Prevalence of etravirine (ETR)-RAMs at NNRTI failure and predictors of resistance to ETR in a large Italian resistance database (ARCA).

The prevalence of drug resistance associated with the failure of non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens and the predictors of resistance to Etravirine (ETR) were assessed in 2854 subjects: 39 < 18 (paediatric) and 2815 ≥ 18 (adult) years old. These subjects failed to respond to their current NNRTI treatment, were three-class experienced and had been exposed to NNRTI for ≥3 months. A total of 1827 adult (64.9%) and 32 paediatric subjects (82.1%) harboured the virus with at least one ETR mutation. V179I, Y181C and G190A were the most frequent mutations in both groups. A significantly increased risk of ETR resistance with all three algorithms (Monogram (MGR) >3, Tibotec (TBT) >2 and enhanced MGR (ENH) ≥4) emerged in the paediatric population. Multivariate analysis revealed an increased risk of developing TBT >2 for NNRTI exposure, ENH ≥4 for NNRTI and EFV exposure in paediatric subjects; NVP exposure and higher (≥3.5 log10) HIV-RNA values for all three algorithms in adult subjects, whereas CD4 ≥ 200/µL appeared to be protective. The risk of being ETR resistant was more than doubled for paediatric vs. adult subjects, probably due to a more extensive use of NNRTI and an incomplete virological control.

Prevalence of transmitted HIV-1 drug resistance in HIV-1-infected patients in Italy: evolution over 12 years and predictors.

OBJECTIVES: Transmitted HIV-1 drug resistance (TDR) can reduce the efficacy of first-line antiretroviral therapy. PATIENTS AND METHODS: A retrospective analysis was performed to assess the prevalence and correlates of TDR in Italy over time. TDR was defined as the presence of at least one of the mutations present in the surveillance drug resistance mutation (SDRM) list. RESULTS: Among 1690 antiretroviral therapy-naive patients, the most frequent HIV subtypes were B (78.8%), CRF02_AG (5.6%) and C (3.6%). Overall, TDR was 15%. TDR was 17.3% in subtype B and 7.0% in non-B carriers (P < 0.001). TDR showed a slight, although not significant, decline (from 16.3% in 1996-2001 to 13.4% in 2006-07, P = 0.15); TDR declined for nucleoside reverse transcriptase inhibitors (from 13.1% to 8.2%, P = 0.003) but remained stable for protease inhibitors (from 3.7% to 2.5%, P = 0.12) and non-nucleoside reverse transcriptase inhibitors (from 3.7% to 5.8%). TDR to any drug was stable in B subtype and showed a decline trend in non-B. In multivariable analysis, F1 subtype or any non-B subtype, compared with B subtype, and higher HIV RNA were independent predictors of reduced odds of TDR. CONCLUSIONS: Prevalence of TDR to nucleoside reverse transcriptase inhibitors seems to have declined in Italy over time. Increased prevalence of non-B subtypes partially justifies this phenomenon.

Effects of cryopreservation on lymphocyte immunophenotype and function.

Cryopreservation of isolated peripheral blood mononuclear cells (PBMCs) for phenotypic and functional analyses is considered a standard procedure in order to minimize operator-dependent inter-assay variability and to optimize the use of available resources. However, only few and somewhat conflicting data are presently available on the effects of cryopreservation on PBMCs, especially in samples from HIV-infected patients in which assessment of lymphocyte phenotype and function is of the outmost importance. In this study, we compared fresh versus frozen/thawed (F/T) samples isolated from 19 healthy individuals and 21 HIV-infected patients, showing that cryopreservation induces: (i) a profound decrease of CD62L expression, with a consequent significant decline of the calculated proportions of "naïve" (CD45RA+CD62L+) and "central memory" (CD45RO+CD62L+) T cells; (ii) an increase of the calculated proportions of "effector" CD8+ T cells (CD45RA+CD62L- and CD45RO+CD62L-) in the healthy subjects, while no changes were observed in the HIV-infected group; (iii) a significant decline of CC chemokine receptor 5 (CCR5) expression; (iv) a loss of proliferative responses to some HIV antigens (i.e. p24) and recall antigens [cytomegalovirus (CMV) and Influenza] in HIV-infected patients. We thus conclude that cryopreservation induces a consistent set of changes in PBMCs from both healthy and HIV-infected individuals, and that certain immunological studies of HIV-infected patients (i.e. studies of immune reconstitution following antiretroviral therapy in HIV-infected patients or studies of HIV-infectivity in vitro using CCR5-tropic strains) should be performed on fresh samples.

Implementation of an Italian multicentric care network for early HIV infection diagnosis: 1999-2001 report.

BACKGROUND: Diagnosis of a new HIV infection during the primary phase (PHI) is sometimes misleading in a primary care setting. Since 1999 the Italian network for the study of acute HIV infection (ISAI) has been operative. At the time of PHI diagnosis the case is reported to the coordinating centre and enrolled in the National Register which records all epidemiological, demographic and clinical information. PATIENTS AND METHODS: From 1999 to September 2001, 51 symptomatic or asymptomatic patients with diagnosis of primary HIV infection were signalled to the coordinating centre. At screening, assessments were: interview to collect demographic and epidemiological data, clinical history (regarding PHI signs and symptoms) and, if available, relevant index case information; physical examination; routine hematology and chemistry; lymphocyte count; plasma HIV-RNA. In a subset of patients PBMC HIV-DNA, HIV-RNA, resistance genotyping and HIV subtype characterization were assessed. RESULTS: 74.5% of patients were males and all but four were Italian. Hetero and homosexual contacts were the prevalent route of HIV transmission. Forty-five patients (89%) were symptomatic and the most frequent signs and symptoms were: fever, lymphadenopathy, malaise and pharyngodinia. Baseline reverse-transcriptase (RT) and protease (PR) genotyping analysis was available for 29 patients. Only one of 29 patients harbored a virus with a resistance-associated mutation in the RT region (215Y); NNRTI mutations were identified in 3 of 29 patients. In the remaining 20 (69%) patients no mutations were found in the RT region. Sequence data from PR region were successfully obtained in 21 patients. Only one of these had a high-level resistance mutation (46L); in an additional 10 cases 1 or more secondary mutations were identified. The remaining 10 patients harbored a PR region wild type virus. One patient presenting two secondary mutations in the PR region, even if highly adherent and tolerant to drug regimen, showed a slow viral load decrease. CONCLUSIONS: Our cohort confirms the uptrend of new infections through unsafe sexual contacts involving both homosexual and heterosexual couples. Genotype sequencing for antiretroviral resistant viral variants describes a low prevalence of RT resistance-associated mutations, as well as primary mutations in the PR region. On the contrary, a higher prevalence of PR gene polymorphisms and mutations is not known with any certainty to confer resistance to NRTI and NNRTI. The identification of antiretroviral drug resistant HIV strains is strategic for clinical and therapeutical intervention, even though from a public health point of view cost-efficacy must be considered.

Highly active antiretroviral therapy induces specific changes in effector and central memory T cell sub-populations.

Highly active antiretroviral therapy (HAART) improves the immunodeficiency of HIV-infected individuals. In this report we show that HAART increases both naive (CD45RA+CD62L+) and central memory (CD45RO+CD62L+) CD4 lymphocytes. On CD8 lymphocytes, HAART induces an increase of naive cells associated with a consistent decrease of effector cells (CD45 RO+CD62L-). No specific differences in phenotypic changes were observed with different HAART regimens, suggesting that, once viral suppression is achieved, the pharmacological class of antiretroviral drugs does not affect immune reconstitution.

Host-specific modulation of the selective constraints driving human immunodeficiency virus type 1 env gene evolution.

To address the evolution of human immunodeficiency virus type 1 (HIV-1) within a single host, we analyzed the HIV-1 C2-V5 env regions of both cell-free genomic-RNA- and proviral-DNA-derived clones. Sequential samples were collected over a period of 3 years from six untreated subjects (three typical progressors [TPs] and three slow progressors [SPs], all with a comparable length of infection except one. The evolutionary analysis of the C2-V5 env sequences performed on 506 molecular clones (253 RNA- and 253 DNA-derived sequences) highlighted a series of differences between TPs and SPs. In particular, (i) clonal sequences from SPs (DNA and RNA) showed lower nucleotide similarity than those from TPs (P = 0. 0001), (ii) DNA clones from SPs showed higher intra- and intersample nucleotide divergence than those from TPs (P < 0.05), (iii) higher host-selective pressure was generally detectable in SPs (DNA and RNA sequences), and (iv) the increase in the genetic distance of DNA and RNA sequences over time was paralleled by an increase in both synonymous (Ks) and nonsynonymous (Ka) substitutions in TPs but only in nonsynonymous substitutions in SPs. Several individual peculiarities of the HIV-1 evolutionary dynamics emerged when the V3, V4, and V5 env regions of both TPs and SPs were evaluated separately. These peculiarities, probably reflecting host-specific features of selective constraints and their continuous modulation, are documented by the dynamics of Ka/Ks ratios of hypervariable env domains.

Dynamic features of human immunodeficiency virus type 1 (HIV-1) viremia: kinetics of cell-free HIV-1 RNA after therapeutic plasma exchange.

To gain insight into the variables that influence the dynamics of human immunodeficiency virus type 1 (HIV-1) viremia levels, HIV-1 RNA molecules were quantified in plasma from an infected patient undergoing therapeutic plasma exchange (TPEx). After each TPEx procedure (2000 mL of fluid exchanged per session), HIV-1 genome molecule levels dropped to 58%-63% of the basal level but rapidly reverted to pre-TPEx values (doubling time = 3.50-4.04 h). Of interest, mobilization of extravascular cell-free virions (on average, 5.15 x 10(4) viral genome molecules/h) had already occurred during TPEx. After three daily TPEx procedures, HIV-1 viremia rebounded to basal values, while HIV-1 proviruses and viral transcripts in peripheral blood lymphocytes constantly tested at stable levels. Overall, this study extends previous analyses of the rate of HIV-1 turnover, using an alternative approach to the use of antiretroviral drugs, and it provides, albeit indirectly, insights into the amount and dynamic features of extravascular cell-free virus.

Changes in CD8 cell subpopulations induced by antiretroviral therapy in human immunodeficiency virus infected patients.

Although CD4+ T cells are the main target of HIV infection, CD8+ cells also play important roles in the interaction between HIV and the host immune system. The aim of this study was to analyze the effect of anti-HIV therapy on the relative proportion of some important CD8+ cell subpopulations. Five HIV-infected patients were enrolled, and blood samples were collected several times, within 90 days from the initiation of therapy. CD4+ cell count and HIV viremia were investigated, as well as the expression of CD38, HLA-DR, CD28, CD57, CD30, CD95 molecules on CD8+ cells. A complex remodeling of CD8+ cell subpopulations took place between week 2 and week 7 of treatment. This remodeling mainly consisted of: i) decrease of CD8+CD38+ and CD8+DR+ cells; ii) increase of CD8+CD28+ cells; and iii) decreased expression of the CD95/Fas molecule on CD8+ cells. Overall, these findings suggest that effective anti-HIV therapy induces changes of CD8+ subpopulations showing the reversal of the state of chronic activation that is caused by viral replication.

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.

Cervical dysplasia in HIV-seropositive women: role of human papillomavirus infection and immune status.

OBJECTIVE: In the present study we investigated the cytologic and colposcopic characteristics of a cohort of HIV-infected women, with the aim to determine a relationship between immunologic status and frequency and/or severity of cervical abnormalities. MATERIALS AND METHODS: 21 women, who tested positive for the HIV antibody and who were admitted as outpatients because of various gynecologic complications or because of an HIV infection that was under regular clinical surveillance. A pelvic examination was performed and Papanicolaou smears were obtained from endocervix and ectocervix before colposcopic examination. Cytologic samples for human papillomavirus (HPV) detection by polymerase chain reaction were also collected. Results obtained in the group of HIV-infected women were compared with findings in a group of 473 seronegative women recruited consecutively from our outpatient population. Serum samples for T lymphocytes were drawn within 2 weeks of cytologic and colposcopic examination. CD4 and CD8 monoclonal antibodies were purchased from Becton Dickinson (Mountain View, Calif., USA). RESULTS: HIV-infected women had a significantly higher percentage of HPV DNA positivity with respect to the outpatient population (67 vs. 7%, respectively, p < 0.001). Analysis of cytologic specimens revealed 9 women (43%) with cytologic evidence of cervical dysplasia in the HIV-seropositive group vs. 23 (5%) of 473 in the outpatient population (p < 0.001). All the HIV-seropositive women with cervical dysplasia presented an associated HPV DNA positivity; in particular, the percentage of associated HPV DNA type 16 in cervical dysplasia was 78% (7/9 cases). In HIV-infected women, the evaluation of T lymphocyte subset distribution suggested a significant relationship between CD4+ cell decrease and severity of cytologic findings (p = 0.03). DISCUSSION: The HIV-infected women had a tenfold higher prevalence of both HPV infection and cervical dysplasia than the outpatient population; this increased risk seems to be limited mainly to those who also had genital HPV infection. The analysis of immunologic status confirmed previous observations that an impaired immune system results in increased cervical disease.

Intercellular adhesion molecules (ICAM)-1 ICAM-2 and ICAM-3 function as counter-receptors for lymphocyte function-associated molecule 1 in human immunodeficiency virus-mediated syncytia formation.

It has been previously demonstrated that lymphocyte function-associated molecule 1 (LFA-1) plays a major role in human immunodeficiency virus (HIV)-mediated syncytia formation. In the present study we investigated the involvement of intercellular adhesion molecule-1 (ICAM-1), ICAM-2 and ICAM-3 in the process. The ability of monoclonal antibodies (mAb) directed against ICAM-1, ICAM-2 and ICAM-3 to block syncytia was analyzed either in phytohemagglutinin (PHA)-activated lymphocytes infected in vitro with primary or laboratory strains of HIV or by coculturing a T cell line stably expressing HIV envelope with PHA-activated lymphocytes. Complete inhibition of syncytia formation was observed only by the simultaneous addition to the cell cultures of all (i.e. anti-ICAM-1, anti-ICAM-2 and anti-ICAM-3) mAb. These results indicate that the interaction between LFA-1 and ICAM is a critical step in HIV-mediated syncytia formation, and that ICAM-1, ICAM-2 and ICAM-3 are the receptor molecules for the LFA-1-dependent syncytia formation.

HIV-1 infection in the lymphoid organs.

AIM: To develop a model of HIV disease progression. METHOD: Comparative analysis of viral burden and replication between peripheral blood and lymphoid organs and of the changes in viral distribution in the lymphoid tissue. RESULTS: In early-stage disease HIV-1-infected cells were sequestered in the lymphoid tissue, and the viral particles were concentrated and trapped in the germinal centers. The dichotomy in viral burden and viral replication between peripheral blood and lymphoid tissue was related to the histopathologic abnormalities associated with different stages of disease. CONCLUSIONS: These histopathologic abnormalities may not only explain the changes in viral distribution observed in the lymphoid tissue in different stages of the disease, but may also reflect different functional states of the immune system during the progression of HIV-1 infection from early- to late-stage disease.

Kinetics of human immunodeficiency virus type 1 (HIV-1) DNA and RNA synthesis during primary HIV-1 infection.

HIV-1 replication and viral burden in peripheral blood mononuclear cells (PBMC) have been reported to be high in primary infection but generally very low during the prolonged period of clinical latency. It is uncertain precisely when this transition occurs during the HIV-1 infection and what the relationship is between the changes in HIV-1 replication versus the clearance of infected cells in the overall control of viral replication. In the present study, the kinetics of viral burden (i.e., frequency of HIV-1-infected cells) and replication during primary and early-chronic infection were analyzed in PBMC of four acutely infected individuals. High frequencies of HIV-1-infected cells and high levels of virus replication were observed in PBMC after primary HIV-1 infection. Down-regulation of virus replication in PBMC was observed in all four patients coincident with the emergence of HIV-1-specific immune responses. Other parameters of virus replication, such as circulating plasma p24 antigen and plasma viremia showed similar kinetics. In contrast, a significant decline in viral burden in PBMC was observed in only one of four patients. These results indicate that the down-regulation in the levels of virus replication associated with the clinical transition from acute to chronic infection does not necessarily reflect a reduction in viral burden, thus suggesting the involvement of additional factors. Identification of these factors will be important in elucidating the host mechanisms involved in the early control of HIV-1 infection and disease.

The RRE of human immunodeficiency virus type 1 contributes to cell-type-specific viral tropism.

As part of a general program investigating the mechanism of the Rev axis of human immunodeficiency virus type 1 (HIV-1) autoregulation, a series of proviral HIV-1 mutants which differ from the parental HXB2 strain at selected positions within the RRE were constructed. All of the mutations were designed to perturb the RRE by introducing local helix disruptions without altering the coding potential of the overlapping envelope open reading frame. Viral replication in various cell types was monitored by a cell supernatant reverse transcriptase assay and Northern (RNA blot) analysis. All proviral RRE mutants displayed at least some impairment in replication. However, the relative impairment varied drastically among the various cell types tested. This suggests that the RRE may contribute to cell-type-specific viral tropism.

HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease.

Primary infection with the human immunodeficiency virus (HIV) is generally followed by a burst of viraemia with or without clinical symptoms. This in turn is followed by a prolonged period of clinical latency. During this period there is little, if any, detectable viraemia, the numbers of infected cells in the blood are very low, and it is extremely difficult to demonstrate virus expression in these cells. We have analysed viral burden and levels of virus replication simultaneously in the blood and lymphoid organs of the same individuals at various stages of HIV disease. Here we report that in early-stage disease there is a dichotomy between the levels of viral burden and virus replication in peripheral blood versus lymphoid organs. HIV disease is active in the lymphoid tissue throughout the period of clinical latency, even at times when minimal viral activity is demonstrated in blood.

Extensive sequence-specific information throughout the CAR/RRE, the target sequence of the human immunodeficiency virus type 1 Rev protein.

The significance and location of sequence-specific information in the CAR/RRE, the target sequence for the Rev protein of the human immunodeficiency virus type 1 (HIV-1), have been controversial. We present here a comprehensive experimental and computational approach combining mutational analysis, phylogenetic comparison, and thermodynamic structure calculations with a systematic strategy for distinguishing sequence-specific information from secondary structural information. A target sequence analog was designed to have a secondary structure identical to that of the wild type but a sequence that differs from that of the wild type at every position. This analog was inactive. By exchanging fragments between the wild-type sequence and the inactive analog, we were able to detect an unexpectedly extensive distribution of sequence specificity throughout the CAR/RRE. The analysis enabled us to identify a critically important sequence-specific region, region IIb in the Rev-binding domain, strongly supports a proposed base-pairing interaction in this location, and places forceful constraints on mechanisms of Rev action. The generalized approach presented can be applied to other systems.

Lymphoid organs function as major reservoirs for human immunodeficiency virus.

The total number of human immunodeficiency virus type 1 (HIV-1)-infected circulating CD4+ T lymphocytes is considered to be a reflection of the HIV burden at any given time during the course of HIV infection. However, the low frequency of HIV-infected circulating CD4+ T lymphocytes and the low level or absence of plasma viremia in the early stages of infection do not correlate with the progressive immune dysfunction characteristic of HIV infection. In this study, we have determined whether HIV-infected circulating CD4+ T lymphocytes are a correct reflection of the total pool of HIV-infected CD4+ T cells (i.e., HIV burden). To this end, HIV burden has been comparatively analyzed in peripheral blood and lymphoid tissues (lymph nodes, adenoids, and tonsils) from the same patients. The presence of HIV-1 DNA in mononuclear cells isolated simultaneously from peripheral blood and lymphoid tissues of the same patients was determined by polymerase chain reaction amplification. We found that the frequency of HIV-1-infected cells in unfractionated or sorted CD4+ cell populations isolated from lymphoid tissues was significantly higher (0.5-1 log10 unit) than the frequency in peripheral blood. Comparable results were obtained in five HIV seropositive patients in the early stages of disease and in one patient with AIDS. These results demonstrate that a heavy viral load does reside in the lymphoid organs, indicating that they may function as major reservoirs for HIV. In addition, the finding of a heavy viral load in the lymphoid organs of patients in the early stages of disease may explain the progressive depletion of CD4+ T lymphocytes and the immune dysfunction associated with the early stages of HIV infection.