Optimierung der therapeutischen Vakzination zur Behandlung chronischer Hepatitis B im präklinischen Model (Murmeltier)

More than 360 million people worldwide are persistently infected with the hepatitis B virus (HBV). The recommended treatment of chronic hepatitis B with interferon-α and/or nucleoside analogues does not lead to satisfactory results. It is well documented that the viral persistence is caused by an absence of the effective virus-specific T cell response. Therefore, the induction of HBV-specific T cells by therapeutic vaccination may be an innovative strategy to overcome virus persistence. Vaccination with commercially available HBV vaccines and DNA vaccines in patients did not result in an induction of the immune response, which would effectively control the HBV infection. Due to this, a more potent therapeutic vaccines are needed. The woodchuck (Marmota monax) is a useful preclinical model for developing new therapeutic approaches in chronic hepadnaviral infections. Several approaches using classical protein and DNA vaccines were tested previously in the woodchuck model without success. The high viral load observed in chronic hepadnaviral infections may have impaired the induction of an effective virus-specific T cell response. Therefore, the application of more potent vaccines, e.g. recombinant viral vectors in combination with antiviral treatment, may be required to achieve sustained antiviral response. In the presented study, an innovative therapeutic strategy, combining vaccination with optimized DNA and recombinant adenoviral vectors (AdV), as well as potent antiviral treatment with entecavir (ETV), was evaluated in the woodchuck model. It was hypothesized that this approach may be an effective strategy to improve WHV-specific immune responses and as a result lead to the resolution of chronic WHV infection. For that purpose, an improved DNA vaccine (pCGWHc), adenoviral vector serotype 5 (Ad5WHc) and chimeric Ad5 displaying Ad35 fiber (Ad35WHc) expressing high levels of woodchuck hepatitis core antigen (WHcAg) were constructed. The efficacy of the improved vaccines was tested first in mice and in naïve woodchucks. The improved WHcAg expression by pCGWHc caused a detection of significantly stronger CD8+ T cell response (interferon-γ production) and higher anti-WHc levels in C57BL/6 mice, compared to the previously used DNA plasmid vaccine. Immunization of mice by pCGWHc prime-AdV boost regimen enhanced CD8+ T cell response even more, compared to immunization with DNA alone. Moreover, the vigorous cytotoxic activity of these WHcAg-specific CD8+ T cells could be demonstrated in vivo. To further evaluate the efficacy of the new prime-boost regimen the vaccination of WHV transgenic mice (1217 WHV Tg) was performed. The immunizations elicited an unexpected anti-WHc and anti-WHs antibodies and WHcAg-specific CD8+ T cell response which led to a significant suppression of WHV replication in those mice. Next, the heterologous Ad5WHc/Ad35WHc immunization was performed in naïve woodchucks. The immunization resulted in the induction of a strong cellular immune response in woodchucks and protected them from WHV infection after the challenge. Altogether, these results clearly showed, that heterologous DNA-AdV immunization induces a more potent WHV-specific immune response than previously investigated strategies in mice and woodchucks. Therefore, this novel DNA prime-AdV boost regimen was used to treat chronically WHV-infected woodchucks in combination with entecavir (ETV) to induce a proper CTL response. Seven animals were treated with ETV for 23 weeks. Starting from week 8, five of them received additional 9 immunizations with DNA plasmids, expressing WHcAg and WHsAg, Ad5WHc and Ad35WHc. For the first time, the significant WHsAg- and WHcAg-specific proliferative responses (CD4) and degranulation responses (CD8) were detected in all chronic carriers that received immunizations in combination with ETV treatment. Moreover, 2 of 4 immunized woodchucks, which completed the ETV and vaccination treatment, demonstrated sustained antiviral response (undetectable viral load and development of anti-WHs). These findings indicate that the therapeutic approach evaluated in this study induces a potent T cell response and seems to be an effective strategy to achieve sustained control of chronic hepadnaviral infections. These results obtained in a preclinical model might be the base for the clinical trials of therapeutic vaccines which induce strong anti-core T cell response in combination with antivirals as a possible immunotherapy in chronically HBV-infected patients.Weltweit sind derzeit mehr als 360 Millionen Menschen mit dem Hepatitis B Virus (HBV) chronisch infiziert. Die empfohlenen Behandlungsstategien bei chronischer Hepatitis B, bestehend aus Interferon-α und/oder Nukleosid-Analoga, zeigen keine zufriedenstellenden Erfolge. In der Literatur ist beschrieben, dass die Viruspersistenz durch das Fehlen einer effektiven virusspezifischen T-Zellantwort mitunter verursacht wird. Daher stellt die Induktion einer HBV-spezifischen T-Zellantwort durch eine therapeutische Vakzinierung eine innovative Strategie zur Behandlung dar, um die Viruspersistenz zu überwinden. Die Schutzimpfung mit kommerziell erhältlichen HBV-Impfstoffen und DNA-Vakzinen in Patienten führt zu keiner ausreichenden Immunantwort, die HBV effektiv kontrolliert. Infolgedessen werden neue wirksame therapeutische Impfstoffe dringend benötigt. Das nordamerikanische Waldmurmeltier (Marmota monax; engl. woodchuck) ist ein nützliches, präklinisches Modell zur Entwicklung neuer therapeutischer Ansätze in chronischen hepadnaviralen Infektionen. Bisherige Therapieansätze im Murmeltier Model, unter Verwendung von klassischen Protein- und DNA-Vakzinen waren ohne Erfolg. Die hohe Viruslast, die in chronischen hepadnaviralen Infektionen beobachtet wird, kann die effektive, virusspezifische T-Zellantwort beeinträchtigen. Aus diesem Grund werden wirksamere Impfstoffe, wie zum Beispiel eine Kombination aus rekombinanten viralen Vektoren und antivirale Behandlung benötigt, um eine andauernde antivirale Antwort zu erzielen. In der vorliegenden Arbeit wurde eine innovative therapeutische Strategie im Murmeltier-Model verfolgt, die in einer kombinierten Vakzinierungsstrategie aus optimierten DNA- und rekombinanten Adenoviralen Vektoren (AdV) sowie aus einer wirksamen, antiviralen Behandlung mit Entecavir (ETV) bestand. Es wurde angenommen, dass dieser Ansatz eine effektive Immunisierungsstrategie darstellt, um eine spezifische Immunantwort gegen das Murmeltier Hepatitis Virus (WHV) zu verbessern und als Ergebnis zu einer Eliminierung der chronischen WHV-Infektion führt. Zu diesem Zweck wurden Vektoren konstruiert, die das Murmeltier Hepatitis Core Antigen (WHcAg) stark exprimieren: ein verbesserter DNA-Impfstoff (pCGWHc), ein Adenoviraler Vektor des Serotyps 5 (Ad5WHc) und einen chimären Ad5 mit präsentierenden Ad35 Fibern. Die Wirksamkeit dieser verbesserten Impfstoffe wurde zuerst in Mäusen und in naiven Murmeltieren getestet. Die durch pCGWHc verbesserte WHcAg Expression bewirkte eine signifikant stärkere CD8+ T-Zellantwort (Interferon γ Produktion) und höhere anti-WHc Level in C57BL/6 Mäusen als in zuvor verwendeten DNA Plasmid-Vakzinen. Im Vergleich zu DNA-Immunisierung konnte eine weitere Verbesserung der CD8+ T-Zellantwort erzielt werden, indem eine pCGWHc „prime-AdV boost“ Immunisierungsstrategie in Mäusen angewandt wurde. Darüber hinaus konnte gezeigt werden, dass diese WHcAg-spezifischen CD8+ T-Zellen eine starke zytotoxische Aktivität in vivo besaßen. Zur weiteren Beurteilung der neuen „prime-boost“ Strategie wurden WHV-transgene Mäuse (1217 WHV Tg) immunisiert. Die Immunisierung rief eine unerwartete anti-WHc und anti-WHs Antikörperproduktion sowei eine WHcAg-spezifische CD8+ T-Zellantwort aus, die zu einer signifikanten Verminderung der WHV Replikation in den Mäusen führte. Im Anschluss wurde die heterologe Ad5/Ad35WHc Immunisierung in naiven Murmeltieren durchgeführt, welche eine starke zelluläre Immunantwort ausgelöste. Des Weiteren bot diese Immunisierungsstrategie effektiven Schutz vor einer WHV-Infektion. Zusammenfassend zeigen diese Ergebnisse, dass eine heterologe DNA-AdV Immunisierung eine weitaus stärkere WHV-spezifische Immunantwort induzieren kann, als die zuvor in Mäusen und Murmeltieren angewandten Immunisierungsstrategien. Zu diesem Zweck wurde diese neuartige DNA „prime-AdV boost“ Strategie dazu verwendet, chronisch WHV-infizierte Murmeltiere in Kombination mit ETV für 23 Wochen zu behandeln. Ausgehend von Woche 8 erhielten 5 der chronisch WHV-infizierten Murmeltiere 9 Immunisierungen mit den DNA-Plasmiden, welche WHcAg und WHsAg sowie Ad5WHc und Ad35WHc exprimieren. Zum ersten Mal konnte eine signifikante WHsAg- und WHcAg-spezifische Proliferationsantwort (CD4) und Degranulationsantwort (CD8) in allen chronisch infizierten Murmeltieren detektiert werden. Des Weiteren konnte gezeigt werden, dass 2 aus 4 immunisierten Murmeltieren, die die Behandlung mit ETV und Vakzine abgeschlossen hatten, eine anhaltende antivirale Immunantwort aufwiesen. Diese Ergebnisse verdeutlichen, dass der gewählte therapeutische Ansatz eine wirksame T-Zellantwort induziert und eine effektive Behandlungsstrategie zu sein scheint, um eine anhaltende Kontrolle von chronischen hepadnaviralen Infektionen zu erzielen. Die in dieser Arbeit beschriebenen Befunde im prä-klinischen Model könnten die Basis für eine klinische Studie zur therapeutischen Vakzinierung zur Immuntherapie chronischer HBV-infizierter Patienten darstellen, die in Kombination mit antiviralen Agentien in der Lage ist eine starke anti-Core T-Zellantwort zu erzielen

Photothermal convection of a magnetic nanofluid in a direct absorption solar collector

Nanofluid-based direct absorption of solar heat results in thermal efficiencies superior to conventional solar thermal technology. In addition, convection of nanofluid can be sustained pump-free in the collector. In this article, we study an aqueous magnetic nanofluid capable to establish the photothermal convection in a lab-scale direct absorption solar collector equipped with a solenoid. The nanofluid consisted of 60-nm Fe2O3 particles dispersed in distilled water at concentration in the range 0.5% wt.-2.0% wt. An empirical model of the photothermal convection was developed based on the experiments. The model accounted for magnetic and thermophoretic forces acting within the nanofluid. The nanofluid with up to 2.0% wt. iron oxide nanoparticles obtained the velocity of ∼5 mm/s under the magnetic field of up to 28 mT. This resulted in the maximum thermal efficiency of the collector equal to 65%.publishedVersio

Photothermal conversion of biodegradable fluids and carbon black nanofluids

The paper is devoted to the topic of direct absorption solar collectors (DASCs). Various kinds of fluids can be used as heat transfer fluid in DASCs, and the main focus of our paper is on comparing nanofluids (water with carbon black nanoparticles, concentrations between 0.25 and 1.00% weight) and biodegradable coffee colloids. At first, these fluids were tested by exposing them to irradiation caused by artificial light in indoor experiments, and the corresponding temperature increase was recorded. The fluids were placed in a beaker with a relatively large size so that most of the fluid was not directly irradiated. In these experiments, the performance of the two studied fluids was similar: the resulting temperature increase varied between 46 and 50 °C. Our next experiments involved a smaller system subjected to irradiation obtained by using a solar collector. As a result, we detected an intense absorption on the nanoparticle surface so that the temperature rise in the nanofluid was higher than in the coffee colloids. Next, the process was analysed using a theoretical analysis that gave good correspondence with the experiments. Finally, we extended the theoretical analysis to a DASC with a flowing fluid. The model was validated against results from the literature, but it also supported our experimental findings.publishedVersio

Theoretical analysis of erosion in elbows due to flows with nano- and micro-size particles

The present paper focuses on the issue of erosion due to fluid flow laden with nano- and microparticles. We investigated the case of a pipe elbow using theoretical analysis and numerical simulations. For the case when the particles were large, that is, of micrometre size, we observed the expected behaviour in which the erosion rate was greater with increasing particle diameter. The same was seen for flow velocity, and higher velocities promoted the erosion process. For small particles, however, the erosion rate increased with decreasing particle size. This was explained by the formation of secondary flows in the elbow that centrifuged the particles towards the walls. For very small particles, the erosion rate decreased again, i.e. the particle distribution towards the wall was insufficient to erode the pipe wall due to the particles low mass.publishedVersio

PepComposer: computational design of peptides binding to a given protein surface

There is a wide interest in designing peptides able to bind to a specific region of a protein with the aim of interfering with a known interaction or as starting point for the design of inhibitors. Here we describe PepComposer, a new pipeline for the computational design of peptides binding to a given protein surface. PepComposer only requires the target protein structure and an approximate definition of the binding site as input. We first retrieve a set of peptide backbone scaffolds from monomeric proteins that harbor the same backbone arrangement as the binding site of the protein of interest. Next, we design optimal sequences for the identified peptide scaffolds. The method is fully automatic and available as a web server at http://biocomputing.it/pepcomposer/webserver

Direct absorption solar collector: Use of nanofluids and biodegradable colloids

In this paper, an experimental and numerical analysis was performed on both carbon black nanofluids and a biodegradable fluid in a novel pump-free direct absorption solar collector (DASC). In the experiments, the nanofluid consisted of carbon black nanoparticles in water with concentrations ranging from 0.005 to 0.020 wt%, while the biodegradable fluid was coffee colloid. The overall findings indicated a concurrence: the nanofluids exhibited the best thermal performance when compared to pure water. The optimum nanoparticle concentration of 0.010 wt% carbon black yielded a 102% thermal enhancement compared to the base fluid. Furthermore, a numerical analysis using computational fluid dynamics (CFD) software was performed to study the experimental rig. According to these simulations, the optimal nanofluid concentration showed a 76.6 - 90.9% increase compared to the base fluid. The biodegradable fluids did not show a significant enhancement in the experiments, which differs from what has been reported in the scientific literature. Nevertheless, from the computer simulations, the biodegradable fluids also slightly outperformed the case when the pure water was used.publishedVersio

Experimental investigation of erosion due to nanofluids

The demand for efficient and sustainable energy is continuously increasing. Among the many technologies with great potential within this field are nanofluids. Nevertheless, there is still a considerable lack of information regarding their erosive effects on systems materials. In this research, the tribological behaviour of aqueous 1.33 wt% TiO2 nanofluid was investigated when jet-impinged with an average velocity of 0.8 m/s at flat targets of various materials (plastic, copper, rubber). The target surfaces were analysed using scanning electron microscopy (SEM), energy dispersive x-ray spectroscopy (EDX) and X-ray diffraction (XRD). It was found that impinging TiO2 nanofluid caused erosion of 282 g/( yr.mm2) for copper and 212 g/( yr.mm2) for plastic. In addition, a deposition of nanoparticles was found for rubber at rate of 2.7 kg/(yr.mm2).publishedVersio

Detection of specific lytic and latent transcripts can help to predict the status of Epstein-Barr virus infection in transplant recipients with high virus load

Epstein-Barr virus (EBV), a member of the family Herpesviridae, is widely spread in the human population and has the ability to establish lifelong latent infection. In immunocompetent individuals the virus reactivation is usually harmless and unnoticeable. In immunocompromised patients productive infection or type III latency may lead to EBV-associated post-transplant lymphoproliferative disorder (PTLD). The aim of our research was to investigate the utility of PCR-based methods in the diagnosis and monitoring of EBV infections in bone marrow transplant recipients. Thirty-eight peripheral blood leukocyte samples obtained from 16 patients were analysed, in which EBV DNA was confirmed by PCR. We used semi-quantitative PCR to estimate the viral load and reverse-transcription PCR (RT-PCR) to differentiate between latent and productive EBV infection. In 14 patients we confirmed productive viral infection. We observed a correlation between higher number of EBV genome copies and the presence of transcripts specific for type III latency as well as clinical symptoms

Multi-component low and high entropy metallic coatings synthesized by pulsed magnetron sputtering

This paper presents the findings of the synthesis of multicomponent (Al, W, Ni, Ti, Nb) alloy coatings from mosaic targets. For the study, a pulsed magnetron sputtering method was employed under different plasma generation conditions: modulation frequency (10 Hz and 1000 Hz), and power (600 W and 1000 W). The processes achieved two types of alloy coatings, high entropy and classical alloys. After the deposition processes, scanning electron microscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy techniques were employed to find the morphology, thickness, and chemical and phase compositions of the coatings. Nanohardness and its related parameters, namely H3.Er2, H.E, and 1.Er2H ratios, were measured. An annealing treatment was performed to estimate the stability range for the selected coatings. The results indicated the formation of as-deposited coatings exhibiting an amorphous structure as a single-phase solid solution. The process parameters had an influence on the resulting morphology-a dense and homogenous as well as a columnar morphology, was obtained. The study compared the properties of high-entropy alloy (HEA) coatings and classical alloy coatings concerning their structure and chemical and phase composition. It was found that the change of frequency modulation and the post-annealing process contributed to the increase in the hardness of the material in the case of HEA coatings

Woodchuck hepatitis virus core antigen-based DNA and protein vaccines induce qualitatively different immune responses that affect T cell recall responses and antiviral effects

AbstractT helper type 1 (Th1) immunity was considered to play a dominant role in viral clearance of hepadnaviral infection. However, pre-primed Th2 type responses were able to efficiently control hepadnaviral infection in animal models. We investigated how pre-primed Th1/2 responses control hepadnaviral replication using the newly established mouse models. DNA (pWHcIm, pCTLA-4-C) and protein vaccines based on the nucleocapsid protein (WHcAg) of woodchuck hepatitis virus (WHV) primed specific immune responses with distinct features. The pre-primed responses determined the characteristics of recall responses if challenged with a WHcAg-expressing adenoviral vector. Vaccination with pWHcIm and pCTLA4-C facilitated viral control in the hydrodynamic injection model and reduced WHV loads by about 3 and 2 logs in WHV-transgenic mice, respectively, despite of different kinetics of specific CD8+ T cell responses. Thus, pre-primed Th2-biased responses facilitate the development of CD8+ T cell responses in mice compared with naïve controls and thereby confer better viral control