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A DNA Immunization Model Study with Constructs Expressing the Tick-Borne Encephalitis Virus Envelope Protein E in Different Physical Forms

Judith H. Aberle^1,*, Stephan W. Aberle^*, Steven L. Allison^*, Karin Stiasny^*, Michael Ecker^*, Christian W. Mandl^*, Rudolf Berger and Franz X. Heinz^*

^* Institute of Virology and Ludwig Boltzmann Institute for Cytokine Research, University of Vienna, Vienna, Austria

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have conducted a DNA immunization study to evaluate how the immune response is influenced by the physical structure and secretion of the expressed Ag. For this purpose, we used a series of plasmid constructs encoding different forms of the envelope glycoprotein E of the flavivirus tick-borne encephalitis virus. These included a secreted recombinant subviral particle, a secreted carboxyl-terminally truncated soluble homodimer, a nonsecreted full-length form, and an inefficiently secreted truncated form. Mice were immunized using both i.m. injection and Gene Gun-mediated application of plasmids. The functional immune response was evaluated by determining specific neutralizing and hemagglutination-inhibiting Ab activities and by challenging the mice with a lethal dose of the virus. As a measure for the induction of a Th1 and/or Th2 response, we determined specific IgG subclasses and examined IFN-, Il-4, and Il-5 induction. The plasmid construct encoding a secreted subviral particle, which carries multiple copies of the protective Ag on its surface, was superior to the other constructs in terms of extent and functionality of the Ab response as well as protection against virus challenge. As expected, the type of Th response was largely dependent on the mode of application (i.m. vs Gene Gun), but our data show that it was also strongly influenced by the properties of the Ag. Most significantly, the plasmid encoding the particulate form was able to partially overcome the Th2 bias imposed by the Gene Gun, resulting in a balanced Th1/Th2 response.

	Introduction

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Deoxyribonucleic acid immunization is an effective means of generating humoral and cellular immune responses against a wide range of infectious agents (1, 2). The nature and effectiveness of this response, however, has been shown to vary considerably and may critically depend on a variety of factors, including the method of application and the physical form in which the Ag is expressed from the DNA constructs and presented to the immune system (3, 4, 5, 6, 7). Conclusive data on the effect of these factors are therefore important for the design of DNA-based vaccines and their further development for practical use.

It has been demonstrated that the delivery of DNA vaccines by different routes and techniques, e.g., by i.m. injection vs Gene Gun-mediated delivery into the skin, has a strong influence on the efficiency and the type of immune response in mice, including the development of distinct Th subsets, with Th1- and Th2-dominated responses favored by i.m. and Gene Gun immunization, respectively (8, 9). Different types of Th responses have also been attributed to whether or not the expressed Ag is secreted (6, 10, 11), but systematic studies on how the immune response to DNA vaccination is influenced by the structure and secretion properties of the expressed Ag are still needed.

Recently it was shown that the envelope protein E of the flavivirus tick-borne encephalitis (TBE)² virus can be expressed from recombinant plasmids in different physical forms depending on the expression cassette used (12). The availability of this model system allows the immune response to secreted vs nonsecreted and soluble vs particulate forms of the same protective viral Ag to be compared and evaluated in the context of DNA vaccination. TBE virus is a human-pathogenic virus that is endemic in large parts of Europe and Northern Asia. As a member of the flavivirus genus in the family Flaviviridae, it is a close relative of yellow fever, dengue, and Japanese encephalitis virus. Flaviviruses are small enveloped positive-stranded RNA viruses containing a capsid protein (C) and two membrane-associated proteins (E-envelope and M-membrane). The E protein has the dual function of receptor-binding and fusion activity and is the major protective immunogen, inducing neutralizing Abs that presumably block these critical functions. Inoculation of mice with plasmids encoding this protein together with the M protein precursor prM has been shown in several studies with different flaviviruses to induce a protective immune response (13, 14, 15, 16).

In earlier plasmid transfection studies (12, 17), it was shown that the TBE virus E protein, when expressed alone, is retained within the cell and that only low levels of secretion can be achieved by introducing a stop codon to truncate the protein before the membrane anchor. On the other hand, if this same truncated protein is coexpressed with the prM protein, it is efficiently secreted in a soluble dimeric form (17). Furthermore, when the full-length E protein is coexpressed with prM, it is secreted in the form of a recombinant subviral particle (RSP), which presents the E protein in a highly ordered structural context similar to that found on the virion surface (18). Recent cryoelectronmicroscopic data suggest that E dimers on the RSP surface, like those on whole virions, form an icosahedral network (I. Ferlenghi, M. Clarke, D. Thomas, T. Rutten, S. L. Allison, J. Schalich, F. X. Heinz, S. C. Harrison, F. A. Rey, and S. D. Fuller, manuscript in preparation), and purified RSPs have been shown to have a much higher specific immunogenicity than the soluble dimer (19).

In the present study, we have compared the immune response in mice after i.m. inoculation or Gene Gun-mediated delivery of constructs that lead to the synthesis of protein E in intracellular, secreted dimeric, or secreted subviral particle form. The efficacy of the immune response was measured by determining Ab titers in ELISA, neutralization, and hemagglutination inhibition (HI) assays, as well as by testing for lethal challenge protection. As an indicator of the type of immune response, we also determined cytokine profiles and IgG subclasses. We show that, under the conditions used, only the construct leading to the secretion of a subviral particle was capable of inducing complete protection against challenge and high-titered neutralizing Abs. We also found that the synthesis of protein E in different physical forms from different constructs had a strong influence on the dominance of Th cell subsets (Th1 and Th2), as well as the ability to achieve a mixed-type Th response.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Pathogen-free female BALB/c mice aged 6 to 8 wk were purchased from Charles River Laboratories (Sulzfeld, Germany) and were maintained under pathogen-free conditions.

Plasmid construction

The plasmids SV-PEwt, SV-PEst, SV-Ewt, and SV-Est, which have been described in detail by Allison et al. (20), were modified by subcloning the TBE-coding region into the corresponding (NotI) site of the related plasmid pCMV-ß (Clontech Laboratories, Palo Alto, CA) to replace the SV40 promoter/enhancer with the CMV immediate early promoter/enhancer. The parent plasmid pCMV-ß, which encodes ß-galactosidase, was used as negative control for both in vitro and in vivo experiments. To test the expression of the recombinant proteins in cell culture, COS-1 cells (ATCC CRL 1650) were transfected with purified plasmid DNA by electroporation using a Bio-Rad (Munich, Germany) Gene Pulser apparatus. Expression of E proteins was detectable with all of the constructs by indirect immunofluorescence and ELISA, using an E protein-specific polyclonal serum (20). The physical state of the secreted proteins was confirmed in a pelleting experiment using cell supernatants (data not shown).

Immunization and virus challenge

Groups of 16 BALB/c mice each received primary and booster immunizations spaced 4 wk apart. Plasmids were purified using a Qiagen Plasmid Mega Kit (Quiagen, Chatsworth, CA) and administered i.m. (100 µg/dose) or by particle bombardment with DNA-coated gold beads (2 µg/dose) using the helium-powered Helios Gene Gun delivery system (Bio-Rad Laboratories, Richmond, CA). Intramuscular inoculation of DNA in saline was done as a 100-µl injection in the right quadriceps. For Gene Gun inoculation, 1.0 µg of the same DNA was coupled to 0.5 mg of 1.0-µm-diameter gold particles, as recommended by the manufacturer (Bio-Rad). DNA-coated microcarriers were delivered into the abdominal epidermis of mice using the Gene Gun at a helium pressure setting of 400 psi. Mice immunized with the commercial TBE vaccine received 0.2 ml s.c. inoculations containing 1 µg formalin-inactivated virus (FSME Immun Inject, Baxter-Immuno, Vienna, Austria). Two weeks after the second immunization, a blood sample was taken from all of the mice. Eight mice from each group were sacrificed for the recovery of spleen cells for cytokine analysis. The other 8 mice were subsequently challenged by i.p. inoculation with 1000 LD₅₀ of the highly mouse-pathogenic TBE virus strain Hypr. At the end of a 4-wk observation period, sera were collected from all of the mice that had survived challenge.

Serological assays

Pools of equal serum aliquots from individual mice of each group were analyzed for the presence of TBE-specific Abs in hemagglutination-inhibition (HI), neutralization (NT) and ELISA assays.

HI assays were conducted at pH 6.4 with goose erythrocytes according to Clarke and Casals (21), using acetone-extracted Ag from mouse brain infected with TBE virus strain Neudoerfl.

Neutralizing Ab titers were determined in a fixed virus concentration/serum dilution format using baby hamster kidney-21 cells grown in microtiter plates as described in detail by Holzmann et al. (22).

Ab titrations in ELISA were performed with purified live TBE virus as a coating Ag and a goat anti-mouse Ig G-HRP conjugate (Nordic, Lausanne, Switzerland) for the detection of bound Abs, as reported previously (23).

The IgG isotype distribution in the sera was determined by ELISA using 1 µg/ml purified TBE virus as Ag and a Mouse Typer SubIsotyping Kit (Bio-Rad) for detection, according to the manufacturer’s instructions. Experiments using purified TBE virus-specific IgG1 and IgG2a mouse mabs indicated that the sensitivities of the IgG1 and IgG2a ELISAs were essentially identical and that the results could therefore be compared directly.

Abs directed against nonstructural proteins were specifically detected by immunofluorescence using baby hamster kidney-21 cells that had been transfected with an RNA replicon encoding only the nonstructural proteins of TBE virus. This replicon, the construction and characterization of which will be described in detail elsewhere, was derived from the TBE virus infectious cDNA clone described by Mandl et al. (24). Briefly, an in-frame deletion, ranging from position 214 to 2391, was introduced into clone pTNd/5' (24), thus removing the entire coding regions for the proteins prM and E and most of protein C. This deletion clone was ligated to plasmid pTNd/3'and used to transcribe RNA as described previously (24). Transfected cells grown on glass coverslips were fixed in a 1:1 mixture of acetone and methanol for 20 min at -20°C. Sera were diluted 10-fold in PBS, and 25 µl of this dilution was added to each coverslip. Cells were then incubated at 37°C for 30 min. After washing twice with PBS, 25 µl of fluorescent goat anti-mouse IgG Ab (Jackson ImmunoResearch, West Grove, PA) diluted 10-fold in PBS + 0.001% naphthalene black was added and incubated with cells for 30 min at 37°C. After two more PBS washes and drying, cells were examined under a fluorescence microscope.

Determination of TBE virus-specific cytokine production

Splenocytes from individual mice were harvested, and RBC were lysed with a 0.84% NH₄Cl solution following standard methods. After several washes in PBS, 10⁶ splenocytes in a volume of 200 µl per well were cultured in RPMI 1640 (10% FCS, 1% L-glutamine, and 1% neomycin) in the presence of 1 µg highly purified TBE virus strain Neudoerfl. Plates were incubated for 48 h at 37°C in a 5% CO₂ atmosphere. Culture supernatants were assayed for IFN-, IL-4, and IL-5 production by the standard ELISA protocol recommended by the manufacturer (Endogen, Woburn, MA). For measurement of IL-4 in the supernatants, IFN- present in the in vitro cultures was neutralized with 1 µg/ml anti-IFN- (R&D Systems, Wiesbaden, Germany) to minimize a possible suppression of IL-4 by the presence of IFN-, as shown by Milich et al. (25).

	Results

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Recombinant plasmids for immunization

To evaluate the immune response after DNA vaccination with constructs that give rise to different physical forms of the same protein, we made use of four plasmids encoding full-length (E) and C-terminally truncated (E*) TBE virus envelope proteins expressed under the control of the CMV immediate early promoter, either alone or together with prM (Fig. 1). In the latter case the ectodomain of prM is proteolytically removed intracellularly before the release of RSPs or E dimers from transfected cells. Its possible role in the immunogenicity of the constructs is therefore indirect, influencing the physical form and secretion of the E protein as indicated in Fig. 1. Transfection of the four plasmids into COS cells yielded proteins with the expected physical form and secretion properties.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Schematic representation of the expression cassettes of the recombinant plasmids used for expressing different forms of TBE virus E protein. The positions of the prM and E genes and the CMV immediate early promoter (CMV) are indicated. The N- and C-termini of the prM and E proteins are generated by signalase cleavage, except where an extra stop codon (*) has been introduced (pCMV-E*, pCMV-prM-E*).

To assess the immune response to the four DNA constructs shown in Fig. 1, groups of mice received either 2 i.m. inoculations or 2 Gene Gun-mediated applications as described in Materials and Methods. As a control, mice were also immunized with a commercial TBE vaccine consisting of formalin-inactivated whole virus and adsorbed onto Al(OH)₃ as an adjuvant. Two weeks after the second inoculation, TBE virus-specific Ab titers were determined by ELISA, HI, and NT (Materials and Methods), and the mice were challenged with a lethal dose of virulent TBE virus. The results of this experiment are shown in Table I.

We further assessed whether the observed immunity against challenge was "sterilizing," i.e., whether replication of the challenge virus was completely inhibited. This was done by testing postchallenge sera for increases in the ELISA titer and for the development of Abs to nonstructural proteins, which are not induced by any of the materials used for immunization. As shown in Table II, postchallenge Ab titers from mice immunized with the RSP plasmid, both i.m. and by Gene Gun, and from mice vaccinated with the TBE vaccine were identical to the corresponding prechallenge titers. The postchallenge sera also displayed no reactivity with nonstructural (NS) proteins, indicating that immunization with the RSP plasmid and with the TBE vaccine prevented replication of the challenge virus. In contrast, postchallenge sera from surviving mice that had been injected with the other constructs exhibited significant increases in anti-E Ab titers and displayed high reactivity with NS proteins as well. This indicates that the immunity, though protective, was not sterilizing in these cases and allowed replication of the challenge virus at least to a certain extent.

To get further information on the quality and type of immune response induced by the different constructs, we investigated the isotype profiles of the TBE-specific Abs induced by the different immunization regimens. As shown in Table III, significant differences were observed both with respect to the method of immunization and the form of the expressed Ag. As expected (8, 9), the use of the Gene Gun tended to cause a Th2-biased response as indicated by higher IgG1 Ab titers and comparatively low IgG2a titers. Nevertheless, the bias toward Th2 imposed by the Gene Gun was partially overcome in the case of the RSP-expressing construct. These mice produced relatively high titers of IgG2a as well as IgG1 Ab. When injected i.m., the RSP-expressing plasmid also induced a mixed but Th1-dominated response as indicated by high IgG2a levels and lower IgG1 levels. The construct encoding the soluble dimer also gave a mixed response, with moderate levels of IgG1 and IgG2a.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

One clear result of the present study is that a DNA construct that gives rise to a secreted particulate form of the Ag (in this case protein E of TBE virus) is far superior to those generating predominantly intracellular or soluble secreted forms of the same Ag. This was shown by the generation of high-titered neutralizing Abs and a sterilizing immunity against virus challenge. In some cases, relatively high-titered Abs were also induced with constructs not leading to the formation of particles. These Abs, however, were deficient in functional activity (NT and HI), and the protection rates in these cases were relatively low.

In addition, the type of immune response was influenced not only by the method of application, as described in previous DNA immunization studies (8, 9), but was also dependent on the form of the expressed Ag. The secretion of particles apparently causes a strong Th1 component, even when the corresponding construct is applied by the Gene Gun, which generally leads to a Th2-dominated response. It is, therefore, a characteristic property of the RSP construct that it induces a mixed Th response that is skewed toward Th2 or Th1, depending on the application method. A similar Th2-dominated mixed immune response is induced by the inactivated whole virus adsorbed onto Al(OH)₃.

The differences observed are reminiscent of those obtained using protein immunization and may be due to at least 2 different factors that have an impact on the extent and quality of the immune response. The first is related to different Ag presentation pathways. In contrast to intracellularly retained Ags, secreted Ags can be presented by transfected cells as well as cells that have taken up the secreted protein from extracellular spaces (including Ag-specific B cells) (30). The form in which the Ag is secreted may also influence the type of APC involved in T cell priming, which may preferentially present Ag to a type 1 or type 2 Th cell population. In a study with different forms of recombinant hepatitis B virus nucleoprotein, Milich et al. (25) have shown that Th cells with the same specificity can develop into Th1 or Th2 cells, depending on the structural form of the immunogen. Their results further indicated that the IgG isotype profile was regulated at the level of Th-B cell interaction.

The second aspect to be considered is that of native antigenic structure of the pathogen, which in many cases cannot be mimicked by isolated proteins alone. This is certainly true for the TBE virus model used in this study, because recent cryoelectronmicroscopic data (I. Ferlenghi, M. Clarke, D. Thomas, T. Rutten, S. L. Allison, J. Schalich, F. X. Heinz, S. C. Harrison, F. A. Rey, and S. D. Fuller, manuscript in preparation), together with previous biochemical and immunological analyses (31), indicate that the dimeric E protein subunit forms a tight icosahedral network on the surface of the RSP and the virion. The native antigenic structure is therefore complex and cannot be mimicked by the isolated E protein alone, which has been shown to lack certain epitopes involved in virus neutralization (19, 32). The finding that Gene Gun-mediated DNA immunization with constructs generating secreted protein E dimers induced high-titered binding Abs (ELISA) with very low functional activity is consistent with these structural considerations. RSPs, on the other hand, carry the E protein in an arrangement similar to that on the surface of whole virions and thus induce a potent functional Ab response. In addition, RSPs, like whole virions, possess characteristics that might allow them to function as T cell-independent Ags, i.e., the highly repetitive nature of the Ags on their surfaces could make them capable of inducing a rapid expansion of specific B cells without the need for prior Th cell induction, as has been observed previously with the hepatitis B core Ag (33). This could greatly enhance an ensuing T cell-dependent B cell stimulation, thus generating a robust IgG response (34).

Our study shows that not only the application method but also the physical form in which a protein Ag is expressed has a strong influence on the type and extent of the immune response to DNA vaccines. The information obtained with the TBE virus model system may be generally applicable for manipulating the immune response and for improving the efficacy of DNA immunization regimens in other viral and nonviral systems.

	Acknowledgments

 
We thank Heide Dippe for her excellent technical assistance.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Judith H. Aberle, Insitute of Virology, University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria. E-mail address:

² Abbreviations used in this paper: TBE, tick-borne encephalitis; HI, hemagglutination inhibition; NT, neutralization; E, full-length TBE virus envelope protein; E*, C-terminally truncated TBE virus envelope protein; NS, nonstructural; prM, M protein precursor; RSP, recombinant subviral particle.

Received for publication May 13, 1999. Accepted for publication September 27, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Donnelly, J. J., J. B. Ulmer, J. W. Shiver, M. A. Liu. 1997. DNA vaccines. Annu. Rev. Immunol. 15:617.[Medline]
2. Lai, W. C., M. Bennett. 1998. DNA vaccines. Crit. Rev. Immunol. 18:449.[Medline]
3. Cohen, A. D., J. D. Boyer, D. B. Weiner. 1998. Modulating the immune response to genetic immunization. FASEB J. 12:1611.[Abstract/Free Full Text]
4. Robinson, H. L., C. A. T. Torres. 1997. DNA vaccines. Semin. Immunol. 9:271.[Medline]
5. Prange, R., M. Werr. 1999. DNA-mediated immunization to hepatitis B virus envelope proteins: preS antigen secretion enhances the humoral response. Vaccine 17:617.[Medline]
6. Boyle, J. S., C. Koniaras, A. M. Lew. 1997. Influence of cellular location of expressed antigen on the efficacy of DNA vaccination: cytotoxic T lymphocytes and antibody responses are suboptimal when antigen is cytoplasmatic after intramuscular DNA immunization. Int. Immunol. 9:1897.[Abstract/Free Full Text]
7. Inchauspe, G., L. Vitivitski, M. E. Major, G. Jung, U. Spengler, M. Maisonnas, C. Trepo. 1997. Plasmid DNA expressing a secreted or a nonsecreted form of hepatitis C virus nucleocapsid: comparative studies of antibody and T-helper responses following genetic immunization. DNA Cell Biol. 16:185.[Medline]
8. Feltquate, D. M., S. Heaney, R. G. Webster, H. L. Robinson. 1997. Different T helper cell types and antibody isotypes generated by saline and gene gun DNA immunization. J. Immunol. 158:2278.[Abstract]
9. Pertmer, T. M., T. R. Roberts, J. R. Haynes. 1996. Influenza virus nucleoprotein-specific immunoglobulin G subclass and cytokine responses elicited by DNA vaccination are dependent on the route of vector DNA delivery. J. Virol. 70:6119.[Abstract]
10. Haddad, D., S. Liljeqvist, S. Stahl, P. Perlmann, K. Berzins, N. Ahlborg. 1998. Differential induction of immunoglobulin G subclasses by immunization with DNA vectors containing or lacking a signal sequence. Immunol. Lett. 61:201.[Medline]
11. Cardoso, A. I., M. Blixenkrone-Moller, J. Fayolle, M. Liu, R. Buckland, T. F. Wild. 1996. Immunization with plasmid DNA encoding for the measles virus hemagglutinin and nucleoprotein leads to humoral and cell-mediated immunity. Virology 225:293.[Medline]
12. Allison, S. L., K. Stadler, C. W. Mandl, C. Kunz, F. X. Heinz. 1995. Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form. J. Virol. 69:5816.[Abstract]
13. Konishi, E., M. Yamaoka, I. Kurane Khin-Sane-Win, P. W. Mason. 1998. Induction of protective immunity against Japanese encephalitis in mice by immunization with a plasmid encoding Japanese encephalitis virus premembrane and envelope genes. J. Virol. 72:4925.[Abstract/Free Full Text]
14. Schmaljohn, C., L. Vanderzanden, M. Bray, D. Custer, B. Meyer, D. Li, C. Rossi, D. Fuller, J. Fuller, J. Haynes, J. Huggins. 1997. Naked DNA vaccines expressing the prM and E genes of Russian spring summer encephalitis virus and Central European encephalitis virus protect mice from homologous and heterologous challenge. J. Virol. 71:9563.[Abstract]
15. Phillpotts, R. J., K. Venugopal, T. Brooks. 1996. Immunization with DNA polynucleotides protects mice against lethal challenge with St. Louis encephalitis virus. Arch. Virol. 141:743.[Medline]
16. Colombage, G., R. Hall, M. Pavy, M. Lobigs. 1998. DNA-based and alphavirus-vectored immunisation with prM and E proteins elicits long-lived and protective immunity against the flavivirus, Murray Valley encephalitis virus. Virology 250:151.[Medline]
17. Allison, S. L., K. Stiasny, K. Stadler, C. W. Mandl, F. X. Heinz. 1999. Mapping of functional elements in the stem-anchor region of the tick-borne encephalitis virus envelope protein E. J. Virol. 73:5605.[Abstract/Free Full Text]
18. Schalich, J., S. L. Allison, K. Stiasny, C. W. Mandl, C. Kunz, F. X. Heinz. 1996. Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions. J. Virol. 70:4549.[Abstract]
19. Heinz, F. X., S. L. Allison, K. Stiasny, J. Schalich, H. Holzmann, C. W. Mandl, C. Kunz. 1995. Recombinant and virion-derived soluble and particulate immunogens for vaccination against tick-borne encephalitis. Vaccine 13:1636.[Medline]
20. Allison, S. L., C. W. Mandl, C. Kunz, F. X. Heinz. 1994. Expression of cloned envelope protein genes from the flavivirus tick-borne encephalitis virus in mammalian cells and random mutagenesis by PCR. Virus Genes 8:187.[Medline]
21. Clarke, D. H., J. Casals. 1958. Techniques for hemagglutination and hemagglutination inhibition with arthropod-borne viruses. Am. J. Trop. Med. Hyg. 7:561.
22. Holzmann, H., K. Kundi, K. Stiasny, J. Clement, P. McKenna, C. Kunz, F. X. Heinz. 1996. Correlation between ELISA, hemagglutination inhibition, and neutralization tests after vaccination against tick-borne encephalitis. J. Med. Virol. 48:102.[Medline]
23. Heinz, F. X., R. Berger, W. Tuma, C. Kunz. 1983. A topological and functional model of epitopes on the structural glycoprotein of tick-borne encephalitis virus defined by monoclonal antibodies. Virology 126:525.[Medline]
24. Mandl, C. W., M. Ecker, H. Holzmann, C. Kunz, F. X. Heinz. 1997. Infectious cDNA clones of tick-borne encephalitis virus European subtype prototype strain Neudoerfl and high virulence strain Hypr. J. Gen. Virol. 78:1049.[Abstract]
25. Milich, D. R., F. Schödel, J. L. Hughes, J. E. Jones, D. L. Peterson. 1997. The hepatitis B virus core and e antigens elicit different Th cell subsets: antigen structure can affect Th cell phenotype. J. Virol. 71:2192.[Abstract]
26. Street, N. E., T. R. Mosmann. 1991. Functional diversity of T lymphocytes due to secretion of different cytokine patterns. FASEB J 5:171.[Abstract]
27. Morein, B. A., A. Helenius, K. Simons, R. Peterson, L. Kääriäinen, V. Schirrmacher. 1978. Effective subunit vaccines against an enveloped virus. Nature (Lond.) 276:715.[Medline]
28. Morein, B., K. Simons. 1985. Subunit vaccines against enveloped viruses: virosomes, micelles and other protein complexes. Vaccine 3:83.[Medline]
29. Crumpton, M. J.. 1974. Protein antigens: the molecular basis of antigenicity and immunogenicity. ed. In The Antigens Vol. 2:1. Academic Press, New York.
30. Constant, S., D. Sant Angelo, T. Pasqualini, T. Taylor, D. Levin, R. Flavell, K. Bottomly. 1995. Peptide and protein antigens require distinct antigen-presenting cell subsets for the priming of CD4⁺ T cells. J. Immunol. 154:4915.[Abstract]
31. Allison, S. L., J. Schalich, K. Stiasny, C. W. Mandl, C. Kunz, F. X. Heinz. 1995. Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH. J. Virol. 69:695.[Abstract]
32. Heinz, F. X., C. W. Mandl, H. Holzmann, C. Kunz, B. Harris, R. Rey, S. C. Harrison. 1991. The flavivirus envelope protein E: isolation of a soluble dimeric form from tick-borne encephalitis virus and its crystallization. J. Virol. 65:5579.[Abstract/Free Full Text]
33. Fehr, T., D. Skrastina, P. Pumpens, R. M. Zinkernagel. 1998. T cell-independent type I antibody response against B cell epitopes expressed repetitively on recombinant virus particles. Proc. Natl. Acad. Sci. USA 95:9477.[Abstract/Free Full Text]
34. Bachmann, M. F., R. M. Zinkernagel. 1996. The influence of virus structure on antibody responses and virus serotype formation. Immunol. Today 12:553.

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