes after treatment of acute HIV-1 infection with triple nucleoside analogue therapy. DESIGN: Immunological and virological parameters were monitored from day 0 to weeks 36-44 in eight patients [median CD4 cells = 451 cells/microl (range: 149-624), viral load = 4.8 log10 copies/ml (range: 6.5-3.3)] who started at time of primary HIV infection (PHI) a therapy including zidovudine (ZDV), didanosine (ddl), and lamivudine (3TC). METHODS: Lymphoid subsets were evaluated on peripheral blood lymphocytes by four-colour flow cytometry using a panel of mAbs directed against differentiation and activation markers. RESULTS: We observed a median -2.1 (range: -1; -3.3) log10 copies/ml viral load decrease and a median +158 cells/microl (range: +7 to +316) CD4 cell count increase at week 4 reaching normal CD4 cell count values of 761 CD4 cells/microl (range: 389-1153) at weeks 36-44. Virus undetectability was obtained at week 24 for all subjects. A rapid CD4 T cell amplification involved both memory and naive CD4 T cells. This was associated with a very rapid and significant decrease in activation markers [human leukocyte antigen-DR (HLA-DR), CD38] on both CD4 and CD8 T cell subsets together with a CD8+CD28+ cell increase as early as week 4. CONCLUSIONS: These results show that early therapy with nucleoside analogues can correct the immunological abnormalities observed in CD4 and CD8 T cell subsets at the time of PHI. This early kinetics in T cell recovery appears to be faster than in established disease.

Reductions in viral load and increases in T lymphocyte numbers in treatment-naive patients with advanced HIV-1 infection treated with ritonavir, zidovudine and zalcitabine triple therapy.

In order to test the hypothesis that a combination of protease inhibitors with nucleoside analogues-agents known to inhibit different steps of the human immunodeficiency virus (HIV) life cycle--is likely to prove more effective in reducing viral loads than either of those modalities alone, we performed a 60 week, open-label trial in 32 HIV-positive patients with depressed CD4 T lymphocyte cell counts but no active AIDS-defining illnesses. For the first 2 weeks, patients received 600 mg twice daily of liquid ritonavir, a protease inhibitor; then zidovudine 200 mg three times daily and zalcitabine 0.75 mg three times daily were added to the treatment regimen. Mononuclear blood cell fractions were analysed for infected cell levels, using a co-culture system. HIV-1 RNA in plasma was measured both by reverse transcriptase-polymerase chain reaction (RT-PCR) and reverse transcriptase quantitative PCR (QcRT-PCR); lymphocyte counts were determined by standard laboratory methods. In the 2 weeks of ritonavir therapy, both the mean count of infectious blood cells and plasma HIV RNA levels decreased dramatically. Mean CD4 cell counts increased from 173 cells/mm3 at baseline to 286 cells/mm3; CD8 cell counts rose from 951 cells/mm3 to 1,141 cells/mm3. With the introduction of the nucleoside analogues, infectious cell counts and plasma virus dropped another log unit to a nadir at 8 weeks, while CD4 T lymphocyte counts continued to rise slowly. By week 28, 12 patients had withdrawn due to adverse events, none of which were life-threatening. At week 36, infectious material could not be detected in the cells of 10 of the 17 remaining patients; by week 60, four of the seven patients with residual viraemia at week 24 had undergone viral relapse. After the introduction of a more palatable capsule formulation of ritonavir at week 52, infectious cells and plasma virus were undetectable in 50-60% of patients. The combination of protease inhibitors and nucleoside analogues significantly reduces HIV load, and in some patients may suppress viral activity for sustained periods.

Restoration of the immune system with anti-retroviral therapy.

Clinical benefits of highly active anti-retroviral treatments (HAART) are increasingly evidenced by resolving opportunistic infections and malignancies, as well as declining hospitalization and mortality rates [1]. This suggests that potent and sustained suppression of viral replication, at least to some extent, is associated with reconstitution of the immune system even in adult patients treated at advanced stages of the disease. Increased susceptibility to opportunistic infections and tumors mainly results from the loss of memory CD4+ T cell reactivity against recall antigens which is an early event in HIV disease progression. Primary responses of naive CD4+ T cells against new pathogens are suppressed even earlier in the course of HIV disease, and the progressive depletion in naive CD4+ T cells reflects profound alterations in T cell regeneration capacities. Previous studies revealed that monotherapy with ritonavir, a protease inhibitor, resulted in a slight improvement in memory CD4+ T cell responses to recall Ags only when detectable prior to onset of therapy, suggesting that the loss of CD4+ T cell reactivity might be irreversible at advanced stages of the disease [2]. In contrast our group demonstrated more recently that restoration in CD4+ T cell reactivity to specific antigens was feasible when HAART was administered in progressors [3]. Here we address some of the questions raised by immune restoration with HAART when administered at advanced stages of the disease.

Frequency of cytokine-producing T cells in HIV-infected patients treated with stavudine, didanosine, and ritonavir.

To assess prospectively the influence of the control of viral replication on the frequency of cytokine-producing T cells, and to correlate these changes with immune activation, we conducted a 15-month follow-up study of IFN-gamma- and IL-2-producing CD4+ and CD8+ T cells at a single-cell level in 12 previously untreated patients receiving highly active antiretroviral therapy (HAART). At baseline we observed a strikingly high proportion of IFN-gamma-producing CD8+ T cells. The treatment-induced decrease in the proportion of IFN-gamma-producing CD8+ T cells ran parallel to the decrease in HLA-DR+ and CD38+CD8+ T cell subsets and was associated with the reduction in HIV RNA level. IL-2-producing cells were mainly CD4+. As a consequence of CD4+ T cell loss, the number of IL-2-producing CD4+ T cells was lower in patients than in control subjects (52 vs. 171 cells/microl), but the proportion of these cells was unchanged (22.4 vs. 19.3). During therapy the proportion of CD4+ IL-2-producing cells was initially stable and then fell markedly at month 5, followed by a gradual return to previous values. The reduction in viral load was associated with the fall in the proportion of CD4+ activated subsets. Intracellular cytokine assays are a new approach to the assessment of T cell function in HIV infection. Our results suggest that the functional capacity of CD4+ T cells is probably less severely altered than previously thought on the basis of conventional assays. CD8+ T cells exhibit an increased capacity to produce IFN-gamma that is associated with an increase in activation marker expression. These alterations decrease partially and in parallel under treatment.

The adult penile urethra is a novel entry site for HIV-1 that preferentially targets resident urethral macrophages.

The penile urethra is routinely targeted by sexually transmitted bacterial and viral pathogens, and also represents a probable site for HIV type-1 (HIV-1) entry. Yet, the mechanisms of urethral HIV-1 transmission are unknown. To describe the initial steps of penile HIV-1 entry, we obtained whole penile tissues from individuals undergoing elective gender reassignment and developed ex vivo polarized explants of different penile epithelia, as well as in vitro immunocompetent reconstructed urethra. In penile explants, 1 h exposure to cell-associated HIV-1 results in higher HIV-1 entry into the urethra, whereas the fossa navicularis and glans are relatively resistant to HIV-1. CCR5+/CD4+ urethral macrophages are the initial cells infected by HIV-1, which exit the epithelial compartment following inoculation with cell-associated HIV-1 that induces decreased CCL2/MCP-1 production. Urethral T cells are mostly CD8+ or naive CD4+, and not infected by HIV-1 on its early entry. In urethral reconstructions, efficient translocation of cell-associated HIV-1 depends on viral tropism (R5>X4) and can be decreased by gp41-specific IgAs. Cell-free HIV-1 is inefficient at urethral penetration. Our results identify the male urethra as a novel entry site for HIV-1 that targets resident urethral macrophages. These results might explain the incomplete prophylactic efficacy of male circumcision in reducing HIV-1 transmission.

[A new plasma exchange technique : coagulation studies (author's transl)]. / Nouvelle technique de plasmaphérèse : étude des anomalies de l'hémostase.

Coagulation studies were performed during and after 60 continuous plasma exchanges in 15 patients with neurologic disorders. One or several plasmapheresis were performed on each patient using a new filtration material "Plasmaflo" from Asahi Medical, Tokyo. During each exchange involving massive heparin infusion, two total plasma volumes were removed and replaced isovolumetrically by a material devoid of clotting factors (albumin and Plasmion). The variation of the platelet count was moderate (mean reduction of 19%). An important and rapid fall of all coagulation factors was observed, and during a single exchange procedure the extent of the fall was comparable for all coagulation factors. The mean reduction during plasmapheresis was 70%. However, a difference in the rapidity of normalisation was observed between the various proteins (F.V. : 24 h, A.T.III : 72 h). A particular property was noted concerning factor VIII. In all cases, a normal or high level of VIII : C as well as of VIII R : Ag was reached 3 hours after exchange. In some cases an increase could be observed even during plasmapheresis. No hemorrhagic diathesis was noted despite massive heparin administration to the patients and a striking decrease of blood procoagulant factors. On the other hand, one patient developed femoral vein thrombosis following the 8th exchange.

Loss of viral fitness associated with multiple Gag and Gag-Pol processing defects in human immunodeficiency virus type 1 variants selected for resistance to protease inhibitors in vivo.

We examined the viral replicative capacity and protease-mediated processing of Gag and Gag-Pol precursors of human immunodeficiency virus (HIV) variants selected for resistance to protease inhibitors. We compared recombinant viruses carrying plasma HIV RNA protease sequences obtained from five patients before protease inhibitor therapy and after virus escape from the treatment. Paired pretherapy-postresistance reconstructed viruses were evaluated for HIV infectivity in a quantitative single-cycle titration assay and in a lymphoid cell propagation assay. We found that all reconstructed resistant viruses had a reproducible decrease in their replicative capacity relative to their parental pretherapy counterparts. The extent of this loss of infectivity was pronounced for some viruses and more limited for others, irrespective of the inhibitor used and of the level of resistance. In resistant viruses, the efficiency of Gag and Gag-Pol precursor cleavage by the protease was impaired to different extents, as shown by the accumulation of several cleavage intermediates in purified particle preparations. We conclude that protease inhibitor-resistant HIV variants selected during therapy have an impaired replicative capacity related to multiple defects in the processing of Gag and Gag-Pol polyprotein precursors by the protease.

Infectious amplification of wild-type human immunodeficiency virus from patients' lymphocytes and modulation by reverse transcriptase inhibitors in vitro.

The relative in vitro potency of nine human immunodeficiency virus (HIV) type 1 reverse transcriptase inhibitors was evaluated in a coculture assay which measures the frequencies of infectious primary cells from HIV-positive patients by the limiting dilution technique and measures their apparent reduction under increasing concentrations of drugs. An advantage of this assay is that it utilizes a variety of wild-type viruses not selected by in vitro propagation. Potency ranking placed the (-)-L-enantiomer of 2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC], an oxathiolane pyrimidine nucleoside analog (90% effective concentration = 55 nM), before 2',3'-dideoxycytidine (DDC) (74 nM), (-)-2',3'-dideoxy-3'-thiacytidine (3TC) (300 nM), 3'-azido-3'-deoxythymidine (AZT) (530 nM), TIBO R82913 (670 nM), and 2',3'-dideoxyinosine (DDI) (6,400 nM). HIV from AZT-naive patients' lymphocytes was more sensitive to the inhibitory effect of (-)-FTC, 3TC, or DDC than was highly AZT-resistant HIV obtained from AZT-treated patients' cells, indicating partial cross-resistance between thymidine and cytidine analogs. Combined inhibitory concentrations of AZT with (-)-FTC, 3TC, DDC, and DDI produced synergistic interactions as determined by the multiple-drug effect analysis. Synergistic interactions were demonstrable with AZT plus (-)-FTC or with AZT plus DDC with cells bearing AZT-resistant HIV. The inhibitory concentrations of AZT established by this cell-to-cell virus transmission assay are closer than those determined by the conventional assay system to the extracellular AZT concentrations required in patients' plasma to achieve comparable levels of HIV inhibition in vivo.

Productive human immunodeficiency virus infection levels correlate with AIDS-related manifestations in the patient.

Mononuclear cells were obtained from 71 human immunodeficiency virus type 1 (HIV-1) seropositive subjects presenting and first visit either as asymptomatic or with minor symptoms and with CD4 lymphocytes greater than 550 per mm3 (group A, 35 patients) or as patients with AIDS, AIDS-related illnesses, or CD4 lymphocytes less than 400 per mm3 (group B, 36 patients). After 1-5 years of follow-up, 13 patients of group A had essentially retained their initial status (asymptomatics); the 22 others had suffered clinical or immunological deterioration (progressors). Frozen cells were thawed and submitted to lethal gamma-irradiation in vitro (4500 rads; 1 rad = 0.01 Gy) before they were cultured with normal phytohemagglutinin-stimulated lymphocytes to determine radiation-resistant HIV expression ex vivo (R-HEV). HIV antigenemia correlated with R-HEV values in 142 samples (r = 0.92, P less than 0.001) but was a less sensitive predictor of disease than R-HEV. R-HEV was detected in all specimens from patients with major AIDS-related illnesses or HIV-associated CD4 lymphopenia. In 77% of the progressors from group A, R-HEV detection preceded the onset of AIDS-associated disease or CD4 lymphopenia by 1 year (average). Conversely, R-HEV was low or was not detected in 36 sequential specimens from the 13 patients who remained asymptomatic over the following 2-5 years. Thus, persistently low HIV expression in vivo predicted a nondiseased state, whereas higher HIV expression levels seemed necessary for disease to occur. These data indicate that R-HEV is related to productive HIV infection in vivo, the latter acting as a determinant of AIDS-related illnesses. In view of this, measurement of HIV expression levels in the patient should be useful in antiviral efficacy trials.

Dynamics and modulation of human immunodeficiency virus type 1 transcripts in vitro and in vivo.

The dynamics of human immunodeficiency virus type 1 (HIV-1) transcription was analyzed in vitro and in vivo by using a specific molecular approach which allows accurate quantitation of the different classes of viral mRNAs. Unspliced (US) and multiply spliced (MS) HIV-1 transcripts were assayed by competitive reverse transcription (cRT)-PCR, using a single competitor RNA bearing in tandem internally deleted sequences of both template species. Acute HIV-1 infection of primary peripheral blood mononuclear cells (PBMCs), monocytes/macrophages cells, and the A3.01 T-lymphocyte-derived cell line was studied; both classes of HIV-1 mRNAs increased exponentially (r2 > 0.98) at days 1 to 3 and 1 to 4 postinfection in HIV(IIIB)-infected A3.01 cells and PBMCs, respectively, whereas monocytes/macrophages infected with monocytotropic HIV(BaL) exhibited a linear (r2 = 0.81 to 0.94) accumulation of US and MS transcripts. Following induction of chronically infected ACH-2 cells, MS transcripts increased 2 h postinduction and peaked at 5 h (doubling time, 58 min), while at 24 h, US mRNAs increased 3,053-fold compared with basal time (doubling time, 137 min). To address the biopathological significance of HIV-1 expression pattern during infection progression, pilot cross-sectional and longitudinal analyses were carried out with samples from untreated and treated HIV-1-infected patients. In almost all untreated (recently infected, long-term nonprogressor, and progressor) patients, MS transcript levels followed the general trend of systemic HIV-1 activity. In patients under treatment with powerful antiretroviral compounds, viral MS transcripts rapidly fell to undetectable levels, indicating that in vivo, levels of MS mRNAs in PBMCs are closely associated with the number of newly infected cells and suggesting a new role for the quantitative analysis of HIV-1 transcription in infected patients.