Heidemarie Holzmann

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.

Molecular epidemiology of tick-borne encephalitis virus: cross-protection between European and Far Eastern subtypes

Tick-borne encephalitis virus isolates from widely separated geographic regions of the USSR, six isolates from Hungary and one from France were compared with the European and Far Eastern prototype viruses. Peptide mapping by limited proteolysis yielded similar patterns for five selected isolates from the USSR. All isolates from Hungary and France exhibited the same reactivity pattern with a panel of 16 protein E-specific monoclonal antibodies, whereas 10 out of 12 isolates from the Soviet Union showed minor differences at certain epitopes. However, no correlation between geographic origin of the isolates and their antigenic structure was observed. No statistically significant difference in the degree of protection was detected when mice were immunized with the European prototype vaccine and challenged with three selected Asian isolates and one from the European part of the USSR.

Antibody-response to three recombinant hepatitis B vaccines: comparative evaluation of multicenter travel-clinic based experience

The immunogenicity of three currently used hepatitis B vaccines was compared in an unselected study population in an every day travel clinical setting. Five hundred and eighteen vaccinees received Engerix-B (EB), 990 received Twinrix (TWX), and 366 were immunised with Gen-HB-Vax (GHB). Overall, 88.6% of the vaccinees, tested within the first 6 months after completion of the vaccination series, developed protective levels of anti-HBs (> or = 10 mIU/ml). However, GHB recipients showed significantly lower seroprotection rates (SPR) than EB and TWX recipients (79.3% vs. 87.7% vs. 92.3%, P < 0.000001). GMTs for anti-HBs, tested within 6 months after the third vaccination, showed the lowest results in the GHB group, followed by EB and TWX (142 vs. 523 vs. 1008 mIU/ml, P < 0.000001). TWX vaccinees, however, showing a higher antibody decline rate than EB recipients within the first years after completion of the full immunisation course (30% vs. 25%; P = 0.0538). This study confirms an overall good immune response to the 20 microg-dose vaccine, in the course of a regular clinical setting. The significant difference in SPRs and GMTs to the 10 microg-dose vaccine, however, may influence future immunisation practices for the elderly.

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 immunogens 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.

In vitro-synthesized infectious RNA as an attenuated live vaccine in a flavivirus model.

Live virus vaccines have in many cases proven to be an extremely effective tool for the prevention of viral diseases. However, the production of conventional live vaccines in eukaryotic cell cultures has many disadvantages, including the potential for contamination with adventitious agents and genetic alterations during propagation, making it necessary to do extensive testing before distribution. Based on results obtained with a flavivirus (tick-borne encephalitis virus) in an experimental animal system, we propose a novel live attenuated virus vaccination strategy consisting of the application of in vitro-synthesized infectious RNA instead of the live virus itself. When administered using the GeneGun, less than 1 ng of RNA was required to initiate replication of virus that was attenuated by a specifically engineered deletion and this induced a protective immunity in laboratory mice. Because this approach uses RNA, it does not have the potential drawbacks of DNA vaccines and thus combines the advantages of conventional live virus vaccines (for example, mimicking natural infection and inducing long-lasting immunity) with those of nucleic acid-based vaccines (for example, ease of production without a requirement for eukaryotic cell culture, stability and purity).

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.

Monitoring the Virus Load Can Predict the Emergence of Drug-Resistant Hepatitis B Virus Strains in Renal Transplantation Patients during Lamivudine Therapy

The development of resistant hepatitis B virus (HBV) strains during lamivudine treatment has been described repeatedly. To investigate whether the development of such resistant HBV strains can be predicted in an early phase of therapy, the HBV loads of 11 renal transplantation patients were screened at 3-month intervals by a quantitative HBV polymerase chain reaction (PCR) assay. Lamivudine resistance was detected by sequence analysis. Five patients developed resistance to lamivudine in the 12-15-month follow-up period. In all of them, a virus load of 1x103 HBV DNA copies still was detectable after 3 months of therapy. This was statistically significantly different from those patients who did not develop lamivudine resistance within the observation period, all of whom had no HBV DNA detectable after 3 months of treatment (P=.0022). Thus, virus load testing by use of a sensitive PCR assay allows the early prediction of the emergence of lamivudine-resistant HBV strains.

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.

Sequence of the genes encoding the structural proteins of the low-virulence tick-borne flaviviruses Langat TP21 and Yelantsev☆

The structural protein coding regions of the genomes of Langat virus (strain TP21) and Yelantsev virus, which was originally described to be a low virulence natural isolate of tick-borne encephalitis virus, were cloned and sequenced. These viruses had both been used as experimental live vaccines against tick-borne encephalitis in Czechoslovakia and Russia, respectively. Peptide mapping and monoclonal antibody binding experiments yielded identical reaction patterns for Langat virus and Yelantsev virus which were distinct, however, from the pattern obtained with tick-borne encephalitis virus. Sequence analysis confirmed this distinctiveness and proved that the vaccine strain Yelantsev was also Langat virus. The envelope protein E of both viruses exhibits an 88% amino acid sequence homology with that of tick-borne encephalitis virus. Assessment of the antigenic reactivity and sequence comparison with the E protein of tick-borne encephalitis virus revealed several differences affecting epitopes involved in virus neutralization. These observations suggest that Langat-like virus-based vaccines may not represent the most effective means to achieve protection against tick-borne encephalitis virus.

Tick-borne encephalitis in dogs: neuropathological findings and distribution of antigen

Abstract Eight dogs originating from different regions of Austria [all of them known as tick-borne encephalitis (TBE) areas] with severe neurological signs were either euthanatized or died spontaneously. Tick-borne encephalitis virus (TBEV) antigen was detected in the brains of five of these dogs by immunohistology, but not in the others. All of the dogs, however, had identical neuropathological changes. There were moderate lymphohistiocytic meningitis, widespread neuronal necroses, karyorrhexis of glial cells, numerous neuronophagic nodules, and extensive microgliosis. In the cerebellum, loss of Purkinje cells and proliferation of microglial cells in the molecular layer were found. All brain regions showed numerous perivascular cuffs consisting of lymphocytes, macrophages, plasma cells and, occasionally, red blood cells. The blood-derived cells were not restricted to the perivascular spaces but diffusely infiltrated the neuropil. The most severe changes were localized in the neuroparenchyma surrounding the fourth ventricle. Lesions were less severe in basal ganglia, thalamus, mesencephalon, nuclei of pons and medulla oblongata. Moderate lesions were found in the gray matter of neocortex and allocortex, hippocampus and molecular and Purkinje cell layers of the cerebellum. White matter was slightly to moderately affected. The choroid plexus was free of inflammation. Due to rapid virus clearance mechanisms in this disease, antigen was not detectable in all cases. Neuropathological changes identical with those of immunohistologically proven cases justified the diagnosis TBE in these cases. In addition, the neuropathological diagnosis was supported by the origin of the affected dogs from endemic areas, the seasonal occurrence of the disease and a clinical history of a highly febrile neurological disease with short duration.