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Priming polyvalent immunity by DNA vaccines expressing chimeric antigens with a stress protein-capturing, viral J-domain¹

Institute of Medical Microbiology and Immunology, University of Ulm, Ulm, Germany

²Correspondence: Institute for Medical Microbiology and Immunology, University of Ulm, Albert Einstein Allee 11, D-89081 Ulm, Germany, E-mail: reinhold.schirmbeck{at}medizin.uni-ulm.de

SPECIFIC AIMS

Fusion constructs containing an amino-terminal hsp73 binding, DnaJ-like domain of SV40 T-Ag and carboxyl-terminal sequences encoding fragments of unrelated viral proteins showed strikingly enhanced and stable expression. We intend to develop new DNA vaccine constructs using this novel expression system for the efficient and selective expression of different antigenic epitopes combined with the innate adjuvanticity of hsp molecules (known to enhance and modulate the immunogenicity of antigens).

PRINCIPIAL FINDINGS

1. Hsp73 binds mutant SV40 T-Ag proteins with an intact NH₂ terminus
Molecular chaperones such as heat shock proteins (hsp) assist in protein folding, degradation, and traffic. Some members of the hsp70 family, the abundantly and constitutively expressed hsp73 (hsc70) in the cytosol of mammalian cells, can stabilize mutant proteins but also facilitate their elimination in a novel lysosomal degradation pathway for intracellular proteins. Expression as an hsp73-associated complex seems an attractive option to enhance the level of fusion protein produced by transfectants. The NH₂ terminus of the large tumor antigens (T-Ag) of papovaviruses (SV40, polyoma) contains a J domain (i.e., sequences homologous to the E. coli DnaJ molecule) with a conserved HPD loop that recruits cellular chaperones, the cytosolic hsp73 chaperone. These viral J domains are functional and stimulate ATPase activity of hsp73. Association of the SV40 T-Ag with cellular hsp73 is facilitated by mutations and truncations of this nucleoprotein that leave the NH₂ terminus intact. We have demonstrated tight binding of hsp73 to different variants of the T-Ag but not to wild-type T-Ag. Mutant T-Ags efficiently bound hsp73 in transfected cell lines of different species (human, mouse, monkey, chicken) and of different tissue origin (kidney, liver, skin, muscle). The large amount and long half-life of mutant, hsp73-associated T-Ag expressed by transfectants was unexpected.

2. Minimal sequence of the T-Ag NH₂ terminus required for hsp73 binding
The NH₂ terminus of SV40 T-Ag contains a J domain homologous to the E. coli DnaJ protein. The J domain of the T-Ag contains the conserved HPD sequence at the top of a loop between two -helices required for its interaction with hsp73. The J domain stimulates ATPase activity of hsp73 and facilitates docking of polypeptide substrates to this chaperone. To confirm the role of this DnaJ structure of the T-Ag NH₂ terminus for hsp73 binding, we reduced it to 77 aa (the minimal sequence containing the intact T-Ag-specific DnaJ structure) and fused different protein fragment-encoding sequences (e.g., a 70 residue HBV core domain) in frame to the T₇₇ fragment (Fig. 1 A). As a control, we fused the same sequences to the T₆₀ fragment (Fig. 1A ). The helical loop structure of the J domain required for the contact of HPD with hsp73 is destroyed in T₆₀ but not in the T₇₇ fragment. We transfected eukaryotic cell lines with the respective expression constructs. The T₇₇ fusion proteins (but not the T₆₀ fusion proteins) were efficiently expressed, associated with hsp73 and accumulated in transfected cells to high steady-state levels (Fig. 1B ). The amino-terminal 77 residue sequence of T-Ag containing the intact J domain is thus required to efficiently express chimeric antigens in tight association with hsp73. Our data suggest that in addition to binding hsp73 through its DnaJ-like domain, mutant T-Ags may expose substrate motif(s) because it has a more unfolded conformation that binds the peptide binding domain of hsp73, locking the hsp/DnaJ structures.

View larger version (32K): [in this window] [in a new window]   Figure 1. Priming HBcAg-specific CTL and antibody responses. A) Maps of the HBV core gen. The C79–149 domain and its fusion constructs to the T60 or the T77. B) LMH cells were transiently transfected with pCI/T60-C70, pCI/T77-C70, labeled with [35S]methionine, extracted, immunoprecipitated with anti T-Ag mAb PAb 108, and processed for SDS-PAGE followed by fluorography of the gels. BALB/c mice were vaccinated C–E) i.m. with 100 µg plasmid DNA or F) i.d. with the gene gun with 2 µg plasmid DNA of the indicated vectors: pCI/T77-C70, pCI/T60-C70, pCI/C, or the control pCI DNA. Serum antibodies were obtained 5–8 wk postimmunization and analyzed for HBcAg-specific IgG serum antibodies in ELISA. The mean titers of antibodies in sera of 3–6 mice per group are shown (C). Spleen cells obtained 11 days postvaccination were restimulated for 5 h with HBcAg-expressing P815/C cells. T cells were surface stained for CD8 and intracellularly stained for IFN-. We determined the frequencies of CD8+ IFN-+ CTL per 105 CD8+ spleen cells by FCM analyses. Restimulation with nontransfected P815 cells was used to determine nonspecific ‘background’ frequencies. The mean number of IFN-+ CD8+ T cells/105 CD8+ spleen cells ± SD of 3–4 individual mice are shown (D, F). Alternatively, spleen cells obtained from immune mice 2–4 wk postvaccination were specifically restimulated in vitro for 5 days with HBcAg-expressing P815 transfectants and tested for specific cytotoxicity in a 4 h 51Cr-release assay. Mean specific lysis values (of triplicates) at an effector/target (E/T) ratio of 20 are shown. The nonspecific lysis of control P815 (<5%) was subtracted (E).

3. Hsp73-capturing, chimeric antigens display enhanced immunogenicity for T and B cells
When hsp73 bound, the chimeric antigens expressed by DNA vaccines showed strikingly enhanced immunogenicity evident in humoral (antibody) and cellular (CTL) responses. We compared the immunogenicity of hsp73-capturing (T77) vs. non-hsp-associated (T60) chimeric antigens using the 70 residue antigenic fragment (C79–149) from the HBV core gene that contained well-defined antibody (e2 determinant) and CTL epitopes (Fig. 1A ). Both expression constructs were used as DNA vaccines by injecting intramuscular (i.m.) 100 µg/mouse of these plasmid DNAs into BALB/c mice. Specific serum antibody and CTL responses were read out at different times postvaccination. Although T₆₀-C70 and hsp73-associated T₇₇-C70 antigens were not secreted from transfected cells, pCI/T₇₇-C70 DNA immunization stimulated the induction of high levels of HBV core-specific serum antibodies (Fig. 1C ). Titers of specific serum antibodies induced by the injection of the DNA vaccine encoding the hsp73 binding T₇₇-C70 chimeric antigen were reproducibly 15- to 25-fold higher than the titers in the serum of mice injected with the DNA vaccine encoding the non-hsp binding T₆₀-C70 variant of the chimeric antigen. The antibody response against the selective e2 determinant was thus strikingly enhanced when the chimeric antigen was bound to hsp73. Mice showed no cross-reactive immune response against the hsp73 autoantigen.

HBV core-specific CTL were primed in mice by injecting plasmid DNA encoding the T₇₇-C70 or T₆₀-C70 antigens. High frequencies CD8⁺ IFN-⁺ CTL specifically stimulated by HBcAg-expressing P815/C cells were detected ex vivo in mice immunized with the pCI/T₇₇-C70 DNA (Fig. 1D ). The frequency of specific CTL elicited by the pCI/T₇₇-C70 encoding plasmid DNA was significantly higher than the CTL frequency elicited by injecting the non-hsp-associated pCI/T₆₀-C70 encoding plasmid DNA (Fig. 1D ) and similar to the CTL frequency elicited by injecting pCI/C plasmid DNA encoding HBcAg (Fig. 1D ). This pattern of CTL induction was confirmed in cytolytic assays in which in vivo primed splenic T cells were cocultured in vitro for 5 days with HBcAg-expressing, syngeneic transfectants and tested for specific cytolytic reactivity using antigen-expressing targets. The pCI/T₇₇-C70 but not the pCI/T₆₀-C70 encoding DNA vaccine efficiently induced HBcAg-specific CTL that lysed P815/C but not control P815 cells (Fig. 1E ). The efficacy of hsp73 binding T₇₇-C70 antigen to specifically prime core-specific CTL precursors was even more striking in gene gun-mediated, intradermal (i.d.) DNA vaccination experiments (Fig. 1F ). Although priming of IFN--producing CD8⁺ CTL was not detectable after injection of DNA vaccines encoding either non-hsp binding T₆₀-C70 antigen or native HBcAg, it was readily detectable after i.d. injection of the hsp73 binding T₇₇-C70 encoding plasmid DNA with the gene gun (Fig. 1F ). The association of antigen with hsp73 thus strikingly enhances its immunogenicity for CTL precursors supporting CTL priming even after i.d. delivery of low doses of DNA vaccines.

CONCLUSIONS AND SIGNIFICANCE

We designed a vector system for efficient expression of hsp73-associated, chimeric proteins in which an amino-terminal, T-Ag-derived J domain is fused to different carboxyl-terminal fragments of unrelated antigens (Fig. 2 ). Many different sequences from heterologous viral antigens have been fused carboxyl terminally to the T-Ag without negatively affecting the binding of hsp73 to the amino-terminal T-Ag J domain. The amino-terminal domain thus is accessible and conformationally intact when fused to carboxyl-terminal fragments of different origin and length. We have not yet found a chimeric construct with an amino-terminal T-Ag fragment that has lost hsp73 binding. The observation that the expression system described can efficiently produce large chimeric protein antigens in stable association with hsp73 is of interest for vaccine designs. We designed vectors that allow expression of up to 800 residue chimeric proteins cloned behind the hsp73 binding DnaJ-like domain of T-Ag. More than 30 different viral antigens have been successfully expressed in this system. Proteins or protein domains that could not be expressed without hsp73 association showed stable expression when fused to the J domain-containing NH₂ terminus (e.g., the HBV preS domain). The system thus facilitates antigen expression by vector DNA.

View larger version (41K): [in this window] [in a new window]   Figure 2.

Hsp molecules also enhance and modulate the immunogenicity of protein and peptide antigens. We have shown that hsp73-bound endogenous antigen is submitted to TAP-independent, endolysosomal processing for MHC class I-restricted epitope presentation, facilitates cross-priming of CTL, and priming of antibody responses to endogenous antigen by DNA vaccines. Here we demonstrate the biochemical basis of these observations and confirm the broad range of potential applications.

Three different approaches have exploited the adjuvant effect of hsp molecules of the hsp70 and hsp90 family. 1) Antigenic peptides can be noncovalently ‘loaded’ (in vitro or in vivo) to purified hsp molecules and injected into mice. Delivered in this way, they display greatly enhanced immunogenicity. 2) Antigenic protein determinants can be fused to hsp molecules and injected as recombinant fusion proteins. These constructs efficiently elicit CD4⁺ helper T cell-independent CD8⁺ T cell responses. 3) Our approach involved the expression of CTL and antibody binding epitopes as chimeric fusion proteins containing an hsp73 binding, amino-terminal viral J domain. This allowed high-level antigen expression in noncovalent but tight association with constitutively expressed cytosolic hsp73 molecules. This strikingly enhanced the immunogenicity of hsp73-associated antigens (vs. nonassociated antigens) delivered by plasmid DNA-based vaccines. The data lay the groundwork for the rational design of DNA vaccines with a broad spectrum of potential applications exploiting insights into the biology of viral antigen expression, hsp-facilitated processing and presentation, and innate adjuvant activities. They contribute to the new field of the biotechnology of rational vaccine designs, an area of increasing importance in molecular medicine.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0993fje; to cite this article, use FASEB J. (May 8, 2002) 10.1096/fj.01-0993fje.

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