John M. Lee

The NS1 protein of tick-borne encephalitis virus forms multimeric species upon secretion from the host cell

Flaviviruses elicit a humoral immune response to two virus-encoded, membrane-associated glycoproteins. One is the major virion surface envelope protein (E), which is recognized by antibody, whereas the other is a secreted, heavily glycosylated non-structural protein (NS1). Inoculation with either protein can give rise to a protective immune response, as can the passive transfer of E and NS1 monospecific monoclonal antibodies. Experiments reported here demonstrate that the secreted form of NS1, whether from cells infected with tick-borne encephalitis virus (TBEV) or from cells infected with a defective recombinant adenovirus containing the NS1 gene, occurs chiefly as a pentamer or hexamer and occasionally as a decamer or dodecamer. Intracellular forms of this protein however occur only as dimers. The higher M(r) forms secreted from the cell are exquisitely sensitive to detergent, suggesting they are held together by hydrophobic bonds. Both intracellular and extracellular forms of the dimer can be dissociated by heat, but at different temperatures. Unlike similar proteins from mosquito-borne viruses. NS1 from TBEV-infected cells cannot be dissociated at ambient temperatures by extremes of pH. Studies on the antigenic structure of this protein show it to have several highly conserved epitopes, confirming similar earlier conclusions from amino acid sequence analyses.

Antigenic Variation among Members of the Tick-borne Encephalitis Complex

The antigenic relationships between seven members of the tick-borne encephalitis complex of flaviviruses (group B arboviruses) were examined by raising a library of 16 monoclonal antibodies against one of them and examining their biological and antigenic properties. These clones reacted with only one of two intracellular, virus-specific polypeptides. One polypeptide [mol. wt. 58 X 10(3) (58K)] is related to the major envelope protein E, but the identity of the other is at present unknown, even though it is a major immunogen in experimental infections and vaccinations. Only those clones specific for the 58K polypeptide contain either neutralizing or haemagglutinin-inhibiting activity, but these epitopes are not identical. In general, epitopes on the 51K polypeptide were more heavily conserved than those on the 58K polypeptide, although both conserved and variant epitopes were found on both polypeptides. One epitope was present on the 51K polypeptide which was conserved on all seven isolates studied and another epitope on the same polypeptide was specific for all the western isolates including one isolate of louping-ill virus. Using the monoclonal antibodies raised in the study, it was shown that louping-ill virus was closely related antigenically to isolates of the Western subtype of tick-borne encephalitis virus.

The Synthesis and Maturation of a Non-structural Extracellular Antigen from Tick-borne Encephalitis Virus and Its Relationship to the Intracellular NS1 Protein

The replication of flaviviruses results in the secretion of four virus-coded proteins into the extracellular environment. Three of these proteins, E, C and M (or pre-M), are found in purified virions. A fourth virus-specified extracellular protein which was not present in either the slowly sedimenting haemagglutinin particles or in virions is described. The relationship of this protein to the intracellular NS1 polypeptide was investigated along with its similarity to the soluble complement-fixing antigen (SCF) reported for mosquito-borne flaviviruses. The difference in the Mr of NS1 and SCF is the result of additional glycosylation of SCF, mostly by the addition of fucose molecules. The synthesis of E and NS1 is sequential but their secretion is simultaneous, suggesting a role for NS1 in virion protein transport or virion release.

Purification and analysis of infectious virions and native non-structural antigens from cells infected with tick-borne encephalitis virus

By employing the techniques of column chromatography and membrane filtration, we have succeeded in purifying flavivirus particles with low particle to infectivity ratios and free from contamination with cellular proteins. Virus particles purified by this method have an average diameter of 53 nm, a particle to infectivity ratio of less than 10, and a KD (partition coefficient) consistent with a molecular weight of 2.63 x 10(7). In addition it has been possible to purify the extracellular form of non-structural protein 1 (NS1), which in its native form appears to be a hexamer. It is also apparent from these studies that the slowly sedimenting haemagglutinin particle (or SHA) is an artifact of purification methods using gradient centrifugation. This technology should not only prove useful in the laboratory for studying the detailed structure of these viruses and the proteins encoded by them, but should also prove useful in industrial vaccine manufacture where large volumes of highly pathogenic material are handled.

Production and purification of murine monoclonal antibodies: aberrant elution from protein A−sepharose 4B

A rapid, one-step method for the efficient purification of murine monoclonal antibodies from tissue culture supernatants is described. This process is based on affinity chromatography on protein A-Sepharose columns. It was found that murine monoclonal antibodies raised against tick-borne encephalitis virus frequently eluted at more than one pH value and these pH values did not always correspond to those of antibodies of the same subclass from polyclonal mouse sera. The two populations of antibody molecule eluting at different pH values showed no variation in molecular weight, isoelectric profiles, specific enzyme-linked immunosorbent assay titer, or antibody subclass.

The synthesis of immunogenic polypeptides encoded by tick-borne encephalitis virus.

Tick-borne encephalitis virus codes for two major immunogenic polypeptides, one of which is the major virion envelope protein E, and the other, NV3, does not have a designated function at present. The intracellular forms of both the E and NV3 polypeptides contain at least four types of sugar residues, i.e. galactose, glucosamine, fucose and mannose. The only glycoprotein in the extracellular virion particles is E. Experiments performed in the presence of tunicamycin have demonstrated that most of these sugars are N-linked. The kinetics of synthesis of E and NV3 have been studied and both show a distinct lag period between initiation of protein synthesis and the appearance of either protein. The kinetics of synthesis of these proteins are consistent with the hypothesis that initiation of protein synthesis starts at the 5 end of a polycistronic genome but the synthesis of the E and NV3 proteins only occurs after translocation of the polyribosome complex to specific areas in the infected cell. No precursors to either the E or NV3 glycoproteins were detected. Synthesis of both glycoproteins can be detected as early as 6 h after infection and rises to a maximum at 15 h after infection.

Rapid vaccination protocols for commercial vaccines against tick-borne encephalitis

Although inactivated viral vaccines have been dramatically successful in controlling many of the worlds most devastating diseases, they frequently need several injections to ensure high levels of protection, and thus their efficacy is reduced in many situations. We have developed several rapid vaccination protocols for two commercial vaccine preparations against tick-borne encephalitis virus and studied their efficacy in an experimental murine model. Vaccination protocols as brief as two doses given over two days elicit efficient protection against challenge with potentially fatal doses of virus and this protection is afforded as soon as 5 or as long as 100 days after the first vaccination. The very rapid induction of protection and the poor antibody responses observed would suggest that cell-mediated immune responses are the most important mechanisms for the protection elicited by conventional inactivated vaccines against tick-borne encephalitis.

Preliminary investigation of the application of on-line membrane extraction of trifluoroacetic acid as an aid to improvement of negative ion electrospray mass spectrometry data

We have recently investigated the biodegradation of a number of acidic aromatic compounds that give excellent chromatography using trifluoroacetic acid (TFA) based HPLC methods. Unfortunately HPLC methods using TFA are not usually compatible with detection by negative ion mass spectrometry as TFA suppresses ionisation of the analyte during the electrospray process. We present a preliminary investigation of the use of an anion-exchange micro-membrane suppressor to remove TFA on-line post column with the aim of improvement of mass spectral data using an aromatic acid as an example, Thus LC-MS using a TFA based HPLC method with negative ion mass spectral detection is shown to be possible with good sensitivity.

The purification of alphavirus virions and subviral particles using ultrafiltration and gel exclusion chromatography

Conventional methods of virus purification using ultracentrifugation frequently result in distorted particles with low levels of biological activity, and are thus unsuitable for preparing samples for high-resolution techniques such as neutron scattering, X-ray scattering in solution, and X-ray crystallography. Moreover, in the event of an instrument failure, ultracentrifugation can also pose a significant hazard when preparing pathogenic viruses or subunits derived from them. By employing exclusively ultrafiltration and gel exclusion chromatography, a method has been developed to prepare highly purified, intact alphavirus particles retaining high levels of biological activity. These procedures have also been adapted to prepare aggregates of viral envelope protein with a defined immunogenic content.

Antigenic relationships between an isolate of the Western Subtype of tick-borne encephalitis virus and an inactivated vaccine derived from it

High mutation rates resulting from the error prone replicases of RNA viruses could lead to antigenic alterations in viral products and pose significant problems during the manufacture of vaccines against RNA viruses. The production of a vaccine against tick-borne encephalitis virus has been monitored using both polyclonal sera and a library of monoclonal antibodies. Only a few antigenic changes were detected during the alteration of host cell from mouse brain to avian fibroblasts and upon subsequent expansion of the virus population during several rounds of replication. In addition, when the formalin inactivation process was monitored for antigenic change, virtually none was detected.