West Nile virus vaccine.

Within the past 5 years, West Nile encephalitis has emerged as an important disease of humans and horses in Europe. In 1999, the disease appeared for the first time in the northeastern United States. West Nile virus (a mosquito-borne flavivirus) has flourished in the North American ecosystem and is expected to expand its geographic range. In this review, the rationale for a human and veterinary vaccine is presented and a novel approach for rapid development of a molecularly-defined, live, attenuated vaccine is described. The technology (ChimeriVax) is applicable to the development of vaccines against all flaviviruses, and products against Japanese encephalitis (a close relative of West Nile) and dengue are in or are nearing clinical trials, respectively. ChimeriVax vaccines utilize the safe and effective vaccine against the prototype flavivirus -yellow fever 17D- as a live vector. Infectious clone technology is used to replace the genes encoding the pre-membrane (prM) and envelope (E) protein of yellow fever 17D vaccine with the corresponding genes of the target virus (e.g., West Nile). The resulting chimeric virus contains the antigens responsible for protection against West Nile but retains the replication efficiency of yellow fever 17D. The ChimeriVax technology is well-suited to the rapid development of a West Nile vaccine, and clinical trials could begin as early as mid-2002. Other approaches to vaccine development are briefly reviewed. The aim of this brief review is to describe the features of West Nile encephalitis, a newly introduced infectious disease affecting humans, horses and wildlife in the United States; the rationale for rapid development of vaccines; and approaches to the development of vaccines against the disease.

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.

Growth characteristics of the chimeric Japanese encephalitis virus vaccine candidate, ChimeriVax-JE (YF/JE SA14--14--2), in Culex tritaeniorhynchus, Aedes albopictus, and Aedes aegypti mosquitoes.

The Japanese encephalitis (JE) virus vaccine candidate, ChimeriVax-JE, which consists of a yellow fever (YF) 17D virus backbone containing the prM and E genes from the JE vaccine strain JE SA14--14--2, exhibits restricted replication in non-human primates, producing only a low-level viremia following peripheral inoculation. Although this reduces the likelihood that hematophagous insects could become infected by feeding on a vaccinated host, it is prudent to investigate the replication kinetics of the vaccine virus in mosquito species that are known to vector the viruses from which the chimera is derived. In this study ChimeriVax-JE virus was compared to its parent viruses, as well as to wild-type JE virus, for its ability to replicate in Culex tritaeniorhynchus, Aedes albopictus, and Aedes aegypti mosquitoes. Individual mosquitoes were exposed to the viruses by oral ingestion of a virus-laden blood meal or by intrathoracic (IT) virus inoculation. ChimeriVax-JE virus did not replicate following ingestion by any of the three mosquito species. Additionally, replication was not detected after IT inoculation of ChimeriVax-JE in the primary JE virus vector, Cx. tritaeniorhynchus. ChimeriVax-JE exhibited moderate growth following IT inoculation into Ae. aegypti and Ae. albopictus, reaching titers of 3.6-5.0 log(10) PFU/mosquito. There was no change in the virus genotype associated with replication in mosquitoes. Similar results were observed in mosquitoes of all three species that were IT inoculated or had orally ingested the YF 17D vaccine virus. In contrast, all mosquitoes either IT inoculated with or orally fed wild-type and vaccine JE viruses became infected, reaching maximum titers of 5.4-7.3 log(10) PFU/mosquito. These results indicate that ChimeriVax-JE virus is restricted in its ability to infect and replicate in these mosquito vectors. The low viremia caused by ChimeriVax-JE in primates and poor infectivity for mosquitoes are safeguards against secondary spread of the vaccine virus.

The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein.

To study the role of the precursor to the membrane protein (prM) in flavivirus maturation, we inhibited the proteolytic processing of the Murray Valley encephalitis (MVE) virus prM to membrane protein in infected cells by adding the acidotropic agent ammonium chloride late in the virus replication cycle. Viruses purified from supernatants of ammonium chloride-treated cells contained prM protein and were unable to fuse C6/36 mosquito cells from without. When ammonium chloride was removed from the cells, both the processing of prM and the fusion activity of the purified viruses were partially restored. By using monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of MVE virus, we found that at least three epitopes were less accessible to their corresponding antibodies in the prM-containing MVE virus particles. Amino-terminal sequencing of proteolytic fragments of the E protein which were reactive with sequence-specific peptide antisera or MAb enabled us to estimate the site of the E protein interacting with the prM to be within amino acids 200 to 327. Since prM-containing viruses were up to 400-fold more resistant to a low pH environment, we conclude that the E-prM interaction might be necessary to protect the E protein from irreversible conformational changes caused by maturation into the acidic vesicles of the exocytic pathway.

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.

A model study of the use of monoclonal antibodies in capture enzyme immunoassays for antigen quantification exploiting the epitope map of tick-borne encephalitis virus.

On the basis of an epitope model, capture enzyme immunoassay systems using monoclonal antibodies have been devised for the detection and quantification of Tick-borne encephalitis virus and compared with a reference system employing polyclonal sera. Monoclonal antibodies were used both as capture and detector antibodies, their suitability depending primarily on their avidity and intrinsic background activity. A considerable increase in sensitivity was achieved by combining antibodies to different non-overlapping epitopes. Biotinylation of the detector antibodies allowed the construction of multiple site simultaneous binding assays. Furthermore the use of monoclonal antibodies of defined serological specificity made virus type identification possible. This assay can therefore be used as a rapid 'test of identity' as required during the manufacture of viral vaccines.

Antibody response to gp E of tick-borne encephalitis virus: comparison between natural infection and vaccination breakdown.

Human sera obtained after tick-borne encephalitis (TBE) without prior vaccination were compared with sera from patients after a vaccination breakdown. Most sera previously shown to have high titers of IgG and IgM against TBE virus as detected in the ELISA and hemagglutination inhibition (HI) tests also reacted in Western blot with TBE virus E protein which is involved in virus neutralization. The serum of a patient with a vaccination breakdown, however, reacted only very weakly with the E protein in the Western blot in spite of a high amount of antibodies detectable in ELISA. Using SDS-denaturated virus as an antigen in ELISA (imitating the blotting condition), this serum revealed a significant reduction in its reactivity with denatured virus compared to the control sera. This indicates that the patient had an insufficient immune response against certain denaturation resistant epitopes which might contribute to development of disease despite vaccination. The analysis of the immune response of human sera at the epitope level revealed a characteristic "fingerprint" for each serum reflecting the genetic control of the production of antibody populations against different antigenic determinants.

The envelope glycoproteins of dengue 1 and dengue 2 viruses grown in mosquito cells differ in their utilization of potential glycosylation sites.

We have previously isolated and characterized two dengue (DEN) 2 viruses mutant in their fusion-from-within (FFWI) phenotype in the insect cell line C6/36. Both viruses lost a potential glycosylation site (Asn-153) in the envelope (E) glycoprotein. To determine whether the change in FFWI phenotype was due to a change in E-glycoprotein glycosylation, we characterized the patterns of glycosylation on the E-glycoprotein of wild-type DEN 1 and DEN 2 viruses. The E-glycoproteins were isolated from purified virus grown in Aedes albopictus C6/36 cells, by use of high-performance size-exclusion chromatography. The tryptic maps of wild-type glycosylated and enzymatically (PNGase F) deglycosylated E-glycoproteins were compared by reverse-phase high-performance liquid chromatography. The DEN 1 virus E-glycoprotein was found to have two peaks in the tryptic map that exhibited shifts after deglycosylation, whereas the DEN 2 virus E-glycoprotein had only one. Besides the potential glycosylation site at Asn-153, both DEN 1 and DEN 2 virus E-glycoproteins have another potential site located at Asn-67. Amino-terminal sequencing of the shifted peaks revealed that DEN 2 virus E-glycoprotein is glycosylated only at Asn-67; however, DEN 1 virus E-glycoprotein is glycosylated at both Asn-67 and Asn-153. These DEN virus serotypes are thus heterogeneous in their use of glycosylation sites. We also determined by a lectin-binding assay that the attached carbohydrates for both viruses were likely to be of the high-mannose type.

Cloning, expression and functional activities of a single chain antibody fragment directed to fusion protein of respiratory syncytial virus.

BACKGROUND: HNK20 is a murine IgA which is currently being investigated in clinical trials against respiratory syncytial virus (RSV) infections in infants and young children. OBJECTIVE: To produce a single chain antibody fragment (scFv) from HNK20 hybridoma cells and assess its functional activities in vitro and in vivo (mouse model). STUDY DESIGN: The V regions of heavy and light chains were cloned and linked by a sequence encoding for (Gly4 Ser)3 and expressed in Escherichia coli. RESULTS: Over 100 mg/l of the HNK20-scFv was produced in shake flasks after induction with isopropyl (beta-D-thiogalactopyranoside (IPTG). ScFv was purified under native conditions on a Ni2+ affinity column and migrated as a single band of 34 kDa on sodium dodecyl sulfate (SDS)-gels. ScFv demonstrated similar affinity as its parent IgA molecule, neutralized RSV in vitro and significantly reduced RSV titers in lungs of mice when administered intranasally shortly before or a day after RSV challenge. CONCLUSION: It is possible that this scFv or its derivatives, when applied by intranasal or pulmonary route, will be useful for treatment of RSV infections in infants and young children.

Adaptation of bluetongue virus in mosquito cells results in overexpression of NS3 proteins and release of virus particles.

Adaptation of bluetongue virus (BTV) to grow in mosquito cells (C6/36) resulted in overexpression of two non-structural proteins (NS3 and NS3a) in infected cells. These proteins also co-purified with BTV particles and were dissociated from the virions upon treatment with an anionic detergent. The expression was not host dependent, since back inoculation of the adapted virus into mammalian cell cultures also resulted in a significant overexpression of these proteins. The BTV-C6/36 produced smaller plaques in Vero cells compared with the parent strain. This is the first report which demonstrates a high level of NS3/NS3a expression in infected cells and subsequent release of infectious BTV particles into the supernatant.

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.

A single amino acid substitution in envelope protein E of tick-borne encephalitis virus leads to attenuation in the mouse model.

We have determined the virulence characteristics of seven monoclonal antibody escape mutants of tick-borne encephalitis virus in the mouse model. One of the mutants with an amino acid substitution from tyrosine to histidine at residue 384 revealed strongly reduced pathogenicity after peripheral inoculation of adult mice but retained its capacity to replicate in the mice and to induce a high-titered antibody response. Infection with the attenuated mutant resulted in resistance to challenge with virulent virus. Assessment of nonconservative amino acid substitutions in other attenuated flaviviruses suggests that a structural element including residue 384 may represent an important determinant of flavivirus virulence in general.

Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions.

The fusion activity of flaviviruses [tick-borne encephalitis (TBE) virus and Japanese encephalitis virus] was assessed by inducing fusion from without of C6/36 mosquito cells with purified virus preparations. Membrane fusion and polykaryocyte formation was observed only after incubating the viruses at acidic pH. Two groups of monoclonal antibodies reacting with distinct non-overlapping antigenic domains on the TBE virus protein E inhibited fusion from without. One of these domains contains the most highly conserved and putative fusion-active sequence of the flavivirus protein E. Of five TBE virus monoclonal antibody escape mutants, each defined by a single amino acid substitution in the envelope protein E, one revealed a reduced fusion activity and another one a lower pH threshold. TBE virus grown in the presence of ammonium chloride as well as Langat virus purified from the supernatant of infected chick embryo cells contained the precursor of protein M (prM) rather than M itself. These 'immature' virions did not cause fusion from without, suggesting that the proteolytic processing of prM may be necessary for the generation of fusion-competent virions.

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.

Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model.

A model of the tick-borne encephalitis virus envelope protein E is presented that contains information on the structural organization of this flavivirus protein and correlates epitopes and antigenic domains to defined sequence elements. It thus reveals details of the structural and functional characteristics of the corresponding protein domains. The localization of three antigenic domains (composed of 16 distinct epitopes) within the primary structure was performed by (i) amino-terminal sequencing of three immunoreactive fragments of protein E and (ii) sequencing the protein E-coding regions of seven antigenic variants of tick-borne encephalitis virus that had been selected in the presence of neutralizing monoclonal antibodies directed against the E protein. Further information about variable and conserved regions was obtained by a comparative computer analysis of flavivirus E protein amino acid sequences. The search for potential T-cell determinants revealed at least one sequence compatible with an amphipathic alpha-helix which is conserved in all flaviviruses sequenced so far. By combining these data with those on the location of disulfide bridges (T. Nowak and G. Wengler, Virology 156:127-137, 1987) and the structural characteristics of epitopes, such as dependency on conformation or on intact disulfide bridges or both, a model was established that goes beyond the location of epitopes in the primary sequence and reveals features of the folding of the polypeptide chain, including the generation of discontinuous protein domains.

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.

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.

Novel intranasal immunization techniques for antibody induction and protection of mice against gastric Helicobacter felis infection.

Intranasal (i.n.) delivery of antigen can be highly effective for generating circulating and secretory antibody responses. Mice were immunized i.n. with two antigens, human IgA, and Helicobacter pylori urease in the presence or absence of mucosal adjuvant. To restrict antigen delivery to the upper airways, protein solutions were administered in a small volume without anesthesia. Repeated daily i.n. administration of antigen without adjuvant elicited high levels of specific IgG in serum and IgA in serum, saliva, and feces. Once weekly i.n. immunization with co-administration of cholera toxin or Escherichia coli heat-labile toxin as adjuvant elicited somewhat lower levels of antibody to urease. When challenged with Helicobacter felis, only mice immunized with urease in the presence of adjuvant were protected against gastric infection.

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.

Selection and partial characterization of dengue 2 virus mutants that induce fusion at elevated pH.

Two types of dengue (DEN) 2 virus mutants were selected either by repeated exposure to acidic pH (acid mutant, AM), or by the addition of ammonium chloride to Aedes albopictus C6/36 cells prior to and during viral infection (fusion mutant, FM). Both mutants grew more slowly than the parent strain and induced smaller plaques in Vero cells. The 50% fusion from within index for both mutants occurred at least 0.65 pH units higher than with the wild-type DEN virus. A single amino acid substitution (Asn-153 to Asp) was found in the envelope (E)-glycoprotein of the AM virus. Three amino acid substitutions were detected on the E-glycoprotein of the FM virus: Ile-6 to Met, Asn-134 to Ser, and Asn-153 to Tyr. No mutations were found in the precursor to the membrane protein, prM. The DEN virus E-glycoprotein has two potential glycosylation sites: Asn-67 and Asn-153. The loss of the potential glycosylation site at Asn-153 or the change in the chemical characteristics resultant from the amino acid substitutions in both mutants implicates these regions of the E-glycoprotein in virus-mediated membrane fusion.