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SARS-CoV-2 spike is evolving to maximize transmissibility and evade the humoral response. The massive genomic sequencing of SARS-CoV-2 isolates has led to the identification of single-point mutations and deletions, often having the recurrence of hotspots, associated with advantageous phenotypes. We report the isolation and molecular characterization of a SARS-CoV-2 strain, belonging to a lineage (C.36) not previously associated with concerning traits, which shows decreased susceptibility to vaccine sera neutralization.

INTRODUCTION Treatment of Diffuse Large B cell lymphoma (DLBCL) in the elderly population is challenging as many patients (pts) are not eligible to receive standard curative therapy, due to comorbid conditions and to a higher susceptibility to the side effects of standard anthracycline containing regimens. Among currently available active drugs, Lenalidomide has been used in the setting of relapsed/refractory DLBCL both as monotherapy and in combination with rituximab, showing a good activity and an acceptable safety profile. We started a prospective, multicenter, single arm, phase II trial to demonstrate activity and safety of a chemo-free combination of lenalidomide + rituximab in older (≥ 70 years) untreated pts with DLBCL who were prospectively defined as frail according to the simplified comprehensive geriatric assessment (sCGA) (Tucci A. et al., Leuk Lymphoma. 2015 Apr). PATIENTS AND METHODS Pts were eligible if they were previously untreated DLBCL patients, older than 69 years and defined as frail according to sCGA. The treatment consisted of a 28-day cycle (R2) combining oral Lenalidomide (20 mg on days 1 to 21) and i.v. Rituximab (375 mg/m2 on day 1); a maximum number of 6 cycles was planned; response assessment was performed after cycles 4 and 6. At the end of the 6 th cycle, patients with partial or complete response continued treatment with Lenalidomide 10mg/d on days 1 to 21 every 28 days, until cycle 12or unacceptable toxicity. Final response was evaluated within 28 days after the last study drug administration. Primary study endpoint was Overall Response Rate (ORR) after 6 R2 cycles, defined according to Lugano 2014 criteria; co-primary endpoint was the rate of extra-hematological toxicity with CTCAE grade >2 and of death for any cause during the treatment The study was planned according to a two stage Simon design. A total of 68 pts had to be enrolled to complete the study. With 34 responses the study would be able to demonstrate the initial hypothesis of an improvement of response rate from 45% (p0) to 65% (P1) RESULTS From August 2018 to June 2021, 68 newly diagnosed frail DLBCL were enrolled in 18 Italian centers. Median age was 83 years (range 70-91) and 73% had stage III/IV; 57% had High risk IPI (i.e. 3-5 risk factors). 64 pts were confirmed eligible and started R2 treatment. The planned 6 courses of R2 were completed in 36 pts (56%). The median number of R2 cycles was 6 (1-6). Treatment was discontinued in 28 pts due to lymphoma progression (11 cases), extra-hematological toxicity (7), hematological toxicity (1), lost after 1 cycle and investigator choice after 5 cycles in CR in 1 case each, death in 7 cases (1 each for infection, pancytopenia, cachexia, ischemia and bowel infarction, and from unknown causes in 2 cases). At the end of 6 th R2 cycle 26 patients achieved a response (ORR 41%), 13 CR and 13 PR. After a median follow-up of 17 months, the overall survival, and progression free survival at 12 months were 69% (95CI 56-79%) and 55% (95%CI 42-66%), respectively. The duration of remission at 12 months was 72%. Regarding safety 46 events were reported including 31 extra-hematological toxicities > CTCAE grade 2: 7 cardio-vascular (1 resulting in death), 4 nervous system disorders (1 resulting in death), 10 gastro-intestinal, 3 infections, 6 skin and subcutaneous tissue disorders, 5 respiratory events (all resolved). The rate of grade 3-4 adverse events was higher than the highest allowed limit of 28. CONCLUSIONS The Reri is the first study to evaluate activity and safety of a chemo-free therapy in patients with diffuse large B-Cell Lymphoma who are not eligible for conventional cytotoxic therapy. Even if this study was not able confirm the initial activity hypothesis, activity of the R2 combination was observed in a significant proportion of cases warranting further exploration of chemo-free approach of elderly frail patient with DLBCL. Disclosures Tucci: janssen: Membership on an entity's Board of Directors or advisory committees; Gentili: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Fabbri: Takeda: Honoraria; Servier/Pfizer: Honoraria; Takeda: Other: Travel, Accomodations, Expenses. Musuraca: janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Zilioli: Takeda: Other: travel expenses, accommodation; Gentili, Takeda, Gilead, Servier: Consultancy, Speakers Bureau; Roche, Italfarmaco: Consultancy, Honoraria; MSD, Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations. Luminari: Roche, Celgene, Teva Pharmaceuticals, Gilead Sciences, and Takeda Pharmaceuticals: Honoraria.

In 54 adult stem cell transplant recipients, the presence and persistence of human rhinoviruses (including the novel lineage C) were evaluated by molecular detection and phylogenetic analysis, independently from respiratory symptoms. In the same group of patients, the presence of other coinfecting respiratory pathogens, including the novel enterovirus 109, was also evaluated.

Maraviroc (MVC) is licensed in clinical practice for patients with R5 virus and virological failure; however, in anecdotal reports, dual/mixed viruses were also inhibited. We retrospectively evaluated the evolution of HIV-1 coreceptor tropism in plasma and peripheral blood mononuclear cells (PBMCs) of an infected adolescent with a CCR5/CXCR4 Trofile profile who experienced an important but temporary immunological and virological response during a 16-month period of MVC-based therapy. Coreceptor usage of biological viral clones isolated from PBMCs was investigated in U87.CD4 cells expressing wild-type or chimeric CCR5 and CXCR4. Plasma and PBMC-derived viral clones were sequenced to predict coreceptor tropism using the geno2pheno algorithm from the V3 envelope sequence and pol gene-resistant mutations. From start to 8.5 months of MVC treatment only R5X4 viral clones were observed, whereas at 16 months the phenotype enlarged to also include R5 and X4 clones. Chimeric receptor usage suggested the preferential usage of the CXCR4 coreceptor by the R5X4 biological clones. According to phenotypic data, R5 viruses were susceptible, whereas R5X4 and X4 viruses were resistant to RANTES and MVC in vitro. Clones at 16 months, but not at baseline, showed an amino acidic resistance pattern in protease and reverse transcription genes, which, however, did not drive their tropisms. The geno2pheno algorithm predicted at baseline R5 viruses in plasma, and from 5.5 months throughout follow-up only CXCR4-using viruses. An extended methodological approach is needed to unravel the complexity of the phenotype and variation of viruses resident in the different compartments of an infected individual. The accurate evaluation of the proportion of residual R5 viruses may guide therapeutic intervention in highly experienced patients with limited therapeutic options.

INTRODUCTION Treatment of Diffuse Large B cell lymphoma (DLBCL) in the elderly population is challenging as many patients (pts) are not eligible to receive standard curative therapy, due to comorbid conditions and to a higher susceptibility to the side effects of standard anthracycline containing regimens. Among currently available active drugs, Lenalidomide has been used in the setting of relapsed/refractory DLBCL both as monotherapy and in combination with rituximab, showing a good activity and an acceptable safety profile. We started a prospective, multicenter, single arm, phase II trial to demonstrate activity and safety of a chemofree combination of lenalidomide + rituximab in elderly (≥ 70 years) untreated pts with DLBCL who were prospectively defined as frail according to a simplified comprehensive geriatric assessment (sCGA) (Tucci A. et al., Leuk Lymphoma. 2015 Apr). PATIENTS AND METHODS Pts were eligible if they were previously untreated DLBCL patients, older than 69 years and defined as frail according to sCGA. The treatment consisted of a 28-day cycle (R2) combining oral Lenalidomide (20 mg on days 1 to 21) and i.v. Rituximab (375 mg/m2 on day 1); a maximum number of 6 cycles was planned; response assessment was performed after cycles 4 and 6. At the end of the 6thcycle, patients with partial or complete response continued treatment with Lenalidomide 10mg/d on days 1 to 21 every 28 days, until cycle 12or unacceptable toxicity. Final response was evaluated within 28 days after the last study drug administration. Primary study endpoint was Overall Response Rate (ORR) after 6 R2 cycles, defined according to Lugano 2014 criteria; co-primary endpoint was the rate of extra-hematological toxicity with CTCAE grade >2 and of death for any cause during the treatment The study was planned according to a two stage Simon design. A total of 68 pts had to be enrolled to complete the study: 23 pts were required in the first stage. Second stage could be activated withat least 12 patients showing a Partial or Complete Response (PR/CR) in stage I. According to the Ray and Rai method less than 15/23 adverse events were also required for the safety coprimary endpoint. RESULTS From January 2017 to December 2017, 24 newly diagnosed frail DLBCL were enrolled in 8 Italian centers. Median age was 83 years (range 76-89) and 79% had stage III/IV; 42% of pts were male, and 44%, had elevated LDH, 45% had High risk IPI (i.e. 3-5 risk factors). All pts were confirmed eligible and started R2 treatment. The planned 6 courses of R2 were completed in 13 pts (54%). The median number of R2 cycles was 6 (1-6). Treatment was discontinued in 11 pts for the following reasons: lymphoma progression in 4 cases, second malignancy in 2, extra-hematological toxicity in 3 cases, consent withdrawal and investigator choice after 4 cycles in CR in 1 case each. Response assessment after 6 R2 cycles showed12 responding pts (ORR 50%), 4 CR and 8 PR, that was higher than by the inferior limit required by the Simon optimal design. Regarding safety coprimary endpoint 13 events were reported including 9 extra-hematological toxicities > grade 2 CTCAE (3 cardio-vascular and 6 respiratory events, all resolved) and 4 deaths (2 patients due visceral arterial ischemia and 2 due infectious disease). The rate of adverse events was lower than the superior limit of 15 allowed the first stage of the study.Since August 2018 the enrollment in the stage II of the trial has been resumed and it is currently ongoing with 45 patients enrolled. CONCLUSIONS The ReRi is the first study to evaluate activity and safety of a chemo-free therapy in patients with diffuse large B-Cell Lymphoma who are not eligible for conventional cytotoxic therapy. The results of the planned interim analysis of our study confirmed the initial efficacy and safety hypotheses of R2 combination in untreated elderly pts with DLBCL. Treatment of elderly frail DLBCL pts with R2 holds promise in terms of both ORR and safety. Disclosures Bassan: Amgen Inc.: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Shire: Honoraria. Merli:Sandoz: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Janssen: Honoraria; Takeda: Honoraria, Other: Travel Expenses; Gilead: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Mundipharma: Honoraria. Liberati:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Clinical trial support; Roche: Other: Clinical trial support; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical trial support; Celgene: Honoraria, Other: Clinical trial support; Bristol-Myers Squibb: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Janssen: Honoraria; Novartis: Other: Clinical trial support. Luminari:ROCHE: Other: Role as Advisor ; CELGENE: Other: Role as Advisor & Travel Grant; TAKEDA: Other: Travel Grant; GILEAD: Other: Lecturer .

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.

ObjectivesAlthough founder viruses in primary HIV-1 infections (PHIs) typically use the CCR5 coreceptor (R5-tropic), 3%–19% of subjects also harbour CXCR4-using viruses (X4-tropic), making tropism determination before CCR5 antagonist usage mandatory. Genotypic methods can be used to accurately determine HIV-1 tropism in chronically infected patients.MethodsWe compared the results of genotypic methods [geno2pheno, PSSMx4r5 including a novel nucleotide-input version (ntPSSM) and distant segments (ds)Kernel] to predict coreceptor usage in a cohort of 67 PHIs. Specimens with discrepant results were phenotypically tested after cloning the V3 gene region into proviral backbones. Recombinant viruses were used to infect U87 indicator cell lines bearing CD4 and either CCR5 or CXCR4.ResultsGeno2pheno10%, PSSMx4r5 and (ds)Kernel gave identical predictions in 85% of cases. Geno2pheno10% predicted the presence of CXCR4 viruses in 18% of patients. Two patients were predicted to carry X4-tropic viruses by all algorithms and X4-tropic viruses were detected in at least one of the recombinant AD8 or NL4-3 backbone-based assays. Ten samples resulted in discordant predictions with at least one algorithm. Full concordance between tropism prediction by using population sequencing and phenotypic assays was observed only with ntPSSM. Geno2pheno prediction and the phenotypic assay gave the same results in a minority of ‘discordant’ patients.ConclusionsCompared with both PSSMx4r5 versions, (ds)Kernel and our phenotypic assay, geno2pheno10% overestimated the frequency of X4-tropic viruses (18% versus 3%). ntPSSM was able to detect one additional X4 virus compared with (ds)Kernel that was confirmed with the phenotypic assay.

A study aiming at cloning and characterizing natural antibodies to human immunodeficiency virus type 1 (HIV-1) targets is described. In particular, we report the molecular cloning of a Fab molecule binding the HIV-1/Tat protein from a seronegative patient. The Fab was characterized for its binding specificity and investigated in regards to its molecular structure. Furthermore, to evaluate the role played by the heavy and light chains in the binding to the antigen, hybrid Fabs were constructed combining the heavy and the light chain of the natural anti-Tat clone with a control high-affinity Fab derived from the repertoire of the same patient. The results indicate that the natural immunoglobulin under study: (i) is a polyreactive antibody of IgG1 isotype, and not an IgM as usually described for anti-HIV natural clones, (ii) shows a pattern of mutations compatible with an antigen-driven mechanisms, (iii) its heavy chain derives from a V-gene subfamily (V3-23) highly represented in fetal life, and (iv) its heavy chain variable region exhibits several characteristics, including an extremely long, hydrophilic CDR3, that are unusual and theoretically important in determining the polyreactive capacity of the molecule.

Background A novel human enterovirus (HEV) type within the species HEV-C, named EV109, was discovered from cases of respiratory illness in Nicaragua in September 2010. The aim of this study, was to retrospectively examine the presence and the role of EV109 in respiratory samples from two patients populations; infants below the age of 2 years, hospitalized for acute respiratory diseases (ARDs) and adult hematopoietic stem cell transplantation recipients. Results A total of 1149 nasopharingeal aspirates were collected and tested for the presence of EV109 by reverse transcription-PCR (RT-PCR). In positive samples, the presence of the most common respiratory viruses was also assayed and clinical symptoms were evaluated. Samples from 2 of the 974 infants tested positive for EV109 RNA (0.2%) and belonged to patients with lower ARDs; co-infection with other viral pathogens under study was observed in both cases. In transplant recipients, one out of the 175 samples analyzed, from a patients with upper respiratory simptoms tested positive for HEV 109 in the absence of co-infecting viruses. Sequence analysis of amplified EV109 genomic regions, showed only a few nucleotide differences when compared with the Nicaraguan strains. Conclusions Overall these results indicate that HEV109 variants have circulated and differentiated in different lineages worldwide. Although more cases and larger studies are needed, HEV109 infection may be associated to ARDs both in infants and in hematopoietic stem cell transplantation recipients. If these preliminary observations will be confirmed, improved molecular methods with a wider panel of potential pathogens will be useful for monitoring these categories of patients.

We report here the synthesis of 2-aminothiazolones along with their biological properties as novel anti-HIV agents. Such compounds have proven to act through the inhibition of the gp120-CD4 protein-protein interaction that occurs at the very early stage of the HIV-1 entry process. No cytotoxicity was found for these compounds, and broad antiviral activities against laboratory strains and pseudotyped viruses were documented. Docking simulations have also been applied to predict the mechanism, at the molecular level, by which the inhibitors were able to interact within the Phe43 cavity of HIV-1 gp120. Furthermore, a preliminary absorption, distribution, metabolism, and excretion (ADME) evaluation was performed. Overall, this study led the basis for the development of more potent HIV entry inhibitors.

BACKGROUND The incidence of non Hodgkin's lymphoma (NHL) increases with age, over one third of NHL cases involves elderly patients >70 years of age. As a matter of fact, many elderly patients (pts) are not enrolled in controlled studies because they do not meet the inclusion criteria. This is the reason why the data from trials on elderly cases are not representative for the whole elderly population. Consequently, many of these patients do not benefit of new therapeutic progresses and the treatment is not yet adequate. Moreover in case of relapse, salvage therapy in the elderly is virtually absent, and the prognosis is extremely poor. Lenalidomide is an immunomodulatory drug with anti-angiogenetic and anti-neoplastic action on cancer cells and also has other anti-tumor activity by acting on the neoplastic microenvironment. Both monotherapy and combination of Lenalidomide with rituximab have shown efficacy in terms of overall response rate (ORR, Overall Response Rate) in the setting of salvage therapy in relapsed/refractory DLBCL, with an acceptable rate of hematologic and extra-hematological toxicities PATIENTS AND METHODS In the period 2013-2014 we consecutively treated 12 elderly patients affected by advanced DLBCL relapsed/refractory. Median age at the start of treatment was 79 years (yrs) (62-86), 5 out 12 was over 80 yrs and 4 out 12 over 75 yrs. The median number of previous treatment was 3 (2-4), 3 pts were transplanted and 4 patients was refractory to previous line of therapy. The treatment scheme included at first cycle: Rituximab 375 mg/m2 i.v. days 1, 8, 15, 22, Desametasone 5 mg p.o. days 1, 8, 15, 22 and Lenalidomide 15 mg/die p.o. from day 2 to 22. From the second to the sixth cycle we administered Rituximab 375 mg/m2 i.v. days 1, 14, Lenalidomide 20 mg/die p.o. from day 2 to 22 RESULTS The overall response rate has been 75% (CR,PR,SD), 3 patients out 12 (25%) achieved a CR and 3 PR (25%). The median PFS was 6 months and with a median follow up of 1 year the overall survival is 25%.(fig.1) All deaths are due to lymphoma progression and the 3 patients in CR are still alive in CCR. CONCLUSIONS In this elderly and heavily pretreated group of patients our scheme containing Lenalidomide-Rituximab has shown an high rate of response with a good rate of CR and a promising trend in overall survival. The treatment appears feasible and safe also in this particular group of frail patients and even if we need more data to confirm we think it will be possible to extend this therapy in frail elderly patients in a most precocious line of treatment. Figure 1. Figure 1. Disclosures Offidani: Celgene, Janssen: Honoraria.

Polyomaviruses KI (KIPyV) and WU (WUPyV) were described recently in children with acute respiratory disease. The pathogenic potential of these human viruses has not been determined completely, but a correlation between immunosuppression and virus reactivation has been suggested. In the present study, the association between KI/WUPyV infection and immunosuppression was investigated using sequential nasopharyngeal aspirates from asymptomatic adult hematopoietic stem cell transplant recipients. In parallel, an investigation on the WU/KIPyV prevalence in children with acute respiratory disease was also carried out. Two of the 126 samples obtained from the 31 hematopoietic transplant recipients were positive for KIPyV (1 sample, 0.79%) and WUPyV (1 sample, 0.79%). Both samples were obtained 15 days after allogeneic transplantation and virus persistence was not observed in subsequent samples. In symptomatic children, 7 of the 486 nasopharyngeal aspirates were positive for WUPyV (1.4%) and 1 for KIPyV (0.2%). Single polyomavirus infection was detected in four patients, whereas the remaining patients were co‐infected with respiratory syncityal virus (three patients) or adenovirus (one patient). The results suggest that WU/KIPyVs have a limited circulation in Italy and a low pathogenic potential in young children. Brief and asymptomatic infection can occur in hematopoietic transplant recipients. J. Med. Virol. 82:153–156, 2010. © 2009 Wiley‐Liss, Inc.

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.

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.

Severe acute respiratory syndrome (SARS) is a systemic disease characterized by both lung pathology and widespread extrapulmonary virus dissemination causing multiple organ injuries. In this regard, renal dysfunction is an ominous sign in patients with SARS. Indeed, clusters of SARS coronavirus (SARS-CoV) particles have been detected in the cytoplasm of renal tubular epithelial cells in postmortem studies, explaining the presence of infectious virus in the urine of SARS patients. In order to investigate the potential SARS-CoV kidney tropism, we have evaluated the susceptibility of human renal cells of tubular and glomerular origin to in vitro SARS-CoV infection. Immortalized cultures of differentiated proximal tubular epithelial cells (PTEC), glomerular mesangial cells (MC), and glomerular epithelial cells (podocytes) were found to express the SARS-CoV receptor angiotensin-converting enzyme 2 on their surface. Productive infection, however, occurred only in PTEC but not in glomerular cells. A transient infection with poor virus production was observed in MC, whereas podocytes were not permissive to SARS-CoV infection. In contrast to the cytopathic infection of the Vero E6 cell line, SARS-CoV did not cause overt cytopathic effects in PTEC or MC. Of interest, PTEC, but not MC, maintained stable levels of SARS-CoV production in serial subcultures, suggesting a persistent state of infection. In this regard, a SARS-CoV variant with increased replication capacity in PTEC was selected after four serial subculture passages. This SARS-CoV variant acquired a single nonconservative amino acid change from glutamic acid (E) to alanine (A) at position 11 in the viral membrane (M) protein. The E11A point mutation was sufficient for enhanced SARS-CoV replication and persistence in PTEC when introduced in a SARS-CoV recombinant infectious clone. These findings indicate that human PTEC may represent a site of SARS-CoV productive and persistent replication favoring the emergence of viral variants with increased replication capacity, at least in these kidney cells.

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

In this single-centre, retrospective study, we analyzed data of 194 patients receiving antiretroviral therapy with

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.

Anthracyclines (AC) still constitute the mainstay of the 1st line treatment in lymphoma: their use, however, is limited by the occurrence of Cardiac Toxicity (CT). The exact prevalence of AC CT occurring after widely used regimens such as R-CHOP or ABVD is unknown and there is uncertainty about the best monitoring method and possible prophylactic or therapeutic interventions. METHODS: We started a prospective observational trial in lymphoma patients undergoing treatment with conventional or liposomal AC. We used a comprehensive approach to monitor for AC CT, using a telemedicine(TM) system integrating echocardiography, ECG and biomarkers (Troponin I - TnI). RESULTS: In this final analysis, 95 patients completed the planned treatment (52 males and 43 females). Median age was 56.03 years (range 19.1 to 78.5 years), and 36 patients were > 65 years. 23 were HL and 72 NHL (DLBCL was the most represented subtype with 47 cases). Liposomal AC was used in 31 patients and classical AC in 64, with mean cumulative doses of 283.33 and 272.76 mg/sqm, respectively. 10/95 patients (11%) developed a TnI rise above 0.08 ng/ml and 39/95 (41%) above 0.03 ng/ml. With both cut-offs, the rises occurred more frequently at cumulative doses >200 mg/sqm. The major arising occurred in the group underwent classical AC, while in the group underwent liposomal AC although the value before first infusion was in 10% more than 0.03 at the cumulative dose of 300 mg /sqm there's a plateau of troponin value. Thanks to this monitoring system we noticed 2 Acute cardiac toxicity events with resolution in 100% of cases. Furthermore in those cases where has registered a subclinical CT has begun a prompt Cardiological therapy by ACE inhibitors and Beta Blocker to reduce the relative risk of Cardiological events. CONCLUSIONS: Even with low cumulative doses, with a median follow up of 13 months, subclinical signs of AC CT were found in at least 11% of patients. The use of liposomal AC allow the safe treatment of patients with a previous heart disease diagnosis rather it seem protective in the higher cumulative doses. A longer follow up will be able to clarify the impact of arising of TnI more than 0.03 and 0.08 on the developing of AC CT in all our series. On the basis of our experience a multicentric trial has begun on behalf Italian Lymphoma Foundation (FIL) In a low-risk setting for AC CT, a monitoring strategy combining clinical, imaging, instrumental and biomarker data seems to enhance the sensitivity of separate methods. This strategy is feasible and resource-saving thanks to the integration in a TM system. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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.