F. X. Heinz

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.

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.

Rapid Diagnosis of Tick-borne Encephalitis by means of Enzyme Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay was applied for determining separately IgM and IgG antibodies against tick-borne encephalitis virus. A micro-modification in microtitre plates proved to be at least as sensitive as the HI test. However, more precise information could be achieved by a macrotest using antigen coated polystyrene balls. False positive results in IgM antibody determinations could be caused by a rheumatoid factor. A high content of IgM antibodies in a serum could impair the determination of its IgG antibodies but not vice versa. Titres were expressed in comparison to a positive control serum.

Formation of Polymeric Glycoprotein Complexes from a Flavivirus: Tick-borne Encephalitis Virus

Treatment of tick-borne encephalitis (TBE) virus with Triton X-100 (TX-100), octylglucoside (OG) or cetyltrimethylammonium bromide (CTAB) caused dissociation of the virus envelope into dimers or monomers of the glycoprotein V3. By centrifugation into detergent-free sucrose density gradients, these subunits were found to reassociate and to form haemagglutinating homogeneous glycoprotein complexes sedimenting at 15 to 16, 16 to 18 and 11 to 23S after TX-100, OG and CTAB treatment, respectively. Glycoprotein complexes obtained after TX-100 solubilization contained less than 1% lipid and detergent by weight.

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

SummaryA 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.

Detectability of IgM antibodies against TBE virus after natural infection and after vaccination

SummaryIgM antibodies againsttick-borne encephalitis (TBE)virus were investigated by means of a three-layer ELISA (antigen bound to the solid phase) and a four-layer ELISA (anti-μ-serum bound to the solid phase) after natural infection as well as after vaccination. In general, the four-layer ELISA detected IgM antibodies more often and for a longer period of time than the three-layer test. In some patients, IgM antibodies were detected for as long as eight months after the disease with the four-layer test but for only six months with the three-layer test. In addition, after the second TBE vaccination, IgM antibodies were found for as long as eight months in 24 sera which were taken within eight months after the second vaccination. Five were positive in the three-layer ELISA and 16 in the four-layer test. IgM antibodies were never detected in specimens taken later than ten months after the second and third vaccination. The results are of diagnostic importance in infections of the CNS which are not caused by TBE virus and which have been preceded by a possibly silent TBE virus infection or by a TBE vaccination.ZusammenfassungIgM-Antikörper gegen dasFrühsommermeningoenzephalitis (FSME)-Virus wurden mittels Drei-Schichten-ELISA (Antigen an die feste Phase gebunden) und Vier-Schichten-ELISA (Anti-μ-Serum an die feste Phase gebunden) nach überstandener Erkrankung sowie nach Impfung untersucht. Generell wurden mit dem Vier-Schichten-ELISA IgM-Antikörper häufiger und für längere Zeit gefunden als mit dem Drei-Schichten-ELISA. Mit ersterem Test wurden bei einigen Patienten IgM-Antikörper bis zu acht, mit letzterem nur bis zu sechs Monaten nach der Erkrankung nachgewiesen. Auch nach zwei FSME-Impfungen wurden IgM-Antikörper bis zu acht Monaten nachgewiesen. Von 24 Sera, die innerhalb von acht Monaten nach der zweiten Impfung abgenommen worden waren, waren fünf im Drei-Schichten-ELISA, aber 16 im Vier-Schichten-ELISA positiv. In Proben, die später als zehn Monate nach der zweiten Impfung abgenommen worden waren, sowie nach der dritten FSME-Impfung konnten keine IgM-Antikörper gefunden werden. Die Befunde haben große Bedeutung bei nicht durch das FSME-Virus hervorgerufenen Infektionen des ZNS, denen eine eventuell stumme FSME-Infektion oder auch Impfung vorangegangen ist.

Evidence for antigenic stability of tick-borne encephalitis virus by the analysis of natural isolates

Strains of tick-borne encephalitis (TBE) virus isolated from ticks in natural foci in Austria were compared to strains isolated from the same foci 14 years previously. Comparative peptide mapping of the envelope (E) glycoproteins as well as analysis of the antigenic structure of the E proteins by the use of 14 monoclonal antibodies defining different epitopes did not provide evidence for antigenic variation. The same also holds true for isolates from a probably newly established natural focus in Western Austria. These results confirm previous data by showing that under natural ecological conditions TBE virus is quite stable and does not undergo major antigenic changes.

Immunogenicity of tick-borne encephalitis virus glycoprotein fragments: epitope-specific analysis of the antibody response.

After digestion with trypsin, alpha-chymotrypsin, or chemical cleavage using CNBr, fragments of the tick-borne encephalitis (TBE) virus glycoprotein were isolated which retained their reactivity with neutralizing monoclonal antibodies defining a denaturation-resistant antigenic domain. Upon immunization of mice, these fragments induced antibodies reactive with the immunizing peptide, the denatured glycoprotein and the native glycoprotein as a constituent of the whole virus. The immune sera revealed the same properties as the monoclonal antibodies that were used to select the fragments for immunization: neutralizing activity; haemagglutination-inhibiting activity; blocking of the binding of antibodies used for selection; enhancement of the binding of other monoclonal antibodies defining a denaturation-sensitive antigenic domain. It was shown that the natural immune response against certain functionally important, denaturation-resistant immunogenic domains on the native protein can be closely mimicked by immunization with defined protein fragments. Antigenic sites present on these fragments may therefore represent essential constituents of a synthetic vaccine. The fine specificities of antibody populations in anti-peptide or anti-protein immune sera were analysed on the basis of single antigenic determinants by blocking assays using radiolabelled monoclonal antibodies that define eight distinct epitopes on the TBE virus glycoprotein. Quantitative differences in the blocking of certain monoclonal antibodies were also observed between human convalescent sera. The establishment of such blocking profiles using a panel of well-characterized monoclonal antibodies may represent a general method for dissecting the specificities of antibody populations present in polyclonal immune sera and could allow investigations on determinant-restricted differences of immune responses and its possible implications for the course of the disease.

Protease treatment and chemical crosslinking of a Flavivirus: Tick borne encephalitis virus

SummaryTick-borne encephalitis virus was treated with pronase or thermolysin. The resulting particles were banded in sucrose gradients and analyzed for polypeptide composition. Both enzymes caused a reduction in particle density from 1.19 to 1.15–1.16 g/cm3. No loss of viral lipid or nucleic acid could be observed. SDS-polyacrylamidegel electrophoresis showed that only the core protein V2 was unchanged whereas the envelope proteins V3 and V1 had disappeared from their original positions in the PAGE profile. Instead a new peptide(s) with molecular weight of 4000–6000 was found in which hydrophobic amino-acids were enriched.Crosslinking by dimethyl-3.3′-dithiobispropionimidate (DTBP) made the virus resistent to solubilization of the envelope proteins by TX-100. This could be interpreted by the formation of a dense envelope protein network around the nucleocapsid preventing its liberation by TX-100. Some data however indicate that direct crosslinking of at least one of the envelope proteins with the core cannot be excluded.

Laboratory diagnosis of tick-borne encephalitis

As isolation of tick-borne encephalitis (TBE) virus is successful only from blood in the first, nonspecific phase of disease or from autopsy material, laboratory diagnosis is done usually by serological means. Classical serologic tests have been replaced by ELISA for detecting IgM antibodies to TBE virus. We tested ELISA formats that demonstrated differences in both sensitivity and susceptibility to interfering factors, e.g., rheumatoid factors or heterophile antibodies. In our experience the anti-μ test using enzyme-labelled antigen proved to be the method best suited for routine laboratory diagnosis of TBE virus infection.