Dynamics of the pool of infected resting CD4 HLA-DR- T lymphocytes in patients who started a triple class five-drug antiretroviral regimen during primary HIV-1 infection

Starting standard antiretroviral therapy within 10 days after the onset of a primary HIV-1 infection cannot prevent the establishment of a reservoir of HIV-1-infected memory CD4 T cells. Here we studied the reservoir of HIV-1-infected memory CD4 T cells in four patients who started a triple class, five-drug regimen during primary HIV-1 infection. There was a strong correlation between the proportion of productively infected CD4 HLA-DR- T lymphocytes and plasma HIV-1 RNA levels (r=0.852; P<0.001) during the first 24 weeks of therapy. Within 45 weeks of treatment, in three of the four patients the proportion of productively infected CD4 HLA-DR- T lymphocytes was reduced below the level of quantification. In the fourth patient the cellular reservoir remained quantifiable. In two patients who stopped therapy 44 weeks after initiation an immediate rebound of the plasma HIV-1 RNA level and the proportion of productively infected CD4 HLA-DR- T lymphocytes occurred. In conclusion, initiation of a potent five-drug, triple class regimen during primary HIV-1 infection does not result in virus-specific immune control upon discontinuation of therapy after 44 weeks. Therefore, longer or even stronger suppression of viral replication might be necessary to achieve this goal in primary HIV-1 infection.     

A stable latent reservoir for HIV-1 in resting CD4(+) T lymphocytes in infected children

HIV-1 persists in a latent state in resting CD4⁺ T lymphocytes of infected adults despite prolonged highly active antiretroviral therapy (HAART). To determine whether a latent reservoir for HIV-1 exists in infected children, we performed a quantitative viral culture assay on highly purified resting CD4⁺ T cells from 21 children with perinatally acquired infection. Replication-competent HIV-1 was recovered from all 18 children from whom sufficient cells were obtained. The frequency of latently infected resting CD4⁺ T cells directly correlated with plasma virus levels, suggesting that in children with ongoing viral replication, most latently infected cells are in the labile preintegration state of latency. However, in each of 7 children who had suppression of viral replication to undetectable levels for 1–3 years on HAART, latent replication-competent HIV-1 persisted with little decay, owing to a stable reservoir of infected cells in the postintegration stage of latency. Drug-resistance mutations generated by previous nonsuppressive regimens persisted in this compartment despite more than 1 year of fully suppressive HAART, rendering untenable the idea of recycling drugs that were part of failed regimens. Thus the latent reservoir for HIV-1 in resting CD4⁺ T cells will be a major obstacle to HIV-1 eradication in children.     

Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-infected cells

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell–mediated clearance of HIV-1–infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity–mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4⁺ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8⁺ T cell clearance of CD4⁺ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected–patient resting CD4⁺ T cells. Moreover, DARTs mediated CD8⁺ T cell clearance of HIV from resting CD4⁺ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals.     

HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir

The persistence of latently infected, resting CD4+ T cells is considered to be a major obstacle in preventing the eradication of HIV-1 even in patients who have received effective antiviral therapy for an average duration of 5 years. Although previous studies have suggested that the latent HIV reservoir in the resting CD4+ T cell compartment is virologically quiescent in the absence of activating stimuli, evidence has been mounting to suggest that low levels of ongoing viral replication persist and in turn, prolong the overall half-life of HIV in patients receiving antiviral therapy. Here, we demonstrate the persistence of replication-competent virus in CD4+ T cells in a cohort of patients who had received uninterrupted antiviral therapy for up to 9.1 years that rendered them consistently aviremic throughout that time. Surprisingly, substantially higher levels of HIV proviral DNA were found in activated CD4+ T cells when compared with resting CD4+ T cells in the majority of patients we studied. Phylogenetic analyses revealed evidence for cross infection between the resting and activated CD4+ T cell compartments, suggesting that ongoing reactivation of latently infected, resting CD4+ T cells and spread of virus by activated CD4+ T cells may occur in these patients. Such events may allow continual replenishment of the CD4+ T cell reservoir and resetting of the half-life of the latently infected, resting CD4+ T cells despite prolonged periods of aviremia.     

Virological success of lopinavir/ritonavir salvage regimen is affected by an increasing number of lopinavir/ritonavir-related mutations

We evaluated the virological outcome of lopinavir/ritonavir (LPV/RTV) in 224 HIV-1-infected and protease inhibitor (PI)-experienced patients showing virological failure to a highly active antiretroviral therapy (HAART) regimen and followed up for at least 3 months. At baseline, the median level of plasma viraemia was 4.61 log10 copies/ml (range 3-6.48) and the median CD4 cell count was 219 cells/mm3 (range 1-836). During a median follow-up of 272 days (range 92-635), we observed an increase in the number of CD4 cells (P=0.02) and a dramatic decrease in plasma viraemia levels (P=0.0001), which became undetectable in 122 patients (54.5%). The closely related predictive factors were baseline plasma viraemia levels and the number of mutations known to reduce susceptibility to LPV/RTV. Thirty-one patients (13.8%) discontinued LPV/RTV during the follow-up, and one AIDS event and three deaths were recorded. Of the 134 patients (59.8%) who underwent a baseline genotype resistance test, 22 (16.4%) had > or = 6 mutations known to reduce LPV/RTV susceptibility; plasma viraemia became undetectable in 76 patients (56.7%), only five of whom harboured > or = 6 mutations at baseline (P=0.0001). The independent predictive factors related to virological success were plasma viraemia levels and the number of mutations reducing susceptibility to LPV/RTV at baseline; each additional log10 copies/ml of HIV RNA reduced the probability of virological success by 34.0% and each extra mutation by 14.5%.     

Lopinavir/ritonavir monotherapy as a simplification strategy in routine clinical practice

OBJECTIVES: The efficacy and safety of lopinavir/ritonavir monotherapy has been explored with promising results in well-controlled, randomized clinical trials. However, less information about its clinical usefulness in routine clinical practice is currently available. The objective of this study was to assess the effectiveness and safety of monotherapy with lopinavir/ritonavir as a treatment simplification strategy in HIV-infected patients with viral suppression outside a clinical trial setting. METHODS: Fifty-one subjects who were switched to lopinavir/ritonavir monotherapy and whose HIV-1 RNA was <50 copies/mL were included in this retrospective study. Data were obtained from a prospectively compiled database. The primary endpoint was the percentage of subjects who maintained viral suppression after 48 weeks of follow-up. Secondary endpoints included the incidence of adverse events and changes in CD4+ T cell count and in lipid profile. RESULTS: Two patients lost viral suppression, seven patients interrupted lopinavir/ritonavir monotherapy because of adverse events and four patients were lost before completing 48 weeks of follow-up. Thus, 38/40 (95.0%) patients maintained viral suppression when only subjects whose outcomes were available up to week 48 were considered and 38/51 (74.5%) patients maintained viral suppression when subjects who discontinued therapy or who were lost to follow-up were considered as treatment failures. The mean CD4+ T cell count significantly increased, from 541 (280) cells/mm3 at baseline to 609 (212) cells/mm3 at week 48 of follow-up (P=0.034). This increase was similar to that observed in the 48 weeks prior to lopinavir/ritonavir monotherapy (P=0.792). Although total cholesterol remained unchanged, there was a significant decrease in triglyceride levels during follow-up (P=0.029). CONCLUSIONS: Monotherapy with lopinavir/ritonavir is safe and effective as a treatment simplification approach in HIV-1-infected patients with sustained viral suppression in routine clinical practice, particularly in those patients already receiving a lopinavir/ritonavir-based antiretroviral regimen.     

Early therapy in HIV-1-infected children: effect on HIV-1 dynamics and HIV-1-specific immune response

BACKGROUND: Perinatal HIV-1 infection is acquired in the milieu of a developing immune system, leading to high levels of uncontrolled viral replication. Few data have been reported that address the viral dynamics and immunological response in infants who initiated aggressive antiretroviral therapy (ART) shortly after birth. METHODS: Six HIV-1-infected infants who started ART within 3 months of age were studied. The median followup was 61 months. Plasma HIV-1 RNA, cell-associated HIV-1 DNA, unspliced and multiply spliced HIV-1 mRNAs, HIV-1 antibodies, and CD4+ and CD8+ T-cell subsets were assessed in sequential peripheral blood samples. HIV-1 cellular immune response was measured by EliSpot assay. RESULTS: All children showed a decline in plasma viraemia to undetectable levels. HIV-1 DNA persisted in four children, but only two of these had detectable HIV-1 mRNA. All viral parameters remained persistently negative in two children. Only two children produced HIV-1 antibodies, while the others, after having lost maternal antibodies, remained seronegative. No HIV-1 cellular immune response was observed in any child. Therapy interruption was performed in two children: one HIV-1-seropositive and one HIV-1-seronegative with persistently undetectable levels of all viral parameters. Rebound of HIV-1 plasma viraemia in the seronegative child was more rapid and higher than that observed in the seropositive child. CONCLUSIONS: Early ART treatment in infants modifies the natural course of infection by controlling HIV-1 replication and reducing viral load to below the threshold levels required for onset of HIV-1 immune response, but does not prevent the establishment of a reservoir of latently infected cells that precludes virus eradication.     

Genetic characterization of rebounding HIV-1 after cessation of highly active antiretroviral therapy

Despite prolonged treatment with highly active antiretroviral therapy (HAART), infectious HIV-1 continues to replicate and to reside latently in resting memory CD4(+) T lymphocytes, creating a major obstacle to HIV-1 eradication. It is therefore not surprising to observe a prompt viral rebound after discontinuation of HAART. The nature of the rebounding virus, however, remains undefined. We now report on the genetic characterization of rebounding viruses in eight patients in whom plasma viremia was undetectable throughout about 3 years of HAART. Taking advantage of the extensive length polymorphism in HIV-1 env, we found that in five patients who did not show HIV-1 replication during treatment, the rebound virus was identical to those isolated from the latent reservoir. In three other patients, two of whom had been free of plasma viremia but had showed some residual viral replication, the rebound virus was genetically different from the latent reservoir virus, corresponding instead to minor viral variants detected during the course of treatment in lymphoid tissues. We conclude that in cases with apparent complete HIV-1 suppression by HAART, viral rebound after cessation of therapy could have originated from the activation of virus from the latent reservoir. In patients with incomplete suppression by chemotherapy, however, the viral rebound is likely triggered by ongoing, low-level replication of HIV-1, perhaps occurring in lymphoid tissues.     

Therapeutic vaccination of HIV-1-infected patients on HAART with a recombinant HIV-1 nef-expressing MVA: safety, immunogenicity and influence on viral load during treatment interruption

The safety and immunogenicity of an HIV-1 nef-expressing modified vaccinia virus Ankara (MVA) was investigated in 14 HIV-1-positive patients (CD4 >400/microl) on highly active antiretroviral therapy (HAART). Patients were vaccinated at weeks 0, 4 and 16, followed by interruption of HAART at week 18. MVA-nef was well-tolerated except for local reactions, with only mild systemic side effects reported in a few patients. Vaccination with MVA-nef was associated with recognition of new HIV-1 T-cell epitopes (cytotoxic T-lymphocyte epitopes in 9/14 patients, CD4 epitope/recombinant Nef protein in 2/14) and an increase in CD4+ and CD8+ T cells. All patients had been vaccinated against smallpox and a strong T-cell and antibody response to MVA was induced in all patients. After interruption of HAART, viral load rebounded in all patients, but after a median time of 36 (4-76) weeks in 9/14 patients, viraemia remained below the pre-HAART viral load and CD4 counts stayed above the pre-HAART levels. While six patients have remained off therapy for a median time of 64 (57-76) weeks, HAART was resumed in 8/14 patients after a median treatment interruption time of 15 (4-38) weeks. This study has demonstrated that MVA-nef is safe and immunogenic in HIV-1-infected subjects and has provided encouraging data on the potential of therapeutic vaccinations.     

The safety and tolerability of Z-100 in patients infected with HIV-1

Z-100 is an extract of the Mycobacterium tuberculosis strain Aoyama B, which contains various polysaccharides. Aoyama B has previously been shown to induce a T helper 1-type cytokine response in various murine oncological models and has also demonstrated inhibitory activity against HIV-1 in vitro. This multicentre study primarily determined the safety of Z-100 in early HIV-1-infected patients who were treatment naive; were treatment experienced, but had elected to discontinue highly active antiretroviral therapy (HAART) 8 weeks or longer before the study; or were stable on their first or second HAART regimen for at least 12 weeks before the study. Thirty-two individuals participated in this study and self-injected either placebo, 20 microg or 40 microg Z-100 twice a week for 8 weeks. Z-100 was well tolerated and the safety profiles of the Z-100 treatment groups were not meaningfully different compared with the placebo group. Plasma levels of HIV-1 RNA were not statistically significantly different in any treatment group at the end of the treatment period. There were no statistically significant differences among the treatment groups in the change from baseline to week 8 for any of the biological endpoints including plasma levels of HIV-1 RNA; CD4+ and CD8+ T-cell counts; levels of macrophage inflammatory protein 1; soluble tumour necrosis factor receptor 1; C-reactive protein; interleukin-6; and granulocyte colony stimulating factor. Consequently, this trial demonstrates the safety of Z-100 in HIV-1 infected patients without evidence of any activity at the doses administered.     

Immunological and virological activity of zalcitabine and zidovudine in combination in HIV-positive people with CD4 cell counts of between 200-500 cells/mm3

We evaluated the effect of combination therapy with zidovudine (AZT) plus zalcitabine (ddC) in human immunodeficiency virus type 1 (HIV-1)-infected patients who had not previously received antiretroviral treatment ('naive' patients). The immunological and virological parameters evaluated were CD4 cell count, syncytium-inducing (SI) viral phenotype and plasma HIV-1 RNA copies/ml (HIV viral load). A total of 75 patients entered the study, with CD4 cell counts between 200 and 500 cells/mm3. All received zidovudine (200 mg) plus zalcitabine (0.75 mg) three times daily for 24 weeks. Treatment was well tolerated. However, four patients presented with anaemia (haemoglobin < 10.0 g/dl) and one patient had both anaemia and neutropenia (0.8 x 10(9) neutrophils/l). Combination therapy with zidovudine plus zalcitabine resulted in a pronounced improvement of virological and immunological markers. Approximately 25% of patients achieved undetectable plasma HIV RNA levels (< 200 copies/ml) at week 24. At the end of the study (24 weeks) a significant reduction (> 0.5 log) of plasma HIV RNA was observed in approximately 70% of patients and in 50% an even greater decrease (> 1 log) was achieved. The most significant decrease in mean plasma HIV RNA levels was observed at week 4, whereas the highest increase in CD4 cell count was found at week 24. Approximately 80% of patients who showed baseline plasma HIV RNA levels below 20000 copies/ml had less than 5000 copies/ml at week 24. The plasma HIV RNA reduction observed at week 4 was significantly maintained at week 24. Therefore, we can rapidly select those who will not respond to therapy and adjust the treatment after a short interval. Our study supports the idea of early therapy because all patients who reached undetectable levels of plasma HIV RNA at week 24 had at baseline a median plasma HIV RNA load of 2560 copies/ml. In conclusion, zidovudine in combination with zalcitabine was well tolerated in the majority of patients and led to a significant reduction in plasma HIV RNA copies in most of the patients with initial viraemia lower than 20000 copies/ml.     

An open-label trial of stavudine, lamivudine and efavirenz in the treatment of HIV-positive, treatment-naive patients, and implications for clinical practice

A phase II, open-label study of stavudine, lamivudine and efavirenz resulted in significant reductions in plasma HIV-1 RNA over a 24 week period in human immunodeficiency virus (HIV)-infected individuals. The trial currently has 68 patients, and this presentation covers data on the first 42 patients enrolled for 24 weeks. The mean plasma HIV-1 RNA on entry was 75858 HIV RNA copies/ml, and the mean CD4 count was 380 cells/mm3. After 24 weeks, the CD4 count increased by 169 cells/mm3 above baseline. plasma HIV-1 RNA was markedly reduced: at 24 weeks, more than 97% of patients had <50 HIV RNA copies/ml based on observed data, and 89% of patients had <50 copies/ml based on strict intent-to-treat analysis (non-completer=failure). The favourable interactions of these agents resulted in no discontinuations owing to adverse effects. This regimen provides an important first-line treatment for antiretroviral-naive patients.     

CD4 T cell measurements in the management of antiretroviral therapy--A review with an emphasis on pediatric HIV-infected patients

The measurement of both the percentage (in pediatric patients aged less than 5 or 6 years) and the absolute number of circulating CD4+ T cells remains the single most important parameter for establishing prognosis and determining when to treat HIV-1 infected infants. The predictive power of CD4+ T cell measurements in HIV-1 infected individuals has resulted in robust guidelines from numerous agencies on the use of CD4+ T cell measurements ranging from pretreatment evaluations to the initial assessment and monitoring of therapeutic responses and treatment failures. The increase in availability of HIV-1 antiretroviral drugs in resource limited setting has led to the urgent need to develop systems and technologies for the accurate and cost-effective measurement of CD4+ T cells. The establishment of standardized guidelines for antiretroviral therapy (including CD4 testing) along with significant advancements in the development of structured access to health care, centralized CD4 testing programs, improved quality assurance programs, and inexpensive CD4 measurement technologies are making CD4 testing more universally available. Recent evidence suggests that a CD4/CD8 ratio of less than 1 may provide a reliable marker of presumptive HIV-1 infection in HIV-1 exposed infants. This review will summarize the current guidelines for the use of CD4 testing in HIV-1 infected infants and the potential for the CD4:CD8 ratio to be used as a surrogate of HIV-1 infection in resource limited settings.     

HIV-1 viral replication below 50 copies/ml in patients on antiretroviral therapy is not associated with CD8+ T-cell activation

OBJECTIVES: To determine if the expression of CD38 on CD8+ T-cells could be used as a marker of viral replication <50 copies/ml in peripheral blood. METHODS: In a cross-sectional study of patients attending a single HIV clinic in London, an ultra-sensitive HIV RNA viral load assay, with a limit of detection of 3 copies/ml, was used to determine HIV-1 replication in plasma in 70 patients who had sustained viral suppression <50 copies/ml by bDNA assays. Immune activation using the expression of CD38 on CD8+ T-cells was also assessed in patients on antiretroviral therapy (ART) with sustained viral suppression, individuals with persistent low-level viraemia <400 copies/ml and subjects failing ART (viral load >400 copies/mi). RESULTS: There was no significant difference in the percentage of CD8+CD38++ T-cells between patients with <50 copies or <3 copies/ml. Immune activation was significantly increased in patients with persistent low-level viraemia and in subjects failing ART. CD4+ T-cell counts in patients on long-term successful ART are inversely associated with CD8+ T-cell activation. CONCLUSIONS: T-cell activation in patients on long-term successful ART is not due to residual low-level viral replication in the blood compartment of HIV-1. CD8+ T-cell activation in this patient group appears to be associated with poor CD4+ T-cell recovery.     

Improvement in immune function due to treatment with indinavir despite severe immune deficiency

To study the virological, immunological and clinical effects of the protease inhibitor indinavir in human immunodeficiency virus (HIV)-infected patients with CD4 counts < 50 cells/mm3, indinavir was added to prior treatment with nucleoside analogues in a prospective open-label study in 23 HIV-infected patients with median CD4 count of 10 cells/mm3 and median serum HIV-1 RNA load of 27,508 copies/ml. Addition of indinavir induced a decrease in HIV-1 RNA levels to < 400 copies/ml in 15 patients that was maintained until week 36 of the study in 8 (35%) patients. The median increase in CD4 cell counts was 92 cells/mm3 (range 55-258 cells/mm3) and in CD8 counts was 245 cells/mm3 (range 51-1552 cells/mm3) at week 30. The treatment induced a significant CD8 T cell expansion, consisting in the first 6 weeks of predominantly memory CD45RO+ cells and followed by expansion of naive cells from week 12 on, and a significant decrease in the proportion of activated CD8/CD38 cells. In addition, significant increases in T cell proliferation following stimulation with phytohaemagglutinin and significant decreases in the rates of spontaneous apoptosis of CD4+ and CD8+ T cells were observed. In conclusion, the addition of indinavir induced restoration of both memory and naive CD8 T cells. Corresponding evidence of improving T cell function, as assessed by enhanced lymphoproliferative capacity and diminished propensity to undergo apoptosis, provides evidence for treatment-induced regeneration of immune function even in patients with severe immunodeficiency.     

Determination of HIV RNA concentration for evaluation of the efficacy of combined therapy of HIV infection

Plasma HIV RNA (viral load) and count of CD4+ T cells were evaluated in 23 patients with HIV infection treated with invirase (1800 mg/day), zidovudine (600 mg/day), and zalcitabine (2.25 mg/day) for 6 months in order to evaluate the efficiency of antiretroviral therapy. Viral load was measured by AMPLICOR HIV-1 Monitor test. The reproducibility of HIV RNA measurements was in line with reported data (CV 15-41%), allowing highly accurate (15%) evaluation of RNA in a standard control sample provided by National Institute for Biological Standards and Control, Great Britain. Plasma HIV RNA concentration decreased to an undetectable level (below 400 RNA copies/ml plasma) after 6 months of treatment in 52.2% patients. In 17.4% the therapy failed, and in 30.4% it resulted in a reduction of viral load to > 1 lg, although HIV RNA was still detected in the plasma after 6-month therapy. The count of CD4+ T cells increased by 9.5%. Changes in the viral load outstripped changes in CD4+ cells. Viral load was in high correlation with the count of CD4+ lymphocytes: -0.53, p = 0.01 before treatment and -0.61, p = 0.002 after 6-month treatment.     

Impact of early viral kinetics on T-cell reactivity during antiviral therapy in chronic hepatitis B

BACKGROUND: The patterns of hepatitis B viral dynamics during different antiviral therapies and the associated changes in HBV-specific T-cell reactivity are not well defined. METHODS: We investigated the impact of early viral load decline on virus-specific T-cell reactivity in 30 hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B randomized to monotherapy with adefovir dipivoxil (ADV) or in combination with emtricitabine (ADV/FTC). Viral kinetics were analysed by mathematical modelling. T-cell reactivity to HBV core and/or surface antigens and natural killer T cell frequency were tested longitudinally, baseline to week 48, using EliSPOT assays and/or flow cytometry. RESULTS: Mathematical modelling of early HBV kinetics identified two subsets of patients: 11 fast responders (undetectable viraemia by week 12; eight on ADV/FTC three on ADV) and 19 slow responders who remained viremic (six on ADV/FTC 13 on ADV). The rate of infected hepatocyte loss was higher in fast than in slow responders (P = 0.0007), and correlated inversely with pre-treatment levels of intrahepatic covalently closed circular HBV DNA. The frequency of HBV core-specific CD4+ T-cells increased significantly only in fast responders, peaking between week 16 and 24, while the HBV surface-specific CD4+ T-cells increased in both subsets. These changes in CD4+ T-cell reactivity were transient however, and no increase in HBV-specific CD8+ T-cells was observed. By week 48, HBeAg seroconversion occurred only in 3/30 (10%) patients. CONCLUSIONS: Early viraemia clearance facilitates recovery of virus-specific CD4+ T-cell reactivity, but appears insufficient to establish clinically relevant antiviral immunity.