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OBJECTIVES To investigate the value of tropism (determined by genotypic testing) to predict CD4 depletion in HIV-infected antiretroviral-naive patients with high CD4 counts. METHODS Viral tropism was determined by geno2pheno (false positive rate = 10%) in 223 HIV-infected subjects naive to antiretrovirals with CD4 count ≥350 cells/μL and HIV-RNA >500 copies/mL enrolled in the ICONA Foundation Study for whom a stored plasma sample (baseline) was retrospectively tested. We monitored CD4 cell count and identified predictors of decline before antiretroviral therapy initiation, applying a mixed linear model with covariates (age, gender, tropism, HIV risk factor, calendar year of HIV infection, months from HIV diagnosis to baseline, hepatitis C virus status, CD4 and HIV-RNA at sample collection and duration of follow-up). RESULTS Two hundred and twenty-three subjects met the eligibility criteria; 137 (61%) were male and the median age was 35 (31-40) years. Median follow-up was 16.4 (3.2-37.2) months. Median CD4 decrease during follow-up was -157 (-278 to -13) cells/μL. At baseline, 192 (86%) subjects were defined as harbouring R5 virus and 31 (14%) non-R5. Median CD4 count was 571 (458-729) cells/μL and median HIV-RNA was 4.08 (3.57-4.55) log(10) copies/mL. At multivariable analysis, a greater mean CD4 decrease was associated with non-R5 viral tropism (-159.9 ± 12.22, P = 0.0002) at baseline. Other significant covariates were female gender, older age, intravenous drug use, longer duration of follow-up, and higher CD4 cell count and higher HIV-RNA at sample collection. CONCLUSIONS In patients with CD4 counts ≥350 cells/μL, non-R5 viral tropism by geno2pheno is predictive of CD4 decrease independent of their viral set point and CD4 counts.

Significance Functional loss of gut barrier integrity with subsequent increased antigen trafficking and occurrence of low-grade intestinal inflammation precede the onset of type 1 diabetes (T1D) in patients and preclinical models, thus suggesting that these events are mechanistically linked to the autoimmune pathogenesis of the disease. However, a causal relationship between increased intestinal permeability and autoimmune diabetes was never demonstrated. Our data show that breakage of gut barrier continuity leads to activation of islet-reactive T cells in the intestine, thus triggering autoimmune diabetes. An important implication of our findings is that restoration of a healthy gut barrier through microbiota and diet modulation in diabetes-prone individuals could reduce intestinal activation of islet-reactive T cells and prevent T1D occurrence., Low-grade intestinal inflammation and alterations of gut barrier integrity are found in patients affected by extraintestinal autoimmune diseases such as type 1 diabetes (T1D), but a direct causal link between enteropathy and triggering of autoimmunity is yet to be established. Here, we found that onset of autoimmunity in preclinical models of T1D is associated with alterations of the mucus layer structure and loss of gut barrier integrity. Importantly, we showed that breakage of the gut barrier integrity in BDC2.5XNOD mice carrying a transgenic T cell receptor (TCR) specific for a beta cell autoantigen leads to activation of islet-reactive T cells within the gut mucosa and onset of T1D. The intestinal activation of islet-reactive T cells requires the presence of gut microbiota and is abolished when mice are depleted of endogenous commensal microbiota by antibiotic treatment. Our results indicate that loss of gut barrier continuity can lead to activation of islet-specific T cells within the intestinal mucosa and to autoimmune diabetes and provide a strong rationale to design innovative therapeutic interventions in “at-risk” individuals aimed at restoring gut barrier integrity to prevent T1D occurrence.

Common features of immune-metabolic and inflammatory diseases such as metabolic syndrome, diabetes, obesity and cardiovascular diseases are an altered gut microbiota composition and a systemic pro-inflammatory state. We demonstrate that active immunization against the outer membrane protein of bacteria present in the gut enhances local and systemic immune control via apoE-mediated immune-modulation. Reduction of western-diet-associated inflammation was obtained for more than eighteen weeks after immunization. Immunized mice had reduced serum cytokine levels, reduced insulin and fasting glucose concentrations; and gene expression in both liver and visceral adipose tissue confirmed a reduced inflammatory steady-state after immunization. Moreover, both gut and atherosclerotic plaques of immunized mice showed reduced inflammatory cells and an increased M2 macrophage fraction. These results suggest that adaptive responses directed against microbes present in our microbiota have systemic beneficial consequences and demonstrate the key role of apoE in this mechanism that could be exploited to treat immune-metabolic diseases. Common features of immune-metabolic and inflammatory diseases such as metabolic syndrome, diabetes, obesity and cardiovascular diseases are an altered gut microbiota composition and a systemic pro-inflammatory state. We demonstrate that active immunization against the outer membrane protein of bacteria present in the gut enhances local and systemic immune control via apoE-mediated immune-modulation. Reduction of western-diet-associated inflammation was obtained for more than eighteen weeks after immunization. Immunized mice had reduced serum cytokine levels, reduced insulin and fasting glucose concentrations; and gene expression in both liver and visceral adipose tissue confirmed a reduced inflammatory steady-state after immunization. Moreover, both gut and atherosclerotic plaques of immunized mice showed reduced inflammatory cells and an increased M2 macrophage fraction. These results suggest that adaptive responses directed against microbes present in our microbiota have systemic beneficial consequences and demonstrate the key role of apoE in this mechanism that could be exploited to treat immune-metabolic diseases.

The gut microbiota modulates the autoimmune pathogenesis of type 1 diabetes (T1D) via mechanisms that remain largely unknown. The inflammasome components are innate immune sensors that are highly influenced by the gut environment and play pivotal roles in maintaining intestinal immune homeostasis. In this study we show that modifications of the gut microbiota induced by oral treatment with Lactobacillaceae-enriched probiotic VSL#3, alone or in combination with retinoic acid (RA), protect NOD mice from T1D by affecting inflammasome at the intestinal level. In particular, we show that VSL#3 treatment inhibits IL-1βexpression while enhancing release of protolerogenic components of the inflammasome, such as indoleamine 2,3-dioxygenase (IDO) and IL-33. Those modifications of the intestinal microenvironment in VSL#3-treated NOD mice modulate gut immunity by promoting differentiation of tolerogenic CD103+DCs and reducing differentiation/expansion of Th1 and Th17 cells in the intestinal mucosa and at the sites of autoimmunity, that is, within the pancreatic lymph nodes (PLN) of VSL#3-treated NOD mice. Our data provide a link between dietary factors, microbiota composition, intestinal inflammation, and immune homeostasis in autoimmune diabetes and could pave the way for new therapeutic approaches aimed at changing the intestinal microenvironment with probiotics to counterregulate autoimmunity and prevent T1D.

To understand the dynamic viral evolution observed during failure on raltegravir-containing regimens, we studied the genotypic and phenotypic patterns of resistance to raltegravir and the residual replication capacity (rRC) of HIV-1 variants selected in vivo.Clonal genotypic analyses were performed on sequential HIV-1 integrase sequences amplified from 11 failing patients and sampled every 4-24 weeks for up to 64 weeks. Fully replicating recombinant viruses were generated using modified vectors in which selected viral integrase genes amplified from patients' plasma were cloned. rRC was measured by a novel multiple cycle competition assay. Resistance to raltegravir and the rRC of resistant HIV-1 variants selected in vivo were evaluated in purified CD4+ T cells.In all of the patients but one, failure was associated with selection of mutations in positions 143, 148 or 155. Unlike mutations at position 143 (Y143S/K/R), identified alone or in combination with others, mutations at position 148 and 155 were always found in combination. A wide range of resistance levels to raltegravir [from 10- to 770-fold change in 50% inhibitory concentration (IC(50)) compared with baseline] was observed using recombinant viral clones. Finally, rRC was not significantly altered in highly resistant variants.Two patterns of viral evolution were observed in the resistant viral populations, driving the variants towards a fast (most of them with G140S + Q148H mutations) or progressive increase in resistance to raltegravir. These results may have implications either for the evaluation of genotypic results, or for the correct clinical use of the compound.

HIV-1 integrase (IN) active site inhibitors are the latest class of drugs approved for HIV treatment. The selection of IN strand-transfer drug-resistant HIV strains in patients supports the development of new agents that are active as allosteric IN inhibitors. Here, a docking-based virtual screening has been applied to a small library of natural ligands to identify new allosteric IN inhibitors that target the sucrose binding pocket. From theoretical studies, kuwanon-L emerged as the most promising binder and was thus selected for biological studies. Biochemical studies showed that kuwanon-L is able to inhibit the HIV-1 IN catalytic activity in the absence and in the presence of LEDGF/p75 protein, the IN dimerization, and the IN/LEDGF binding. Kuwanon-L also inhibited HIV-1 replication in cell cultures. Overall, docking and biochemical results suggest that kuwanon-L binds to an allosteric binding pocket and can be considered an attractive lead for the development of new allosteric IN antiviral agents. Docking simulations exploring a small library of natural compounds, together with biological studies, allowed kuwanon-L to be identified as a new HIV-1 integrase (IN) inhibitor with an allosteric mode of action. Kuwanon-L can thus be considered an attractive lead for the development of new allosteric IN antiviral agents.

Most RNA viruses have evolved mechanisms to avoid neutralizing antibody responses, and it is generally believed that variability of envelope-encoding regions is the major molecular basis of this phenomenon. However, it has been hypothesized that other mechanisms can be involved. Recent experimental data indicate that in hepatitis C virus (HCV) infection, the anti-envelope humoral response includes cross-reactive antibody clones able to neutralize vesicular stomatitis virus (VSV) pseudotypes containing HCV E1 and E2 glycoproteins (HCV/VSV pseudotype) as well as other clones devoid of such activity. In this work, we demonstrate that natural infection with a large variety of HCV isolates belonging to different genotypes elicits HCV/VSV pseudotype-neutralizing cross-reactive anti-envelope antibodies together with clones unable to neutralize this pseudovirus. This was shown by designing a novel strategy for quantitation of serum antibodies binding selectively to single viral cross-reactive conformational epitopes. These data can be useful not only for a better understanding of the virus–host interplay in important viral diseases, but also for the development of an effective anti-HCV vaccine.

During the recent severe acute respiratory syndrome (SARS) outbreak, the etiologic agent was identified as a new coronavirus (CoV). We have isolated a SARS-associated CoV (SARS-CoV) strain by injecting Vero cells with a sputum specimen from an Italian patient affected by a severe pneumonia; the patient traveled from Vietnam to Italy in March 2003. Ultrastructural analysis of infected Vero cells showed the virions within cell vesicles and around the cell membrane. The full-length viral genome sequence was similar to those derived from the Hong-Kong Hotel M isolate. By using both real-time reverse transcription-polymerase chain reaction TaqMan assay and an infectivity plaque assay, we determined that approximately 360 viral genomes were required to generate a PFU. In addition, heparin (100 mg/mL) inhibited infection of Vero cells by 50%. Overall, the molecular and biologic characteristics of the strain HSR1 provide evidence that SARS-CoV forms a fourth genetic coronavirus group with distinct genomic and bioloaic features.

"Coronary atherosclerosis, the main condition predisposing to acute myocardial infarction, has an inflammatory component caused by stimuli that are yet unknown. We molecularly investigated the nature of the immune response within human coronary lesion in four coronary plaques obtained by endoluminal atherectomy from four patients. We constructed phage-display libraries containing the IgG1\/kappa antibody fragments produced by B-lymphocytes present in each plaque. By immunoaffinity, we selected from these libraries a monoclonal antibody, arbitrarily named Fab7816, able to react both with coronary and carotid atherosclerotic tissue samples. We also demonstrated by confocal microscopy that this monoclonal antibody recognized human transgelin type 1, a cytoskeleton protein involved in atherogenesis, and that it co-localized with fibrocyte-like cells transgelin+, CD68+, CD45+ in human sections of coronary and carotid plaques. In vitro fibrocytes obtained by differentiating CD14+ cells isolated from peripheral blood mononuclear cells also interacted with Fab7816, thus supporting the hypothesis of a specific recognition of fibrocytes into the atherosclerotic lesions. Interestingly, the same antibody, cross-reacted with the outer membrane proteins of Proteus mirabilis and Klebsiella pneumoniae (and possibly with homologous proteins of other enterobacteriaceae present in the microbiota). From all the other three libraries, we were able to clone, by immunoaffinity selection, human monoclonal antibodies cross-reacting with bacterial outer membrane proteins and with transgelin. These findings demonstrated that in human atherosclerotic plaques a local cross-reactive immune response takes place."

Objectives: It is currently debated whether patients with residual viraemia are at higher risk of virological failure than those attaining ,1 HIV RNA copy/mL. We therefore investigated the effect of residual viraemia on virological rebound. Methods: We used a prospective, non-interventional, single-centre, study. This analysis was based on HIVinfected patients with two consecutive HIV RNA viral loads (VLs) of ,50 copies/mL as tested by Versant bDNA, followed by two HIV RNA VLs of ,50 copies/mL as tested using the Versant kinetic PCR molecular system (kPCR; limit of quantification ¼ 1 copy/mL). Virological rebound was defined as two consecutive HIV RNA values of .50 copies/mL after baseline, and the time to virological rebound was calculated using the Kaplan ‐ Meier method. Results: There were 739 eligible patients; 446 (60.4%) had HIV RNA ,1 copy/mL (group A) and 293 (39.6%) had residual viraemia (1 ‐49 HIV RNA copies/mL; group B). After a follow-up (median 48.9 weeks), virological rebound occurred in four patients in group A (0.9%) and six patients in group B (2%); the time to virological rebound was similar in the two groups (log-rank test P ¼ 0.231). CD4+ cell recovery (slope) was significantly less in the patients with residual viraemia; +14.3 (27.7, 43.9) cells/mm 3 per year versus +21.2 (22.5, 53.2) cells/mm 3 per year; P ¼0.036. Conclusions: Residual viraemia assessed by kPCR was not associated with virological rebound during 1 year of follow-up. However, the patients attaining ,1 HIV RNA copy/mL showed a small but statistically significant improvement in CD4+ cell recovery.

Although, the antiviral activity, tolerability and convenience of protease inhibitors have improved significantly in recent years, toxicity-associated adverse events including diarrhea, lipid alterations, disturbance of glucose homeostasis and liver enzyme elevations still remain a major concern during treatment of HIV-1 patients. We have recently shown that the covalent attachment of the NO moiety to the HIV-1 protease inhibitor saquinavir (Saq–NO) reduces its toxicity. In this study, we evaluated in vitro the anti-HIV activity of Saq–NO vs. its parental compound Saq. Site directed mutants with the most frequently identified Saq associated resistance mutations and their combinations were generated on proviral AD8-based backbones. Phenotypic assays were conducted using wild type clinical isolates and fully replicating recombinant viruses with Saq and Saq–NO in parallel on purified CD4+ T cells. The following recombinant viruses were generated and tested: L33F, M46I, G48V, I54V, I84V + L90M, M46I + L90M, G48V + L90M, M46I + I54V + L90M, L33F + M46I + L90M. The fold change resistance compared to the wild type viruses was between 1.3 and 7 for all single mutants, between 3.4 and 20 for double mutants and between 16.7 and 28.5 for viruses carrying three mutations for both compounds. The results clearly demonstrate that Saq–NO maintains an anti-HIV-1 profile very similar to that of Saq. The possibility to reduce Saq associated side effects and to increase the concentration of the drug in vivo may allow a higher and possibly more effective dosage of Saq–NO in HIV-1-infected patients and to increase the genetic barrier of this PI thus impairing the selection of resistant clones.

The new H1N1 swine-origin influenza virus (S-OIV) strain is a global health problem. The elucidation of the virus–host relationship is crucial for the control of the new infection. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned before the emergence of S-OIV from a patient who had a broad-range H1N1 serum neutralizing activity. The two HMabs neutralized all tested H1N1 strains, including S-OIV and a swine strain with IC50 ranging from 2 to 7 μg/ml. Data demonstrate that infection with previously circulating H1N1 strains can elicit antibodies neutralizing S-OIV. Finally, the human genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered being potentially useful in the prophylaxis and the treatment of this human infection.

Recent data indicated that adaptive immunity is involved in the process of atherogenesis. Oligoclonal recruitment of T lymphocytes has been described in coronary plaques of patients with acute coronary syndrome. However, the nature of immune response remains to be determined. In the present study, we examined the Ab response in six coronary plaques obtained by endoluminal directional atherectomy. The IgG1/κ-coding gene repertoires of B lymphocytes present in circulating blood and in coronary plaques were cloned and analyzed. In all of the six plaques, we observed 1) a skewed usage of heavy and light IgG1/κ Ab-coding genes, 2) an oligoclonal distribution of VK, JK, and VH, DH, and JH genes with overrepresentation of some rarely used IgG genes, and 3) the unequivocal signs of Ag-driven clonal expansion and evolution of B cells. The data document for the first time the presence of a local Ag-driven clonal evolution of B cells in human atherosclerotic plaques.

Objective : Evaluate HIV-1 subtype B integrase gene evolution in patients failing raltegravir (RAL)-based savage regimens by clonal analysis of the replicating viral quasispecies. Design : Seven triple class failure HIV-1 (subtype B)-infected patients, followed at San Raffaele Hospital and enrolled in the RAL Expanded Access Program (MK0518-023), were evaluated. Patients were followed up for 24-48 weeks and due to the absence of other active drugs, RAL was maintained in their regimens even if resistance mutations were detected. Methods : Immunologic and virologic parameters were recorded every 4 weeks, and amplification and clonal analysis of viral populations were performed at baseline and every 4-12 weeks in all patients. Results : Resistance to RAL appeared initially associated with selection of single variants (Y143R, Q148R N155H) in the majority of patients; however, in three patients, complex patterns of viral mutations were observed. The clonal analysis of viral quasispecies allowed to describe the evolution of each viral population and the progressive accumulation of RAL resistance-associated mutations and polymorphisms associated with therapy failure. Conclusion : The complex patterns of resistance mutations observed, including novel variants evolved under continuous RAL pressure, suggesting that they are the result of the equilibrium between drug resistance and enzyme function. Despite the efficacy of this compound, our data discourage its use in a functional monotherapy and maintaining RAL even in presence of RAL resistance-associated mutations may lead to the progressive formation of viral reservoirs with multiple integrase inhibitor-resistant variants that may limit the future efficacy of other integrase inhibitors due to cross-resistance.

The characteristics of intra-host human immunodeficiency virus type 1 (HIV-1) env evolution were evaluated in untreated HIV-1-infected subjects with different patterns of disease progression, including 2 normal progressor [NP], and 5 Long term non-progressor [LTNP] patients. High-resolution phylogenetic analysis of the C2-C5 env gene sequences of the replicating HIV-1 was performed in sequential samples collected over a 3–5 year period; overall, 301 HIV-1 genomic RNA sequences were amplified from plasma samples, cloned, sequenced and analyzed. Firstly, the evolutionary rate was calculated separately in the 3 codon positions. In all LTNPs, the 3rd codon mutation rate was equal or even lower than that observed at the 1st and 2nd positions (p = 0.016), thus suggesting strong ongoing positive selection. A Bayesian approach and a maximum-likelihood (ML) method were used to estimate the rate of virus evolution within each subject and to detect positively selected sites respectively. A great number of N-linked glycosylation sites under positive selection were identified in both NP and LTNP subjects. Viral sequences from 4 of the 5 LTNPs showed extensive positive selective pressure on the CD4-binding site (CD4bs). In addition, localized pressure in the area of the IgG-b12 epitope, a broad neutralizing human monoclonal antibody targeting the CD4bs, was documented in one LTNP subject, using a graphic colour grade 3-dimensional visualization. Overall, the data shown here documenting high selective pressure on the HIV-1 CD4bs of a group of LTNP subjects offers important insights for planning novel strategies for the immune control of HIV-1 infection.

Antibodies against conserved epitopes on HIV-1 envelope glycoproteins (Env), such as the gp120 CD4-binding site (CD4bs), could contribute to protection against HIV-1. Env-based immunogens inducing such a response could be a major component of future anti-HIV-1 strategies. In this proof-of-concept study we describe the generation of two anti-idiotype (AI) murine antibodies mimicking the CD4bs epitope. Sera were collected from long-term non-progressor patients to obtain CD4bs-directed IgG, through sequential purification steps. The purified IgG were then used as Fab fragments to immunize mice for hybridoma generation. Two hybridomas (P1 and P2), reacting only against the CD4bs-directed IgG, were identified and characterized. The P1 and P2 antibodies were shown to recognize the idiotype of the broadly neutralizing anti-CD4bs human mAb b12. Both P1 and P2 Fabs were able to induce a strong anti-gp120 response in rabbits. Moreover, the rabbits' sera were shown to neutralize two sensitive tier 1 strains of HIV-1 in an Env-pseudotype neutralization assay. In particular, 3/5 rabbits in the P1 group and 1/5 in the P2 group showed greater than 80% neutralizing activity against the HXB2 pseudovirus. Two rabbits also neutralized the pseudovirus HIV-MN. Overall, these data describe the first anti-idiotypic vaccine approach performed to generate antibodies to the CD4bs of the HIV-1 gp120. Although future studies will be necessary to improve strength and breadth of the elicited neutralizing response, this proof-of-concept study documents that immunogens designed on the idiotype of broadly neutralizing Abs are feasible and could help in the design of future anti-HIV strategies.

A prospective 2‐year analysis including 322 infant patients with acute respiratory disease (ARD) hospitalized in a pediatric department in northern Italy was carried out to evaluate the role as respiratory pathogens or co‐pathogens of recently identified viruses. The presence of respiratory syncitial virus (RSV), human Metapneumoviruses (hMPVs), human Bocaviruses (hBoVs), and human Coronaviruses (hCoVs) was assayed by molecular detection and clinical symptoms evaluated. Nasopharyngeal aspirates from 150 of the 322 infants (46.6%) tested positive for at least one pathogen. Ninety samples (28.0%) tested positive for RSV RNA (61.5% genotype A and 38.5% genotype B), 46 (14.3%) for hMPV RNA (71.7% subtype A and 28.3% subtype B), 28 (8.7%) for hCoV RNA (39.3% hCoV‐OC43, 35.7% hCoV‐NL63, 21.4% hCoV‐HKU1, and 3.6% hCoV‐229E), and 7 (2.2%) for hBoV DNA (of the 6 typed, 50% subtype 1 and 50% subtype 2); 21/150 samples revealed the presence of 2 or more viruses. Co‐infection rates were higher for hMPVs, hCoVs, and hBoV (38.3%, 46.4%, and 57.1%,) and lower for RSV (23.3%). RSV was associated with the presence of complications (P

A real-time PCR assay targeting the highly specific erm34 sequence of Bacillus clausii DNAwas developed and optimized. The quantitative assay showed a sensitivity level of 10 2 CFU/Al of sample. The method may represent a useful tool for monitoring the role of B. clausii as probiotic in vivo. D 2005 Elsevier B.V. All rights reserved.

Sequential nasopharyngeal aspirates from patients without respiratory symptoms undergoing hematopoietic stem cell transplantation (HSCT) were tested for genomic RNA of human metapneumovirus (hMPV). Persistent hMPV infection was documented in most of them and confirmed by virus isolation. hMPV infection etiology was also evaluated during the same period in samples from pediatric patients with acute respiratory diseases (ARDs). Sequence analysis of hMPV in HSCT recipients documented infection by hMPV genotype A and strong interhost similarity; this pattern differs from that observed in pediatric patients with ARDs. The data indicate that HSCT recipients may frequently develop symptomless hMPV infection.

To understand the evolutionary characteristics of HIV- 1 variants resistant to protease inhibitors ( PI), the replicating plasma virus was analyzed in three patients shifted to a PI- sparing regimen after virological failure. The dynamic features of carryover of mutations associated with PI resistance in the absence of selective pressure on the protease gene indicate that viral variants resistant to reverse transcriptase inhibitors and bearing mutations of the protease sequence can maintain efficient replication capacity in vivo.

The objective of this study was to address the evolution of human immunodeficiency virus type 1 (HIV-1) mutations resistant to the integrase inhibitor raltegravir after drug interruption. Thirteen HIV-1 infected patients undergoing virological failure due to the selection of raltegravir-resistant variants, who had interrupted raltegravir treatment, were enrolled. For all patients, the virological failure was associated with the selection of variants, with mutations conferring resistance to all of the drugs present in their regimens. Patients were prospectively monitored at baseline (raltegravir interruption) and every 4–24 weeks for clinical, virological and immunological parameters, including HIV-1 viraemia, CD4 + T-cell counts, and sequence analysis of the HIV-1 integrase sequence. Reversion to the wild-type HIV-1 integrase sequence genotype was observed between 4 and 36 weeks after raltegravir withdrawal in eight out of the 13 patients. Reversion was not observed in three patients. In two patients, reversion was partial at week 24 from raltegravir interruption. These results highlight that in eight out of 13 patients under treatment with raltegravir and experiencing a virological failure, HIV-1 variants harbouring mutations associated with raltegravir resistance become undetectable after drug interruption within a few weeks (in some cases, very rapidly). This occurs under different therapy regimens and in patients receiving 3TC mono-therapy. In the other patients, complete reversion of the integrase sequence is not observed, and either primary or secondary resistance mutations are fixed in the replication competent viral population in vivo also for long time, suggesting that other factors may influence this dynamic process.