Multiepitope Trojan antigen peptide vaccines for the induction of antitumor CTL and Th immune responses

We describe in this study a strategy to produce synthetic vaccines based on a single polypeptide capable of eliciting strong immune responses to a combination CTL and Th epitopes with the purpose of treating malignancies or preventing infectious diseases. This strategy is based on the capacity of Trojan Ags to deliver exogenous Ags into the intracellular compartments, where processing into MHC-binding peptides takes place. Our previous work demonstrated that Trojan Ags containing a CTL epitope localized to intracellular compartments, where MHC class I-binding peptides were generated in a TAP-independent fashion by the action of various exopeptidases and the endopeptidase furin. In this study, we report that Trojan Ags containing several CTL epitopes joined via furin-sensitive linkers generated all of the corresponding MHC class I-binding peptides, which were recognized by CTL. However, Trojan Ags prepared with furin-resistant linkers failed to produce the MHC class I-binding peptides. We also present data indicating that Trojan Ags bearing both CTL and Th epitopes can generate the corresponding MHC class I- and II-binding peptides, which are capable of stimulating T cell responses. Most significantly, in vivo vaccination of mice with a single injection of multiepitope Trojan Ags resulted in strong CTL and Th responses that translated into significant antitumor responses in a model of malignant melanoma. The overall results indicate that Trojan Ags prepared with furin-sensitive linkers are ideal candidates for producing synthetic multiepitope vaccines for the induction of CTL and Th responses that could be used against a variety of diseases, including cancer.     

Exogenous peptides delivered by ricin require processing by signal peptidase for transporter associated with antigen processing-independent MHC class I-restricted presentation

In this study we demonstrate that a disarmed version of the cytotoxin ricin can deliver exogenous CD8(+) T cell epitopes into the MHC class I-restricted pathway by a TAP-independent, signal peptidase-dependent pathway. Defined viral peptide epitopes genetically fused to the N terminus of an attenuated ricin A subunit (RTA) that was reassociated with its partner B subunit were able to reach the early secretory pathway of sensitive cells, including TAP-deficient cells. Successful processing and presentation by MHC class I proteins was not dependent on proteasome activity or on recycling of MHC class I proteins, but rather on a functional secretory pathway. Our results demonstrated a role for signal peptidase in the generation of peptide epitopes associated at the amino terminus of RTA. We showed, first, that potential signal peptide cleavage sites located toward the N terminus of RTA can be posttranslationally cleaved by signal peptidase and, second, that mutation of one of these sites led to a loss of peptide presentation. These results identify a novel MHC class I presentation pathway that exploits the ability of toxins to reach the lumen of the endoplasmic reticulum by retrograde transport, and suggest a role for endoplasmic reticulum signal peptidase in the processing and presentation of MHC class I peptides. Because TAP-negative cells can be sensitized for CTL killing following retrograde transport of toxin-linked peptides, application of these results has direct implications for the development of novel vaccination strategies.     

Efficient delivery of Antennapedia homeodomain fused to CTL epitope with liposomes into dendritic cells results in the activation of CD8+ T cells.

The in vivo induction of a CTL response using Antennapedia homeodomain (AntpHD) fused to a poorly immunogenic CTL epitope requires that the Ag is given in presence of SDS, an unacceptable adjuvant for human use. In the present report, we developed a hybrid CTL epitope delivery system consisting of AntpHD peptide vector formulated in liposomes as an alternative approach to bypass the need for SDS. It is proposed that liposomes will prevent degradation of the Ag in vivo and will deliver AntpHD recombinant peptide to the cytosol of APCs. We show in this work that dendritic cells incubated with AntpHD-fused peptide in liposomes can present MHC class I-restricted peptide and induce CTL response with a minimal amount of Ag. Intracellular processing studies have shown that encapsulated AntpHD recombinant peptide is endocytized before entering the cytosol, where it is processed by the proteasome complex. The processed liposomal peptides are then transported to the endoplasmic reticulum. The increase of the CTL response induced by AntpHD-fused peptide in liposomes correlates with this active transport to the class I-processing pathway. In vivo studies demonstrated that positively charged liposomes increase the immunogenicity of AntpHD-Cw3 when injected s.c. in mice in comparison to SDS. Moreover, addition of CpG oligodeoxynucleotide immunostimulatory sequences further increase the CD8+ T cell response. This strategy combining lipid-based carriers with AntpHD peptide to target poorly immunogenic Ags into the MHC class I processing pathway represents a novel approach for CTL vaccines that may have important applications for development of cancer vaccines.     

Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help

The unique ether glycerolipids of ARCHAEA: can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8(+) T cell response, as measured by IFN-gamma production and cytolytic activity toward the immunodominant CTL epitope OVA(257-264). In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8(+) T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4(+) T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8(+) T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8(+) T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8(+) T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.     

HIV envelope protein inhibits MHC class I presentation of a cytomegalovirus protective epitope

CTL recognize peptides that derive from viral protein Ags by proteolytic processing and are presented by MHC class I molecules. In this study we tested whether coexpression of viral Ags in the same cell leads to competition between them. To this end, two L(d)-restricted epitopes derived from HIV-1 envelope gp160 (ENV) and from CMV pp89 phosphoprotein were coexpressed. HIV ENV strain IIIB, but not MN variant, impaired recognition by specific CTL of CMV pp89 epitope 9pp89. Susceptibility to inhibition after ENV coexpression was inversely related to the amount of antigenic 9pp89 peptide processed from different antigenic constructs. In line with it, competition decreased the yield of naturally processed antigenic 9pp89 peptide bound to MHC class I molecules in coinfected cells. Also, point mutants of the presenting MHC class I molecule differed in their competition pattern. Collectively, the data imply that competition operates at the step of MHC-peptide complex assembly or stabilization. We conclude that, although not the rule, in certain combinations there is interference between different Ags expressed in the same cell and presented by the same MHC class I allele. These studies have implications for vaccine development and for understanding immunodominance.     

Presentation of endogenously synthesized MHC class II-restricted epitopes by MHC class II cancer vaccines is independent of transporter associated with Ag processing and the proteasome

Cell-based vaccines consisting of invariant chain-negative tumor cells transfected with syngeneic MHC class II (MHC II) and costimulatory molecule genes are prophylactic and therapeutic agents for the treatment of murine primary and metastatic cancers. Vaccine efficacy is due to direct presentation of endogenously synthesized, MHC II-restricted tumor peptides to CD4+ T cells. Because the vaccine cells lack invariant chain, we have hypothesized that, unlike professional APC, the peptide-binding groove of newly synthesized MHC II molecules may be accessible to peptides, allowing newly synthesized MHC II molecules to bind peptides that have been generated in the proteasome and transported into the endoplasmic reticulum via the TAP complex. To test this hypothesis, we have compared the Ag presentation activity of multiple clones of TAP-negative and TAP-positive tumor cells transfected with I-Ak genes and the model Ag hen egg white lysozyme targeted to the endoplasmic reticulum or cytoplasm. Absence of TAP does not diminish Ag presentation of three hen egg white lysozyme epitopes. Likewise, cells treated with proteasomal and autophagy inhibitors are as effective APC as untreated cells. In contrast, drugs that block endosome function significantly inhibit Ag presentation. Coculture experiments demonstrate that the vaccine cells do not release endogenously synthesized molecules that are subsequently endocytosed and processed in endosomal compartments. Collectively, these data indicate that vaccine cell presentation of MHC II-restricted endogenously synthesized epitopes occurs via a mechanism independent of the proteasome and TAP complex, and uses a pathway that overlaps with the classical endosomal pathway for presentation of exogenously synthesized molecules.     

Anti-peptide antibody blocks peptide binding to MHC class I molecules in the endoplasmic reticulum

The finding that MHC class I molecules are physically associated with the TAP transporter has suggested that peptides may be directly transported into the binding groove of the class I molecules rather than into the lumen of the endoplasmic reticulum (ER) where they subsequently would encounter class I molecules by diffusion. Such a mechanism would protect peptides from peptidases in the ER and/or escaping back into the cytoplasm. However, we find that an anti-peptide Ab that is cotranslationally transported into the ER prevents TAP-transported peptides from being presented on class I molecules. The Ab only blocks the binding of its cognate peptide (SIINFEKL) but not other peptides (KVVRFKDL, ASNENMETM, and FAPGNYPAL). Therefore, most TAP-transported peptides must diffuse through the lumen of the ER before binding stably to MHC class I molecules.     

Retrovirally transduced mouse dendritic cells require CD4+ T cell help to elicit antitumor immunity: implications for the clinical use of dendritic cells

Presentation of MHC class I-restricted peptides by dendritic cells (DCs) can elicit vigorous antigen-specific CTL responses in vivo. It is well established, however, that T cell help can augment CTL function, raising the question of how best to present tumor-associated MHC class I epitopes to induce effective tumor immunity. To this end, we have examined the role of MHC class II peptide-complexes present on the immunizing DCs in a murine melanoma model. To present MHC class I- and II-restricted Ags reliably on the same cell, we retrovirally transduced bone marrow-derived DCs with the model Ag OVA encoding well-defined class I- and II-restricted epitopes. The importance of CD4+ T cells activated by the immunizing DCs in this model is demonstrated by the following findings: 1) transduced DCs presenting class I and class II epitopes are more efficient than class I peptide-pulsed DCs; 2) MHC class II-deficient DCs fail to induce tumor protection; 3) CD4+ T cell depletion abolishes induction of tumor protection; and 4) DCs presenting bovine serum Ags are more effective in establishing tumor immunity than DCs cultured in syngeneic serum. When MHC class II-deficient DCs were directly activated via their CD40 receptor, we indeed observed a moderate elevation of OVA-specific CTL activity. However, this increase in CTL activity was not sufficient to induce in vivo tumor rejection. Thus, our results demonstrate the potency of genetically modified DCs that express both MHC class I and II epitopes, but caution against the use of DCs presenting only the former.     

Phosphorylated peptides can be transported by TAP molecules, presented by class I MHC molecules, and recognized by phosphopeptide-specific CTL.

CTL recognize short peptide fragments presented by class I MHC molecules. In this study, we examined the effect of phosphorylation on TAP transport, binding to class I MHC molecules, and recognition by CTL of peptide fragments from known phosphorylated oncogene proteins or virus phosphoproteins. We show that phosphopeptides can be efficiently transported from the cytosol to the endoplasmic reticulum by the TAP. Furthermore, we show that phosphorylation can have a neutral, negative, or even a positive effect on peptide binding to class I MHC. Finally, we have generated phosphopeptide-specific CTL that discriminate between the phosphorylated and the nonphosphorylated versions of the peptide. We conclude that phosphopeptide-specific CTL responses are likely to constitute a subset of the class I MHC-restricted CTL repertoire in vivo.     

The murine cytomegalovirus pp89 immunodominant H-2Ld epitope is generated and translocated into the endoplasmic reticulum as an 11-mer precursor peptide

The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-L(d) 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.     

Intramembrane proteolysis of signal peptides: an essential step in the generation of HLA-E epitopes.

Signal sequences of human MHC class I molecules are a unique source of epitopes for newly synthesized nonclassical HLA-E molecules. Binding of such conserved peptides to HLA-E induces its cell surface expression and protects cells from NK cell attack. After cleavage from the pre-protein, we show that the liberated MHC class I signal peptide is further processed by signal peptide peptidase in the hydrophobic, membrane-spanning region. This cut is essential for the release of the HLA-E epitope-containing fragment from the lipid bilayer and its subsequent transport into the lumen of the endoplasmic reticulum via the TAP.     

New genes in the MHC that encode proteins for antigen processing

Most cells process proteins into short peptides that are displayed on the cell surface bound to class I or class II proteins encoded by the major histocompatibility complex (MHC). These protein-peptide complexes can then be recognized by the circulating lymphocytes of the immune system. Several genes found recently in the MHC encode proteins with possible roles in the supply of peptides to class I molecules. The results imply that the peptides are produced in the cytoplasm by proteasomes and are translocated into the endoplasmic reticulum by 'peptide transporters' related to the multidrug resistance proteins. While there is little biochemical evidence to validate these ideas, Robert DeMars and Thomas Spies discuss here the arguments supporting this view. New data indicate that there may also be factors for class II peptide-processing hidden in the MHC.     

A modular and combinatorial view of the antigen cross-presentation pathway in dendritic cells

Major histocompatibility complex class I (MHC I) presentation of exogenous antigens (cross-presentation) by dendritic cells (DC) is essential for CD8 T-cell immunity. Most cells use MHC I molecules to present peptides derived from endogenous proteins processed in the cytosol by the proteasome. The resulting peptides are translocated into the endoplasmic reticulum for loading onto MHC I molecules, and these complexes are then transported to the cell surface. In cross-presenting DC, these steps have been proposed to occur along two major tracks. In the 'endocytic' track, exogenous antigen processing and loading occur within endosomal compartments, using MHC I molecules recycled from the plasma membrane and transported back to the surface. In the 'cytosolic' track, antigens are translocated from endosomes to the cytosol, accessing the endogenous MHC I presentation pathway. This dichotomy now appears too simplistic. Some steps may occur in locations belonging to the endosomal track and others in the cytosolic track, or in hybrid compartments combining features of both. We propose a 'modular' view of cross-presentation, whereby processing, loading and MHC I transport represent modules that can occur in one or more locations. Cross-presentation of each MHC I-peptide complex may result from combining one or more options for each of these modules.     

Dendritic cells transduced with protein antigens induce cytotoxic lymphocytes and elicit antitumor immunity

Dendritic cell (DC)-based vaccines are being developed for treatment of patients with cancer, in part because DC are potent inducers of CD8(+) CTL. DC MHC class I:antigenic peptide complexes that are required for CTL elicitation are most often generated by incubating DC with peptides or by transfecting (or transducing) DC with cDNAs (or viral vectors) that encode protein Ags. The former approach is feasible when MHC class I Ags and relevant peptides are known. The latter approach may be hampered by inefficient DC transfection (transduction) and/or difficulties associated with preparation and use of viral vectors. Herein we demonstrate that a bacterial recombinant model tumor-associated Ag (OVA) that contains the HIV TAT protein transduction domain (PTD) was readily engineered and purified, efficiently transduced murine lymphocytes and DC, and was processed by proteasomes for MHC class I-restricted presentation to CTL. In addition, PTD-containing rOVA was processed and presented by DC to CD4 T cells as efficiently as native OVA or rOVA lacking the PTD. PTD-OVA-transduced DC induced CTL in vivo in a Th cell-independent fashion and vaccinated against OVA-expressing tumors. In contrast, rOVA lacking the PTD did not enter the DC MHC class I presentation pathway and DC treated with this protein did not prime OVA-specific CTL in vivo. Treatment of mice harboring clinically apparent OVA-expressing tumors with PTD-OVA-transduced DC resulted in tumor regression in some animals. This straightforward vaccination strategy may translate into DC-based treatments for patients with cancer and other serious diseases.     

Tumor cells present MHC class II-restricted nuclear and mitochondrial antigens and are the predominant antigen presenting cells in vivo

MHC class II-restricted tumor Ags presented by class II(+) tumor cells identified to date are derived from proteins expressed in the cytoplasm or plasma membrane of tumor cells. It is unclear whether MHC class II(+) tumor cells present class II-restricted epitopes derived from other intracellular compartments, such as nuclei and/or mitochondria, and whether class II(+) tumor cells directly present Ag in vivo. To address these questions, a model Ag, hen egg lysozyme, was targeted to various subcellular compartments of mouse sarcoma cells, and the resulting cells were tested for presentation of three lysozyme epitopes in vitro and for presentation of nuclear Ag in vivo. In in vitro studies, Ags localized to all tested compartments (nuclei, cytoplasm, mitochondria, and endoplasmic reticulum) are presented in the absence invariant chain and H-2M. Coexpression of invariant chain and H-2M inhibit presentation of some, but not all, of the epitopes. In vivo studies demonstrate that class II(+) tumor cells, and not host-derived cells, are the predominant APC for class II-restricted nuclear Ags. Because class II(+) tumor cells are effective APC in vivo and probably present novel tumor Ag epitopes not presented by host-derived APC, their inclusion in cancer vaccines may enhance activation of tumor-reactive CD4(+) T cells.     

Antigen processing by proteasomes: insights into the molecular basis of crypticity

Eight to eleven amino acid residues are the sizes of predominant peptides found to be associated with MHC class I molecules. Proteasomes have been implicated in antigen processing and generation of such peptides. Advanced methodologies in peptide elution together with sequence determination have led to the characterisation of MHC class I binding motifs. More recently, screening of random peptide phage display libraries and synthetic combinatorial peptide libraries have also been successfully used. This has led to the development and use of predictive algorithms to screen antigens for potential CTL epitopes. Not all predicted epitopes will be generated in vivo and the emerging picture suggests differential presentation of predicted CTL epitopes ranging from cryptic to immunodominant. The scope of this review is to discuss antigen processing by proteasomes, and to put forward a hypothesis that the molecular basis of immunogenicity can be a function of proteasomal processing. This may explain how pathogens and tumours are able to escape immunosurveillance by altering sequences required by proteasomes for epitope generation. Abbreviations: CTL – cytotoxic T lymphocytes; DRiPs – defective ribosomal products; ER – endoplasmic reticulum; Hsps – heat shock proteins; LMP – low molecular weight peptide; MHC – major histocompatibility complex; TAP – transporter associated with antigen processing.     

The varicellovirus-encoded TAP inhibitor UL49.5 regulates the presentation of CTL epitopes by Qa-1b1

Impairment of MHC class I Ag processing is a commonly observed mechanism that allows viruses and tumors to escape immune destruction by CTL. The peptide transporter TAP that is responsible for the delivery of MHC class I-binding peptides into the endoplasmic reticulum is a pivotal target of viral-immune evasion molecules, and expression of this transporter is frequently lost in advanced cancers. We recently described a novel population of CTL that intriguingly exhibits reactivity against such tumor-immune escape variants and that recognizes self-peptides emerging at the cell surface due to defects in the processing machinery. Investigations of this new type of CTL epitopes are hampered by the lack of an efficient inhibitor for peptide transport in mouse cells. In this article, we demonstrate that the varicellovirus protein UL49.5, in contrast to ICP47 and US6, strongly impairs the activity of the mouse transporter and mediates degradation of mouse TAP1 and TAP2. Inhibition of TAP was witnessed by a strong reduction of surface MHC class I display and a decrease in recognition of conventional tumor-specific CTL. Analysis of CTL reactivity through the nonclassical molecule Qa-1(b) revealed that the presentation of the predominant leader peptide was inhibited. Interestingly, expression of UL49.5 in processing competent tumor cells induced the presentation of the new category of peptides. Our data show that the varicellovirus UL49.5 protein is a universal TAP inhibitor that can be exploited for preclinical studies on CTL-based immune intervention.     

The importance of exogenous antigen in priming the human CD8+ T cell response: lessons from the EBV nuclear antigen EBNA1

Mouse models suggest that the processing of exogenous Ag by dendritic cells can be important for priming the CD8(+) CTL response. To study the situation in humans, we have exploited the CTL response to EBV infection. In this context EBV expresses eight latent proteins, of which EBV-encoded nuclear Ag (EBNA) 3A, 3B, and 3C appear to be immunodominant for CTL responses, whereas another nuclear Ag, EBNA1, which is completely protected from endogenous presentation via the MHC class I pathway, is thought to induce responses rarely, if ever. Here, using EBNA1 peptides and/or EBNA1 protein-loaded dendritic cells as in vitro stimuli, we have identified memory CTL responses to HLA-B*3501, -B7, and -B53-restricted EBNA1 epitopes that can be as strong as those seen in immunodominant epitopes from the "conventionally processed" EBNA3 Ags. Furthermore, we used HLA-peptide tetramers to show that the primary response to one such EBNA1 epitope constituted up to 5% of the CD8(+) T cells in infectious mononucleosis blood, the strongest latent Ag-specific response yet detected in this setting. We conclude that exogenous protein represents a significant source of Ag for priming the human CTL response.