Farshad Guirakhoo

Epitope model of tick-borne encephalitis virus envelope glycoprotein E: Analysis of structural properties, role of carbohydrate side chain, and conformational changes occurring at acidic pH

A panel of monoclonal antibodies (MAbs) was prepared to analyze the antigenic structure of the tick-borne encephalitis (TBE) virus glycoprotein E. Nineteen different epitopes were identified and characterized with respect to serological specificity, functional activity, structural properties, and topological relationships. Except for 3 isolated epitopes (i1, i2, and i3), these cluster to form three non-overlapping domains termed A, B, and C. The structural properties of epitopes were assessed by analyzing the effect of different treatments (SDS denaturation, reduction and carboxymethylation, performic acid oxidation, exposure to pH 5.0, CNBr, and trypsin cleavage) on the antigenic reactivities of each epitope. Only 3 epitopes of domain A as well as i2 were sensitive to SDS alone, whereas all others were SDS resistant. Reduction and carboxymethylation, however, destroyed the antigenic reactivity of all epitopes of domain B and also that of two SDS-resistant epitopes of domain A, indicating the role of disulfide bridges in stabilizing the conformation of these epitopes. Deglycosylation by N-Glycanase abolished the SDS resistance of domain C, providing evidence of the role of the carbohydrate side chain in stabilizing these epitopes. A conformational change induced by acid pH was revealed by differences in protease (proteinase K) cleavage maps before and after acid pH treatment. The conformational change involved the epitopes of domain A and occurred between pH 6.0 and 5.5 with the the threshold at pH 7.0.

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.

Characterization of antigenic variants of tick-borne encephalitis virus selected with neutralizing monoclonal antibodies

Antigenic variants of tick-borne encephalitis virus were selected by the use of six neutralizing monoclonal antibodies (MAbs), each defining a different epitope of the envelope glycoprotein E. These variants were characterized with respect to antigenic changes by analysing the binding of each of 18 precisely mapped MAbs in ELISA and haemagglutination inhibition tests. The results yielded information about interrelations between epitopes exceeding that obtained previously from competitive binding studies. In addition, variants were tested for their specific haemagglutination activities, which revealed a significant reduction of this functional activity in one of the variants.

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.

Separation of flavivirus membrane and capsid proteins by multistep high-performance liquid chromatography optimized by immunological monitoring

Complete separation of the three structural proteins, E, C and M, of an enveloped virus (tick-borne encephalitis virus) was achieved by means of a two-step high-performance liquid chromatography (HPLC) technique in less than 1 h. The hydrophobically associated membrane proteins E and M were successfully separated by high-performance gel permeation chromatography (TSK-3000 SW column) in the presence of sodium dodecyl sulphate (SDS), whereas the separation of M and C as well as desalting and removal of SDS was achieved by subsequent reversed-phase chromatography on a C3 column. With regard to further characterization by peptide mapping, analysis of the amino acid composition and aminoterminal sequencing, the second step was performed with volatile buffer systems. Quality control of the separation was achieved by a combination of HPLC with a highly sensitive dot immunoassay by the use of polyclonal as well as monoclonal antibodies. This method proved extremely sensitive and revealed strong tailing effects and cross-contaminations of peaks well-separated in reversed-phase chromatography, which were neither apparent in the absorbance curve at 214 nm nor in the analysis by SDS-polyacrylamide gel electrophoresis. By visualization of the peak-tailing effect, the chromatographic conditions could be modified in order to achieve an optimum separation of proteins.

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.

The relationship between the flaviviruses Skalica and Langat as revealed by monoclonal antibodies, peptide mapping and RNA sequence analysis

The flavivirus Skalica was isolated from a bank vole in Czechoslovakia in 1976. It can be serologically distinguished from prototype strains of tick-borne encephalitis (TBE) virus and has a decreased virulence for adult mice. We have further defined the relationship of Skalica virus to other members of the TBE serocomplex (TBE European and Far Eastern subtypes, Langat and louping ill virus) by using a panel of 22 monoclonal antibodies, peptide mapping and RNA sequence analyses. By these criteria Skalica virus proved to be distinct from TBE virus and to be very closely related to Langat virus, differing by only two bases among a total of 416 nucleotides compared. The sequence of 22% of the Langat genome was determined and the encoded amino acid sequences were derived. Comparison of these with the corresponding amino acid sequences of TBE virus revealed a similarity of 85%, as opposed to 93% similarity between the European and Far Eastern subtypes of TBE virus.