Induction of MHC class I-restricted CTL response by DNA immunization with ubiquitin-influenza virus nucleoprotein fusion antigens

DNA vaccines have been shown to be an effective means of inducing cytotoxic T-lymphocyte (CTL) responses in both young and aged mice. Better understanding of the pathways by which antigens encoded by DNA vaccines are processed and presented to CTL may allow for improvements in CTL responses in older animals. Since CTL recognize short peptides presented by MHC class I molecules, and since ubiquitin-dependent proteolysis is widely believed to be responsible for degradation of endogenously synthesized antigens and generation of these peptide ligands, we sought to use ubiquitin (Ub) conjugation to target influenza virus nucleoprotein (NP) antigen into the Ub-proteasome degradation pathway for MHC class I-restricted antigen processing and presentation. However, the addition of the Ub moiety did not affect the half-life of Ub-NP protein in transiently transfected human rhabdomyosarcoma (RD) cells. Moreover, the modifications of NP DNA vaccine with Ub conjugation did not affect their ability to induce a CTL response specific for the H-2Kd-restricted NP147-155 epitope, as assessed by both percent cytolysis in bulk CTL culture and by CTL precursor (CTLp) frequency in limiting dilution analysis (LDA). In contrast, the anti-NP antibody (Ab) responses were dramatically reduced in mice immunized with low doses (1 microgram) of Ub-NP constructs, compared with mice immunized with wild-type NP DNA. These results demonstrate that Ub conjugation alone does not guarantee targeting of endogenously synthesized antigens for rapid degradation by proteasomes. Furthermore, the ability of ubiquintination to reduce Ab responses to NP without affecting CTL responses suggests that the Ub modifications result in a lower availability of full-length NP from transfected cells in vivo. The implications of these data on antigen presentation and cross-priming are discussed.     

Neonatal DNA immunization with a plasmid encoding an internal viral protein is effective in the presence of maternal antibodies and protects against subsequent viral challenge

Conventional vaccines are remarkably effective in adults but are much less successful in the very young, who are less able to initiate a mature immune response and who may carry maternal antibodies which inactivate standard vaccines. We set out to determine whether DNA immunization might circumvent these problems. We have previously shown that intramuscular injection of plasmid DNA encoding the nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) is capable of inducing immune responses and protecting 50% of adult mice against lethal and sublethal challenge with LCMV. Here we demonstrate that mouse pups injected with the same plasmid hours or days after birth produce major histocompatibility complex-restricted, NP-specific cytotoxic T lymphocytes (CTL) that persist into adulthood; 48% of vaccinated pups responded to subsequent sublethal viral challenge by the accelerated production of anti-NP LCMV-specific CTL, indicating that these animals had been successfully immunized by the plasmid DNA. In addition, these mice showed a >95% reduction in splenic viral titers 4 days postinfection compared to control mice, demonstrating a more rapid control of infection in vivo. Furthermore, pups born of and suckled on LCMV-immune dams (and therefore containing passively acquired anti-LCMV antibodies at the time of DNA inoculation) responded to the DNA vaccine in a similar manner, showing that maternally derived anti-LCMV antibodies do not significantly inhibit the generation of protective immune responses following DNA vaccination. These findings suggest that, at least in this model system, DNA immunization circumvents many of the problems associated with neonatal immunization.     

Early postoperative gadolinium-DTPA-enhanced MR imaging after successful lumbar discectomy

Cytotoxic T lymphocytes (CTLs) recognise antigenic peptides in the context of major histocompatibility complex (MHC) class I molecules on virus-infected cells. The formation and transportation of antigenic peptides to class I MHC in the cells are multi-step reactions known as antigen processing. In order to design a good DNA vaccine, it is important to dissect the specificity of antigen processing. Here we describe the construction of an epitope-based plasmid vector as a device to investigate antigen processing in transfected cells. The epitope-based plasmid vector was constructed by insertion of an epitope-encoding minigene into the lacZ gene. We used a CTL epitope on influenza A virus nucleoprotein (NP366-374 epitope) as a model. Upon transfection, the epitope-based plasmid vector induced the expression of NP epitope antigenically as well as immunogenically. Immunization of mice with plasmid-transfected cells was able to induce NP epitope-specific CTLs in vivo. Moreover, the plasmid vector functioned as a gene vaccine; NP epitope-specific CTLs were primed in vivo upon transfection of the vector into dermis by electroporation. The results suggest that this epitope-based DNA delivery system may provide a new strategy for in vivo induction of epitope-specific CTLs to investigate antigen processing and presentation.     

Priming of CD8+ CTL effector cells in mice by immunization with a stress protein-influenza virus nucleoprotein fusion molecule

Literature is accumulating which suggests the potential for stress proteins to form the basis of a novel vaccine technology. Immunization with mammalian tumor-derived stress proteins and their associated peptides promote anti-tumor immunity. Vaccination with HIV-1 p24 antigen fused to mycobacterial heat shock protein (Hsp) Hsp71 enhances p24-specific immunity, as measured by p24-specific antibody production and in vitro cell proliferation and cytokine induction. An ovalbumin-Hsp71 fusion protein primes ovalbumin-specific CTL activity and resistance to challenge with an ovalbumin-expressing tumor. We have extended these observations by using a mycobacterial Hsp65 fusion molecule to prime CTL specific for a viral antigen. Gene fusion constructs were generated from DNA encoding Mycobacterium bovis strain BCG Hsp65 and individual fragments of influenza virus nucleoprotein (NP) encompassing H-2Kd- and H-2Db-restricted CTL epitopes. The ability of these purified recombinant fusion proteins to prime NP-specific CTL was assessed in mice of appropriate H-2 haplotypes. We observed that adjuvant-free immunization with either fusion protein elicited significant CTL activity when administered at doses of 10-100 micrograms per mouse. An NP fusion protein made with glutathione-S-transferase failed to elicit NP-specific CTL, indicating that the phenomenon requires Hsp65 sequences. A single immunization with the Hsp65-NP fusion protein elicited CTL activity which persisted for a minimum of 4 months post-immunization, at which time it could be boosted by a second immunization. To our knowledge, this is the first report of a member of the Hsp60 family priming for antigen-specific CTL activity when employed as a fusion protein partner.     

Intraductal papillary mucinous tumors of the pancreas: imaging studies and treatment strategies

Heat shock proteins (hsp's) isolated from murine cancer cells can elicit protective immunity and specific cytotoxic T lymphocytes (CTLs) by channeling tumor-derived peptides bound to hsp's to the major histocompatibility class I antigen presentation pathway. Here we have investigated if hsp70 can be used in a novel peptide vaccine for the induction of protective antiviral immunity and memory CTLs. A CTL epitope from the well-defined lymphocytic choriomeningitis virus (LCMV) system was mixed with recombinant hsp70 in vitro under conditions that optimize peptide binding to hsp70. Mice were immunized with the hsp70-peptide mixture and challenged with LCMV. Virus titers were reduced 10-100-fold in these mice compared to control mice. Immunization with the hsp70-peptide mixture resulted in the development of CTL memory cells that could be reactivated during LCMV infection, and that in a 51Cr-release assay could lyse cells pulsed with the same peptide, but not cells pulsed with another LCMV peptide. These results show that hsp70 can be used with CTL epitopes to induce efficient protective antiviral immunity and the generation of peptide-specific CTLs. The results also demonstrate the usefulness of hsp70 as an alternative to adjuvants and DNA vectors for the delivery of CTL epitopes to antigen-presenting cells.     

Identification of Db- and Kb-restricted subdominant cytotoxic T-cell responses in lymphocytic choriomeningitis virus-infected mice

Antiviral cytotoxic T-cells are critical for control of lymphocytic choriomeningitis virus (LCMV) infection in mice. In H-2b mice, the antiviral response is directed against three Db-restricted epitopes in the viral nucleoprotein (NP396-404) and glycoprotein (GP276-286 and GP33-41). Our present data revealed a clear hierarchy among these three epitopes, in which NP396-404 is immunodominant, followed by GP33-41 and GP276-286, respectively. In order to identify additional CTL epitopes in the LCMV nucleoprotein and glycoprotein, we used the motifs for Db2- and Kb-binding peptides, combined with MHC class I-binding assays. Out of 23 Db motif-fitting peptides, we identified 4 Db binders, one of which (GP92-101) turned out to be a new CTL epitope. Among 28 Kb motif-fitting peptides, 12 bound Kb, and one of these (NP205-212) was a CTL epitope. Both newly identified CTL peptides were recognized by LCMV-immune splenocytes after secondary in vitro stimulation. Both peptides bound their MHC class I molecules with intermediate affinity (470 and 170 nM for GP92-101 and NP205-212, respectively). Responses against these peptides were weaker than the responses against the three major epitopes. None of the high affinity binders were new epitopes, suggesting that high affinity binders are either immunodominant epitopes or no epitopes at all. Thus, analysis of 51 Kb and Db motif-fitting peptides yielded 2 new, subdominant epitopes. Immunization of C57BL/6 mice with these peptides, or vaccinia virus recombinants expressing these epitopes as minigenes, protected against chronic LCMV infection, demonstrating that immunization with subdominant epitopes can confer protection against chronic viral infection.     

The mode of presentation and route of administration are critical for the induction of immune responses to p53 and antitumor immunity

We have examined the immune response to full-length wild-type human p53 presented by a recombinant canarypox vector (ALVAC) and by plasmid DNA. For the ALVAC recombinant, intravenous, but not subcutaneous, intramuscular or intradermal administration, induced CD8+ CTLs that lysed tumor cells transfected with human mutant p53. Intrasplenic administration also induced CTLs. Biodistribution studies showed that intravenously injected ALVAC localized primarily in the lung, liver and spleen, whereas intramuscularly injected virus remained predominantly at the injection site. Intradermal and intramuscular immunization with naked plasmid DNA encoding human wild-type p53 also induced a specific CTL response. DNA immunization induced complete protection against challenge with a mouse embryo fibroblast transfected with human mutant p53 and partial, but significant, protection against a transfected mastocytoma. The ALVAC recombinant induced partial protection in both models. These results suggest that recombinant ALVAC and DNA might be interesting presentation platforms for p53 to be tested in clinical studies.     

Modelling the dynamics of LCMV infection in mice: conventional and exhaustive CTL responses

Lymphocytic choriomeningitis virus (LCMV) infection in mice provides an example of an extraordinarily dynamic process with an extreme sensitivity of phenotype of infection to parameters of virus/host interaction. A mathematical model is developed to examine the dynamics of virus-specific cytotoxic T lymphocyte (CTL) response for LCMV infection in mice. The model, formulated by a system of nonlinear delay-differential equations, considers the interacting populations of viruses, precursor CTLs, terminally differentiated effector CTLs and total virus antigen load. Clonal elimination of virus-specific cytotoxic T cells in high-dose LCMV-Docile infection represents an example of the classical phenomenon--high zone tolerance. To describe both conventional and exhaustive CTL responses in the acute phase of LCMV-D infection two mechanisms are invoked: the high virus antigen load inhibition of T-cells proliferation via energy induction and the activation-induced cell death by apoptosis. Parameters of the model, characterizing the rates of virus and CTL production and elimination in spleen, are estimated by assimilating with the model data on the LCMV-D infection in C57BL/6 mice for low-, moderate- and high-dose infections. It is suggested that not only the clonal expansions have to be described in mathematical models as being virus regulated but also the later phases of primary immune response. Down-regulation of the primary CTL response is controlled by a network of mechanisms inducing anergy and apoptosis in activated T cells. The model is used to investigate the effect of variations in virus and CTL response parameters on LCMV infection outcome and suggest predictions for experimental studies, in particular the phenotype of LCMV-WE infection in C57BL/6 as a function of initial virus doses.     

Differential presentation of the same MHC class I epitopes by fibroblasts and dendritic cells

Ag is presented to CTL as peptide associated with MHC class I molecules, which are present on most types of cells. We have investigated the presentation of Db-restricted lymphocytic choriomeningitis virus (LCMV) peptides by a fibroblast line (MC57) and a dendritic cell line (JawsII) to splenocytes from LCMV-immune C57BL/6 mice. We found that when LCMV-infected MC57 were used to restimulate the spleen cells, the resulting CTL line lost its ability to respond to the two dominant epitopes of the immune response to LCMV glycoprotein (gp)33 and nucleoprotein (np)396 but remained strongly lytic for targets coated with the subdominant gp276 epitope. In contrast, when LCMV-infected JawsII cells were used to restimulate the splenocytes, the resulting line continued to target gp33 and np396 but lost reactivity to gp276. When uninfected JawsII or MC57 cells were coated with peptides and used as stimulators, the resulting CTL lines continued to recognize all three epitopes, indicating that costimulatory or other potential innate differences in Ag presentation between the two cell lines are unlikely to account for the selective expansion of CTL specificities. When infected, both cell types produce similar levels of infectious LCMV, have similar levels of the NP and GP proteins from which np396 and gp33 are derived, and can be recognized by CTL specific for each of the three epitopes. These data indicate that in the generation of peptides for MHC-I binding and presentation to CTL, MC57 and JawsII process the same set of virus proteins in quantitatively different ways.     

Iron-dependent oxidation, ubiquitination, and degradation of iron regulatory protein 2: implications for degradation of oxidized proteins

The ability of iron to catalyze formation of reactive oxygen species significantly contributes to its toxicity in cells and animals. Iron uptake and distribution is regulated tightly in mammalian cells, in part by iron regulatory protein 2 (IRP2), a protein that is degraded efficiently by the proteasome in iron-replete cells. Here, we demonstrate that IRP2 is oxidized and ubiquitinated in cells before degradation. Moreover, iron-dependent oxidation converts IRP2 into a substrate for ubiquitination in vitro. A regulatory pathway is described in which excess iron is sensed by its ability to catalyze site-specific oxidations in IRP2, oxidized IRP2 is ubiquitinated, and ubiquitinated IRP2 subsequently is degraded by the proteasome. Selective targeting and removal of oxidatively modified proteins may contribute to the turnover of many proteins that are degraded by the proteasome.     

A nonimmunodominant nucleoprotein-derived peptide is presented by influenza A virus-infected H-2b cells

Influenza A virus-infected H-2b mice mount a CTL response directed against the nucleoprotein (NP) 366-374 but not against the NP 55-63 peptide, although both peptides fulfill the prerequisites for having high binding affinity toward the Db molecule. Two hypotheses have been proposed to explain the inability of B6 mice to respond to the NP 55-63 peptide: 1) B6 mice are tolerant to the NP 55-63 peptide and 2) NP 55-63 peptide is not naturally processed by H-2b cells. Our results show that 1) B6 mice possess a NP 55-63-specific CTL repertoire because their immunization with the NP 55-63 peptide itself recruits specific CTL; 2) NP 55-63 peptide is naturally processed by the virus-infected H-2b cells but its efficient presentation by the Db molecule requires higher amounts of NP than presentation of the NP 366-374 peptide; 3) NP 55-63 peptide is naturally presented in virus infected B6 mice, however, the quantity of Db/NP 55-63 complexes at the cell surface is sufficient to tolerize but not to recruit and stimulate specific CTL; and 4) NP 55-63 peptide binds to the Db molecule with a lower affinity than NP 366-374 does and this difference could explain the inefficient presentation of the NP 55-63 peptide by B6 cells. The involvement of the self-protein-derived nonimmunodominant peptides in self-tolerance and the possibility of using nonimmunodominant peptides of viral proteins for peptide vaccination are discussed.     

A protective cytotoxic T cell response to a subdominant epitope is influenced by the stability of the MHC class I/peptide complex and the overall spectrum of viral peptides generated within infected cells

This study identifies instability of MHC class I/peptide complexes and intermolecular competition for MHC class I presentation as factors responsible for the subdominance of cytotoxic T lymphocyte (CTL) epitopes. This evidence is based on the characterization of a new CTL epitope derived from the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV). This epitope, peptide GP117-125 (GP117) is presented to T cells by the mouse MHC class I molecule, H-2Db. In short-term experiments induction of GP117-specific CTL by vaccination rendered C57BL/6 mice only partially resistant to infection with wild-type LCMV (LCMV-WE) but completely resistant to challenge with a previously described LCMV variant. The variant virus, LCMV-8.7B23, bears point mutations within both known LCMV-GP, H-2 Db-restricted epitopes GP33-41 (GP33) and GP276-286 (GP276) resulting in a valine to leucine change at position 35 in peptide GP33 (V35L) and an asparagine to serine change at position 280 in peptide GP276 (N280S). Although variant peptide GP33/V35L stimulates a weak CTL response, GP276/N280S does not. Elution of peptide GP117 from both LCMV-WE- and LCMV-8.7B23-infected cells revealed that the difference in the capacity of GP117-specific CTL to protect against LCMV-WE and the virus variant LCMV-8.7B23 was due to differences in the level of GP117 presentation on the surface of both types of cells. Thus, it appears that the protective capacity of CTL specific for the subdominant epitope GP117 is influenced by the extent of presentation of other immunodominant peptide epitopes present within infected cells.     

Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745-1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4+ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8+ T cells and cytokine-secreting CD4+ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.     

Self antigens expressed by solid tumors Do not efficiently stimulate naive or activated T cells: implications for immunotherapy

Induction and maintenance of cytotoxic T lymphocyte (CTL) activity specific for a primary endogenous tumor was investigated in vivo. The simian virus 40 T antigen (Tag) expressed under the control of the rat insulin promoter (RIP) induced pancreatic beta-cell tumors producing insulin, causing progressive hypoglycemia. As an endogenous tumor antigen, the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) was introduced also under the control of the RIP. No significant spontaneous CTL activation against GP was observed. However, LCMV infection induced an antitumor CTL response which efficiently reduced the tumor mass, resulting in temporarily normalized blood glucose levels and prolonged survival of double transgenic RIP(GP x Tag2) mice (137 +/- 18 d) as opposed to control RIP-Tag2 mice (88 +/- 8 d). Surprisingly, the tumor-specific CTL response was not sustained despite the facts that the tumor cells continued to express MHC class I and LCMV-GP-specific CTLs were present and not tolerized. Subsequent adoptive transfer of virus activated spleen cells into RIP(GP x Tag2) mice further prolonged survival (168 +/- 11 d), demonstrating continued expression of the LCMV-GP tumor antigen and MHC class I. The data show that the tumor did not spontaneously induce or maintain an activated CTL response, revealing a profound lack of immunogenicity in vivo. Therefore, repetitive immunizations are necessary for prolonged antitumor immunotherapy. In addition, the data suggest that the risk for induction of chronic autoimmune diseases is limited, which may encourage immunotherapy against antigens selectively but not exclusively expressed by the tumor.     

Ubiquitin-mediated regulation of 3-hydroxy-3-methylglutaryl-CoA reductase

Regulation of the sterol-synthesizing mevalonate pathway occurs in part through feedback-regulated endoplasmic reticulum degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R). In yeast, the Hmg2p isozyme of HMG-R is regulated in this manner. We have tested the involvement of ubiquitination in the regulated degradation of Hmg2p, by using both genetic and direct biochemical approaches. Hmg2p degradation required the UBC7 gene, and Hmg2p protein was directly ubiquitinated. Hmg2p ubiquitination was dependent on UBC7 and was specific for the degraded yeast Hmg2p isozyme. Furthermore, Hmg2p ubiquitination was regulated by the mevalonate pathway in a manner consistent with regulation of Hmg2p stability. Thus, regulated ubiquitination appeared to be the mechanism by which Hmg2p stability is controlled in yeast. Finally, our data indicated that the feedback signal controlling Hmg2p ubiquitination and degradation was derived from farnesyl diphosphate, and thus implied conservation of an HMG-R degradation signal between yeast and mammals.     

Dissecting the immune response to moloney murine sarcoma/leukemia virus-induced tumors by means of a DNA vaccination approach

The intramuscular inoculation of Moloney murine sarcoma/leukemia (M-MSV/M-MuLV) retroviral complex gives rise to sarcomas that undergo spontaneous regression due to the induction of a strong immune reaction mediated primarily by cytotoxic T lymphocytes (CTL). We used a DNA-based vaccination approach to dissect the CTL response against the Gag and Env proteins of M-MSV/M-MuLV in C57BL/6 (B6) mice and to evaluate whether plasmid DNA-immunized mice would be protected against a subsequent challenge with syngeneic tumor cells expressing the viral antigens. Intramuscular DNA vaccination induced CTL against both Gag and Env proteins. A detailed analysis of epitopes recognized by CTL generated in mice inoculated with the whole virus and with the Gag-expressing plasmid confirmed the presence of an immunodominant peptide in the leader sequence of Gag protein (Gag85-93, CCLCLTVFL) that is identical to that described in B6 mice immunized with Friend MuLV-induced leukemia cells. Moreover, CTL generated by immunization with the Env-encoding plasmid recognized a subdominant Env peptide (Env189-196, SSWDFITV), originally described in the B6.CH-2(bm13) mutant strain. B6 mice immunized with the Gag-expressing plasmid were fully protected against a lethal tumor challenge with M-MuLV-transformed MBL-2 leukemia cells, while vaccination with the Env-expressing plasmid resulted in rejection of the tumor in 44% of the mice and in increased survival of an additional 17% of the animals. Taken together, these results indicate the existence of a hierarchy in the capacity of different structural viral proteins to induce a protective immune response against retrovirus-induced tumors.     

The role of macrophages in the induction and regulation of immunity elicited by exogenous antigens

Different delivery vehicles may target to different antigen presenting cells (APC) because of their composition, size and/or physical properties. In this study, we examined the priming of cytotoxic T lymphocyte (CTL) responses to a soluble exogenous protein in vivo, using various delivery vehicles. In addition, we determined the role of macrophages as APC in vivo for each of these delivery vehicles by comparing the induction of antigen-specific CTL and serum antibodies in normal and macrophage-depleted mice. Influenza A virus-derived virosomes, liposomes and monophosphoryl lipid A/squalene (MPLSQ) efficiently induced antigen-specific CTL as well as antibody responses, of which virosomes proved to be the most efficient inducers. In mice that were immunized with cell-associated antigen, strong CTL responses but no antigen-specific antibodies were detectable, while aluminium hydroxide and aluminium phosphate elicited antigen-specific antibodies but no CTL responses. Elimination of macrophages in vivo before immunization abrogated CTL responses induced with liposomes and MPL/SQ, but did not affect induction of antigen-specific CTL with virosomes or cell-associated antigen. Importantly, serum antibody levels were not altered after macrophage depletion, regardless of the delivery vehicle used, suggesting that in the absence of macrophages, other APC may phagocytose the exogenous antigens for major histocompatibility complex (MHC) class II processing and presentation. These results suggest that soluble exogenous antigens delivered in different carrier systems may be processed differently by different APC in vivo for MHC class I- or class II-restricted presentation.     

A welder with chemical pneumonitis caused by inhalation of zinc fume]

The PowderJect system, a device that uses compressed helium gas to accelerate microscopic particles into the skin, was used as a delivery system for DNA vaccines to elicit a virus-specific cytotoxic T cell response (CTL) in mice. Transient expression of beta-galactosidase (beta-Gal) was observed in the epidermis when gold particles coated with beta-Gal expression plasmid were delivered to mouse skin with the device. When DNA encoding the nucleoprotein gene (NP) of influenza A virus was used to coat gold particles, a strong and specific anti-NP CTL response was elicited by immunizations with nanogram amounts of the NP DNA vector. This study shows the potential for application of the PowderJect system to intradermal delivery of DNA in order to elicit an immune response.     

Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Development of CD8 alphabeta CTL epitope-based vaccines requires an effective strategy capable of co-delivering large numbers of CTL epitopes. Here we describe a DNA plasmid encoding a polyepitope or "polytope" protein, which contained multiple contiguous minimal murine CTL epitopes. Mice vaccinated with this plasmid made MHC-restricted CTL responses to each of the epitopes, and protective CTL were demonstrated in recombinant vaccinia virus, influenza virus, and tumor challenge models. CTL responses generated by polytope DNA plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering a gene for granulocyte-macrophage CSF, and appeared to be induced in the absence of CD4 T cell-mediated help. The ability to deliver large numbers of CTL epitopes using relatively small polytope constructs and DNA vaccination technology should find application in the design of human epitope-based CTL vaccines, in particular in vaccines against EBV, HIV, and certain cancers.     

Antigen expressed on tumor cells fails to elicit an immune response, even in the presence of increased numbers of tumor-specific cytotoxic T lymphocyte precursors

We have used T-cell receptor (TCR) transgenic mice to analyze the interaction of tumors with the immune system. We show that the tumor cell line Lewis lung-lymphocytic choriomeningitis virus (LL-LCMV), genetically manipulated to express an H-2 Db-restricted epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV33-41), can grow progressively in TCR transgenic mice, where approximately 50% of CD8+ T cells are specific for LCMV33-41. TCR transgenic T cells were not deleted in tumor-bearing mice, and their surface phenotype and cytokine secretion patterns remained typical of naive T cells. Also, TCR transgenic T cells from tumor-bearing mice had undiminished capacity to proliferate to antigen in vitro. Tumor-protective immune responses could be elicited in TCR transgenic mice by immunization with LCMV33-41 peptide-loaded dendritic cells. Tumor resistance correlated with the switch of TCR transgenic T cells from a CD44low to a CD44high phenotype and increased capacity to produce IFNgamma in vitro. Results similar to those obtained in TCR transgenic mice were also obtained using an adoptive transfer system, where small numbers of TCR transgenic T cells were injected into normal C57BL/6 hosts. These data indicate that even large tumors may not induce specific immunization, tolerance, or anergy to tumor antigens, and that high numbers of tumor-specific CTL precursors are not sufficient to provide tumor resistance.