Biomedical subjects

K Stiasny

Publications and source records attributed to K Stiasny.

32 records · Page 2Linked to original sources

PubMed1997

Characterization of monoclonal antibody-escape mutants of tick-borne encephalitis virus with reduced neuroinvasiveness in mice.

Escape mutants of tick-borne encephalitis (TBE) virus were selected using neutralizing monoclonal antibodies (MAbs) that react with three different and previously unrecognized epitopes in the envelope protein E of TBE virus. Two of these variants (V-IC3 and V-IE3) exhibited a significantly reduced reactivity with their selecting MAbs, as determined by ELISA, whereas with one variant (V-IO3), reactivity was completely unchanged. Comparative sequence analyses demonstrated that each of the variants differed from the wild-type virus by a single amino acid substitution located at exposed positions within domains I, II and III of protein E. In the mouse model, all three mutants were still neuro-virulent but exhibited a significantly reduced neuro-invasiveness after subcutaneous inoculation. Virus replication, however, was sufficient to induce a specific antibody response. The observed alterations in virulence properties were not associated with reduced growth rates in vertebrate cell cultures, but one variant (V-IE3) exhibited a small plaque phenotype. The mutation of variant V-IO3 resulted in a temperature-sensitive phenotype and a significant elevation of the pH-threshold of the conformational change necessary for fusion activity.

Animals↗

PubMed1996

Correlation between ELISA, hemagglutination inhibition, and neutralization tests after vaccination against tick-borne encephalitis.

The significance of IgG antibody levels determined by a binding assay (ELISA) was investigated as a surrogate marker for the presence of neutralizing and hemagglutination inhibiting antibodies in sera from individuals vaccinated against tick-borne encephalitis (TBE). To assess the extent of interference by flavivirus cross-reactive antibodies, sera from persons with a proven or suspected history of other flavivirus infections and/or vaccinations were also examined. An excellent and highly significant correlation was found between ELISA IgG units and the antibody titers obtained by the hemagglutination inhibition (HI) as well as by the neutralization test (NT), provided that there was no other exposure to flavivirus antigens except TBE vaccination. Yellow fever vaccination and/or dengue virus infections induced significant levels of antibodies reactive in the TBE ELISA and HI test, which did not exhibit, however, neutralizing activity against TBE virus. The phenomenon and problem of "original antigenic sin" was demonstrated in a TBE vaccinee with a history of previous flavivirus infections. TBE vaccination first induced a booster reaction resulting in a rise in the level of cross-reactive antibodies only, whereas TBE virus-neutralizing antibodies became detectable only after the third vaccination. It is concluded that the level of IgG antibodies determined by ELISA is a good marker for predicting the presence of neutralizing antibodies after TBE vaccination, but only in the absence of flavivirus cross-reactive antibodies. Otherwise, a neutralization assay is necessary for assessing immunity.

Animals↗

PubMed1996

Characterization and complete genome sequences of high- and low- virulence variants of tick-borne encephalitis virus.

The entire genomic sequences of two strains (Hypr and 263) of the flavivirus tick-borne encephalitis (TBE) virus differing in virulence from the prototypic strain Neudoerfl were determined. Strain Hypr is a human isolate of TBE virus with a high laboratory passage history which exhibits a significantly higher neuro-invasiveness in mice compared to the prototype strain. Strain 263 is a low-passage tick-isolate with a temperature-sensitive and attenuated phenotype. Except for the heterogeneous 3' non-coding regions strains Hypr and 263 share, respectively, 97.2% and 97.6% nucleotide sequence identity with strain Neudoerfl, and differ by a total of 42 and 36 amino acids from the prototypic strain. Of these, only 12 amino acids for each of the two strains represent non-conservative differences unique to an individual strain and some of these are located at positions highly conserved among flaviviruses. Based on these observations, the potential biological significance of particular sequence differences is discussed in the context of the current knowledge about molecular determinants of flavivirus virulence.

Animals↗

PubMed1996

Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus.

The exposure of the flavivirus tick-borne encephalitis (TBE) virus to an acidic pH is necessary for virus-induced membrane fusion and leads to a quantitative and irreversible conversion of the envelope protein E dimers to trimers. To study the structural requirements for this oligomeric rearrangement, the effect of low-pH treatment on the oligomeric state of different isolated forms of protein E was investigated. Full-length E dimers obtained by solubilization of virus with the detergent Triton X-100 formed trimers at low pH, whereas truncated E dimers lacking the stem-anchor region underwent a reversible dissociation into monomers without forming trimers. These data suggest that the low-pH-induced rearrangement in virions is a two-step process involving a reversible dissociation of the E dimers followed by an irreversible formation of trimers, a process which requires the stem-anchor portion of the protein. This region contains potential amphipathic alpha-helical and conserved structural elements whose interactions may contribute to the rearrangements which initiate the fusion process.

Amino Acid Sequence↗

PubMed1996

Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions.

Recombinant subviral particles (RSPs) obtained by coexpression of the envelope (E) and premembrane (prM) proteins of tick-borne encephalitis virus in COS cells (S. L. Allison, K. Stadler, C. W. Mandl, C. Kunz, and F. X. Heinz, J. Virol. 69:5816-5820, 1995) were extensively characterized and shown to be ordered structures containing envelope glycoproteins with structural and functional properties very similar to those in the virion envelope. The particles were spherical, with a diameter of about 30 nm and a buoyant density of 1.14 g/cm3 in sucrose gradients. They contained mature E proteins with endoglycosidase H-resistant glycans as well as fully cleaved mature M proteins. Cleavage of prM, which requires an acidic pH in exocytic compartments, could be inhibited by treatment of transfected cells with ammonium chloride, implying a common maturation pathway for RSPs and virions. RSPs incorporated [14C]choline but not [3H]uridine, demonstrating that they contain lipid but probably lack nucleic acid. The envelope proteins of RSPs exhibited a native antigenic and oligomeric structure compared with virions, and incubation at an acidic pH (pH <6.5) induced identical conformational changes and structural rearrangements, including an irreversible quantitative conversion of dimers to trimers. The RSPs were also shown to be functionally active, inducing membrane fusion in a low-pH-dependent manner and demonstrating the same specific hemagglutination activity as whole virions. Tick-borne encephalitis virus RSPs thus represent an excellent model system for investigating the structural basis of viral envelope glycoprotein functions.

Animals↗

PubMed1996

[Therapy of idiopathic and uremic restless legs syndrome].

Sensory and motor symptoms of the limbs, motor restlessness and an urge to move only at rest are the characteristics of the restless legs syndrome (RLS), which often leads to severe sleep disturbances. The clinical diagnosis can be made on the basis of the typical history, normal neurological findings and, in some cases, a positive family history, and can be confirmed by polysomnography. The indication for treatment depends on the patient's discomfort and the severity of the sleep disturbances. L-DOPA is the treatment of first choice both in idiopathic and uremic RLS. A bedtime dose of 100-200 mg L-DOPA standard plus decarboxylase inhibitor is effective against mild and moderate sleep disturbances in RLS. Titration of the dosage and additional treatment with sustained-release preparations of L-DOPA should be applied individually. Opioids and dopamine agonists are effective alternative treatments in idiopathic RLS. Benzodiazepines are indicated only in individual cases. Besides L-DOPA, uremic RLS patients can be treated with opioids and benzodiazepines. Various approaches in the treatment of idiopathic and uremic RLS are reviewed and the practical management of therapy is outlined.

Central Nervous System Agents↗

PubMed1995

Tick-borne encephalitis virus envelope protein E-specific monoclonal antibodies for the study of low pH-induced conformational changes and immature virions.

A set of ten monoconal antibodies (mabs) specific for the tick-borne encephalitis (TBE) virus envelope protein E were prepared and characterized with respect to their functional activities, the location of their binding sites on protein E and the involvement of their epitopes in acid pH-induced conformational changes and interactions with the precursor to the membrane protein (prM) in immature virions. The majority of these mabs mapped to the previously defined antigenic domain A. All of the mabs recognize parts of the E protein which undergo low pH-induced structural rearrangements believed to be necessary for the fusion activity of the virus, and six of the mabs define epitopes which are affected by the prM-E interaction in immature virions. They are therefore of potential value as specific reagents for studying the structure and function of protein E, as well as the function of the prM-E association. Five of the mabs exhibited neutralizing activity, and can therefore be used for the selection of escape mutants.

Animals↗

PubMed1995

Recombinant and virion-derived soluble and particulate immunogens for vaccination against tick-borne encephalitis.

Using different forms of the envelope glycoprotein E from tick-borne encephalitis virus we investigated the influence of physical and antigenic structure on the efficacy of vaccination. Different protein E-containing preparations were either derived from purified virions or were produced as recombinant proteins in COS cells. These included soluble dimeric forms (virion-derived protein E dimers with and without membrane anchor; recombinant protein E dimers without membrane anchor), micellar aggregates of protein E (rosettes), and recombinant subviral particles (RSPs). The structural differences between these immunogens were verified by sedimentation analysis, immunoblotting and epitope mapping with a panel of monoclonal antibodies. Specific immunogenicities were determined in mice in comparison to formalin-inactivated whole virus. Rosettes and RSPs were excellent immunogens and exhibited similar efficacies as inactivated virus in terms of antibody induction and protection against challenge, whereas all of the soluble forms were much less immunogenic. These data emphasize the importance of the immunogen's antigenic and physical structure for an effective stimulation of the immune system and indicate that RSPs represent an excellent candidate for a recombinant vaccine against tick-borne encephalitis.

Animals↗

PubMed1995

Laboratory acquired tick-borne meningoencephalitis: characterisation of virus strains.

BACKGROUND: The handling of tick-borne encephalitis (TBE) virus is potentially hazardous, as indicated by a number of laboratory-acquired infections in the prevaccination era. OBJECTIVES: (1) To reemphasize the hazard of handling TBE virus without being vaccinated by describing the case of a laboratory-acquired full-blown TBE in a microbiologist who isolated the virus from a blood sample. (2) To molecularly characterize the causative virus strain isolated in Slovenia in comparison with the European prototype strain Neudoerfl. STUDY DESIGN: The virological diagnosis of the laboratory infection was established by serology and virus isolation. The virus was characterized by restriction fragment analysis of PCR products of amplified genomic sequences and a panel of monoclonal antibodies reacting with the major envelope protein. RESULTS: The laboratory infection, most probably acquired by aerosol, resulted in a biphasic course of the disease with a severe meningoencephalitis in the second phase. Both by restriction fragment and monoclonal antibody analysis the Slovenian virus strains involved were indistinguishable from the European prototype strain. CONCLUSIONS: This report confirms the potential hazard of handling TBE virus in the laboratory without being vaccinated. The similarity of the virus isolates from Slovenia with the European prototype strain confirms the previously observed homogeneity of TBE virus strains from different European countries.

Journal Article↗

PubMed1995

L-dopa therapy of uremic and idiopathic restless legs syndrome: a double-blind, crossover trial.

We report the effects of a single bedtime dose of L-dopa 100-200 mg on sleep quality, frequency of periodic leg movements (PLM) and daily living in patients with idiopathic and uremic restless legs syndrome (RLS). Seventeen patients with idiopathic and 11 with uremic (on continuous hemodialysis) RLS were evaluated comparatively by polysomnography, actigraphy and subjective ratings in a randomized, controlled and double-blind crossover trial with L-dopa and placebo for 4 weeks each. Neurophysiologic assessments showed significant reduction of the number of periodic leg movements (p = 0.003) and the PLM-index (p = 0.005) most pronounced during the first 4 hours of bedtime after L-dopa (p = 0.001). Subjective evaluation confirmed improvement of sleep quality (p = 0.002) and showed significantly higher quality of life during daytime (p = 0.030) while the patients received L-dopa therapy. We conclude that L-dopa 100-200 mg proved to be effective in idiopathic RLS and for the first time under controlled conditions in uremic RLS without any severe side effects.

Adult↗

PubMed1995

Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH.

The flavivirus envelope protein E undergoes irreversible conformational changes at a mildly acidic pH which are believed to be necessary for membrane fusion in endosomes. In this study we used a combination of chemical cross-linking and sedimentation analysis to show that the envelope proteins of the flavivirus tick-borne encephalitis virus also change their oligomeric structure when exposed to a mildly acidic environment. Under neutral or slightly alkaline conditions, protein E on the surface of native virions exists as a homodimer which can be isolated by solubilization with the nonionic detergent Triton X-100. Solubilization with the same detergent after pretreatment at an acidic pH, however, yielded homotrimers rather than homodimers, suggesting that exposure to an acidic pH had induced a simultaneous weakening of dimeric contacts and a strengthening of trimeric ones. The pH threshold for the dimer-to-trimer transition was found to be 6.5. Because the pH dependence of this transition parallels that of previously observed changes in the conformation and hydrophobicity of protein E and that of virus-induced membrane fusion, it appears likely that the mechanism of fusion with endosomal membranes involves a specific rearrangement of the proteins in the viral envelope. Immature virions in which protein E is associated with the uncleaved precursor (prM) of the membrane protein M did not undergo a low-pH-induced rearrangement. This is consistent with a protective role of protein prM for protein E during intracellular transport of immature virions through acidic compartments of the trans-Golgi network.

Animals↗

PubMed1995

[Treatment of idiopathic and uremic restless legs syndrome with L-dopa--a double-blind cross-over study].

In a randomized, controlled and double-blind cross-over trial with L-Dopa and placebo we evaluated the effects of a single bedtime dose of L-Dopa 100 to 200 mg on sleep quality and frequency of periodic leg movements (PLM) in 17 patients with idiopathic and in 11 patients with uremic restless legs syndrome (RLS) by polysomnography, actigraphy, and subjective ratings. Treatment with L-Dopa showed significant reduction of the number of periodic leg movements and the PLM-index most pronounced during the first 4 hours of bedtime. Subjective evaluation confirmed improvement of sleep quality. L-Dopa proved to be effective in idiopathic and uremic RLS without any severe side effects.

Administration, Oral↗

PubMed1994

Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM.

We have used tick-borne encephalitis virus to study the involvement of acidic compartments during the entry and release phases of flavivirus infection and to elucidate the role of protein prM in immature virions. Elevation of the pH in acidic intracellular compartments by either bafilyomycin A1, a specific inhibitor of the vacuolar type H(+)-ATPase or by NH4Cl had a strong inhibitory effect during virus penetration and also prevented the cleavage of prM when added in the late phase of the viral life cycle. In the latter case the release of virus particles was not impaired. These immature (prM-containing) virions exhibited a 20- to 50-fold lower specific infectivity and HA activity than mature virions and in contrast to these did not undergo low pH-triggered aggregation. The presence of prM also affected the binding of monoclonal antibodies to protein E, especially at sites which have been shown to undergo acid pH-induced conformational changes in mature virions. Crosslinking, solubilization, and sedimentation analyses revealed the existence of prM-E heterooligomeric complexes, suggesting that the function of prM is to protect protein E from undergoing the irreversible conformational changes in acidic compartments of the secretory pathway that are necessary for triggering fusion activity in the endosome during virus entry.

Ammonium Chloride↗

PubMed1994

The interactions of the flavivirus envelope proteins: implications for virus entry and release.

Viral membrane proteins play an important role in the assembly and disassembly of enveloped viruses. Oligomerization and proteolytic cleavage events are involved in controlling the functions of these proteins during virus entry and release. Using tick-borne encephalitis virus as a model we have studied the role of the flavivirus envelope proteins E and prM/M in these processes. Experiments with acidotropic agents provide evidence that the virus is taken up by receptor-mediated endocytosis and that the acidic pH in endosomes plays an important role for virus entry. The envelope glycoprotein E undergoes irreversible conformational changes at acidic pH, as indicated by the loss of several monoclonal antibody-defined epitopes, which coincide with the viral fusion activity in vitro. Sedimentation analysis reveals that these conformational changes lead to aggregation of virus particles, apparently by the exposure of hydrophobic sequence elements. None of these features are exhibited by immature virions containing E and prM rather than E and M. Detergent solubilization, sedimentation, and crosslinking experiments provide evidence that prM forms a complex with protein E which prevents the conformational changes necessary for fusion activity. The functional role of prM before its endoproteolytic cleavage by a cellular protease thus seems to be the protection of protein E from acid-inactivation during its passage through acidic trans Golgi vesicles in the course of virus release.

Acids↗