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Serological testing for the detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as an important component of the clinical management of patients with coronavirus disease 2019 (COVID-19) as well as the epidemiological assessment of SARS-CoV-2 exposure worldwide. In addition to molecular testing for the detection of SARS-CoV-2 infection, clinical laboratories have also needed to increase testing capacity to include serological evaluation of patients with suspected or known COVID-19. While regulatory approved serological immunoassays are now widely available from diagnostic manufacturers globally, there is significant debate regarding the clinical utility of these tests, as well as their clinical and analytical performance requirements prior to application. This document by the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Taskforce on COVID-19 provides interim guidance on: (A) clinical indications and target populations, (B) assay selection, (C) assay evaluation, and (D) test interpretation and limitations for serological testing of antibodies against SARS-CoV-2 infection. These evidence-based recommendations will provide practical guidance to clinical laboratories in the selection, verification, and implementation of serological assays and are of the utmost importance as we expand our pandemic response from initial case tracing and containment to mitigation strategies to minimize resurgence and further morbidity and mortality.

The present study focused on inhibition of HSV-1 and -2 replication and pathogenesis in vitro and in vivo, through the selective targeting of the envelope glycoprotein D. Firstly, a human monoclonal antibody (Hu-mAb#33) was identified that could neutralise both HSV-1 and -2 at nM concentrations, including clinical isolates from patients affected by different clinical manifestations and featuring different susceptibility to acyclovir in vitro. Secondly, the potency of inhibition of both infection by cell-free viruses and cell-to-cell virus transmission was also assessed. Finally, mice receiving a single systemic injection of Hu-mAb#33 were protected from death and severe clinical manifestations following both ocular and vaginal HSV-1 and -2 lethal challenge. These results pave the way for further studies reassessing the importance of HSV entry as a novel target for therapeutic intervention and inhibition of cell-to-cell virus transmission.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system (CNS), observed in immunodeficient patients and caused by JC virus ((JCV), also called JC polyomavirus (JCPyV)). After the HIV pandemic and the introduction of immunomodulatory therapy, the PML incidence significantly increased. The correlation between the use of natalizumab, a drug used in multiple sclerosis (MS), and the PML development of particular relevance. The high incidence of PML in natalizumab-treated patients has highlighted the importance of two factors: the need of PML risk stratification among natalizumab-treated patients and the need of effective therapeutic options. In this review, we discuss these two needs under the light of the major viral models of PML etiopathogenesis.

Background: The cellular adaptive response directed against herpesviruses is widely described in the scientific literature as a pivotal component of the immune system able to control virus replication. The role of humoral immunity remains unclear and controversial. Aims: Discussing the role of adaptive immunity in herpesvirus infection control, highlighting the potential role of the humoral branch of immunity through the description of human monoclonal antibodies directed against herpesviruses. Sources: PubMed search for relevant publications related to protective immunity against Herpesviridae. Content: This review describes the role of adaptive immunity directed against Herpesviridae, focusing on the human humoral response naturally elicited during their infections. Given the ever-increasing interest in monoclonal antibodies as novel therapeutics, the contribution of humoral immunity in controlling productive infection, during both primary infection and reactivations, is discussed. Implications: Human monoclonal antibodies directed against the different Herpesviridae species may represent novel molecular probes to further characterize the molecular machinery involved in herpesvirus infection; and allow the development of novel therapeutics and effective vaccine strategies. N. Clementi, CMI 2017;▪:1 © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved

The emergence of new influenza strains causing pandemics represents a serious threat to human health. From 1918, four influenza pandemics occurred, caused by H1N1, H2N2 and H3N2 subtypes. Moreover, in 1997 a novel influenza avian strain belonging to the H5N1 subtype infected humans. Nowadays, even if its transmission is still circumscribed to avian species, the capability of the virus to infect humans directly from avian reservoirs can result in fatalities. Moreover, the risk that this or novel avian strains could adapt to inter-human transmission, the development of resistance to anti-viral drugs and the lack of an effective prevention are all incumbent problems for the world population. In this scenario, the identification of broadly neutralizing monoclonal antibodies (mAbs) directed against conserved regions shared among influenza isolates has raised hopes for the development of monoclonal antibody-based immunotherapy and “universal” anti-influenza vaccines.

The global burden of herpes simplex virus (HSV) legitimates the critical need to develop new prevention strategies, such as drugs and vaccines that are able to fight either primary HSV infections or reactivations. Moreover, the ever-growing number of patients receiving transplants increases the number of severe HSV infections that are unresponsive to current therapies. Finally, the high global incidence of genital HSV-2 infection increases the risk of perinatal transmission to newborns, in which disseminated infection or central nervous system (CNS) involvement is frequent, with associated high morbidity and mortality rates. There are several key features shared by novel anti-HSV drugs, from currently available optimized drugs to small molecules able to interfere with various virus replication steps. However, several virological aspects of the disease and associated clinical needs highlight why an ideal anti-HSV drug has yet to be developed. © 2016 Elsevier Ltd. All rights reserved.

Objective The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). Design Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3 zeta domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). Results In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon gamma, interleukin 2 and tumour necrosis factor a. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. Conclusions Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool.

JC virus (JCPyV) has gained novel clinical importance as cause of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease recently associated to immunomodulatory drugs, such as natalizumab used in multiple sclerosis (MS) cases. Little is known about the mechanisms leading to PML, and this makes the need of PML risk stratification among natalizumab-treated patients very compelling. Clinical and laboratory-based risk-stratification markers have been proposed, one of these is represented by the JCPyV-seropositive status, which includes about 54% of MS patients. We recently proposed to investigate the possible protective role of neutralizing humoral immune response in preventing JCPyV reactivation. In this proof-of-concept study, by cloning the first human monoclonal antibody (GRE1) directed against a neutralizing epitope on JCPyV/VP1, we optimized a robust anti-JCPyV neutralization assay. This allowed us to evaluate the neutralizing activity in JCPyV-positive sera from MS patients, demonstrating the lack of correlation between the level of anti-JCPyV antibody and anti-JCPyV neutralizing activity. Relevant consequences may derive from future clinical studies induced by these findings; indeed the study of the serum anti-JCPyV neutralizing activity could allow not only a better risk stratification of the patients during natalizumab treatment, but also a better understanding of the pathophysiological mechanisms leading to PML, highlighting the contribution of peripheral versus central nervous system JCPyV reactivation. Noteworthy, the availability of GRE1 could allow the design of novel immunoprophylactic strategies during the immunomodulatory treatment.Copyright © 2014 Elsevier B.V. All rights reserved.

"\"Monoclonal antibodies (mAbs) have been used successfully both in research and for clinical purposes. The possible use of protective mAbs directed against different microbial pathogens is currently being considered. The fine definition of the epitope recognized by a protective mAb is an important aspect to be considered for possible development in epitope-based vaccinology. The most accurate approach to this is the X-ray resolution of mAb\\\/antigen crystal complex. Unfortunately, this approach is not always feasible. Under this perspective, several surrogate epitope mapping strategies based on the use of bioinformatics have been developed. In this article, we review the most common, freely accessible, bioinformatic tools used for epitope characterization and provide some basic examples of molecular visualization, editing and computational analysis.\""

"\"\\\"\\\\\\\"\\\\\\\\\\\\\\\"In the last two decades, several phage display-selected monoclonal antibodies (mAbs) have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV) Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses.\\\\\\\\\\\\\\\"\\\\\\\"\\\"\""

Background Biobanks are imperative infrastructures, particularly during outbreaks, when there is an obligation to acquire and share knowledge as quick as possible to allow for implementation of science-based preventive, diagnostic, prognostic and therapeutic strategies. Methods We established a COVID-19 biobank with the aim of collecting high-quality and well-annotated human biospecimens, in the effort to understand the pathogenic mechanisms underlying COVID-19 and identify therapeutic targets (COVID-BioB, NCT04318366). Here we describe our experience and briefly review the characteristics of the biobanks for COVID-19 that have been so far established. Results A total of 46,677 samples have been collected from 913 participants (63.3% males, median [IQR] age 62.2 [51.2 - 74.0] years) since the beginning of the program. Most patients (66.9%) had been admitted to hospital for COVID-19, with a median length of stay of 15.0 (9.0 - 27.0) days. A minority of patients (13.3% of the total) had been admitted for other reasons and subsequently tested positive for SARS-CoV-2. The remainder were managed at home after being seen at the Emergency Department. Conclusions Having a solid research infrastructure already in place, along with flexibility and adaptability to new requirements, allowed for the quick building of a COVID-19 biobank that will help expand and share the knowledge of SARS-CoV-2.

Both emerging viruses and well-known viral pathogens endowed with neurotropism can either impair directly the neuronal functions or induce physio-pathological changes by diffusing from the periphery through neurosensory-epithelial connections. However, the current lack of anin vitrosystem modeling the connectivity between human neurons and peripheral tissues excludes the analysis of viral latency and reactivation and the assessment of natural/artificial induced anti-viral immunity. In this study, we developed the first stable topographic neurosensory-epithelial connection on-a-chip using human stem cell derived dorsal root ganglia (DRG) sensory neurons. Bulk and single cell transcriptomics showed that different combinations of key receptors for Herpes Simplex Virus 1 (HSV-1) are expressed by each sensory neuronal cell type. This neuronal-epithelial circuitry enabled a detailed analysis of the HSV infectivity faithfully modeling its dynamics and cell type specificity. The reconstitution of an organized connectivity between human sensory neurons and keratinocytes into microfluidic chips provides for the first time a powerfulin vitroplatform to model viral latency and reactivation of human viral pathogens.

Several viruses target the human respiratory tract, causing different clinical manifestations spanning from mild upper airway involvement to life-threatening acute respiratory distress syndrome (ARDS). As dramatically evident in the ongoing SARS-CoV-2 pandemic, the clinical picture is not always easily predictable due to the combined effect of direct viral and indirect patient-specific immune-mediated damage., SUMMARY Several viruses target the human respiratory tract, causing different clinical manifestations spanning from mild upper airway involvement to life-threatening acute respiratory distress syndrome (ARDS). As dramatically evident in the ongoing SARS-CoV-2 pandemic, the clinical picture is not always easily predictable due to the combined effect of direct viral and indirect patient-specific immune-mediated damage. In this review, we discuss the main RNA (orthomyxoviruses, paramyxoviruses, and coronaviruses) and DNA (adenoviruses, herpesviruses, and bocaviruses) viruses with respiratory tropism and their mechanisms of direct and indirect cell damage. We analyze the thin line existing between a protective immune response, capable of limiting viral replication, and an unbalanced, dysregulated immune activation often leading to the most severe complication. Our comprehension of the molecular mechanisms involved is increasing and this should pave the way for the development and clinical use of new tailored immune-based antiviral strategies.

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.

Effective and economical measures are needed to either prevent or inhibit the replication of SARS-CoV-2, the causative agent of COVID-19, in the upper respiratory tract. As fumigation of vinegar at low concentration (0.34%) ameliorated the symptoms of mild SARS-CoV-2 infection, we tested in vitro the potential antiviral activity of vinegar and of its active component, acetic acid. We here demonstrate that both vinegar and acetic acid indeed strongly inactivate SARS-CoV-2 infectivity in Vero cells. Furthermore, vinegar treatment caused a 90% inhibition of the infectious titer when directly applied to a nasopharyngeal swab transfer medium of a COVID-19 patient. These effects were potentiated if conduced at a temperature of 45 °C vs. 37 °C, a condition that is transiently generated in the upper respiratory tract during fumigation. Our findings are consistent and extend the results of studies performed in the early and mid-20th century on the disinfectant capacity of organic acids and can provide an affordable home-made aid to prevent or contain SARS-CoV-2 infection of the upper respiratory tract.

Background Platelet activation and thrombotic events characterizes COVID‐19. Objectives To characterize platelet activation and determine if SARS‐CoV‐2 induces platelet activation. Patients/Methods We investigated platelet activation in 119 COVID‐19 patients at admission in a university hospital in Milan, Italy, between March 18 and May 5, 2020. Sixty‐nine subjects (36 healthy donors, 26 patients with coronary artery disease, coronary artery disease, and seven patients with sepsis) served as controls. Results COVID‐19 patients had activated platelets, as assessed by the expression and distribution of HMGB1 and von Willebrand factor, and by the accumulation of platelet‐derived (plt) extracellular vesicles (EVs) and HMGB1+ plt‐EVs in the plasma. P‐selectin upregulation was not detectable on the platelet surface in a fraction of patients (55%) and the concentration of soluble P‐selectin in the plasma was conversely increased. The plasma concentration of HMGB1+ plt‐EVs of patients at hospital admission remained in a multivariate analysis an independent predictor of the clinical outcome, as assessed using a 6‐point ordinal scale (from 1 = discharged to 6 = death). Platelets interacting in vitro with SARS‐CoV‐2 underwent activation, which was replicated using SARS‐CoV‐2 pseudo‐viral particles and purified recombinant SARS‐CoV‐2 spike protein S1 subunits. Human platelets express CD147, a putative coreceptor for SARS‐CoV‐2, and Spike‐dependent platelet activation, aggregation and granule release, release of soluble P‐selectin and HMGB1+ plt‐EVs abated in the presence of anti‐CD147 antibodies. Conclusions Hence, an early and intense platelet activation, which is reproduced by stimulating platelets in vitro with SARS‐CoV‐2, characterizes COVID‐19 and could contribute to the inflammatory and hemostatic manifestations of the disease.

Severe COVID-19 is characterized by overproduction of immune mediators, but the role of interferons (IFNs) of the type I (IFN-I) or type III (IFN-III) families remains debated. We scrutinized the production of IFNs along the respiratory tract of COVID-19 patients and found that high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity. Production of specific IFN-III, but not IFN-I, members, denotes patients with a mild pathology and efficiently drives the transcription of genes that protect against SARS-CoV-2. In contrast, compared to subjects with other infectious or non-infectious lung pathologies, IFNs are over-represented in the lower airways of patients with severe COVID-19 that exhibit gene pathways associated with increased apoptosis and decreased proliferation. Our data demonstrate a dynamic production of IFNs in SARS-CoV-2-infected patients and show IFNs play opposing roles at distinct anatomical sites., An in-depth analysis of interferons in COVID-19 reveals differences in their roles based on anatomical location, viral load, age and disease severity. In the upper respiratory tract, high levels of IFN-III are protective and result in mild disease in spite of higher SARS-CoV-2 viral burden while the lower airways of patients with severe COVID-19 demonstrate elevated IFN-I and III, cell death and a reduction in interferon stimulated genes.

Hepatitis C virus (HCV) is a major causative agent of acute and chronic hepatitis. It is estimated that 400,000 people die every year from chronic HCV infection, mostly from severe liver-related diseases such as cirrhosis and liver cancer. Although HCV was discovered more than 30 years ago, an efficient prophylactic vaccine is still missing. The HCV glycoprotein complex, E1/E2, is the principal target of neutralizing antibodies (NAbs) and, thus, is an attractive antigen for B-cell vaccine design. However, the high genetic variability of the virus necessitates the identification of conserved epitopes. Moreover, the high intrinsic mutational capacity of HCV allows the virus to continually escape broadly NAbs (bNAbs), which is likely to cause issues with vaccine-resistant variants. Several studies have assessed the barrier-to-resistance of vaccine-relevant bNAbs in vivo and in vitro. Interestingly, recent studies have suggested that escape substitutions can confer antibody resistance not only by direct modification of the epitope but indirectly through allosteric effects, which can be grouped based on the breadth of these effects on antibody susceptibility. In this review, we summarize the current understanding of HCV-specific NAbs, with a special focus on vaccine-relevant bNAbs and their targets. We highlight antibody escape studies pointing out the different methodologies and the escape mutations identified thus far. Finally, we analyze the antibody escape mechanisms of envelope protein escape substitutions and polymorphisms according to the most recent evidence in the HCV field. The accumulated knowledge in identifying bNAb epitopes as well as assessing barriers to resistance and elucidating relevant escape mechanisms may prove critical in the successful development of an HCV B-cell vaccine.

Plenty of serologic tests for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) have been developed so far, thus documenting the importance of evaluating the relevant features of the immune response to this viral agent. The performance of these assays is currently under investigation. Amongst them, LIAISON® SARS‐CoV‐2 S1/S2 IgG by DiaSorin and Elecsys Anti‐SARS‐CoV‐2 cobas® by Roche are currently used by laboratory medicine hospital departments in Italy and many other countries. In the present study, we firstly compared two serologic tests on serum samples collected at two different time points from 46 laboratory‐confirmed coronavirus disease‐2019 (COVID‐19) subjects. Secondly, 85 negative serum samples collected before the SARS‐CoV‐2 pandemic were analyzed. Thirdly, possible correlations between antibody levels and the resulting neutralizing activity against a clinical isolate of SARS‐CoV‐2 were evaluated. Results revealed that both tests are endowed with low sensitivity on the day of hospital admission, which increased to 97.8% and 100% for samples collected after 15 days for DiaSorin and Roche tests, respectively. The specificity evaluated for the two tests ranges from 96.5% to 100%, respectively. Importantly, a poor direct correlation between antibody titers and neutralizing activity levels was evidenced in the present study. These data further shed light on both potentials and possible limitations related to SARS‐CoV‐2 serology. In this context, great efforts are still necessary for investigating antibody kinetics to develop novel diagnostic algorithms. Moreover, further investigations on the role of neutralizing antibodies and their correlate of protection will be of paramount importance for the development of effective vaccines., Highlights A comparative analysis between two serologic assays for the detection of antibodies directed against SARS‐CoV‐2 was carried out on sera from subjects testing positive for SARS‐CoV‐2 nasopharyngeal swabs.Results revealed that both tests are endowed with low sensitivity on the day of hospital admission, which increased for samples collected after 15 days.A poor direct correlation between antibody titers and neutralizing activity levels was evidenced in the present study.

Routine biochemical and hematological tests have been reported to be useful in the stratification and prognostication of pediatric and adult patients with diagnosed coronavirus disease (COVID-19), correlating with poor outcomes such as the need for mechanical ventilation or intensive care, progression to multisystem organ failure, and/or death. While these tests are already well established in most clinical laboratories, there is still debate regarding their clinical value in the management of COVID-19, particularly in pediatrics, as well as the value of composite clinical risk scores in COVID-19 prognostication. This document by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on COVID-19 provides interim guidance on: (A) clinical indications for testing, (B) recommendations for test selection and interpretation, (C) considerations in test interpretation, and (D) current limitations of biochemical/hematological monitoring of COVID-19 patients. These evidence-based recommendations will provide practical guidance to clinical laboratories worldwide, underscoring the contribution of biochemical and hematological testing to our collective pandemic response.

Background: The role of microbiota in Lynch syndrome (LS) is still under debate. We compared oral and fecal microbiota of LS saliva and stool samples with normal healthy controls (NHC). Methods: Total DNA was purified from feces and saliva to amplify the V3–V4 region of the 16s rRNA gene. Sequences with a high-quality score and length >250 bp were used for taxonomic analysis with QIIME software. Results: Compared to NHC, LS fecal samples demonstrated a statistically significant increase of Bacteroidetes and Proteobacteria and a significant decrease of Firmicutes at the phylum level and of Ruminococcaceae at the family level. Moreover, LS oral samples exhibited a statistically significant increase of Veillonellaceae and Leptotrichiaceae and a statistically significant decrease of Pasteurellaceae. A beta-diversity index allowed differentiation of the two groups. Conclusions: A peculiar microbial signature is associated with LS, similar to that of sporadic colorectal cancer and Crohn’s disease. These data suggest a possible role of proinflammatory bacteria in tumor development in a condition of genetic predisposition, such as LS.

Comprehensive characterization of the urinary and urothelium-bound microbiomes in bladder cancer (BCa) and healthy state is essential to understand how these local microbiomes may play a role in BCa tumorigenesis and response to therapy, as well as to explain sex-based differences in BCa pathobiology. Performing 16 s rDNA microbiome analysis on 166 samples (urine and paired bladder tissues) from therapy-naive BCa patients undergoing radical cystectomy and healthy controls, we defined (1) sex-specific microbiome differences in the urine and bladder tissue, and (2) representativeness of the tissue microenvironment by the voided urinary microbiome. The genus Klebsiella was more common in the urine of female BCa patients versus healthy controls, while no clinically relevant bacteria were found differently enriched in men. In tissues, the genus Burkholderia was more abundant in the neoplastic versus the non-neoplastic tissue in both sexes, suggesting a potential role in BCa pathobiology. Lastly, we found that the urinary microbiome shares >80% of the bacterial families present in the paired bladder tissue, making the urinary microbiome a fair proxy of the tissue bacterial environment. Patient summary We identified specific bacteria present in the urine and tissues of male and female bladder cancer patients. These novel data represent a first step toward understanding the influence of the bladder microbiome on the development of bladder cancer and on the response to intravesical and systemic therapies.

While the SARS-CoV-2 pandemic is heavily hitting the world, it is of extreme importance that significant in vitro observations guide the quick set up of clinical trials. In this study, we evidence that the anti-SARS-CoV2 activity of a clinically achievable hydroxychloroquine concentration is maximized only when administered before and after the infection of Vero E6 and Caco-2 cells. This suggests that only a combined prophylactic and therapeutic use of hydroxychloroquine may be effective in limiting viral replication in patients.

Objectives A milder clinical course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been anecdotally reported over the latest phase of COVID-19 pandemic in Italy. Several factors may contribute to this observation, including the effect of lockdown, social distancing, lower humidity, lower air pollution, and potential changes in the intrinsic pathogenicity of the virus. In this regard, the clinical severity of COVID-19 could be attenuated by mutations in SARS-CoV-2 genome that decrease its virulence, as well as by lower virus inocula. Methods In this pilot study, we compared the reverse transcription polymerase chain reaction (RT-PCR) amplification profile of 100 nasopharyngeal swabs consecutively collected in April, during the peak of SARS-CoV-2 epidemic, to that of 100 swabs collected using the same procedure in May. Results The mean Ct value of positive samples collected in May was significantly higher than that of samples collected in the previous period (ORF 1a/b gene: 31.85 ± 0.32 vs. 28.37 ± 0.5, pE gene: 33.76 ± 0.38 vs. 29.79 ± 0.63, p Conclusions This pilot study prompts further investigation on the correlation between SARS-CoV-2 load and different clinical manifestation of COVID-19 during different phases of the pandemic. Laboratories should consider reporting quantitative viral load data in the molecular diagnosis of SARS-CoV-2 infection.

Background Vaginal and seminal microbiomes have gained increasing interest for their involvement in reproductive health and fertility. However, their role in reproductive outcome is not fully understood yet. In this study, we aimed to correlate the vaginal and the seminal microbiome of 23 couples with idiopathic infertility to the clinical pregnancy rate after intrauterine insemination (IUI). Methods Vaginal swabs and seminal fluids were collected on the day of IUI procedure and analyzed through polymerase chain reaction amplification of variable regions 3 and 4 (V3–V4) of 16S ribosomal ribonucleic acid genes and Illumina MiSeq sequencing. The taxonomic data were then correlated to IUI success. Results Idiopathic infertile women showed a different average composition of vaginal microbiome compared with control sequences, whereas for seminal counterpart no relevant differences were observed. Furthermore, among idiopathic infertile women, different patterns of Lactobacillus species dominations were observed, with a predominance either of Lactobacillus crispatus, a marker of a healthy vaginal ecosystem, or of Lactobacillus iners and Lactobacillus gasseri, associated with a more dysbiosis-prone environment. More important, considering all investigated variables, vaginal L crispatus domination was the only factor strongly associated to IUI success (P = .0002). Conclusions Our results strengthen the potential role of L crispatus in promoting a favorable environment for pregnancy and suggest that microbiome characterization could be useful, together with standard clinical and laboratory assessments, in the pre-IUI evaluation of infertile couples.

STUDY QUESTION: Does the presence of human papillomavirus (HPV) in semen impact seminal parameters and sperm DNA quality in white European men seeking medical help for primary couple's infertility? SUMMARY ANSWER: HPV seminal infections involving high-risk (HR) genotypes are associated with impaired sperm progressive motility and sperm DNA fragmentation (SDF) values. WHAT IS KNOWN ALREADY: HPV is commonly present in semen samples. However, whether the presence of HPV in semen is actually associated with impaired sperm parameters and SDF values have yet to be elucidated. STUDY DESIGN, SIZE, DURATION: In this cross-sectional study, complete demographic, clinical and laboratory data from 729 infertile men were analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Health-significant comorbidities were scored with the Charlson comorbidity index (CCI). Serum hormones and SDF index (measured by the sperm chromatin structure assay [SCSA]) were measured in every patient (SDF ≥30% was defined as pathological). Semen analysis was based on 2010 World Health Organisation reference criteria. Amplification by nested PCR was used to detect HPV-DNA sequences in semen samples. Descriptive statistics and linear regression models were used to test the association between the presence of HPV and clinical and seminal characteristics in the whole cohort. MAIN RESULTS AND THE ROLE OF CHANCE: The overall rate of HPV positivity was 15.5% (113/729). Overall, 78/729 (10.7%) and 35/729 (4.8%) patients had HR HPV+ and low-risk HPV+, respectively. HPV16 was the most prevalent type (22.1%), followed by HPV43 (10.6%), HPV56 and HPV42 (both 8.8%). No differences were found in terms of clinical and hormonal characteristics between patients with or without seminal HPV. Sperm progressive motility was significantly lower (P = 0.01) while SDF values were higher (P = 0.005) in HPV+ men compared to those with no HPV. In particular, HR HPV+ men had lower sperm progressive motility (P = 0.007) and higher SDF values (P = 0.003) than those with a negative HPV test. Univariable analysis showed that HR HPV+ was associated with impaired sperm progressive motility (P = 0.002) and SDF values (P = 0.003). In the multivariable analysis, age, FSH levels and testicular volume were significantly associated with impaired sperm progressive motility (all P ≤ 0.04). Conversely BMI, CCI, smoking habits and HPV status were not. Only age (P = 0.02) and FSH (P = 0.01) were significantly associated with SDF, after accounting for BMI, CCI, testicular volume, smoking habits and HPV status. LIMITATIONS, REASONS FOR CAUTION: Main limitations are the cross-sectional design of our study and the relatively small sample size of the subgroups. Additional limitations are the lack of a control group of normal fertile men and the lack of follow-up testing to check the clearance or the persistence of HPV in semen after a 6-12 months. WIDER IMPLICATIONS OF THE FINDINGS: Overall, these observations point out the importance of an accurate investigation of seminal HPV presence in everyday clinical practice in the diagnostic work-up of infertile men. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used. There are no competing interests.

Key Points The interplay between tumour cells and the extracellular microenvironment has been recognized as one of the key determinants of cancer development and progressionInteraction between extracellular matrix components and bacterial products controls tissue homeostasis; its dysregulation might prepare protumorigenic environmental niches, which could also favour disease relapseBladder colonization with specific bacterial genera throughout an individual's lifetime might influence the propensity for bladder pathology and partly explain the gender differences in the rate of urinary diseasesExtracellular-matrix-based and microbiota-based biomarkers might be new prognostic factors in bladder cancer, exploitable for risk stratification, tumour staging and for predicting relapse and outcomeTargeting the bladder extracellular matrix or the associated microbiota might bypass treatment resistance mechanisms of tumour cells Supplementary information The online version of this article (doi:10.1038/nrurol.2015.292) contains supplementary material, which is available to authorized users., The interaction between tumour cells and their microenvironment has an important role in cancer pathogenesis. Alfano et al. review how dysregulation of the extracellular matrix and microbiota associated with the human epithelium might influence the development and progression of urothelial carcinomas. Supplementary information The online version of this article (doi:10.1038/nrurol.2015.292) contains supplementary material, which is available to authorized users., Many pathological changes in solid tumours are caused by the accumulation of genetic mutations and epigenetic molecular alterations. In addition, tumour progression is profoundly influenced by the environment surrounding the transformed cells. The interplay between tumour cells and their microenvironment has been recognized as one of the key determinants of cancer development and is being extensively investigated. Data suggest that both the extracellular matrix and the microbiota represent microenvironments that contribute to the onset and progression of tumours. Through the introduction of omics technologies and pyrosequencing analyses, a detailed investigation of these two microenvironments is now possible. In urological research, assessment of their dysregulation has become increasingly important to provide diagnostic, prognostic and predictive biomarkers for urothelial bladder cancer. Understanding the roles of the extracellular matrix and microbiota, two key components of the urothelial mucosa, in the sequelae of pathogenic events that occur in the development and progression of urothelial carcinomas will be important to overcome the shortcomings in current bladder cancer treatment strategies. Supplementary information The online version of this article (doi:10.1038/nrurol.2015.292) contains supplementary material, which is available to authorized users.

PN-SIA28 is a human monoclonal antibody (Hu-MAb) targeting highly conserved epitopes within the stem portion of the influenza virus hemagglutinin (HA) (N. Clementi, et al, PLoS One 6:e28001, 2011, http://dx.doi.org/10.1371/journal.pone.0028001). Previous in vitro studies demonstrated PN-SIA28 neutralizing activities against phylogenetically divergent influenza A subtypes. In this study, the protective activity of PN-SIA28 was evaluated in mice inoculated with lethal influenza A/WSN/33 (H1N1), A/Quebec/144147/09 (H1N1)pdm09, and A/Victoria/3/75 (H3N2) viruses. At 24 h postinoculation (p.i.), animals received PN-SIA28 intraperitoneally (1 or 10 mg/kg of body weight) or 10 mg/kg of unrelated Hu-MAb (mock). Body weight loss and mortality rate (MR) were recorded for 14 days postinfection (p.i.). Lung viral titers (LVT) were determined at day 5 p.i. In A/WSN/33 (H1N1)-infected groups, all untreated and mock-receiving mice died, whereas MRs of 87.5% and 25% were observed in mice that received PN-SIA28 1 and 10 mg/kg, respectively. In influenza A(H1N1) pdm09-infected groups, an MR of 75% was recorded for untreated and mock-treated groups, whereas the PN-SIA28 1-mg/kg and 10-mg/kg groups had rates of 62.5% and 0%, respectively. In A/Victoria/3/75 (H3N2)-infected animals, untreated and mock-treated animals had MRs of 37.5% and 25%, respectively, and no mortalities were recorded after PN-SIA28 treatments. Accordingly, PN-SIA28 treatments significantly reduced weight losses and resulted in a ≥1-log reduction in LVT compared to the control in all infection groups. This study confirms that antibodies targeting highly conserved epitopes in the influenza HA stem region, like PN-SIA28, not only neutralize influenza A viruses of clinically relevant subtypes in vitro but also, more importantly, protect from a lethal influenza virus challenge in vivo. Copyright © 2015, American Society for Microbiology. All Rights Reserved. PN-SIA28 is a human monoclonal antibody (Hu-MAb) targeting highly conserved epitopes within the stem portion of the influenza virus hemagglutinin (HA) (N. Clementi, et al, PLoS One 6:e28001, 2011, http://dx.doi.org/10.1371/journal.pone.0028001). Previous in vitro studies demonstrated PN-SIA28 neutralizing activities against phylogenetically divergent influenza A subtypes. In this study, the protective activity of PN-SIA28 was evaluated in mice inoculated with lethal influenza A/WSN/33 (H1N1), A/Quebec/144147/09 (H1N1)pdm09, and A/Victoria/3/75 (H3N2) viruses. At 24 h postinoculation (p.i.), animals received PN-SIA28 intraperitoneally (1 or 10 mg/kg of body weight) or 10 mg/kg of unrelated Hu-MAb (mock). Body weight loss and mortality rate (MR) were recorded for 14 days postinfection (p.i.). Lung viral titers (LVT) were determined at day 5 p.i. In A/WSN/33 (H1N1)-infected groups, all untreated and mock-receiving mice died, whereas MRs of 87.5% and 25% were observed in mice that received PN-SIA28 1 and 10 mg/kg, respectively. In influenza A(H1N1) pdm09-infected groups, an MR of 75% was recorded for untreated and mock-treated groups, whereas the PN-SIA28 1-mg/kg and 10-mg/kg groups had rates of 62.5% and 0%, respectively. In A/Victoria/3/75 (H3N2)-infected animals, untreated and mock-treated animals had MRs of 37.5% and 25%, respectively, and no mortalities were recorded after PN-SIA28 treatments. Accordingly, PN-SIA28 treatments significantly reduced weight losses and resulted in a ≥1-log reduction in LVT compared to the control in all infection groups. This study confirms that antibodies targeting highly conserved epitopes in the influenza HA stem region, like PN-SIA28, not only neutralize influenza A viruses of clinically relevant subtypes in vitro but also, more importantly, protect from a lethal influenza virus challenge in vivo.

Febrile neutropenia and sepsis are common and life-threatening complications in hematological diseases. This study was performed retrospectively in 514 patients treated for febrile neutropenia at our institute, to investigate the clinical usefulness of a molecular tool, LightCycler® SeptiFast test (SF), to promptly recognize pathogens causing sepsis in hematological patients. We collected 1837 blood samples of 514 consecutive hematological patients. The time of processing is short. Overall, 757 microorganisms in 663 episodes were detected by molecular test and standard blood cultures (BC): 73.6% Gram-positive bacteria, 23.9% Gram-negative bacteria, and 2.5% fungal species. This large analysis demonstrated a significant episode-to episode agreement (71.9%) between the two methods, higher in negative samples (89.14%), and a specificity of 75.89%. Clinical variables that gave a statistically significant contribution to their concordance were absolute neutrophil count, ongoing antimicrobial therapy, timing of test execution, and organ localization of infection. The large analysis highlights the potential of molecular-based assays directly performed on blood samples, especially if implementing the detection of antibiotic resistance genes, which was lacking in the used study.

STUDY QUESTION Given the relevant role of the extracellular microenvironment in regulating tissue homeostasis, is testicular bacterial microbiome (BM) associated with germ cell aplasia in idiopathic non-obstructive azoospermia (iNOA)? SUMMARY ANSWER A steady increase of dysbiosis was observed among testis with normal spermatogenesis vs. iNOA with positive sperm retrieval and iNOA with complete germ cell aplasia. WHAT IS KNOWN ALREADY Tissue-associated BM has been reported to be a biologically important extracellular microenvironment component for numerous body habitats, but not yet for the human testis. STUDY DESIGN, SIZE, DURATION Cross-sectional study, investigating tissue-associated BM in the testis of (i) five men with iNOA and negative sperm retrieval at microdissection testicular sperm extraction (microTESE); (ii) five men with iNOA and positive sperm retrieval at microTESE; and (iii) five normozoospermic men upon orchiectomy. Every testicular specimen was histologically classified and analyzed in terms of bacterial community. PARTICIPANTS/MATERIALS, SETTING, METHODS Massive ultra-deep pyrosequencing was applied to investigate testis microbiome. Metagenome was analyzed using Quantitative Insights Into Microbial Ecology (QIIME). Tissue-associated bacterial load was quantified by digital droplet PCR. MAIN RESULTS AND THE ROLE OF CHANCE Normozoospermic men showed small amounts of bacteria in the testis, with Actinobacteria, Bacteroidetes, Firmicutes Proteobacteria as the dominating phyla; iNOA individuals had increased amounts of bacterial DNA (P = 0.02), associated with decreased taxa richness due to the lack of Bacteroidetes and Proteobacteria (P = 2 × 10−5). Specimens with negative sperm retrieval at microTESE depicted complete germ cell aplasia and a further decrease in terms of Firmicutes and Clostridia (P < 0.05), a complete lack of Peptoniphilus asaccharolyticus, but increased amount of Actinobacteria. LIMITATIONS, REASONS FOR CAUTION The limited number of specimens analyzed in this preliminary study deserves external validation. The paraneoplastic microenvironment could have an impact on the residential bacterial flora. WIDER IMPLICATION OF THE FINDINGS Human testicular microenvironment is not microbiologically sterile, containing low amounts of Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. A dysbiotic bacterial community was associated with iNOA and complete germ cell aplasia. Novel findings on testicular BM could support future translational therapies of male-factor infertility. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by URI-Urological Research Institute free funds. Authors declared no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

Background Infections and graft-vs-host disease (GvHD) still represent major, not easily predictable complications in allogeneic hematopoietic stem cell transplant (allo-HSCT). Both conditions have been correlated to altered enteric microbiome profiles during the peritransplant period. The main objective of this study was to identify possible early microbiome-based markers useful in pretransplant risk stratification. Methods Stool samples were collected from 96 consecutive patients at the beginning of the pretransplant conditioning regimen (T0) and at 10 (T1) and 30 (T2) days following transplant. When significant in univariate analysis, the identified microbiome markers were used in multivariate regression analyses, together with other significant clinical variables for allo-HSCT-related risk stratification. Four main outcomes were addressed: (1) septic complications, (2) GvHD, (3) relapse of the underlying disease, and (4) mortality. Results The presence of >5% proinflammatory Enterobacteriaceae at T0 was the only significant marker for the risk of microbiologically confirmed sepsis. Moreover, ≤10% Lachnospiraceae at T0 was the only significant factor for increased risk of overall mortality, including death from both infectious and noninfectious causes. Finally, a low bacterial alpha-diversity (Shannon index ≤ 1.3) at T1 was the only variable significantly correlating with an increased risk of GvHD within 30 days. Conclusions Microbiome markers can be useful in the very early identification of patients at risk for major transplant-related complications, offering new tools for individualized preemptive or therapeutic strategies to improve allo-HSCT outcomes.

BK virus (BKV) associated nephropathy (BKVAN) is still an important cause of allograft dysfunction after kidney transplantation (KT). Recent data have shown that the new interferon (IFN)-λ family has been ascribed antiviral properties similar to IFNα, and that the response to IFNλ in kidney is restricted to epithelial cells, suggesting that the IFNλ system evolves as specific protection of the epithelia. We aimed to test the hypothesis of correlation between a single nucleotide polymorphism (C/T dimorphism rs12979860) in the genomic region of IL28B and BKVAN, in patients after KT. Fifty kidney-transplanted patients were included as follow: Group 1 (BKV+/BKVAN+): 11 patients with active BKV− replication and biopsy-proven BKVAN; Group 2 (BKV+/BKVAN−): 22 patients with active BKV− replication but without evidence of BKVAN; Group 3 (BKV−/BKVAN−): 17 patients without evidence of BKV− replication (control group). Here we show that the C/C genotype was statistically higher in group 2 than in group 1 and BKVAN was detected significantly more frequently in patients with C/T and T/T genotypes than in patients with C/C genotype. We therefore propose IL28B polymorphism (rs12979860), as a predictor-marker to differentiate between patients with self-limited, even if persistent, BKV− reactivation and patients with a high risk of progression towards BKVAN, and to modulate the clinical management of these patients accordingly.

Objectives: The cross-resistance profiles of elvitegravir and dolutegravir on raltegravir-resistant variants is still controversial or not available in macrophages and lack extensive evaluations on wide panels of clonal variants. Thus, a complete evaluation in parallel with all currently available integrase inhibitors (INIs) was performed. Methods: The integrase coding region was RT –PCR-amplified from patient-derived plasma samples and cloned into an HIV-1 molecular clone lacking the integrase region. Twenty recombinant viruses bearing mutations to all primary pathways of resistance to raltegravir were phenotypically evaluated with each integrase inhibitor in freshly purified CD4+ T cells or monocyte-derived macrophages. Results: Y143R single mutants conferred a higher level of raltegravir resistance in macrophages [fold change (FC) 47.7 –60.24] compared with CD4+ T cells (FC 9.55 –11.56). All other combinations had similar effects on viral susceptibility to raltegravir in both cell types. Elvitegravir displayed a similar behaviour both in lymphocytes and macrophages with all the tested patterns. When compared with raltegravir, none to modest increases in resistance were observed for the Y143R/C pathways. Dolutegravir maintained its activity and cross-resistance profile in macrophages. Only Q148H/R variants had a reduced level of susceptibility (FC 5.48–18.64). No variations were observed for the Y143R/C (+/2T97A) or N155H variants. Conclusions: All INIs showed comparable antiretroviral activity in both cell types even if single mutations were associated with a different level of susceptibility in vitro to raltegravir and elvitegravir in macrophages. In particular, dolutegravir was capable of inhibiting with similar potency infection of raltegravir-resistant variants with Y143 or N155 pathways in both HIV-1 major cell reservoirs.

The design of vaccine strategies and the development of drugs targeting the early stages of Hepatitis C virus (HCV) infection are hampered by the lack of structural information about its surface glycoproteins E1 and E2, the two constituents of HCV entry machinery. Despite the recent crystal resolution of limited versions of both proteins in truncated form, a complete picture of the E1E2 complex is still missing. Here we combined deep computational analysis of E1E2 secondary, tertiary and quaternary structure with functional and immunological mutational analysis across E1E2 in order to propose an in silico model for the ectodomain of the E1E2 heterodimer. Our model describes E1-E2 ectodomain dimerization interfaces, provides a structural explanation of E1 and E2 immunogenicity and sheds light on the molecular processes and disulfide bridges isomerization underlying the conformational changes required for fusion. Comprehensive alanine mutational analysis across 553 residues of E1E2 also resulted in identifying the epitope maps of diverse mAbs and the disulfide connectivity underlying E1E2 native conformation. The predicted structure unveils E1 and E2 structures in complex, thus representing a step towards the rational design of immunogens and drugs inhibiting HCV entry.

Context: Increasing evidences suggest a correlation between gut and type 1 diabetes (T1D). Objective: The objective of this study is to evaluate the gut inflammatory profile and microbiota in patients with T1D compared with healthy control (CTRL) subjects and patients with celiac disease (CD) as gut inflammatory disease controls. Design/Setting/Participants: The inflammatory status and microbiome compositionwere evaluated in biopsies of the duodenal mucosa of patients with T1D (n = 19), in patients with CD (n = 19), and CTRL subjects (n = 16) recruited at San Raffaele Scientific Institute, in Milan, Italy, between 2009 and 2015. Main Outcome Measures: Inflammation was evaluated by gene expression study and immunohistochemistry. Microbiome composition was analyzed by 16S ribosomal RNA gene sequencing. Results: An increased expression of CCL13, CCL19, CCL22, CCR2, COX2, IL4R, CD68, PTX3, TNFa, and VEGFA was observed in patientswith T1D compared with CTRL subjects and patientswith CD. Immunohistochemical analysis confirmed T1D-specific inflammatory status compared with healthy and CD control tissues, mainly characterized by the increase of the monocyte/macrophage lineage infiltration. The T1D duodenal mucosal microbiome results were different from the other groups, with an increase in Firmicutes and Firmicutes/Bacteroidetes ratio and a reduction in Proteobacteria and Bacteroidetes. The expression of genes specific for T1D inflammation was associated with the abundance of specific bacteria in the duodenum. Conclusions: This study shows that duodenal mucosa in T1D presents disease-specific abnormalities in the inflammatory profile and microbiota. Understanding the mechanisms underlying these features is critical to disentangle the complex pathogenesis of T1D and to gain new perspectives for future therapies targeting the intestine. (J Clin Endocrinol Metab 102: 1468-1477, 2017).

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.

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.

Human herpesvirus 6 (HHV-6) is increasingly recognized as a potentially life-threatening pathogen in allogeneic hematopoietic stem cell transplantation (alloSCT). We retrospectively evaluated 54 adult patients who developed positivity to HHV-6 after alloSCT. The median time from alloSCT to HHV-6 reactivation was 34 days. HHV-6 was present in plasma samples from 31 patients, in bone marrow (BM) of 9 patients, in bronchoalveolar lavage fluid and liver or gut biopsy specimens from 33 patients, and in cerebrospinal fluid of 7 patients. Twenty-nine patients developed acute graft-versus-host disease (GVHD), mainly grade III-IV, and 15 had concomitant cytomegalovirus reactivation. The median absolute CD3+ lymphocyte count was 207 cells/µL. We reported the following clinical manifestations: fever in 43 patients, skin rash in 22, hepatitis in 19, diarrhea in 24, encephalitis in 10, BM suppression in 18, and delayed engraftment in 11. Antiviral pharmacologic treatment was administered to 37 patients; nonetheless, the mortality rate was relatively high in this population (overall survival [OS] at 1 year, 38% ± 7%). A better OS was significantly associated with a CD3+ cell count ≥200/µL at the time of HHV-6 reactivation (P = .0002). OS was also positively affected by the absence of acute GVHD grade III-IV (P = .03) and by complete disease remission (P = .03), but was not significantly influenced by steroid administration, time after alloSCT, type of antiviral prophylaxis, plasma viral load, or organ involvement. Although HHV-6 detection typically occurred early after alloSCT, better T cell immune reconstitution seems to have the potential to improve clinical outcomes. Our findings provide new insight into the interplay between HHV-6 and the transplanted immune system.

The association between natalizumab and progressive multifocal leukoencephalopathy (PML) is established, but a reliable clinical risk stratification flow-chart is lacking. New risk factors are needed, such as the possible role of the anti-JC polyomavirus (JCPyV) neutralizing antibody. In this pilot study, we analyzed this parameter during natalizumab treatment. Sequential sera of 38 multiple sclerosis patients during their first year of natalizumab treatment were collected, and grouped according to the number of infusions. For 11 patients, samples were also available after 24 infusions (T24), when progressive multifocal leukoencephalopathy (PML) risk is higher. The reactivity against VP1, the main JCPyV surface protein, and the anti-JCPyV neutralizing activity were evaluated. During the first year, a lack of correlation between anti-JCPyV antibody response and its neutralizing activity was observed: a significant decrease in anti-JCPyV antibody response was observed (p = 0.0039), not paralleled by a similar trend in the total anti-JCPyV neutralizing activity (p = 0.2239). This lack of correlation was even more evident at T24 when, notwithstanding a significant increase in the anti-JCPyV response (p = 0.0097), a further decrease of the neutralizing activity was observed (p = 0.0062). This is the first study evidencing, prospectively, the lack of correlation between the anti-JCPyV antibody response and its neutralizing activity during natalizumab treatment.

The role of hepatitis C virus (HCV) infection in the induction of type II mixed cryoglobulinemia (MCII) and the possible establishment of related lymphoproliferative disorders, such as B-cell non-Hodgkin lymphoma (B-NHL), is well ascertained. However, the molecular pathways involved and the factors predisposing to the development of these HCV-related extrahepatic complications deserve further consideration and clarification. To date, several host- and virus-related factors have been implicated in the progression to MCII, such as the virus-induced expansion of selected subsets of B-cell clones expressing discrete immunoglobulin variable (IgV) gene subfamilies, the involvement of complement factors and the specific role of some HCV proteins. In this review, we will analyze the host and viral factors taking part in the development of MCII in order to give a general outlook of the molecular mechanisms implicated.

The suggested HCV escape mechanism consisting in the elicitation of antibody (Ab) subpopulations interfering with the neutralizing activity of other Abs has recently been questioned. In particular, it was originally reported that Abs directed against the 436-447 region (epitope II) of HCV/E2 glycoprotein may interfere with the neutralizing Abs directed against the 412-423 region (epitope I) involved in the binding to CD81. In this paper, we investigate on the molecular features of this phenomenon describing an anti-HCV/E2 monoclonal Ab (mAb) (e509) endowed with a weak neutralizing activity, and whose epitope is centered on epitope II. Interestingly, e509 influenced the potent neutralizing activity of AP33, one of the best characterized anti-HCV/E2 mAb, whereas it did not show any interfering activity against two other broadly neutralizing mAbs (e20 and e137), whose epitopes partially overlap with that of e509 and which possibly displace it from the antigen. These data may give a possible clue to interpret the conflicting studies published to date on the mechanism of interference, suggesting the existence of at least two groups of broadly neutralizing anti-HCV/E2 Abs: (i) those whose epitope is focused on the 412-423 CD81-binding region and whose activity may be hampered by other Abs directed against the 436-447 region, and (ii) those directed against CD81-binding regions but whose epitope contains also residues within the 436-447 region recognized by interfering mAbs, thus competing with them for binding. The conflicting results of previous studies may therefore depend on the relative amount of each of these two populations in the polyclonal preparations used. Overall, a better comprehension of this phenomenon may be of importance in the set up of novel mAb-based anti-HCV therapeutic strategies.