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Background Previous reports highlighted the efficacy of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific monoclonal antibodies (mAbs) against coronavirus disease 2019. Methods We conducted a prospective study on the clinical outcome and antiviral effects of mAbs added to standard of care therapy in SARS-CoV-2-infected patients with primary antibody defects. Results Median time of SARS-CoV-2 quantitative polymerase chain reaction (qPCR) positivity was shorter in 8 patients treated with mAbs (22 days) than in 10 patients treated with standard of care therapy only (37 days, P=.026). Median time of SARS-CoV-2 qPCR positivity from mAb administration was 10 days. Conclusions The SARS-CoV-2 mAbs treatment was effective and well tolerated in patients with primary antibody defects.

The immunological mechanisms underlying the clinical presentation of SARS-CoV-2 infection and those influencing the disease outcome remain to be defined. Myeloid-derived suppressor cells (MDSC) have been described to be highly increased during COVID-19, however, their role remains elusive. We performed an in depth analysis of MDSC in 128 SARS-CoV-2 infected patients. Polymorphonuclear (PMN)-MDSC expanded during COVID-19, in particular in patients who required intensive care treatments, and correlated with IL-1β, IL-6, IL-8, and TNF-α plasma levels. PMN-MDSC inhibited T-cells IFN-γ production upon SARS-CoV-2 peptides stimulation, through TGF-β- and iNOS-mediated mechanisms, possibly contrasting virus elimination. Accordingly, a multivariate regression analysis found a strong association between PMN-MDSC percentage and fatal outcome of the disease. The PMN-MDSC frequency was higher in non-survivors than survivors at the admission time, followed by a decreasing trend. Interestingly, this trend was associated with IL-6 increase in non-survivors but not in survivors. In conclusion, this study indicates PMN-MDSC as a novel factor in the pathogenesis of SARS-CoV2 infection, and open up to new therapeutic options.

Limited data are available on risks and benefits of anti‐SARS‐CoV2 vaccination in solid organ transplant recipients, and weaker responses have been described. At the Italian National Institute for Infectious Diseases, 61 liver transplant recipients underwent testing to describe the dynamics of humoral and cell‐mediated immune response after two doses of anti‐SARS‐CoV2 mRNA vaccines and compared with 51 healthy controls. Humoral response was measured by quantifying both anti‐spike and neutralizing antibodies; cell‐mediated response was measured by PBMC proliferation assay with IFN‐γ and IL‐2 production. Liver transplant recipients showed lower response rates compared with controls in both humoral and cellular arms; shorter time since transplantation and multi‐drug immunosuppressive regimen containing mycophenolate mofetil were predictive of reduced response to vaccination. Specific antibody and cytokine production, though reduced, were highly correlated in transplant recipients.

Approximately 71 million people worldwide are infected with the hepatitis C virus (HCV). Injectable drug use represents the most common route of transmission in Europe and other developed countries. We studied the molecular characteristics of the HCV infection among mono-infected people who used drugs (PWUD) in Italy. Among 208 PWUD with anti-HCV antibodies, 101 (48.6%) were HCV RNA-positive, the majority (47%) were infected with the HCV genotype (Gt)1a, followed by Gt3a (34.9%), Gt4 (9.1%), Gt1b (4.5%), and Gt2 (4.5%). Bayesian phylogenetic analyses of clustered HCV NS5B sequences from 66 HCV-positive PWUDs with available plasma samples indicated age and neighborhood proximity as the most common characteristics between closely related HCV strains. Population dynamics, as measured by a coalescent Bayesian skyline analysis, revealed an increase in HCV Gt1a infections from the mid-1980s to mid-1990s. While HCV Gt3a infections were first detected in the 1980s, patient numbers with this genotype subtype remained relatively constant. For both Gt1a and Gt3a, Birth–Death Bayesian Skyline analyses produced higher reproduction numbers post 2014. For earlier time intervals, slow growths were observed for both Gt1a and Gt3a with reproduction numbers (Re) of approximately 1. The evolutionary rates for Gt1a and Gt3a were estimated as 2.23 × 10−4 and 3.85 × 10−4, respectively.

Liver injury in COVID-19 patients has progressively emerged, even in those without a history of liver disease, yet the mechanism of liver pathogenicity is still controversial. COVID-19 is frequently associated with increased serum ferritin levels, and hyperferritinemia was shown to correlate with illness severity. The liver is the major site for iron storage, and conditions of iron overload have been established to have a pathogenic role in development of liver diseases. We presented here six patients who developed severe COVID-19, with biochemical evidence of liver failure. Three cases were survived patients, who underwent liver biopsy, the other three were deceased patients, who were autopsied. None of the patients suffered underlying liver pathologies. Histopathological and ultrastructural analyses were performed. The most striking finding we demonstrated in all patients was iron accumulation into hepatocytes, associated with degenerative changes. Abundant ferritin particles were found enclosed in siderosomes, and large aggregates of hemosiderin were found, often in close contact with damaged mitochondria. Iron-caused oxidative stress may be responsible for mitochondria metabolic dysfunction. In agreement with this, association between mitochondria and lipid droplets was also found. Overall, our data suggest that hepatic iron overload could be the pathogenic trigger of liver injury associated to COVID-19.

Autophagy is a lysosomal-dependent degradative mechanism essential in maintaining cellular homeostasis, but it is also considered an ancient form of innate eukaryotic fighting against invading microorganisms. Mounting evidence has shown that HIV-1 is a critical target of autophagy that plays a role in HIV-1 replication and disease progression. In a special subset of HIV-1-infected patients that spontaneously and durably maintain extremely low viral replication, namely, long-term nonprogressors (LTNP), the resistance to HIV-1-induced pathogenesis is accompanied, in vivo, by a significant increase in the autophagic activity in peripheral blood mononuclear cells. Recently, a new player in the battle of autophagy against HIV-1 has been identified, namely, tripartite motif protein 5α (TRIM5α). In vitro data demonstrated that TRIM5α directly recognizes HIV-1 and targets it for autophagic destruction, thus protecting cells against HIV-1 infection. In this paper, we analyzed the involvement of this factor in the control of HIV-1 infection through autophagy, in vivo, in LTNP. The results obtained showed significantly higher levels of TRIM5α expression in cells from LTNP with respect to HIV-1-infected normal progressor patients. Interestingly, the colocalization of TRIM5α and HIV-1 proteins in autophagic vacuoles in LTNP cells suggested the participation of TRIM5α in the autophagy containment of HIV-1 in LTNP. Altogether, our results point to a protective role of TRIM5α in the successful control of the chronic viral infection in HIV-1-controllers through the autophagy mechanism. In our opinion, these findings could be relevant in fighting against HIV-1 disease, because autophagy inducers might be employed in combination with antiretroviral drugs.

Objectives: To investigate the association between sex hormones and the severity of coronavirus disease 2019 (COVID-19). Furthermore, associations between sex hormones and systemic inflammation markers, viral shedding and length of hospital stay were studied. Design and methods: This case–control study included a total of 48 male patients with COVID-19 admitted to an Italian reference hospital. The 24 cases were patients with PaO2/FiO2 300 mmHg at all times and who may have required low-flow oxygen supplementation during hospitalization (mild COVID-19). For each group, sex hormones were evaluated on hospital admission. Results: Patients with severe COVID-19 (cases) had a significantly lower testosterone level compared with patients with mild COVID-19 (controls). Median total testosterone (TT) was 1.4 ng/mL in cases and 3.5 ng/mL in controls (P = 0.005); median bioavailable testosterone (BioT) was 0.49 and 1.21 in cases and controls, respectively (P = 0.008); and median calculated free testosterone (cFT) was 0.029 ng/mL and 0.058 ng/mL in cases and controls, respectively (P = 0.015). Low TT, low cFT and low BioT were correlated with hyperinflammatory syndrome (P = 0.018, P = 0.048 and P = 0.020, respectively) and associated with longer length of hospital stay (P = 0.052, P = 0.041 and P = 0.023, respectively). No association was found between sex hormone level and duration of viral shedding, or between sex hormone level and mortality rate. Conclusions: A low level of testosterone was found to be a marker of clinical severity of COVID-19.

European Association of the Study of the Liver (EASL) guidelines specify HEV RNA, as well as anti-HEV IgG and IgM as positive markers for acute HEV infection. HEV RNA assay sensitivity limitations may lead to false negative test results in patients with low levels of viremia. Moreover, anti-HEV IgM positivity is not a reliable indicator for distinguishing between acute and resolved infections given the ability of this antibody to persist several months after a resolved infection. Our study aims were to assess HEV IgG avidity for diagnosing acute and resolved infections, regardless of the anti-HEV IgM serostatus, and examine assay reliability when evaluating different genotype 3 (GT3) HEV subtypes. Patient serum samples (n = 104) were tested for HEV IgG avidity by utilizing the DIA.PRO kit on a DSX automated instrument. Among patients identified with acute HEV infections, 32 were infected with GT3: GT3c (n = 5), GT3e (n = 8), 3f (n = 17) and GT3-unsubtyped (n = 2). Avidity sensitivity was 91.2% and specificity was 100%. For patients with long-lasting anti-HEV IgM persistence, an Avidity Index &gt, 70% was observed. Thus, the DIA.PRO avidity assay may be utilized to distinguish between recently acquired and resolved HEV GT3 infections. However, for equivocal results (Avidity Index &gt, 40–70%), HEV RNA molecular testing will be required to confirm a recent infection.

Patients with HCV who achieve a sustained virological response (SVR) on direct-acting antiviral (DAA) therapy still need to be monitored for signs of liver disease progression. To this end, the identification of both disease biomarkers and therapeutic targets is necessary.Extracellular vesicles (EVs) purified from plasma of 15 healthy donors (HDs), and 16 HCV-infected patients before (T0) and after (T6) DAA treatment were utilized for functional and miRNA cargo analysis. EVs purified from plasma of 17 HDs and 23 HCV-infected patients (T0 and T6) were employed for proteomic and western blot analyses. Functional analysis in LX2 cells measured fibrotic markers (mRNAs and proteins) in response to EVs. Structural analysis was performed by qPCR, label-free liquid chromatography-mass spectrometry and western blot.On the basis of observations indicating functional differences (i.e. modulation of FN-1, ACTA2, Smad2/3 phosphorylation, collagen deposition) of plasma-derived EVs from HDs, T0 and T6, we performed structural analysis of EVs. We found consistent differences in terms of both miRNA and protein cargos: (i) antifibrogenic miR204-5p, miR181a-5p, miR143-3p, miR93-5p and miR122-5p were statistically underrepresented in T0 EVs compared to HD EVs, while miR204-5p and miR143-3p were statistically underrepresented in T6 EVs compared to HD EVs (p0.05); (ii) proteomic analysis highlighted, in both T0 and T6, the modulation of several proteins with respect to HDs; among them, the fibrogenic protein DIAPH1 was upregulated (LogTaken together, these results highlight structural EV modifications that are conceivably causal for long-term liver disease progression in patients with HCV despite DAA-mediated SVR.Direct-acting antivirals lead to virological cure in the majority of patients with chronic hepatitis C virus infection. However, the risk of liver disease progression or complications in patients with fibrosis and cirrhosis remains in some patients even after virological cure. Herein, we show that extracellular vesicle modifications could be linked to long-term liver disease progression in patients who have achieved virological cure; these modifications could potentially be used as biomarkers or treatment targets in such patients.

Increased production of inflammatory cytokines and myeloid-derived suppressor cells occurs in patients with coronavirus disease 2019. These inversely correlated with perforin-expressing natural killer (NK) and CD3+ T cells. We observed a lower number of perforin-expressing NK cells in intensive care unit (ICU) patients compared with non-ICU patients, suggesting an impairment of the immune cytotoxic arm as a pathogenic mechanism.

The sinusoidal endothelial cells present in the liver (LSECs) are tipically characterized by the presence of pores (fenestrae). During some pathological conditions LSECs undergo “capillarization”, a process characterized by loss of fenestrations and acquisition of a vascular phenotype. In chronic liver disease capillarization has been reported to precede the development of fibrosis. LSECs modification in the setting of HCV infection is currently poorly investigated. Considering that HCV accounts for important changes in hepatocytes and in view of the intimate connection between hepatocytes and LSECs, here we set out to study in great detail the LSECs modifications in individuals with HCV-dependent chronic hepatitis. Electron microscopy analysis, and evaluation of CD32, CD31 and caveolin-1 expression showed that in HCV infection LSECs display major morphological changes but maintain their phenotypical identity. Capillarization was observed only in cases at initial stages of fibrosis. Our findings showed that the severity of LSECs modifications appears to be correlated with hepatocytes damage and fibrosis stage providing novel insight in the pathogenesis of HCV-chronic hepatitis.

Background Daclatasvir (DCV) is an approved NS5A inhibitor with potent anti-HCV activity and broad genotype coverage. DCV resistance-associated variants (RAVs) have been described for patients infected with genotype (GT) 1, but increased GT4 prevalence in European countries as a result of immigration has boosted interest in this genotype. Objectives Establishment of NS5A variability in treatment-naive patients with HCV genotype 4 infection and a case study of the dynamics of resistance-associated variants in a virologic failure receiving pIFN/RBV + DCV, as assessed by ultra-deep sequencing. Study design Five treatment-naive GT4 patients (GT4a [n = 1], GT4d [n = 3], GT4o [n = 1]) were evaluated for inclusion in the COMMAND-4 study and treatment with pIFN/RBV ± DCV. Results Patient (Pt) 1 received pIFN/RBV; Pts2–4 received pIFN/RBV + DCV; Pt5 was a screening failure. Pt1 relapsed; Pt2 experienced breakthrough at Wk4; Pts3 and 4 achieved a sustained virologic response. No substitutions associated with DCV-resistance were detected at baseline. In terms of viremic time points for Pts1 and 2, the extent of NS5A diversity pre-treatment was not significantly related to viral load (r = −0568; p = 0.035). In Pt2, multiple substitutions associated with DCV-resistance were observed after breakthrough at NS5A amino acid positions 28, 31 and 93. These substitutions were frequently observed on the same haplotype (L28S + M31I = 55.52, 82.50, and 99.36% at Wk4, 8 and 9; L28S + M31I + Y93H = 11.77, 5.01 and Conclusions This is the first report to describe DCV-resistance in patients infected with GT4d, supporting a possible role for a recently described RAV (L28S), and presenting the dynamics of HCV quasispecies during therapy failure, with indications of changes of diversity and association of mutations.

Hepatitis C virus (HCV) genotype (GT)3 is associated with increased risk of steatosis, development of cirrhosis and hepatocellular carcinoma. Limited data are available regarding genetic variability and use of direct-acting antiviral agents in these patients. non-structural protein 5A (NS5A) and non-structural protein 5B (NS5B) sequencing was performed on 45 HCV GT3-infected Italian patients subsequently treated with sofosbuvir ± daclatasvir (SOF ± DCV). Novel GT3a polymorphisms were observed by Sanger sequencing in three NS5A (T79S, T107K, and T107S) and three NS5B (G166R, Q180K, and C274W) baseline sequences in patients who achieved sustained virological response (SVR). Baseline NS5A resistance-associated substitutions (RASs) A30K and Y93H were detected in 9.5% of patients; one patient with A30K did not achieve SVR. Phylogenetic analyses of sequences showed no distinct clustering. Genetic heterogeneity of NS5A and NS5B was evaluated using ultra-deep pyrosequencing (UDPS) in samples longitudinally collected in patients not achieving SVR. Some novel NS5A and NS5B polymorphisms detected at baseline may not impact treatment outcome, as they were not enriched in post-failure samples. In contrast, the clinically novel GT3 NS5A-L31F RAS emerged in one treatment failure, and I184T, G188D and N310S, located on the same NS5B haplotype, became predominant after failure. These findings suggest a potential impact of these novel substitutions on the treatment outcome; however, their significance requires further investigation.

Antiviral therapy has revolutionized treatment of chronic HBV infections. First generation compounds, lamivudine and adefovir, displayed a high rate of treatment failures, and have been replaced by more potent compounds with high genetic barrier to resistance. However, the evolution of the virus towards resistance due the use of first generation compounds may still provide useful information for a better management of current antivirals. A single center sequence database including 705 HBV reverse transcriptase sequences from patients failing antiviral treatments (2002-2012) has been statistically analyzed to highlight viral evolution in relationship to the use of antiviral compounds and to their associations/sequencing in those years. The influence of viral genotypes and polymorphisms on resistance-related mutational patterns was also investigated. This study documents how, after the first years of antiviral therapy, the use of adefovir as an add-on strategy allowed a consistent reduction treatment failures. It also documents the effects of the initial misuse of entecavir in lamivudine experienced patients. In the latest years, the correct use of entecavir and the introduction of tenofovir allowed further curbing of resistance-related treatment failures, which virtually disappeared. Furthermore, the study allows a better understanding of how viral genotype (A vs D) conditions specific mutational pathways to resistance against lamivudine and entecavir, and demonstrates that the use of adefovir in lamivudine experienced patients is associated to peculiar mutational patterns, in particular A181V + F/Y221L. Despite some concern may arise for patients previously treated with lamivudine/adefovir, in sequence or combination, where the virus may have developed a lower genetic barrier against resistance to tenofovir, the outlook of antiviral treatment of HBV infection should be quite optimistic.

Background. Phase I/II studies in human immunodeficiency virus (HIV)–infected patients receiving antiretroviral therapy have shown that a single cycle of 3 weekly subcutaneous (s/c) injections of recombinant human interleukin 7 (r-hIL-7) is safe and improves immune CD4 T-cell restoration. Herein, we report data from 2 phase II trials evaluating the effect of repeated cycles of r-hIL-7 (20 µg/kg) with the objective of restoring a sustained CD4 T-cell count >500 cells/µL. Methods. INSPIRE 2 was a single-arm trial conducted in the United States and Canada. INSPIRE 3 was a 2 arm trial with 3:1 randomization to r-hIL-7 versus control conducted in Europe and South Africa. Participants with plasma HIV RNA levels 63% of their follow-up time with a CD4 T-cell count >500 cells/µL. Conclusions. Repeated cycles of r-hIL-7 were well tolerated and achieved sustained CD4 T-cell restoration to >500 cells/µL in the majority of study participants. Clinical Trials Registration. INSPIRE II: clinicaltrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT01190111","term_id":"NCT01190111"}}NCT01190111) and INSPIRE III: EudraCT (No. 2010-019773-15) and clinicaltrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT01241643","term_id":"NCT01241643"}}NCT01241643).

The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials based on its role in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune response with the asymptomatic stage as well as on its sequence conservation among HIV clades. A randomized, double blind, placebo-controlled phase I study (ISS P-001) was conducted in healthy adult volunteers without identifiable risk of HIV infection. Tat was administered 5 times monthly, subcute in alum or intradermic alone at 7.5 μg, 15 μg or 30 μg, respectively (ClinicalTrials.gov identifier: NCT00529698 ). Vaccination with Tat resulted to be safe and well tolerated (primary endpoint) both locally and systemically. In addition, Tat induced both Th1 and Th2 type specific immune responses in all subjects (secondary endpoint) with a wide spectrum of functional antibodies that are rarely seen in natural infection, providing key information for further clinical development of the Tat vaccine candidate.

The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials in both uninfected (ClinicalTrials.gov identifier: NCT00529698) and infected volunteers (ClinicalTrials.gov identifier: NCT00505401). The rationale was based on the role of Tat in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune responses with the asymptomatic stage and slow-progression rate as well as on its sequence conservation among HIV clades (http://www.hiv1tat-vaccines.info/). The parallel conduction in the same clinical centers of randomized, double blind, placebo-controlled phase I studies both in healthy, immunologically competent adults and in HIV-infected, clinically asymptomatic, individuals represents a unique occasion to compare the vaccine-induced immune response in both the preventive and therapeutic setting. In both studies, the same lot of the native Tat protein was administered 5 times, every four weeks, subcute (SC) with alum adjuvant or intradermic (ID), in the absence of adjuvant, at 7.5 microg, 15 microg or 30 microg doses, respectively. The primary and secondary endpoints of these studies were the safety and immunogenicity of the vaccine candidate, respectively. The study lasted 52 weeks and monitoring was conducted for on additional 3 years. The results of both studies indicated that the Tat vaccine is safe and well tolerated both locally and systemically and it is highly immunogenic at all the dosages and by both routes of administration. Vaccination with Tat induced a balanced immune response in uninfected and infected individuals. In particular, therapeutic immunization induced functional antibodies and partially reverted the marked Th1 polarization of anti-Tat immunity seen in natural infection, and elicited a more balanced Th1/Th2 immune response. Further, the number of CD4 T cells correlated positively with anti-Tat antibody titers. Based on these results, a phase II study is ongoing in infected drug-treated individuals (http://www.hiv1tat-vaccines.info/).

Monocyte differentiation into dendritic cells (DCs) depends on microenvironmental conditions. In this study, the capacity of human monocytes to differentiate into mature DCs and their ability to induce an antiviral immune response was investigated in HIV-infected patients. In healthy subjects, monocytes differentiate into CD1a DCs in the presence of granulocyte macrophage colony-stimulating factor and interleukin (IL)-4 and matured in the presence of lipopolysaccharide. Here, we found that in 30% and 45% of HIV-infected white and African subjects, respectively, monocytes gave rise to a homogeneous CD1a DC population. In the patients who gave rise only to the CD1a DCs, this population spontaneously produced IL-10 but not IL-12, and induced a T helper 2-like immune response when cultured with human T cells isolated from cord blood mononuclear cells. In patients with monocytes differentiated into CD1a DCs, a high percentage of HIV-specific CD4 T cells producing IL-4 were seen in the peripheral blood. Furthermore, differentiation of monocytes into DCs with CD1a phenotype correlated with low CD4 T-cell counts and high viral loads in HIV-infected subjects. These results suggest that the differentiation of monocytes into CD1a DCs may be a phenotypic marker associated with progression of the disease. © 2007 Lippincott Williams & Wilkins, Inc.

Several abnormalities of lipid metabolism, including hypo-beta-lipoproteinemia and liver steatosis are associated with infection by hepatitis C virus (HCV). The aim of this study was to determine whether circulating lipoproteins of patients with HCV infection could directly cause alterations of lipid cellular metabolism. To this end the metabolic response of human monocyte-derived macrophages (HMDM) to very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), measuring the cholesteryl ester (CE) and triglyceride (TG) production was analyzed. Lipoproteins were isolated from 18 patients infected with hepatitis C virus (HCV-VLDL and HCV-LDL) and from normal healthy donors (ct-VLDL and ct-LDL). In comparison to ct-lipoproteins, HCV-lipoproteins induced significant differences in HMDM CE and TG production. HCV-VLDL decreased CE and TG production; while HCV-LDL induced an increased TG synthesis. The present findings suggest that HCV infection modifies VLDL and LDL molecular composition, affecting cellular lipid metabolism, thus promoting intracellular lipid accumulation and hypo-beta-lipoproteinemia.

This study aimed to define clinical and virological parameters associated with spontaneous control of HIV replication in patients having initiated HAART during primary HIV infection, who underwent structured therapy interruption by two protocols with either fixed or HIV viremia-guided scheme. At the end of the protocol all patients were changed to viremia-guided scheme and observed for 12 months (follow-up). Patients maintaining HIV viremia below the indications for resumption of HAART during the follow-up, were defined controllers, those who had to resume HAART were defined non-controllers. The following parameters were examined: pre-interruption therapy duration, CD4+, HIV RNA, proviral DNA, evolution of viral quasispecies. No specific advantage was conferred by either interruption of structured therapy in the proportion of controllers and non-controllers. Pre-HAART and zenith CD4+, pre-therapy interruption, HAART duration, but not pre-HAART HIV RNA, were significantly higher in controllers as compared to non-controllers. HIV RNA levels after the first interruption cycle of therapy were significantly lower in controllers than in non-controllers. Proviral DNA levels were also lower in controllers at this time point. HIV RNA and proviral DNA levels associated with the last interruption of therapy cycle were not different from those associated with the first cycle, and, in spite of multiple waves of virus rebound, very few gag quasispecies variants emerged in each patient. The data suggest that pre-treatment clinical parameters and virological events associated with the first viral rebound are crucial factors in determining the ability to control viral replication after multiple cycles of interruption of treatment. J. Med. Virol. 79: 1047–1054, 2007. © 2007 Wiley-Liss, Inc.

The impact of pre-existing variants in hepatitis C virus (HCV) quasispecies, carrying resistance-associated mutations (RAMs), on the outcome of treatment with direct acting antiviral agents (DAA) is debated and it is complicated by the lack of knowledge of quasispecies distribution between the viral reservoir (liver) and the circulating compartment.To evaluate NS3 protease heterogeneity and presence of RAMs on baseline plasma and liver biopsy samples. Plasma dynamics were also analyzed during therapy and after its suspension. Study design Ultra-deep pyrosequencing (UDPS) was performed in two HCV genotype 1a patients who received telaprevir (TVR)-based therapy and developed treatment failure due to TVR-resistance.In both patients the baseline diversity of NS3 quasispecies in plasma was higher than in liver (183.6×10(-4) vs 47.8×10(-4) and 246.0×10(-4) vs 55.0×10(-4) nt substitution/site, respectively, p0.0001), but phylogenetic trees did not evidence compartmentalization between the two compartments. At baseline RAMs (i.e. V36A, T54A) were detected very low levels (range: 0.31-0.52%) in both specimen types. However, phylogenetic analyses revealed that the viral variants carrying these mutations at baseline were different from those that became fixed at breakthrough, when combined V36M+R155K, conferring high-level resistance to TVR, were observed. The frequency of resistance-associated variants declined after withdrawal of drug selective pressure.UDPS allowed extensive evaluation of quasispecies compartmentalization and of their dynamics after withdrawal of TVR. Plasma and liver NS3 quasispecies, including low level RAMs, do not show significant difference.

Gut-associated immune system has been identified as a major battlefield during the early phases of HIV infection. γδ T-cells, deeply affected in number and function after HIV infection, are able to act as a first line of defence against invading pathogens by producing antiviral soluble factors and by killing infected cells. Despite the relevant role in mucosal immunity, few data are available on gut-associated γδ T-cells during HIV infection. Aim of this work was to evaluate how primary (P-HIV) and chronic (C-HIV) HIV infection affects differentiation profile and functionality of circulating and gut-associated Vδ1 and Vδ2 T-cells. In particular, circulating and mucosal cells were isolated from respectively whole blood and residual gut samples from HIV-infected subjects with primary and chronic infection and from healthy donors (HD).Differentiation profile and functionality were analyzed by multiparametric flow cytometry. P-HIV and C-HIV were characterized by an increase in the frequency of effector Vδ1-T cells both in circulating and mucosal compartments. Moreover, during PHIV mucosal Vδ1 T-cells expressed high levels of CD107a, suggesting a good effector cytotoxic capability of these cells in the early phase of infection that was lost in C-HIV. P-HIV induced an increase in circulating effector Vδ2 T-cells in comparison to C-HIV and HD. Notably, P-HIV as well as HD were characterized by the ability of mucosal Vδ2 T-cells to spontaneously produce IFN-γ that was lost in C-HIV. Altogether, our data showed for the first time a functional capability of mucosal Vδ1 and Vδ2 T-cells during P-HIV that was lost in C-HIV, suggesting exhaustion mechanisms induced by persistent stimulation. Copyright

Background/Aims Hepatitis C virus (HCV) causes a chronic infection that can lead to fibrosis and carcinoma. Immune responses mediated by cytotoxic T lymphocytes (CTLs) could be involved in viral clearance or persistence, and therefore in determining the course of the disease. Methods Intrahepatic and peripheral blood CD8+T cells were obtained from 32 HCV-chronically infected patients and analysed by flow-cytometry for surface markers of differentiation, IFNγ and TNFα production, degranulation capacity and perforin content, after CD3 triggering. Results were compared with those obtained from 13 patients with a non-viral liver disease. Results Intrahepatic CD8+T cells of HCV-infected patients, despite their phenotype of pre-terminally and terminally differentiated effectors (CCR7–CD45RA−/+), are poorly responsive to T cell receptor (TCR)-mediated stimulation compared with those obtained from uninfected subjects. This defect correlates with the severity of fibrosis, is more pronounced in patients with ALT 1.5×NU/ml, and is not evident after mitogen stimulation. Conclusions The present study describes the accumulation of hypo-responsive CD8+T cells in the liver of patients with chronic HCV infection. Understanding the mechanisms underlying this impairment may be helpful in the design of innovative strategies for HCV treatment.

Transaminase elevation is frequently seen in hepatitis C virus (HCV)-HIV-coinfected patients receiving antiretroviral therapy (ART), representing an increase in the immune response against HCV and being one of the mechanisms proposed to be involved. There is a report claiming that HCV genotype 3 is an independent risk factor. Our objectives were to assess the incidence of liver toxicity in an HIV-HCV-coinfected population with relatively preserved cellular immunity, and the role of HCV genotypes in the elevation of liver enzymes, both at baseline and after initiating ART. All HIV(+) patients with positive anti-HCV serology and CD4(+) cell counts above 100/mm(3) who began triple ART were identified, and their HCV-RNA levels and HCV genotype were determined. Liver enzymes were determined at baseline and bimonthly during follow-up. Of anti-HCV patients 147 were included, 128 (87.1%) of whom had detectable plasma HCV-RNA. HCV-1 and HCV-4 genotypes were found to confer an increased probability of having at baseline transaminases within normal limits over the other genotypes. Severe transaminase elevations (grades 3 and 4) occurred in 5/124 patients (4.0%), all with high pre-HAART ALT and positive HCV-RNA levels. Multivariate analysis showed that patients with genotype HCV-3 had a 3.27 times higher risk of developing HAART-related transaminase elevations of any grade. In conclusion, subjects with the HCV-1 genotype more often had transaminases within normal limits at baseline. The incidence of severe transaminase elevation after initiating ART was very low (4%) in this HIV(+) population with relatively preserved cellular immunity. HCV genotype 3 was identified as a risk factor for the development of transaminase elevation of any grade.

Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. Since human Vgamma9Vdelta2 T lymphocytes play a critical role in the immune response against viruses, we analyzed their antiviral functions on Huh7 hepatoma cells carrying the subgenomic HCV replicon (Rep60 cells). In a transwell culture system, Rep60 cells were co-cultured with either PBMCs or highly purified gammadelta T cells stimulated by non-peptidic antigens. Vgamma9Vdelta2 T cell activation was associated with a dramatic reduction of HCV RNA levels. Neutralizing antibodies targeting IFN-gamma revealed a critical role for this cytokine in the inhibition of HCV replication. Interestingly, drugs already in clinical use, such as Phosphostim and Zoledronate, known to activate gammadelta T cells, were shown to induce the inhibition of HCV replication mediated by Vgamma9Vdelta2 T cells of HCV patients. Our data suggest that the therapeutic activation of Vgamma9Vdelta2 T lymphocytes may represent an additional strategy to inhibit HCV replication and to restore a Th1-oriented immune response in HCV-infected patients.

Alterations in NK cell numbers and function have been repeatedly shown during HIV infection. In this study, NK cell number and MHC class I expression on CD4+ T cells were studied in HIV patients at different stages of disease progression. An increased expression of HLA-E was seen on CD4+ T cells. In parallel, a reduced number of CD94+ NK cells was observed in advanced disease stages. Moreover, a decline in CD94 expression on NK cells was observed at the HIV replication peak in patients undergoing antiretroviral treatment interruption, suggesting a role of viral replication on NK cells alterations. In vitro HIV infection induced a rapid down-regulation of HLA-A,B,C expression, paralleled by an increased expression of HLA-E surface molecules, the formal ligands of CD94 NK receptors. HIV-infected HLA-E expressing cells were able to inhibit NK cell cytotoxicity through HLA-E expression, since cytotoxicity was restored by antibody masking experiments. These data indicate that the CD94/HLA-E interaction may contribute to NK cell dysfunction in HIV infection, suggesting a role of HIV replication in this process.

Structured treatment interruption (STI) may help to alleviate the problems associated with long-term antiretroviral therapy (ART) in human immunodeficiency virus (HIV)-infected patients. We analyzed the role that baseline levels of cytokines in plasma play as markers of a favorable outcome of STI. Two groups of patients were defined: STI responders and STI nonresponders. STI responders showed a higher baseline concentration of interleukin (IL)-15 in plasma than did STI nonresponders and showed lower levels of tumor necrosis factor (TNF)-alpha during STI. No differences were observed in levels of IL-2, IL-7, or interferon-alpha in plasma. Our data show that (1) levels of TNF-alpha in plasma correlate with HIV viremia and (2) monitoring baseline levels of IL-15 in plasma allows for the identification of a favorable outcome of STI.

Objectives Integrase drug resistance monitoring deserves attention because of the increasing number of patients being treated with integrase strand-transfer inhibitors. Therefore, we evaluated the integrase genotyping success rate at low-level viraemia (LLV, 51–1000 copies/mL) and resistance in raltegravir-failing patients. Methods An integrase genotypic resistance test (GRT) was performed on 1734 HIV-1 samples collected during 2006–13. Genotyping success rate was determined according to the following viraemia levels: 51–500, 501–1000, 1001–10 000, 10 001–100 000 and >100 000 copies/mL. The reproducibility of integrase GRT was evaluated in 41 plasma samples processed in duplicate in two reference centres. The relationship between LLV and resistance prevalence was evaluated in a subset of 120 raltegravir-failing patients. Results Overall, the integrase genotyping success rate was 95.7%. For viraemia levels 51–500 and 501–1000 copies/mL, the rate of success was 82.1% and 94.0%, respectively. GRT was reproducible, producing sequences with a high similarity and an equal resistance profile regardless of the sequencing centre or viraemia level. Resistance was detected both at LLV and at viraemia >1000 copies/mL (51–500 copies/mL = 18.2%; 501–1000 = 37.5%; 1001–10 000 = 53.7%; 10 001–100 000 = 30.0%; and >100 000 = 30.8%). At viraemia ≤500 copies/mL, Q148H/K/R and N155H had the same prevalence (9.1%), while the Y143C/H/R was completely absent. At early genotyping (within 3 months of raltegravir treatment), Q148H/K/R and N155H mutations were detected regardless of the viraemia level, while Y143C/H/R was observed only in samples with viraemia >1000 copies/mL. Conclusions Our findings prove the reliability of HIV-1 integrase genotyping and reinforce the concept that this assay may be useful in the management of failures even at LLV.

HIV and hepatis C virus (HCV) coinfection is frequently associated with rapid progression of HCV-related disease, resulting in a higher risk of cirrhosis. Data suggest that natural T cells expressing the Vdelta1 T cell receptor rearrangement are recruited in the liver of chronically HCV-infected patients and are increased in the peripheral blood of HIV-infected persons. We studied gammadelta T cell distribution in the peripheral blood and liver of HCV-infected and HIV/HCV-coinfected patients in the presence and absence of antiretroviral therapy. We observed that Vdelta1+ T cells releasing helper T cell type 1 cytokines are compartmentalized not only in the liver of HCV+ patients, but also of HIV/HCV-coinfected persons. HIV/HCV patients showed an increased frequency of both peripheral and intrahepatic Vdelta1 natural T lymphocytes, resulting in a higher degree of hepatic inflammation when compared with patients with other liver diseases. Finally, highly active antiretroviral therapy (HAART) was unable to restore Vdelta1T cell circulation to normal levels in chronically HIV-infected persons. We conclude that gammadelta T lymphocytes released from tissue to the bloodstream circulation under the influence of chronic HIV infection may contribute to intrahepatic Vdelta1 compartmentalization and progression of liver disease, independently of HAART.

Objectives Identifying the predictive factors of Sustained Virological Response (SVR) represents an important challenge in new interferon-based DAA therapies. Here, we analyzed the kinetics of antiviral response associated with a triple drug regimen, and the association between negative residual viral load at different time points during treatment. Methods Twenty-three HCV genotype 1 (GT 1a n = 11; GT1b n = 12) infected patients were included in the study. Linear Discriminant Analysis (LDA) was used to establish possible association between HCV RNA values at days 1 and 4 from start of therapy and SVR. Principal compo- nent analysis (PCA) was applied to analyze the correlation between HCV RNA slope and SVR. A ultrasensitive (US) method was established to measure the residual HCV viral load in those samples which resulted "detected

SUMMARYIn vitro lymphoproliferative responses to HIV-1 recombinant antigens (gp160, p24, and Rev protein) were studied in 83 patients with asymptomatic HIV-1 infection (CDC groups 11 and III) and circulating CD4 lymphocyte numbers > 400/mm3. Significant response to at least one of the three antigens was detected in 52.4% of the subjects, but the responses were weak, and concordance of the response to the three antigens was rare, the frequency of individuals responding to each antigen not exceeding 22.4%. Increasing frequencies of response were observed when recall antigens (tetanus toxoid and Candida albicans glycomannoprotein) (65.5%) and anti-CD3 MoAb (76.6%) were used as stimuli. Although a significant association between lymphocyte response to p24, but not gp160, and steadiness of CD4 lymphocyte numbers before the assay was observed, no predictive value for lack of CD4 cell decrease was confirmed for either antigen, and fluctuation of the responses to HIV antigens was seen during subsequent follow up. The panel of T cell assays used could be regarded as appropriate for monitoring both HIV-specific responses and T lymphocyte function during immunotherapy with soluble HIV antigens.

In chronic HCV infection, treatment failure and defective host immune response highly demand improved therapy strategies. Vγ9Vδ2 T-cells may inhibit HCV replication in vitro through IFN-γ release after Phosphoantigen (PhAg) stimulation. The aim of our work was to analyze Vγ9Vδ2 T-cell functionality during chronic HCV infection, studying the role of IFN-α on their function capability. IFN-γ production by Vγ9Vδ2 T-cells was analyzed in vitro in 24 HCV-infected patients and 35 healthy donors (HD) after PhAg stimulation with or without IFN-α. The effect of in vivo PhAg/IFN-α administration on plasma IFN-γ levels was analyzed in M. fascicularis monkeys. A quantitative analysis of IFN-γ mRNA level and stability in Vγ9Vδ2 T-cells was also evaluated. During chronic HCV infection, Vγ9Vδ2 T-cells showed an effector/activated phenotype and were significantly impaired in IFN-γ production. Interestingly, IFN-α was able to improve their IFN-γ response to PhAg both in vitro in HD and HCV-infected patients, and in vivo in Macaca fascicularis primates. Finally, IFN-α increased IFN-γ-mRNA transcription and stability in PhAg-activated Vγ9Vδ2 T-cells. Altogether our results show a functional impairment of Vγ9Vδ2 T-cells during chronic HCV infection that can be partially restored by using IFN-α. A study aimed to evaluate the antiviral impact of PhAg/IFN-α combination may provide new insight in designing possible combined strategies to improve HCV infection treatment outcome. © 2012 Cimini et al.

Objectives: HIV-1 V3 quasispecies was analyzed by ultra-deep pyrosequencing, in early HIV-infected patients, to assess possible correlations between quasispecies diversity, frequency of variants predicted to use CXCR4 and need for early antiretroviral treatment. Methods: Twenty patients were retrospectively enrolled: 10 patients (group A) required HAART within 6 months from seroconversion and 10 (group B) remained free of therapy during this period. V3 quasispecies was assessed on plasma viral RNA and in peripheral blood mononuclear cell-associated proviral DNA. Prediction of coreceptor usage was performed by position-specific score matrix analysis. Results: Variants predicted to use CXCR4 were detected (frequency >0.3%) in the plasma of 50% of early infected patients (60% from group A and 40% from group B). Intrapatient frequency of these variants was highly variable (0.3-56.3%). A positive correlation was observed between the proportion of X4 variants and intrapatient quasispecies diversity. Quasispecies diversity and absolute numbers of X4 variants were significantly higher in patients from group A. The analysis of proviral DNA quasispecies, performed in a subgroup of five patients, showed that X4 variants were not detected in patients with RNA frequency below 0.3%, and detected at 3.6% in the patient with 56.3% of X4 plasma variants. Conclusion: Our findings show that X4 variants may be frequently found, at variable intrapatient frequency, in early infected patients, and that quasispecies diversity and absolute numbers of X4 variants are significantly higher in patients undergoing early antiretroviral treatment. Further studies are mandatory to explore the clinical relevance of X4 variants present during early infection with respect to clinical progression and possible therapeutic implications.

Background/aims The life cycle of hepatitis C virus (HCV) is intimately linked to the lipid metabolism of the host. In particular, HCV exploits the metabolic machinery of the lipoproteins in several steps of its life cycle such as circulation in the bloodstream, cell attachment and entry, assembly and release of viral particles. However, the details of how HCV interacts with and influences the metabolism of the host lipoproteins are not well understood. A study was undertaken to investigate whether HCV directly affects the protein composition of host circulating lipoproteins. Methods A proteomic analysis of circulating very low-, low- and high-density lipoproteins (VLDL, LDL and HDL), isolated from either in-treatment naive HCV-infected patients or healthy donors (HD), was performed using two-dimensional gel electrophoresis and tandem mass spectrometry (MALDI-TOF/TOF). The results obtained were further investigated using in vitro models of HCV infection and replication. Results A decreased level of apolipoprotein A-I (apoA-I) was found in the LDL fractions of HCV-infected patients. This result was confirmed by western blot and ELISA analysis. HCV cellular models (JFH1 HCV cell culture system (HCVcc) and HCV subgenomic replicons) showed that the decreased apoA-I/LDL association originates from hepatic biogenesis rather than lipoprotein catabolism occurring in the circulation, and is not due to a downregulation of the apoA-I protein concentration. The sole non-structural viral proteins were sufficient to impair the apoA-I/LDL association. Functional evidence was obtained for involvement of apoA-I in the viral life cycle such as RNA replication and virion production. The specific siRNA-mediated downregulation of apoA-I led to a reduction in both HCV RNA and viral particle levels in culture. Conclusions This study shows that HCV induces lipoprotein structural modification and that its replication and production are linked to the host lipoprotein metabolism, suggesting apoA-I as a new possible target for antiviral therapy.

It was estimated that up to 70% of hepatitis C virus (HCV)-infected subjects became chronically infected, and these patients have an increased risk of developing cirrhosis and hepatocellular carcinoma.1 The standard treatment for HCV-infected patients is pegylated interferon α (IFNα) in combination with ribavirin; however, data on the correlation between treatment outcome and the activation state of components of the immune system are missing. A very interesting paper by Mengshol et al was published in Gut regarding the effect of anti-HCV treatment on the activation state of circulating dendritic cells (DCs).2 The authors showed that levels of CXCR3 and CXCR4 on plasmacytoid DC (pDCs) were higher at baseline compared with controls, and decreased with treatment. Pretreatment levels of the co-stimulatory marker CD40 and the maturation marker CD83 were higher in pDCs of patients chronically infected with HCV compared with healthy controls; moreover, levels of both markers dropped significantly with treatment in the sustained virological responder (SVR) group only. Finally, baseline pDCs chemotaxis to CXCL12 …

In this work we evaluated plasmacytoid (pDC) and myeloid dendritic (mDC) cells activation before and during anti-HCV treatment in HCV+/HIV+ individuals.HCV+/HIV+ patients received Peg-IFN-α2b subcutaneously for 28. days, followed by oral weight-based ribavirin. DCs activation was evaluated by flow cytometry.Baseline pDC CD80 and CD86 expression was correlated with HIV, but not with HCV viral load. A transient decrease of HIV RNA was found not associated with DC activation. When patients were grouped according to early/sustained virological response (EVR/SVR) to anti-HCV treatment, baseline pDC CD80 and CD86 expression was higher in non-EVR and non-SVR compared to EVR and SVR. Moreover, in responder patients CD80 and CD86 were upregulated by IFN-α. Our data suggest a correlation between DCs activation and response to therapy. These findings could be helpful to better understand the mediators of IFN-α action in HCV+/HIV+ patients and to explore possible exploitation of this knowledge to improve therapeutic response. © 2010 Elsevier Inc.

A randomized, double blind, placebo-controlled phase I vaccine trial based on the native Tat protein was conducted in HIV-infected asymptomatic individuals. The vaccine was administered five times subcute with alum or intradermally without adjuvant at 7.5microg, 15microg or 30microg doses, respectively. The Tat vaccine was well tolerated both locally and systemically and induced and/or maintained Tat-specific T helper (Th)-1 T-cell responses and Th-2 responses in all subjects with a wide spectrum of functional anti-Tat antibodies, rarely seen in HIV-infected subjects. The data indicate the achievement of both the primary (safety) and secondary (immunogenicity) endpoints of the study.

The use of HIV-1 DNA quantitation in cellular reservoirs to predict disease progression and treatment outcome in infected patients is hampered by the lack of standardization among the available methods. In the present study, real-time PCR methods used commonly for HIV-1 proviral DNA evaluation were compared, showing strong differences in the results, probably as a consequence of genome variability in the target regions. Standardization of HIV-1 proviral DNA quantitation assays is needed for use in clinical management of patients with HIV-1.

Background Virus-associated cell membrane proteins acquired by HIV-1 during budding may give information on the cellular source of circulating virions. In the present study, by applying immunosorting of the virus and of the cells with antibodies targeting monocyte (CD36) and lymphocyte (CD26) markers, it was possible to directly compare HIV-1 quasispecies archived in circulating monocytes and T lymphocytes with that present in plasma virions originated from the same cell types. Five chronically HIV-1 infected patients who underwent therapy interruption after prolonged HAART were enrolled in the study. The analysis was performed by the powerful technology of ultra-deep pyrosequencing after PCR amplification of part of the env gene, coding for the viral glycoprotein (gp) 120, encompassing the tropism-related V3 loop region. V3 amino acid sequences were used to establish heterogeneity parameters, to build phylogenetic trees and to predict co-receptor usage. Results The heterogeneity of proviral and viral genomes derived from monocytes was higher than that of T-lymphocyte origin. Both monocytes and T lymphocytes might contribute to virus rebounding in the circulation after therapy interruptions, but other virus sources might also be involved. In addition, both proviral and circulating viral sequences from monocytes and T lymphocytes were predictive of a predominant R5 coreceptor usage. However, minor variants, segregating from the most frequent quasispecies variants, were present. In particular, in proviral genomes harboured by monocytes, minority variant clusters with a predicted X4 phenotype were found. Conclusion This study provided the first direct comparison between the HIV-1 quasispecies archived as provirus in circulating monocytes and T lymphocytes with that of plasma virions replicating in the same cell types. Ultra-deep pyrosequencing generated data with some order of magnitude higher than any previously obtained with conventional approaches. Next generation sequencing allowed the analysis of previously inaccessible aspects of HIV-1 quasispecies, such as co-receptor usage of minority variants present in archived proviral sequences and in actually replicating virions, which may have clinical and therapeutic relevance.

Replication-competent HIV, as well as HIV-1 DNA, has been detected in CD4 T cells and in monocytes during antiretroviral therapy (ART), indicating that these cells could represent an important viral reservoir. We measured HIV-1 DNA in monocytes and CD4 T cells in patients undergoing transient therapy interruption (TTI), to establish the dynamic of HIV-1 DNA burden and to find possible correlations with immune restoration and re-establishment of virological control after ART resumption. In most patients CD4 depletion and viral load rebound followed TTI. Rapid resumption of virological and immunological control was achieved after ART reintroduction. After TTI, in most cases a transient increase of both monocyte and CD4 HIV-1 DNA burden was observed. After ART reintroduction, both CD4 T cell and monocyte HIV-1 DNA copy number decreased, reaching baseline levels at the end of observation. At this time monocyte HIV-1 DNA burden was always undetectable, while CD4 T cell HIV-1 DNA burden was lower than at baseline. As CD4 T cell HIV-1 DNA values are independently associated with CD4 depletion, the increase of HIV-1 DNA burden in these cells after TTI is presumably due to acute infection, causing cell death. This is also supported by the pattern of 2-LTR appearance in these cells after TTI. HIV-1 DNA burden in monocytes and CD4 T cells show high correlation, suggesting reciprocal re-feeding of two cell populations. Repopulation by HIV these cells after TTI is temporary, and no significant changes of HIV-1 DNA burden were observed after ART resumption respect to pre-TTI period.

In drug-naive HIV + patients, we analyzed the effects of highly active antiretroviral therapy (HAART) on the reconstitution of the T-cell receptor (TCR) repertoire. We followed 2 groups of patients for 1 year: 18 individuals who experienced acute HIV infection and 24 patients who had HIV infection for many years but never took HAART. They were compared with 10 healthy controls who were longitudinally analyzed for the same period. We performed cytofluorometric analysis of the Vβ TCR repertoire and detected the clonality of different Vβ families by the spectratyping method. A new statistical approach based on the use of mixed models was then employed to analyze the data. Before the beginning of therapy, the repertoire of patients with acute or chronic infection was significantly different from that of healthy controls. After therapy, patients with acute HIV infection showed an improvement of the repertoire among either CD4 + or CD8 + T lymphocytes. Conversely, patients with chronic infection were capable of changing their repertoire among CD8 + but not CD4 + T lymphocytes. Our results indicate that HAART can restore the T-cell repertoire in individuals whose immune system is not severely compromised by the infection.

A rebound in the plasma and seminal viral load was detected in 12 patients after the interruption of effective highly active antiretroviral therapy. The viral rebound was generally higher in plasma, although the highest level observed during interruption was higher in semen in two patients. The reintroduction of therapy was followed by an overall decrease in HIV-RNA in plasma and semen. One to 2 months after restarting treatment, four out of seven patients showed undetectable HIV-RNA in semen.

During the last few years, highly active antiretroviral therapy has considerably reduced human immunodeficiency virus (HIV) disease progression (11). However, the treatment outcome is not always satisfactory (7). This can depend on different virological, immunological, behavioral, or pharmacological factors (5). In the framework of aspects, the interaction between P-glycoprotein (P-gp) and antiretroviral drugs has been evidenced (8, 13, 15). P-gp is a transmembrane phosphoglycoprotein belonging to the ATP-binding cassette superfamily that is able to transport several substrates through the cell membrane acting as a cationic pump (2, 3, 4, 6, 9, 14). Specifically, studies on protease inhibitors (PI) have evidenced that the transporter activity of P-gp may contribute to the reduced bioavailability of these drugs, which can act as substrates for P-gp (4, 15, 16). However, the influence of HIV infection on P-gp expression is still a matter of debate (1, 9), and as yet, no data are available on the effects of highly active antiretroviral therapy on its expression.

Objective: The influence of structured treatment interruption on effector/memory CD8 T cell dynamics was analysed in chronic HIV-infected patients showing a rapid or delayed viral rebound. Design: Structured treatment interruption consisted of at least one month of discontinuation, followed by highly active antiretroviral therapy (HAART) resumption. Two groups of HIV structured treatment interruption patients were selected on the basis of plasma viral HIV-RNA value (> 30 000 copies/ml, branched DNA): group A (n = 14), patients with a rapid viral rebound (within one month) and group B (n = 6), patients with a delayed or no viral rebound (after a minimum of 4 months). Methods: A clinical and immunological follow-up was performed at HAART suspension (t 0), one month from suspension (t 1), at HAART resumption (t 2), and 30 days from resumption (t 3). Results: A sustained viral rebound was observed in group A patients, showing a rapid expansion of circulating CD8 T lymphocytes. In this group, the frequencies of CD8 T cells releasing IFN-y after mitogen-induced or Gag-specific stimulation were highly increased after HAART discontinuation. Nevertheless, these CD8 T lymphocytes were mainly composed of pre-terminally differentiated cytotoxic T lymphocytes (CTL) expressing a CCR7 - CD27 +/- CD45RA - phenotype and a reduced amount of perforin. In contrast, group B patients showed no significant changes in immunological parameters during a prolonged drug-free period. Conclusion: These data indicate that monitoring CD8 T cell dynamics during structured treatment interruption could be clinically relevant, and new therapeutic strategies should aim qualitatively to restore CTL effector functions.

We report a 36-year-old HIV-infected woman who developed primary Toxoplasma gondii infection during pregnancy that was treated with spiramycin and antiretroviral drugs. There was no vertical transmission of toxoplasmosis and HIV.