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Background Notwithstanding the efforts of direct-acting antivirals (DAAs) for the treatment of chronically infected hepatitis C virus (HCV) patients, concerns exist regarding the emergence of resistance-associated substitutions (RAS) related to therapy failure. Sanger sequencing is still the reference technique used for the detection of RAS and it detects viral variants present up to 15%, meaning that minority variants are undetectable, using this technique. To date, many studies are focused on the analysis of the impact of HCV low variants using next-generation sequencing (NGS) techniques, but the importance of these minority variants is still debated, and importantly, a common data analysis method is still not defined. Methods Serum samples from four patients failing DAAs therapy were collected at baseline and failure, and amplification of NS3, NS5A and NS5B genes was performed on each sample. The genes amplified were sequenced using Sanger and NGS Illumina sequencing and the data generated were analyzed with different approaches. Three different NGS data analysis methods, two homemade in silico pipeline and one commercially available certified user-friendly software, were used to detect low-level variants. Results The NGS approach allowed to infer also very-low level virus variants. Moreover, data processing allowed to generate high accuracy data which results in reduction in the error rates for each single sequence polymorphism. The results improved the detection of low-level viral variants in the HCV quasispecies of the analyzed patients, and in one patient a low-level RAS related to treatment failure was identified. Importantly, the results obtained from only two out of the three data analysis strategies were in complete agreement in terms of both detection and frequency of RAS. Conclusions These results highlight the need to find a robust NGS data analysis method to standardize NGS results for a better comprehension of the clinical role of low-level HCV variants. Based on the extreme importance of data analysis approaches for wet-data interpretation, a detailed description of the used pipelines and further standardization of the in silico analysis could allow increasing diagnostic laboratory networking to unleash true potentials of NGS.

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 fine characterization of protective B cell epitopes plays a pivotal role in the development of novel vaccines. The development of epitope-based vaccines, in fact, cannot be possible without a clear definition of the antigenic regions involved in the binding between the protective antibody (Ab) and its molecular target. To achieve this result, different epitope-mapping approaches have been widely described (Clementi et al. Drug Discov Today 18(9-10):464-471, 2013). Nowadays, the best way to characterize an Ab bound region is still the resolution of Ab-antigen (Ag) co-crystal structure. Unfortunately, the crystallization approaches are not always feasible. However, different experimental strategies aimed to predict Ab-Ag interaction and followed by in silico analysis of the results may be good surrogate approaches to achieve this result. Here, we review few experimental techniques followed by the use of "basic" informatics tools for the analysis of the results.

"\"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.\""

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.

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.

Bacteriophage therapy dates back almost a century, but the discovery of antibiotics led to a rapid decline in the interests and investments within this field of research. Recently, the novel threat of multidrug-resistant bacteria highlighted the alarming drop in research and development of new antibiotics: 16 molecules were discovered during 1983–87, 10 new therapeutics during the nineties, and only 5 between 2003 and 2007. Phages are therefore being reconsidered as alternative therapeutics. Phage display technique has proved to be extremely promising for the identification of effective antibodies directed against pathogens, as well as for vaccine development. At the same time, conventional phage therapy uses lytic bacteriophages for treatment of infections and recent clinical trials have shown great potential. Moreover, several other approaches have been developed in vitro and in vivo using phage-derived proteins as antibacterial agents. Finally, their use has also been widely considered for public health surveillance, as biosensor phages can be used to detect food and water contaminations and prevent bacterial epidemics. These novel approaches strongly promote the idea that phages and their proteins can be exploited as an effective weapon in the near future, especially in a world which is on the brink of a “postantibiotic era.”

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

The 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.

Viral fitness, the replicative adaptation of a virus to its environment, has viral and environmental determinants. Presently, growth competition assays of recombinant viruses can supply a precise evaluation of this parameter of potential clinical interest. On the basis of the data obtained by monitoring viral infections under treatment with antiviral compounds and by sequence-function analysis of drug-resistant variants, it has recently been proposed that intentional selection of viral mutants with reduced fitness could be an efficient antiviral strategy in several important viral infections of humans. From this point of view, specific antiviral compounds as well as human monoclonal antibodies targeting crucial viral proteins could be effective in modifying the viral replication capacity and the pathogenic potential of rapidly evolving RNA viruses that are normally able to escape under the pressure of treatments with single or multiple antiviral drugs.

The aim of the present in vitro study was to evaluate bacterial microleakage from inside to outside the implant-abutment assembly in a new design of internal conical connection compared to eight different internal connections. The design of this connection should prevent or limit microbiologic leakage into the surrounding implant tissue, that could contribute to infections without bone loss (mucositis) or with bone loss (peri-implantits). In order to investigate bacterial microleakage, the inner part of each system was inoculated with an Escherichia coli suspension. Eight different groups were considered; each group was composed of 10 dental implants, for a total of 80 implants. Groups 1-7 were considered controls, while group 8 was the test connection (an internal connection characterized by a double taper principle). Results showed that in control implants (Group 1 to 7), little microleakage was observed after the first 6 hours (500 CFU/ μl) and, after 24 hours of incubation, they showed a significant bacterial contamination in all samples (>100.000 CFU/ μl). In group 8 (test connection) no contamination was found in the first 6 hours, with 7 out of 10 implants showing no contamination even after 96 hours. Statistically significant differences were found between Group 8 and the other groups (p

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.

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

We evaluated the costs and clinical outcomes of episodes of suspected sepsis in hematological patients. A propensity score-matched study was planned, comparing a retrospective cohort managed with standard assays and a prospective cohort managed with the addition of a molecular assay. Diagnostic procedures and therapy were considered as costs variables. The primary clinical endpoint was sepsis-related mortality, whereas the length of each suspected sepsis episode was investigated as a secondary endpoint. A total of 137 and 138 episodes in the prospective and the retrospective cohorts were studied, respectively; 101 pairs of highly matched episodes were analyzed, evidencing a trend of higher mortality in the retrospective cohort. No difference in length of suspected sepsis episode was observed. Significant savings were observed in the prospective cohort, especially due to reduced costs in antifungal therapy. The apparently more expensive molecular assay favored a more rational use of economic resources without influencing, and probably improving, the clinical outcome.

Clinical and experimental evidence indicates that the hepatitis C virus (HCV) E2 glycoprotein (HCV/E2) is the most promising candidate for the development of an effective anti-HCV vaccine. Identification of the human epitopes that are conserved among isolates and are able to elicit protective antibodies would constitute a significant step forward. This work describes the mapping of the B-cell epitopes present on the surface of HCV/E2, as recognized by the immune system during infection, by the analysis of the reciprocal interactions of a panel of human recombinant Fabs derived from an HCV-infected patient. Three unrelated epitopes recognized by antibodies with no neutralization-of-binding (NOB) activity were identified; a fourth, major epitope was defined as a clustering of minor epitopes recognized by Fabs endowed with strong NOB activity.

Evidence from clinical and experimental studies indicates that hepatitis C virus E2 (HCV/E2) glycoprotein is the major target of a putatively protective immune response. However, even in the presence of a vigorous production of anti-HCV/E2 antibodies, reinfection can occur. Dissection of the human immune response against HCV/E2 indicated that blocking of binding of HCV/E2 to target cells [neutralization of binding (NOB) activity] varies widely among antibody clones. Moreover, in vivo, simultaneous binding of antibodies to distinct epitopes can induce conformational changes and synergies that may be relevant to understanding the anti-HCV immune response. In this study, human recombinant Fabs were generated by affinity-selecting a phage display repertoire library with antibody-coated HCV/E2. These Fabs, which share the same complementarity-determining region DNA sequences, had higher affinity than other anti-HCV/E2 Fabs but showed no NOB activity even at the highest concentrations. Binding of Fabs to HCV/E2 caused conformational changes modifying Fab-binding patterns and reducing, with a negative synergistic effect, Fab-mediated NOB activity. These data suggest that some antibody clones have the potential to modify HCV/E2 conformation and that, in this state, binding of this glycoprotein to its cellular target is less prone to inhibition by some antibody clones. This can explain why high anti-HCV/E2 antibody titers do not directly correlate with protection from infection. Information on the interactions among different antibody clones can contribute to understanding virus–host interplay and developing more effective vaccines.

Background: The course of chronic pancreatitis is often unpredictable and many factors are likely to be involved in the progression of the disease. In physiological condition, pancreatic juice exerts significant antibacterial activity, which is impaired in patients with chronic pancreatitis. Aim: Hypothesizing that Helicobacter pylori could, in these conditions, lead to an ascending infection, we aimed to assess the presence of H. pylori sequences in pancreatic juices of patients with chronic pancreatitis. Methods: 40 patients (mean age 52±3 yr) with alcoholic chronic pancreatitis and H. pylori infection were examined. Pancreatic juices were collected during endoscopic retrograde cholangiopancreatography. Using polymerase chain reaction (PCR) with two primers homologous to a portion of urease-C gene, H. pylori DNA was detected. Gastric biopsies, microscopically positive to H. pylori were used as positive controls. Results: All gastric biopsies produced H. pylori-specific DNA products. Conversely, no H. pylori urease-C gene sequences have been detected in any of the pancreatic juices. Conclusion: Our data suggest that the impaired antibacterial activity of pancreatic juices in patients affected by chronic pancreatitis does not have a permissive role for a superimposingH. pyloriinfection in the pancreas. The possibility that Helicobacter species other than pylori may be involved in a superimposing infection requires further investigation.

We evaluated the Verigene Gram-negative blood culture (BC-GN) test, a microarray that detects Gram-negative bacteria and several resistance genes. A total of 102 positive blood cultures were tested, and the BC-GN test correctly identified 97.9% of the isolates within its panel. Resistance genes (CTX-M, KPC, VIM, and OXA genes) were detected in 29.8% of the isolates, with positive predictive values of 95.8% (95% confidence interval [CI], 87.7% to 98.9%) in Enterobacteriaceae and 100% (95% CI, 75.9% to 100%) in Pseudomonas aeruginosa and negative predictive values of 100% (95% CI, 93.9% to 100%) and 78.6% (95% CI, 51.0% to 93.6%), respectively.

Molecular cloning of the antibody repertoire in phage display combinatorial vectors is a powerful method enabling the dissection of the immunoresponse against a given pathogen. In this paper we describe the construction of a combinatorial library displayed on phage surface, containing the antibody repertoire of a patient with high serological response against hepatitis C virus (HCV) antigens. Following selection of the library against solid-phase-bound antigen, sixteen human antibody Fab fragments able to bind to HCV-specific antigens were generated and studied for binding characteristics. The majority of them appeared to have specificity for the HCV c33 peptide. All the clones reacting with the c33 peptide shared the same heavy-chain CDR3 sequence. This is the first report of molecular cloning in a combinatorial phage display vector of the antibody repertoire of an anti-HCV-positive patient.

The concept of using “magic bullets” in the fight against infectious diseases was originally proposed by Paul Ehrlich, one of the founding fathers of immunology and of the basis of antiinfective therapy [1]. These “magic” compounds should have been able to target microbes without harming the infected host. Actually, this concept is the mainstay of antiinfective therapy, as we still know it today. What Dr. Ehrlich did not and could not foresee was the microbial ability of using “magic tricks” against therapeutic compounds, that is, of developing resistance mechanisms. This is true for all groups of pathogens and all classes of antiinfective drugs, making the need of reliable alternatives everyday more compelling [2–5].

We present three cases of pre-term low-weight infants with suspected necrotizing enterocolitis (NEC) [one eventually recognized as a connatal cytomegalovirus (CMV) infection], microbiologically monitored using a molecular assay detecting bacterial and fungal DNA in blood. The detection of DNA from enteric pathogens in blood was interpreted as a sign of ongoing perforation, and represented a useful complement in the management of the presented cases. Moreover, these cases suggest the opportunity for larger future studies to assess the possible role of a molecular approach in the close monitoring of infants with suspected NEC or with other conditions at-risk for intestinal perforation.

Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA) glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49) that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5). Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections.

The association between hepatitis C virus (HCV) infection and type II mixed cryoglobulinemia (MCII) is well established, but the role played by distinct HCV proteins and by specific components of the anti-HCV humoral immune response remains to be clearly defined. It is widely accepted that HCV drives the expansion of few B-cell clones expressing a restricted pool of selected immunoglobulin variable (IgV) gene subfamilies frequently endowed with rheumatoid factor (RF) activity. Moreover, the same IgV subfamilies are frequently observed in HCV-transformed malignant B-cell clones occasionally complicating MCII. In this paper, we analyze both the humoral and viral counterparts at the basis of cryoglobulins production in HCV-induced MCII, with particular attention reserved to the single IgV subfamilies most frequently involved.

The role of humoral response in the effective control of infection by influenza viruses is well known, but the protection is usually limited to the infecting or vaccinating isolate and to few related strains. Recent studies have evidenced the existence of B-cell epitopes broadly conserved among different influenza subtypes recognized by monoclonal antibodies endowed with unprecedented broad activity. In this review, all major monoclonal antibodies directed against different influenza virus proteins are reported and their potential in the design of new anti-influenza prophylactic or therapeutic strategies is discussed.

SUMMARYSepsis, a leading cause of morbidity and mortality throughout the world, is a clinical syndrome with signs and symptoms relating to an infectious event and the consequent important inflammatory response. From a clinical point of view, sepsis is a continuous process ranging from systemic inflammatory response syndrome (SIRS) to multiple-organ-dysfunction syndrome (MODS). Blood cultures are the current “gold standard” for diagnosis, and they are based on the detection of viable microorganisms present in blood. However, on some occasions, blood cultures have intrinsic limitations in terms of sensitivity and rapidity, and it is not expected that these drawbacks will be overcome by significant improvements in the near future. For these principal reasons, other approaches are therefore needed in association with blood culture to improve the overall diagnostic yield for septic patients. These considerations have represented the rationale for the development of highly sensitive and fast laboratory methods. This review addresses non-culture-based techniques for the diagnosis of sepsis, including molecular and other non-culture-based methods. In particular, the potential clinical role for the sensitive and rapid detection of bacterial and fungal DNA in the development of new diagnostic algorithms is discussed.

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

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

Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Law M, Maruyama T, Lewis J, Giang E, Tarr AW, Stamataki Z, Gastaminza P, Chisari FV, Jones IM, Fox RI, Ball JK, McKeating JA, Kneteman NM, Burton DR. A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable. [Abstract reproduced by permission of Nat Med 2008;14:25–27].

http://hdl.handle.net/20.500.11768/96973 An increase in the incidence of fungal infections has highlighted the need for rapid and precise detection and identification methods in clinical mycology. This report describes the data obtained on corneal samples from 24 patients with suspected keratomycosis using a conventional cultural approach in parallel with PCR amplification and sequencing of the internal transcribed spacers (ITSs) of the rDNA regions. Using the cultural approach, seven samples (58.3% of the 12 samples positive for an infectious pathogen) tested positive for a fungal aetiology, with final identification taking a mean time of more than 5 days. In two cases, diagnosis required 10 days. Using the ITS-based molecular approach, a direct diagnosis was obtained in only five of the seven fungus-positive cases (71(.)4%) starting from the clinical samples, but identification was still possible in all seven cases within 24 h (by using 16 h cultures for the two remaining cases). Despite the less-than-optimal sensitivity when working directly on clinical samples, the obtained data indicate that the molecular strategy used in this study is a useful complement to the conventional diagnostic approaches used for keratomycosis and, in particular, allows precise and fast fungal identification, in response to the clinical requirements. Similar studies on larger panels of patients and on different clinical samples are required for further investigation of the clinical potential of ITS-based approaches in the diagnosis of mycotic infections.

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

In the last decade an increasing number of antibodies have made their way from the research benchtops into the clinics and many more are currently under clinical trial. Among monoclonal antibody-producing techniques, phage-display is undoubtedly the most effective and versatile. Cloning of the entire humoral repertoire derived from an infected patients into a phage display vector allows not only the simple generation of monoclonal antibodies of desired specificity, but also the molecular dissection of the antibody response itself. Generation of large panels of human monoclonal antibodies against human pathogens could open new perspectives in understanding the interplay between the infectious agent and the infected host providing tools for the prevention and the therapy of human communicable diseases. In this paper the basic principles of the phage-display approach as well as its most recent applications are reviewed.

A complete understanding of the molecular features of humoral immune response could be of pivotal importance in the management of persistent viruses as HCV. In this study, 24 HCV-positive samples, characterized by classical virological parameters, are evaluated using a new assay for the quantitation of antibody subpopulations directed against discrete epitopes on surface glycoprotein E2, a key viral protein. The results, besides confirming the usefulness of this new approach, highlight the extreme heterogeneity of anti-HCV/E2 response as far as single epitopes are concerned. The specific epitopes under study are also demonstrated to be widely shared among different genotypes.