Covalent coupling of HIV-1 glycoprotein trimers to biodegradable calcium phosphate nanoparticles via genetically encoded aldehyde-tags.

The usage of antigen-functionalized nanoparticles has become a major focus in the field of experimental HIV-1 vaccine research during the last decade. Various molecular mechanisms to couple native-like trimers of the HIV-1 envelope protein (Env) onto nanoparticle surfaces have been reported, but many come with disadvantages regarding the coupling efficiency and stability. In this study, a short amino acid sequence ("aldehyde-tag") was introduced at the C-terminus of a conformationally stabilized native-like Env. The post-translational conversion of a tag-associated cysteine to formylglycine creates a site-specific aldehyde group without alteration of the Env antigenicity. This aldehyde group was further utilized for bioconjugation of Env trimers. We demonstrated that the low acidic environment necessary for this bioconjugation is not affecting the trimer conformation. Furthermore, we developed a two-step coupling method for pH-sensitive nanoparticles. To this end, we conjugated aldehyde-tagged Env with Propargyl-PEG3-aminooxy linker (oxime ligation; Step-one) and coupled these conjugates by copper-catalyzed azide-alkyne cycloaddition (Click reaction; Step-two) to calcium phosphate nanoparticles (CaPs) functionalized with terminal azide groups. CaPs displaying orthogonally arranged Env trimers on their surface (o-CaPs) were superior in activation of Env-specific B-cells (in vitro) and induction of Env-specific antibody responses (in vivo) compared to CaPs with Env trimers coupled in a randomly oriented manner. Taken together, we present a reliable method for the site-specific, covalent coupling of HIV-1 Env native-like trimers to the surface of nanoparticle delivery systems. This method can be broadly applied for functionalization of nanoparticle platforms with conformationally stabilized candidate antigens for both vaccination and diagnostic approaches. STATEMENT OF SIGNIFICANCE: During the last decade antigen-functionalized nanoparticles have become a major focus in the field of experimental HIV-1 vaccines. Rational design led to the production of conformationally stabilized HIV-1 envelope protein (Env) trimers - the only target for the humoral immune system. Various molecular mechanisms to couple Env trimers onto nanoparticle surfaces have been reported, but many come with disadvantages regarding the coupling efficiency and stability. In this paper, we describe a highly selective bio-conjugation of Env trimers to the surface of medically relevant calcium phosphate nanoparticles. This method maintains the native-like protein conformation and has a broad potential application in functionalization of nanoparticle platforms with stabilized candidate antigens (including stabilized spike proteins of coronaviruses) for both vaccination and diagnostic approaches.

Laboratory diagnosis of congenital CMV infection in newborns: Impact of pre-analytic factors.

BACKGROUND: To identify infants with congenital cytomegalovirus (cCMV) saliva polymerase chain reaction (PCR) is an ideal screening method. However, there are only few data on the influence of pre-analytic factors on the analytical sensitivity of the CMV PCR. OBJECTIVES: This study aimed to evaluate the performance of different swabbing materials, transport time and initial virus concentration regarding to the efficacy of recovery of CMV-DNA. STUDY DESIGN: Two CMV suspensions containing a high or low concentration of the laboratory strain AD 169 were prepared as test samples. Sampling was simulated by immersion of different swabs in these CMV suspensions and storing the swabs dry or in specified transport media. Transport conditions were modeled by storing the samples for defined time periods prior to DNA extraction and quantitative PCR analyses. Parallel analyses in two different laboratories allowed determination of lab to lab consistency. RESULTS: The duration of storage under the conditions analysed did not have a major effect on the recovery efficiency for the swabbing materials tested. With exception of flocked dry swabs, all tested swabbing materials demonstrated good recovery of CMV DNA. The flocked swab/eNAT system showed the best overall performance. CONCLUSIONS: All tested swabbing materials (with exception of the flocked dry swabs) seem to be well suited for recovery of CMV DNA and appropriate for use for the diagnosis of cCMV infection in symptomatic cases and in general cCMV screening programs of newborns.

Mucosal expression of DEC-205 targeted allergen alleviates an asthmatic phenotype in mice.

Considering the rising incidence of allergic asthma, the symptomatic treatments that are currently applied in most cases are less than ideal. Specific immunotherapy is currently the only treatment that is able to change the course of the disease, but suffers from a long treatment duration. A gene based immunization that elicits the targeting of allergens towards dendritic cells in a steady-state environment might have the potential to amend these difficulties. Here we used a replication deficient adenovirus to induce the mucosal expression of OVA coupled to a single-chain antibody against DEC-205. A single intranasal vaccination was sufficient to mitigate an OVA-dependent asthmatic phenotype in a murine model. Invasive airway measurements demonstrated improved lung function after Ad-Dec-OVA treatment, which was in line with a marked reduction of goblet cell hyperplasia and lung eosinophilia. Furthermore OVA-specific IgE titers and production of type 2 cytokines were significantly reduced. Together, the here presented data demonstrate the feasibility of mucosal expression of DEC-targeted allergens as a treatment of allergic asthma.

Clinically significant CMV (re)activation during or after radiotherapy/chemotherapy of the brain : Correlation with neurological deterioration and improvement upon antiviral treatment.

INTRODUCTION: For both patients with high-grade gliomas and multiple cerebral metastases, radio(chemo)therapy is the standard therapy. Neurological decline during treatment is rarely attributed to infections of the brain but to tumor progression or side effects of radiotherapy. CASE REPORTS: We present 4 cases of cytomegalovirus (CMV) viremia associated with neurological deterioration, which occurred during or shortly after radiotherapy and/or chemotherapy of the brain (brain metastases 2, high-grade glioma 1, carcinoma infiltrating brain 1). In all cases, neurological decline was sudden and unexpected, and causes such as increased intracranial pressure or tumor progression could be excluded radiologically. Treatment with dexamethasone and mannitol had no or only very short-term effects. General infections were either excluded or receding before the neurological symptoms occurred. All patients presented with decreasing levels of thrombocytes. In all cases, CMV (re)activation could be proven using blood test for CMV-DNA. The anti-CMV-IgG status suggested reactivation rather than a primary infection. One patient died within 72 h of onset of the symptoms (results of CMV tests were received postmortem). Diagnosis of 3 patients allowed successful administration of antiviral treatment, which greatly improved the general and neurological conditions of the patients within 48 h. DISCUSSION: Neurological deterioration during RT is hardly ever attributed to viral infections. These cases suggest that CMV reactivation and subsequent infection might actually be causative and has to be considered and treated. CONCLUSION: Further prospective studies verifying and investigating this observation in terms of frequency and clinical relevance seem indicated.

Codon-optimization of the hemagglutinin gene from the novel swine origin H1N1 influenza virus has differential effects on CD4(+) T-cell responses and immune effector mechanisms following DNA electroporation in mice.

DNA electroporation is a powerful vaccine strategy that could be rapidly adapted to address emerging viruses. We therefore compared cellular and humoral immune responses in mice vaccinated with DNA expression plasmids encoding either the wildtype or a codon-optimized sequence of hemagglutinin from the novel swine origin H1N1 influenza virus. While expression of HA from the wildtype sequence was hardly detectable, the H1N1 hemagglutinin was well expressed from the codon-optimized sequence. Despite poor expression of the wildtype sequence, both plasmids induced similar levels of CD4(+) T-cell responses. However, CD8(+) T-cell and antibody responses were substantially higher after immunization with the codon-optimized DNA vaccine. Thus, efficient induction of immune effector mechanisms against HA of the novel H1N1 influenza virus requires codon-optimization of the DNA vaccines. Since DNA vaccines and several viral vector vaccines employ the same cellular RNA-Polymerase II dependent expression pathway, the poor expression levels from wildtype HA sequences might also limit the induction of immune effector mechanisms by such viral vector vaccines.

Adjuvant properties of AcF1, an immunostimulant fraction of Alchornea cordifolia extract.

As a result of strong experimental data supporting effectiveness and safety, herb-based immunomodulators are paving way as alternative sources of potent adjuvants for vaccines. In this study, the immunostimulatory and adjuvant properties of AcF1, a flavonoids-rich fraction of Alchornea cordifolia extract, was evaluated. In vitro, AcF1 was shown to activate total splenocytes, CD4+ T cells, and B cells, inducing remarkable increases in CD69 expression, profound proliferation, and increased IL-4 and IFN-gamma expression by the naïve splenic cells in a concentration-dependent manner. Lympho-activation and proliferation induced by AcF1 was partially inhibited by U0126, a selective mitogen activated protein kinase kinase (MKK) inhibitor. Additionally, AcF1 was shown to induce structural and functional maturation of bone marrow-derived dendritic cells (BM-DCs) and their specific-antigen presentation functions. Used as an adjuvant in a homologous prime-boost OVA immunisation in C57BL/6 mice, AcF1 significantly (P<0.05) increased the level of OVA-specific antibody titres in the sera of immunised mice, compared to the control group immunised with OVA alone. The results of this study show AcF1 as a potent immunostimulant and a potential adjuvant for further study in combination with other vaccine antigens.

Immunogenicity and efficacy of intradermal tattoo immunization with adenoviral vector vaccines.

Since intradermal delivery of DNA vaccines via tattoo device is an efficient strategy to induce antigen-specific immune responses, we evaluated this route of application for adenoviral vector vaccines in mice. Although expression levels were comparable after i.d. injection and i.d. tattoo immunization of adenoviral vectors, the tattoo application confined antigen expression to the upper layers of the dermis. Both delivery approaches resulted in strong CD8+ T-cell and humoral immune responses to three different antigens and conferred protection against mucosal challenge with respiratory syncytial virus. However, in contrast to results obtained with DNA vaccines, intradermal tattoo immunization did not provide any obvious advantage in comparison to simple intradermal injection of the adenoviral vector vaccines.

[Detection of respiratory viruses--how, when, where and why?]. / Nachweis von Atemwegsviren--Wie, warum, wann und wo?

Respiratory viruses trigger the majority of common colds, acute respiratory illnesses in children during the cold season as well as acute exacerbations of asthma and chronic obstructive pulmonary disease. They also play a role in community acquired pneumonia. Unfortunately their detection is still difficult. The aim of this review is therefore to introduce the methods of detection and to present the current knowledge of the clinical role of respiratory viruses in different diseases.

Broad spectrum antiviral fractions from the lichen Ramalina farinacea (L.) Ach.

BACKGROUND: An ethylacetate-soluble fraction (ET4) from the lichen Ramalina farinacea has previously been shown to inhibit the infectivity of lentiviral and adenoviral vectors, as well as wild-type HIV-1. We now determined the antiviral activity of ET4 against other wild-type viruses, including the herpes simplex virus type 1 (HSV-1) and the respiratory syncytial virus (RSV). METHODS: Wild-type HIV-1, HSV-1 or RSV were pre-incubated with various concentrations of ET4 for 30 min at 37 degrees C before adding to P4CCR5 indicator cell line (HIV-1), ELVIS TM indicator cell line (HSV-1) or HEp2 cell line (RSV) in 96-well microtitre plates. Controls contain virus alone without ET4. The anti-HIV and anti-HSV activities were quantified by estimating beta-galactosidase expression of the respective indicator cell lines while the anti-RSV activity was determined via an immunofluorescent technique, employing monoclonal mouse antibody against the P-protein of RSV. Toxicity of ET4 to cell lines was evaluated in parallel using either the BrdU incorporation method or the MTT method. The effect of ET4 on the enzymatic activity of HIV-1 reverse transcriptase was also evaluated using a chemiluminescent reverse transcriptase assay. Bioassay-guided fractionation of the whole methanol extract of R. farinacea involved sequential screening of HPLC fractions using a vector-based assay technique. RESULTS: ET4 inhibited HSV-1 and RSV potently (IC(50)=6.09 and 3.65 microg/ml, respectively). Time-of-addition studies suggest that both entry and post-entry steps of the HIV-1 replication cycle and the entry step of the RSV replication cycle are targeted. Furthermore, ET4 inhibited HIV-1 reverse transcriptase with an IC(50) of 0.022 microg/ml. Bioassay-guided fractionation of ET4 led to the identification sub-fraction rfO, with activity against lentiviral vector and HIV-1 (RNA viruses) but not against HSV-1 (DNA virus) and sub-fraction rfM, with activity against HSV-1 but not against the lentiviral vector. CONCLUSIONS: ET4 represents a novel fraction from the lichen R. farinacea with broad spectrum antiviral activity against DNA viruses (adenovirus and HSV-1) and RNA viruses (HIV-1 and RSV). The effect against DNA and RNA viruses is mediated by different sub-fractions within R. farinacea.

Potential anti-respiratory syncytial virus lead compounds from Aglaia species.

Although the global prevalence of respiratory syncytial virus (RSV) infection, especially among infants and young children is on the increase, there are only limited therapeutic options for treatment of this disease. Therefore, the search for novel antiviral inhibitors of RSV has become more intensive. In a pilot screening of eighteen compounds from various Aglaia species for anti-RSV activity, we identified dammarenolic acid (ignT1), aglaiol (dupT1) and niloticin (cucT1) as potential anti-RSV compounds, with ignT1 being the most potent. Methylation of ignT1 results in a complete loss of anti-RSV activity. Time of addition studies reveal that both ignT1 and dupT1 target the RSV replication at a post-entry stage, although ignT1 was more potent. Dammarenolic acid (ignT1) was also more cytotoxic than aglaiol (dupT1). By carrying out parallel anti-RSV screening with aphidicolin (a highly cytotoxic diterpenoid) and ignT1, we showed that although aphidicolin was more cytotoxic than ignT1, it had virtually no anti-RSV activity. Therefore, dammarenolic acid, aglaiol and niloticin demonstrate potent anti-RSV activity that shouldbe explored further in the current search for anti-RSV therapeutic agents.

The central nervous system in mucosal vaccination of rhesus macaques with simian immunodeficiency virus Deltanef.

We studied the central nervous system (CNS) of rhesus macaques during series of vaccination experiments in which attenuated simian immunodeficiency virus (SIV), SIVmac239Deltanef, was applied to the tonsils and the animals were later challenged with pathogenic SIVmac251 or SHIV/89.6P via tonsils or rectum. The pathologic lesions were graded on a scale of 0-5. The lesions were in general very mild, with a score of 0.5, except for one case, in which the animal had progressed to simian AIDS (SAIDS) and had severe lesions of grade 4. Except for the SAIDS case, the most common lesions were meningitis, ependymitis, inflammation of choroid plexus, and astrocytosis. Invasion of the challenge virus, SIVmac251, and pathologic lesions were detected 4 days post infection. The main features of the pathological lesions were similar during short-term follow-up (4 days to 2 weeks) and long-term follow-up (23 to 56 weeks) after challenge. No significant difference was found between unvaccinated controls infected with the challenge viruses and vaccinated and challenged animals. The pathological lesions in the one SAIDS case consisted of extensive lesions of the white matter in connection with confluent ependymitis, indicating an invasion through the choroid plexus. The lesions were characterized by a myriad of multinucleated giant cells of macrophage origin, which showed, together with individual macrophages, strong labelling for viral RNA and proteins. Productive infection of astrocytes was a very rare finding. In three cases infected via tonsils with SIVmac239Deltanef without challenge, we detected expression of Nef-derived peptides, indicating a selective pressure for Nef functions in the CNS.

In vitro pharmacodynamic evaluation of antiviral medicinal plants using a vector-based assay technique.

AIMS: Medicinal plants are increasingly being projected as suitable alternative sources of antiviral agents. The development of a suitable in vitro pharmacodynamic screening technique could contribute to rapid identification of potential bioactive plants and also to the standardization and/or pharmacokinetic-pharmacodynamic profiling of the bioactive components. METHODS AND RESULTS: Recombinant viral vectors (lentiviral, retroviral and adenoviral) transferring the firefly luciferase gene were constructed and the inhibition of viral vector infectivity by various concentrations of plant extracts was evaluated in HeLa or Hep2 cells by measuring the changes in luciferase activity. Cytotoxicity of the extracts was evaluated in parallel on HeLa or Hep2 cells stably expressing luciferase. Amongst the 15 extracts screened, only the methanol (ME) and the ethyl acetate (ET) fractions of the lichen, Ramalina farinacea specifically reduced lentiviral and adenoviral infectivity in a dose-dependent manner. Further, chromatographic fractionation of ET into four fractions (ET1-ET4) revealed only ET4 to be selectively antiviral with an IC50 in the 20 microg ml(-1) range. Preliminary mechanistic studies based on the addition of the extracts at different time points in the viral infection cycle (kinetic studies) revealed that the inhibitory activity was highest if extract and vectors were preincubated prior to infection, suggesting that early steps in the lentiviral or adenoviral replication cycle could be the major target of ET4. Inhibition of wild-type HIV-1 was also observed at a 10-fold lower concentration of the extract. CONCLUSIONS: The vector-based assay is a suitable in vitro pharmacodynamic evaluation technique for antiviral medicinal plants. The technique has successfully demonstrated the presence of antiviral principles in R. farinacea. SIGNIFICANCE AND IMPACT OF STUDY: Potential anti-HIV medicinal plants could rapidly be evaluated with the reported vector-based technique. The lichen, R. farinacea could represent a lead source of antiviral substances and is thus worthy of further studies.

Investigation of arthritic joint destruction by a novel fibroblast-based model.

The key pathologic mechanism in rheumatoid arthritis (RA) is the destruction of cartilage by fibroblasts. In a severe combined immunodeficient (SCID) mouse model, this process can be modulated by gene transfer using invasive LS48 fibroblasts. This study aims to investigate the effect of interleukins (IL) -11 and -12 on cartilage destruction when transferred into LS48, and of IL-15 when transfected into non-invasive 3T3 cells; to compare three transduction systems (a lentiviral vector system, a retroviral vector system, and a particle-mediated gene transfer); and to establish an in vitro cartilage destruction system based on LS48 cells. Transduced fibroblasts were injected into SCID mice knee joints, and disease progression assessed microscopically. Distinctive morphologic pattern revealed invasion of fibroblasts into the articular cartilage by transfected, as well as non-transfected, LS48 cells. IL-12 and IL-15 did not alter swelling or cartilage destruction. Animals treated with IL-11-transfected cells showed reduced cartilage damage but no changes in swelling. Efficacy of gene transfer to establish transfected fibroblasts was shown to be >85% for lentiviral transfer, compared to <10% for retroviral transfer and gene gun. Furthermore, cells were co-incubated with porcine cartilage. Transduction of IL-11 led to a reduction of apoptosis in chondrocytes. These findings suggest that cartilage destruction by invasive fibroblasts can be modulated by gene transfer. Lentiviral vector systems offer the most effective approach for gene transduction. In vitro fibroblast/cartilage co-cultures present a convenient system for the assessment of novel therapeutic strategies toward reduction of articular destruction.

[Cost-of-illness Study for the Treatment of COPD in Germany]. / Krankheitskosten von COPD in Deutschland.

The present cost-of-illness study is focused on the costs of COPD in Germany. In a pre-study, data on 814 randomly selected patients were collected to achieve reliable figures for the distribution of COPD severity grades and the frequencies of exacerbations. The main study was performed on 321 randomly selected patients from the pre-study. Data on resource use were collected in a face-to-face interview with the respective physicians using the patient records as a basis. Costs associated with resource consumption were weighted with the frequencies of COPD severity grades as assessed in the pre-study to determine the costs of COPD. Annual COPD-related costs per patient were 3,027 from the societal perspective. Main cost components were hospitalisations (26 %), medication (23 %) and early retirement (17 %). Annual COPD-related costs from the perspective of the German health insurance system (GKV) were 1,944 euros per patient.

[The competence network "acute and chronic leukemias"]. / Das Kompetenznetzwerk "Akute und chronische Leukämien".

Leukemias are a challenge and a cost factor to society because of their frequency in all age groups. They also serve as a model for a variety of diseases and possess exemplary relevance for basic research and patient care. Leukemia research and therapy have achieved high standards and even a leading position in Germany with regard to clinical trials, standardization of diagnostics and molecular studies of prognostic factors, signal transduction and gene expression. Progress is hampered, however, by fragmentation of leukemia trial groups, diagnostic approaches and treatment research activities. A network was therefore created to integrate the leading leukemia trial groups on chronic myeloid leukemia (CML), acute myeloid leukemia (AML), acute lymphatic leukemia (ALL), myelodysplastic syndromes (MDS) and chronic myeloproliferative diseases (CMPD) and their interdisciplinary partners (diagnostics, treatment research, biometry) in cooperation with basic research and pharmaceutical industry to foster advancements in leukemia-related research and health care through clinical trials, promotion of translational research, introduction of standards for diagnostics and therapy, and development of evidence-based guidelines. Achievements include establishment of central information, communication and management structures, creation of an AML intergroup comprising five study groups, formation of a myelodysplastic syndromes study group and establishment of platforms for diagnostics, genomics and proteomics, and medical informatics. Exchange of scientific progress is mediated by intra- and internet, bi-annual newsletters, regular project group meetings and annual network symposia. Already now, the network structures leukemia therapy and research in Germany and supports the spread of scientific excellence in the field of leukemias.

[Clinical research in the "acute and chronic leukemias"competence network ]. / Klinische Forschung im Kompetenznetz "Akute und chronische Leukämien"

Goal of the network is the construction of an exemplary cooperative leukemia network for the improvement of medical care and of health related research in acute and chronic leukemias. This is achieved by improved mechanisms of cooperation among all major groups in Germany that deal with the leukemias in research and in patient care. In practice, cooperation between clinical groups and scientists in research institutes is mediated by various instruments that improve communication, flow of information and interdisciplinary cooperation and also increase information transfer from top research institutions to clinical translation. The network comprises more than 1400 participants in about 400 university centers, large community hospitals and specialty practices with functional communication structures, interdisciplinary cooperation and nation-wide logistics. The improved cooperation and the accelerated information transfer from the bench to the "bedside" results in an added value that ultimately results in improved survival results of patients and in superior competitiveness of involved research workers and clinicians. Sustainability is addressed by establishing a leukemia foundation to support long term financial coverage of the network and by negotiating a proposal for a European Network of Excellence against leukemia within the Sixth Framework Programme of the European Union.

Perceived risk of bovine spongiform encephalopathy and dietary behavior.

The German BSE crisis in early 2001 can be considered as a natural experiment with strong behavioral consequences. The present study investigated psychological and other factors associated with reduced meat consumption compared to the first months of the previous year. As expected, all types of meat, with the exception of poultry and game, were eaten less often. The effect was strongest in beef, where almost half of the sample reported reduced meat consumption. As predicted by the health belief model, perceived threat was associated with subjective vulnerability. It was not, however, strongly associated with perceived seriousness of BSE, probably due to the ubiquitous public discussion of the topic. Reduced beef consumption has three to four times more frequent in the event of subjective threat.

[Evalutation of health monitoring in Bavaria by computer-assisted telephone interviews (CATI) in comparison to the German National Health Examination Survey conducted in 1998 by the Robert Koch Institute]. / Evaluation des Gesundheitsmonitorings in Bayern mit computer-assistierten Telefoninterviews (CATI) durch den Vergleich mit dem Bundes-Gesundheitssurvey 1998 des Robert Koch-Instituts.

Between June 1999 and May 2000, 2051 residents of Bavaria were interviewed on health and health-related behaviour. The objective of the study was to assess the representativeness and validity of data acquired by computer-assisted telephone interviewing (CATI) as compared to results of the German National Health Examination Survey 1998. The following variables showed excellent agreement: subjective health status, lifetime disease prevalences, such as myocardial infarction, stroke, cancer, asthma, diabetes, hypertension, hypercholesterolaemia, utilisation of medical services, and smoking habits (prevalence of present and former smoking, average cigarette consumption per day). Minor differences were found with respect to the frequency of consulting naturopathic doctors. Self-reported anthropometric data from the present study differed considerably from the results of the objective measurements during the German Health Survey. We conclude that, in terms of data quality, CATI compares well with traditional methods such as face-to-face interviewing or mailed questionnaires. In view of several practical advantages of the method, we recommend CATI as a future basic methodology of a German health surveillance system consisting of different coordinated health data collections.