Biomedical subjects

R Schirmbeck

Publications and source records attributed to R Schirmbeck.

At least 19 recordsLinked to original sources

PubMed2000

Efficient vaccination by intradermal or intramuscular inoculation of plasmid DNA expressing hepatitis B surface antigen under desmin promoter/enhancer control.

The small surface antigen of the hepatitis B virus (HB5Ag) was cloned into expression plasmid pCI under either a viral (CMV) promoter;enhancer sequence control (plasmid pCI/S), or a human desmin promoter/enhancer sequence control (plasmid pDes/S). Cells of different species and tissue origin transiently transfected in vitro with pCI/S or pDes/S plasmid DNA expressed readily detectable amounts of HBsAg, either intracellularly (precipitated from cell lysates), or as secreted products (detectable in ELISA). When these plasmids were used in DNA vaccination, both efficiently primed humoral and/or cellular immune responses to HBsAg after a single injection in Balb/c mice. Intramuscular injection of a high dose of DNA (100 rig/mouse) of both plasmids primed MHC-I-restricted cytotoxic T lymphocyte (CTL) responses and Thi serum antibody responses (IgGlIgG2a ratio O.4C0.7) of comparable magnitude in all vaccinated mice. Intradermal injection of low doses of (particle-coated) DNA (1 microgm/mouse) of both plasmids with the gene gun primed Th2 serum antibody responses (IgGl/IgG2a ratio > 100) but no CTL responses. The data indicate that antigens can be efficiently expressed under viral or eukaryotic promoter/enhancer control for immunogenic in vivo presentation, but that the technique, dose and/or route of DNA injection have a decisive role in determining the type of immune response elicited.

Animals↗

PubMed2000

Ongoing murine T1 or T2 immune responses to the hepatitis B surface antigen are excluded from the liver that expresses transgene-encoded hepatitis B surface antigen.

Different protein- or DNA-based vaccination techniques are available that prime potent humoral and cellular, T1 or T2 immune responses to the hepatitis B surface Ag (HBsAg) in mice. T1 and T2 are immune responses with isotype profile indicating Th1 and Th2 immunoregulation. We tested whether HBsAg-specific immune responses can be established in transgenic mice that express HBsAg in the liver (HBs-tg mice) using either these different vaccination techniques or an adoptive transfer system. HBsAg-specific responses could not be primed in HBs-tg mice with the established, potent vaccine delivery techniques. In contrast, adoptive transfers of T1- and T2-type HBsAg-immune spleen cells into congenic HBs-tg hosts (that were not conditioned by pretreatment) suppressed HBsAg antigenemia and gave rise to HBsAg-specific serum Ab titers. The establishment of continuously rising anti-HBsAg serum Ab levels with alternative isotype profiles (reflecting T1 or T2 polarization) in transplanted HBs-tg hosts required donor CD4+ T cell-dependent restimulation of adoptively transferred immune cells by transgene-derived HBsAg. Injections of HBsAg-specific Abs into HBs-tg mice did not establish stable humoral immunity. The expanding T1 or T2 immune responses to HBsAg in HBs-tg hosts did not suppress transgene-directed HBsAg expression in the liver and did not induce liver injury. In addition to priming functional antiviral effector cells, the conditioning of the liver microenvironment to enable delivery of antiviral effector functions to this organ are therefore critical for effective antiviral defense. A major challenge in the development of a therapeutic vaccine against chronic hepatitis B or C virus infection is thus the efficient targeting of specifically induced immune effector specificities to the liver.

Adoptive Transfer↗

PubMed1999

Priming MHC-I-restricted cytotoxic T lymphocyte responses to exogenous hepatitis B surface antigen is CD4+ T cell dependent.

MHC-I (Ld)-restricted, S28-39-specific CTL responses are efficiently primed in H-2d BALB/c mice injected with low doses of native hepatitis B surface Ag (HBsAg) lipoprotein particles without adjuvants. Priming of this CTL response by exogenous HBsAg required CD4+ T cell "help" and IL-12: this CTL response could be neither induced in mice depleted of CD4+ T cells by in vivo Ab treatment, nor in (CD4+ T cell-competent or CD4+ T cell-depleted) IL-12-unresponsive STAT4-/- knockout BALB/c mice. Codelivery of oligonucleotides (ODN) with immunostimulating CpG sequences (ISS) with exogenous HBsAg reconstituted the CTL response to exogenous HBsAg in CD4+ T cell-depleted normal mice and in CD4+ T cell-competent and CD4+ T cell-depleted STAT4-/- BALB/c mice. Injection (by different routes) of "naked" pCI/S plasmid DNA encoding HBsAg into IL-12-responsive or -unresponsive BALB/c mice efficiently primed the MHC-I-restricted, HBsAg-specific CTL response. CTL priming was not detectable when CD4+ T cell-depleted animals were subjected to genetic immunization. In vivo priming of the well-characterized CD8+ CTL response to HBsAg in "high responder" BALB/c mice either by exogenous surface lipoprotein particles or by DNA vaccination is thus CD4+ T cell dependent. CTL priming by exogenous HBsAg, but not by genetic immunization, is IL-12 dependent. The dependence of CTL priming by exogenous HBsAg on CD4+ T cells can be overcome by codelivering ODN with ISS motifs, and this "adjuvants effect" operates efficiently in IL-12-unresponsive mice. The data characterize a feature of the adjuvant effect of ISS-containing ODN on CTL priming that may be of major interest for the design of CTL-stimulating vaccines with efficacy in immunodeficiency conditions.

Adjuvants, Immunologic↗

PubMed1999

DNA immunization confers systemic, but not mucosal, protection against enteroinvasive bacteria.

Naked plasmid DNA (pRc/Y-hsp60) with a cytomegalovirus promoter and a sequence encoding Yersinia enterocolitica 60-kDa heat shock protein (Y-HSP60) was used for vaccination. After intramuscular injection of pRc/Y-hsp60, Y-hsp60 mRNA could be detected by reverse transcription-PCR in muscle, liver and spleen. A single immunization with pRc/Y-hsp60 induced significant Y-HSP60-specific T cell responses after 1 week. IFN-gamma production by spleen cells upon stimulation with Y-HSP60 was strictly dependent on the presence of CD4+ T cells, indicating the generation of a Th1 response upon DNA immunization. DNA immunization in addition induced strong Y-HSP60-specific IgG2a, weak IgG1, but not IgA antibodies. Immunization of BALB/c and C57BL/6 mice with pRc/Y-hsp60 conferred protection against disseminated Y. enterocolitica infection in spleen, but not at the site of mucosal entry, the Peyer's patches. Furthermore, pRc/Y-hsp60 vaccination did not induce cross-protection against related pathogens. Vaccination of beta2-microglobulin- and H2-I-Abeta-deficient mice was not protective, suggesting that both CD4+ and CD8+ T cells are required for protective immunity induced by DNA vaccination.

Animals↗

PubMed1999

Truncated or chimeric endogenous protein antigens gain immunogenicity for B cells by stress protein-facilitated expression.

Truncated variants of the SV40 large T antigen (T-Ag) with an intact N terminus are as efficiently expressed in eukaryotic transfectants as wild-type (wt) T-Ag. Coprecipitation of N-terminal T-Ag fragments with the constitutively expressed, cytosolic stress protein hsp73 suggests that this chaperone stabilized expression of the truncated T-Ag fragments. In contrast to T-Ag, the 163-residue N-terminal preS domain of the hepatitis B surface antigen (HBsAg) is difficult to express. When the preS domain is C-terminally fused to a hsp73-binding cytoplasmic T-Ag (cT-Ag) fragment its stable expression as a chimeric cT-preS protein is obtained. DNA-based vaccination with plasmid DNA encoding either wt or hsp-associated mutant T-Ag elicited potent MHC class I-restricted, T-Ag-specific T cell responses. In contrast, DNA vaccination with hsp73-binding (mutant or chimeric) T-Ag variants, but not with wt T-Ag elicited T-Ag-specific antibody responses. Furthermore, vaccination with cT-preS-encoding plasmid DNA induced antibodies binding to the preS domain of the large HBsAg. Hence, hsp73-bound endogenous antigens efficiently stimulate antibody responses. These findings may be relevant for tumor immunology and autoimmunity.

Animals↗

PubMed1999

Purification and characterization of hepatitis B virus surface antigen particles produced in Drosophila Schneider-2 cells.

The small surface antigen of hepatitis B virus (HBV) was produced in Drosophila melanogaster Schneider-2 (DS-2) cells transfected stably using an inducible Drosophila metallothionein promoter. Selected clonal DS-2 cell-lines expressed and secreted large quantities of HBsAg particles consisting exclusively of non-glycosylated 25 kDa proteins. HBsAg produced by DS-2 cells had physical and biochemical properties very similar to 22 nm particles derived from the human hepatoma cell-line PLC/PRF/5. DS-2 cell-derived HBsAg particles were purified near homogeneity by a strategy based on protein concentration, precipitation and ultracentrifugation. The resulting HBsAg product was < 98% pure. A single immunisation of BALB/c mice with both DS-2 and yeast-cell derived purified HBsAg particles without adjuvants elicited a substantial humoral antibody and class-I restricted cytotoxic T lymphocyte (CTL) response. Adsorption of HBsAg particles to aluminium hydroxide resulted in increased levels of HBsAg-specific antibodies. However, CTLs were not elicited by HBsAg/Alum combinations. Thus, stably transfected DS-2 cells provide a useful source for the production of HBV subviral particles for diagnostic and research purposes as well as for novel vaccine development.

Animals↗

PubMed1999

Adjuvants that enhance priming of cytotoxic T cells to a Kb-restricted epitope processed from exogenous but not endogenous hepatitis B surface antigen.

Intramuscular (i.m.) or s.c. injection of plasmid DNA encoding hepatitis B small surface antigen (HBsAg) primes potent MHC I-restricted cytotoxic T lymphocyte (CTL) responses in H-2(d) (BALB/c) and H-2(b) (C57BL/6) mice. In contrast, i.m. or s.c. injection of exogenous HBsAg particles without adjuvants primes CTL responses in 'high responder' H-2(d) but not 'low responder' H-2(b) mice. We have shown that processing of exogenous but not endogenous HBsAg generates the Kb-binding S208-215 peptide ILSPFLPL. This system allowed us to optimize conditions for stimulating murine CTL responses to exogenous antigen by identifying adjuvants that facilitate priming of Kb-restricted CTL by injecting recombinant HBsAg particles into 'low responder' H-2(b) mice. Synthetic oligodeoxynucleotides with immunostimulating sequences or the recombinant cytokine IL-12 efficiently enhanced priming of CTL to exogenous HBsAg. Hence, the adjuvanticity of DNA sequences that induce Th1 cytokines facilitate priming of MHC I-restricted T cell responses to exogenous antigen and are therefore of potential value in formulating vaccines designed to enhance CTL priming to exogenous antigen.

Adjuvants, Immunologic↗

PubMed1999

Alternative pathways for processing exogenous and endogenous antigens that can generate peptides for MHC class I-restricted presentation.

The concept of distinct endogenous and exogenous pathways for generating peptides for MHC-I and MHC-II-restricted presentation to CD4+ or CD8+ T cells fits well with the bulk of experimental data. Nevertheless, evidence is emerging for alternative processing pathways that generate peptides for MHC-I-restricted presentation. Using a well characterized, particulate viral antigen of prominent medical importance (the hepatitis B surface antigen), we summarize our evidence that the efficient, endolysosomal processing of exogenous antigens can lead to peptide-loaded MHC-I molecules. In addition, we describe evidence for endolysosomal processing of mutant, stress protein-bound, endogenous antigens that liberate peptides binding to (and presented by) MHC-I molecules. The putative biological role of alternative processing of antigens generating cytotoxic T-lymphocyte-stimulating epitopes is discussed.

Animals↗

PubMed1999

Immunization of woodchucks with plasmids expressing woodchuck hepatitis virus (WHV) core antigen and surface antigen suppresses WHV infection.

DNA vaccination can induce humoral and cellular immune response to viral antigens and confer protection to virus infection. In woodchucks, we tested the protective efficacy of immune response to woodchuck hepatitis core antigen (WHcAg) and surface antigen (WHsAg) of woodchuck hepatitis virus (WHV) elicited by DNA-based vaccination. Plasmids pWHcIm and pWHsIm containing WHV c- or pre-s2/s genes expressed WHcAg and WHsAg in transient transfection assays. Pilot experiments in mice revealed that a single intramuscular injection of 100 microgram of plasmid pWHcIm DNA induced an anti-WHcAg titer over 1:300 that was enhanced by boost injections. However, two injections of 100 microgram of pWHcIm did not induce detectable anti-WHcAg in woodchucks. With an increase in the dose to 1 mg of pWHcIm per injection, transient anti-WHcAg response and WHcAg-specific proliferation of peripheral mononuclear blood cells (PMBCs) appeared in woodchucks after repeated immunizations. Four woodchucks vaccinated with pWHcIm were challenged with 10(4) or 10(5) of the WHV 50% infective dose. They remained negative for markers of WHV replication (WHV DNA and WHsAg) in peripheral blood and developed anti-WHs in week 5 after challenge. In contrast, woodchucks not immunized or immunized with the control vector pcDNA3 developed acute WHV infection. Two woodchucks immunized with 1 mg of pWHsIm developed WHsAg-specific proliferative response of PBMCs but no measurable anti-WHsAg response. A rapid anti-WHsAg response developed during week 2 after virus challenge. Neither woodchuck developed any signs of WHV infection. These data indicate that DNA-based vaccination with WHcAg and WHsAg can elicit immunity to WHV infection.

Amino Acid Sequence↗

PubMed1999

Enhancing the immunogenicity of exogenous hepatitis B surface antigen-based vaccines for MHC-I-restricted T cells.

Vaccination with either exogenous hepatitis B surface antigen (HBsAg) lipoprotein particles without adjuvants, or plasmid DNA encoding secreted small HBsAg stimulate long-lasting, potent antibody responses in H-2d (BALB/c) and C57Bl/6 (H-2b) mice. Vaccination with exogenous HBsAg primes MHC-I restricted cytotoxic T lymphocyte (CTL) responses to HBsAg in H-2d but not H-2b mice, while DNA vaccination primes HBsAg-specific CTL responses in both mouse strains. We defined vaccination strategies that could elicit CTL responses to exogenous HBsAg in 'low responder' C57Bl/6 mice. We found that the bacterial plasmid DNA itself, synthetic oligodeoxynucleotides containing immunostimulating sequences, or recombinant Th1 cytokines (IL12, IFNgamma) efficiently support priming of CTL responses to exogenous HBsAg in 'low responder' H-2b mice, but have only minor effects on CTL priming in 'high responder' H-2d mice in the high dose range tested. These molecularly well defined adjuvants can thus efficiently support priming of anti-viral T cell responses under 'low responder' conditions.

Animals↗

PubMed1999

Immunostimulatory CpG motifs trigger a T helper-1 immune response to human immunodeficiency virus type-1 (HIV-1) gp 160 envelope proteins.

Bacterial DNA sequences containing unmethylated CpG motifs have recently been proposed to exhibit immunostimulatory effects on B-, T- and NK cells, leading to the induction of humoral and cell-mediated immune responses. In the present study we investigated the immunomodulatory effects of a CpG-containing oligodeoxynucleotide (CpG ODN) to the HIV-1 gp 160 envelope (Env) protein in the BALB/c mouse model. Priming and boosting of mice with gp 160 adsorbed to aluminium hydroxide (Alum) induced a typical T helper-2 (Th2)-dominated immune response with high titers of gp 160-specific immunoglobulin (Ig)G1 isotypes but a weak IgG2a response. Specifically re-stimulated splenocytes from these mice predominantly secreted interleukin (IL)-5 but only minute amounts of interferon-gamma (IFN-gamma) upon specific re-stimulation. In contrast, a boost immunisation of gp 160/Alum primed mice with a gp 160/Alum/CpG combination resulted in a seven times higher production of IgG2a antibodies, without affecting the titers of IgG1 isotypes. Furthermore, approximately 10-fold increased levels of IFN-gamma, but significantly reduced amounts of IL-5, were secreted from gp 160-restimulated splenic cells. A further greater than 30-fold increase in the levels of specific IgG2a responses and a substantially elevated secretion of IFN-gamma were observed when the mice received gp160/Alum/CpG combinations for priming and boost injections. Thus, CpG ODNs are useful as an adjuvant to induce a typical Th0/Th1 response to HIV gp 160 proteins. However, despite the induction of a more Th1-like immune response, gp 16O/Alum/CpG combinations were not sufficient to prime an Env-specific cytotoxic T-cell (CTL) response.

AIDS Vaccines↗

PubMed1998

T cell-mediated, IFN-gamma-facilitated rejection of murine B16 melanomas.

The murine melanoma cell line B16.F10 (H-2b) was used to study specific T cell responses that reject tumors. Stable B16 transfectants were established that express viral Ags, either the hepatitis B surface Ag (HBsAg) or the large tumor Ag (T-Ag) of SV40. B16 cells and their transfected sublines were CD40+ CD44+ but expressed no (or low levels of the) costimulator molecules CD154 (CD40L), CD48, CD54, CD80, and CD86. Surface expression of MHC class I (Kb, Db) and class II (I-Ab) molecules by B16 cells was low, but strikingly up-regulated by IFN-gamma. CD95 (Fas) and CD95 ligand (CD95L (FasL)) were "spontaneously" expressed by B16 cells growing in vitro in serum-free medium; these markers were strikingly up-regulated by IFN-gamma. B16 cells coexpressing CD95 and CD95L were irreversibly programmed for apoptosis. In vitro, noninduced B16 transfectants stimulated a specific IFN-gamma release response, but no cytolytic response (in a 4-h assay) in MHC class I-restricted CTL; in contrast, IFN-gamma-induced B16 targets were efficiently and specifically lysed by CTL. In vivo, B16 transfectants were specifically rejected by DNA-vaccinated syngeneic hosts through a T-dependent immune effector mechanism. The tumors showed evidence of massive apoptosis in vivo during the rejection process. The data suggest that CTL-derived IFN-gamma enhances an intrinsic suicide mechanism of these tumor cells in addition to facilitating lytic interactions of effectors with tumor targets.

Animals↗

PubMed1998

The hepatitis B virus e antigen cannot pass the murine placenta efficiently and does not induce CTL immune tolerance in H-2b mice in utero.

The function of the secretory core gene product (HBeAg) of the human hepatitis B virus is unclear. It has been discussed that this protein may be passed from the mother to the fetus, where it might induce immunologic tolerance. Here we have examined this possibility with transgenic mice expressing high levels of HBeAg. Analysis of serum samples obtained from nontransgenic fetuses which developed in HBeAg-positive mothers showed no evidence that the HBeAg can pass the placenta. Moreover, direct examination of the HBeAg- and HBcAg-specific cytotoxic T-cell immune response of H-2b mice which developed in either transgenic or nontransgenic mothers revealed no indication that mice which could have been exposed to the HBeAg in utero become tolerant to HBV core gene products. From these data we conclude that the placenta represents an efficient barrier for HBeAg transfer and that the HBeAg does not tolerize cytotoxic T cells, at least in mice of the H-2b haplotype.

Animals↗

PubMed1998

Similar as well as distinct MHC class I-binding peptides are generated by exogenous and endogenous processing of hepatitis B virus surface antigen.

Murine MHC class I-restricted cytotoxic T lymphocyte (CTL) responses can be primed by exogenous as well as endogenous hepatitis B surface antigen (HBsAg). Immunodominant CTL-defined epitopes of this viral envelope protein are the Ld-binding 12-mer S28-39 peptide IPQSLDSWWTSL in H-2d mice, and the Kb-binding 8-mer S208-215 peptide ILSPFLPL in H-2b mice. We tested if CTL recognizing these epitopes can be primed in vivo by HBsAg delivered as either an exogenous antigen (native HBsAg lipoprotein particles), or an endogenous antigen (plasmid DNA encoding HBsAg). Primed T cells were restimulated in vitro prior to the cytotoxicity assay with cells presenting the H-2 class I-binding epitopes generated by either exogenous or endogenous processing of HBsAg. The data indicate that the Ld-binding peptide S28-39 is generated during exogenous as well as endogenous processing of HBsAg. In contrast, the Kb-binding peptide S208-215 is generated during exogenous but not endogenous processing of HBsAg. Hence, some but not all MHC class I-binding, immunogenic peptides are generated during endogenous and exogenous processing of HBsAg but there also exists a repertoire of immunogenic peptides of viral origin that is only revealed after exogenous processing of viral proteins.

Animals↗

PubMed1998

Cytokine and hepatitis B virus DNA co-immunizations enhance cellular and humoral immune responses to the middle but not to the large hepatitis B virus surface antigen in mice.

Genetic immunization is a potentially useful strategy to prevent or treat hepatitis B virus (HBV) infection. We have previously shown that HBV envelope proteins are highly immunogenic using this technique. The large envelope protein (LHBs), however, induced significantly weaker humoral and cellular immune responses when compared with the middle envelope protein (MHBs). We studied the effect of co-immunizations with cytokine DNA expression constructs encoding for interleukin (IL)-2 and (GM-CSF) on the immunogenicity of LHBs at the B-and T-cell level. Co-immunizations of mice with plasmids encoding for MHBs and IL-2 or GM-CSF increased anti-HBs responses, helper T-cell proliferative activity, and cytotoxic T lymphocyte (CTL) killing. In contrast, co-immunizations of plasmids encoding for LHBs and IL-2 or GM-CSF had no effect on humoral and cellular immune responses. LHBs did not inhibit the production or secretion of IL-2 and GM-CSF. In addition, IL-2, tumor necrosis factor alfa (TNF-alpha), and interferon gamma (IFN-gamma) had no suppressive effect on HBV envelope protein expression in vitro. Based on these data, MHBs, but not LHBs, genetic immunization can be augmented by IL-2 or GM-CSF cytokines.

Animals↗

PubMed1998

Polyvalent vaccination against hepatitis B surface and core antigen using a dicistronic expression plasmid.

Genes encoding the small (S) surface antigen (HBsAg) or the core (C) antigen (HBcAg) of hepatitis B virus (HBV) were cloned into the monocistronic expression vectors pCMV-1 or pCMV-2 under HCMV-IE promoter control. Coding fragments of these vectors were fused to generate a dicistronic expression construct pCMV/C-S in which the antigens HBcAg and HBsAg are coexpressed. Transient in vitro transfection studies demonstrated that HBcAg and HBsAg are coexpressed from this construct. Vaccination of mice of different H-2 haplotypes with mono- or dicistronic expression plasmids induced humoral and cellular immune responses to HBsAg and the HBcAg. In particular, intramuscular injection of 'naked' dicistronic plasmid DNA into mice elicited polyvalent humoral and cytotoxic T lymphocyte responses to HBsAg and HBcAg. The studies demonstrate that dicistronic expression plasmids are a novel way to construct a polyvalent vaccine against HBV that comprises HBsAg and HBcAg as immunogens.

Animals↗

PubMed1997

Recombinant human immunodeficiency Pr55gag virus-like particles presenting chimeric envelope glycoproteins induce cytotoxic T-cells and neutralizing antibodies.

Very recently, we demonstrated that the replacement of the human immunodeficiency virus type-1 (HIV-1) gp41 transmembrane protein by an Epstein-Barr virus gp220/350-derived membrane anchor resulted in the incorporation of chimeric envelope (Env) oligomers into Pr55gag virus-like particles (VLPs), exceeding that of wild-type gp160 by a factor of 10. In this study, we examined the immunostimulatory properties of Pr55gag VLPs to both (i) chimeric HIV-1 gp120 external envelope proteins and (ii) full-length gp160 presented on the outer surface of the particles. Immunization studies carried out with VLPs presenting different derivatives of the chimeric and wild-type Env proteins elicited a consistent anti-Pr55gag as well as anti-Env antibody response in complete absence of additional adjuvants. In both cases, the immune sera exhibited an in vitro neutralizing activity against homologous HIV-1 infection in MT4 cells. Noteworthy, these VLPs were also capable of inducing a strong CD8+ cytotoxic T-cell (CTL) response in immunized BALB/c mice that was directed toward a known CTL epitope in the third variable domain V3 of the gp120 external glycoprotein. However, the induction of V3-loop-specific CTLs critically depended on the amounts of Env proteins that were presented by the Pr55gag VLPs. Moreover, the CD8+ CTL response was not significantly altered by adsorbing the VLPs to alum or by repeated booster immunizations. These results illustrate that Pr55gag VLPs provide a safe and effective means of enhancing neutralizing humoral responses to particle-entrapped gp120 proteins and are also capable of delivering these proteins to the MHC class I antigen processing and presentation pathway. Therefore, antigenically expanded Pr55gag VLPs represent an attractive approach in the design of vaccines for which specific stimulation of neutralizing antibodies and cytotoxic effector functions to complex glycoproteins is desired.

AIDS Vaccines↗

PubMed1997

Stress protein (hsp73)-mediated, TAP-independent processing of endogenous, truncated SV40 large T antigen for Db-restricted peptide presentation.

Transporter associated with antigen processing (TAP)-competent and TAP-deficient cell lines were transfected with expression plasmids encoding either the wild-type (wt) large tumor antigen (T-Ag) of SV40, or a truncated cytoplasmic variant (cT-Ag) of this viral protein. Stable expression of comparable levels of both forms of the viral protein was observed in different transfectants. The truncated cT-Ag variant, but not the wtT-Ag was stably associated with the constitutively expressed, cytosolic heat shock protein (hsp)73 chaperone. Two Db-binding peptides and one Kb-binding peptide of T-Ag were presented to cytotoxic T lymphocyte lines (CTLL) by TAP-competent transfectants expressing either wtT-Ag or cT-Ag. TAP-deficient transfectants expressing the wtT-Ag did not present any of these epitopes to CTLL. In contrast, TAP-deficient transfectants expressing the truncated hsp73-associated cT-Ag, presented the two Db-binding epitopes, but not the Kb-binding T-Ag epitope to CTLL. Regurgitation of peptides by transfectants was not detectable. The described data indicate that a pool of post-Golgi Db molecules is available for 2-3 h in TAP-deficient transfectants for loading with peptides released during endolysosomal processing of hsp73-associated, endogenous antigen.

Animals↗