Fusion proteins created by chromosomal translocations in tumors can create neoantigenic determinants at the breakpoint, which are unique to the tumor cells but shared by the vast majority of tumors of that histologic type. If the fusion protein is responsible for the malignant transformation, its expression cannot be lost by the tumor to escape immune responses against this tumor antigen. Here, we identify such a fusion protein breakpoint epitope in the PAX-FKHR fusion protein created by the t(2;13) translocation present in 80% of cases of alveolar rhabdomyosarcoma, a highly aggressive pediatric soft-tissue sarcoma. We use autologous dendritic cells pulsed with the RS10 breakpoint fusion peptide to raise a human CTL line from a normal healthy HLA-B7+ blood donor specific for this peptide. These CTLs are CD8+ (CD4-CD56-) and restricted by HLA-B7. These human peptide-specific CTL lyse human HLA-B7+ rhabdomyosarcoma tumor cells. Therefore, the fusion protein is endogenously processed to produce this natural epitope presented by HLA-B7 and thus this peptide is a bone fide human tumor antigen. We also define a substitution that increases the affinity for HLA-B7 without loss of antigenicity. This epitope-enhanced peptide may serve as a candidate cancer vaccine for HLA-B7+ patients with alveolar rhabdomyosarcoma.

Purpose To determine the ability to induce tumor-specific immunity with individual mutant K-ras- or p53-derived peptides and to monitor clinical outcome.Patients and Methods Patients in varying stages of disease underwent genetic analysis for mutations in K-ras and p53. Thirty-nine patients were enrolled. Seventeen-mer peptides were custom synthesized to the corresponding mutation. Baseline immunity was assessed for cytotoxic T-lymphocyte (CTL) response and interferon gamma (IFN-gamma) release from mutant peptide-primed lymphocytes. Patients' peripheral-blood mononuclear cells were pulsed with the corresponding peptide, irradiated, and applied intravenously. Patients were observed for CTL, IFN-gamma, interleukin (IL)-2, IL-5, and granulocyte-macrophage colony-stimulating factor responses, for treatment-related toxicity, and for tumor response.Results No toxicity was observed. Ten (26%) of 38 patients had detectable CTL against mutant p53 or K-ras, and two patients were positive for CTL at baseline. Positive IFN-gamma responses occurred in 16 patients (42%) after vaccination, whereas four patients had positive IFN-gamma reaction before vaccination. Of 29 patients with evident disease, five experienced a period of stable disease. Favorable prognostic markers were detectable CTL activity and a positive IFN-gamma reaction but not IL-5 release. Median survival times of 393 v 98 days for a positive versus negative CTL response (P = .04), respectively, and of 470 v 88 days for a positive versus negative IFN-gamma response (P = .02), respectively, were detected.Conclusion Custom-made peptide vaccination is feasible without any toxicity. CTL and cytokine responses specific to a given mutation can be induced or enhanced with peptide vaccines. Cellular immunity to mutant p53 and K-ras oncopeptides is associated with longer survival.

Vaccine therapy for prostate and breast cancer may have potential for treating these major causes of death in males and females, respectively. Critical to the development of tumor-specific vaccines is finding and characterizing novel antigens to be recognized by CD8+ T cells. To define new CD8+ T-cell tumor antigens, we determined two wild-type HLA-A2 epitopes from a recently found tumor-associated protein, TARP (T-cell receptor γ alternate reading frame protein), expressed in prostate and breast cancer cells. We were also able to engineer epitope-enhanced peptides by sequence modifications. Both wild-type and enhanced epitopes induced peptide-specific CD8+ T-cell responses in A2Kb transgenic mice. In vitro restimulation of human CD8+ T cells from a prostate cancer patient resulted in CD8+ T cells reactive to the peptide epitopes that could lyse HLA-A2+ human breast cancer cells (MCF-7) expressing TARP. Epitope-specific human CD8+ T cells were also enumerated in patients’ peripheral blood by tetramer staining. Our data suggest that HLA-A2-binding TARP epitopes and enhanced epitopes discovered in this study could be incorporated into a potential vaccine for both breast and prostate cancer.

HIV epitopes may have developed to be poor immunogens. As a counterapproach HIV vaccine strategy, we used epitope enhancement of a conserved HIV reverse transcriptase (RT) epitope for induction of antiviral protection in HLA-A2-transgenic mice mediated by human HLA-A2-restricted CTLs. We designed two epitope-enhanced peptides based on affinity for HLA-A2, one substituted in anchor residues (RT-2L9V) and the other also with tyrosine at position 1 (RT-1Y2L9V), and examined the balance between HLA binding and T cell recognition. CTL lines and bulk cultures in two HLA-A2-transgenic mouse strains showed that RT-2L9V was more effective in inducing CTL reactive with wild-type Ag than RT-1Y2L9V, despite the higher affinity of the latter, because the 1Y substitution unexpectedly altered T cell recognition. Accordingly, RT-2L9V afforded the greatest protection in vivo against a surrogate virus expressing HIV-1 RT mediated by HLA-A2-restricted CTL in a mouse in which all CTL are restricted to only the human HLA molecule. Such antiviral protection has not been previously achieved with an HLA epitope-enhanced vaccine. These findings define a critical balance between MHC affinity and receptor cross-reactivity required for effective epitope enhancement and also demonstrate construction and efficacy of such a component of a new generation vaccine.

Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5' region of one gene with the 3' region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and may be required for persistence, thereby serving as targets for immunotherapy. It was hypothesized that the fusion breakpoint sequences associated with SS, CCS, and DSRCT can serve as tumor-specific neoantigens. To test this, peptides corresponding to the fusion breakpoints were designed and assessed for ability to bind to various class I HLA molecules. Two peptides derived from the SS breakpoint specifically bind the HLA-B7 antigen, and a 10-amino acid minimal epitope was identified for this interaction. Specific binding of a SS peptide and a CCS peptide to HLA-B27 molecule was also observed. Finally, a peptide designed from the DSRCT breakpoint specifically binds the HLA-A3 molecule, and a 9-amino acid optimal epitope was identified for this interaction. The physiological/immunological relevance of these peptide/MHC interactions was demonstrated by the induction of SS-specific CTLs from normal donor lymphocytes using in vitro stimulation with autologous, peptide-pulsed dendritic cells and by the ability of these CTLs to lyse human SS tumor cells endogenously expressing the full-length fusion protein. These results suggest that sequences in the fusion region of sarcoma-associated chimeras can bind class I HLA molecules and serve as neoantigens. These may be useful for the development of novel immunotherapies for sarcoma patients with appropriate HLA molecules and tumors bearing these translocations.

To improve the safety of recombinant vaccinia virus vaccines, modified vaccinia virus Ankara (MVA) has been employed, because it has a replication defect in most mammalian cells. Here we apply MVA to human immunodeficiency virus type 1 (HIV-1) vaccine development by incorporating the envelope protein gp160 of HIV-1 primary isolate strain 89.6 (MVA 89.6) and use it to induce mucosal cytotoxic-T-lymphocyte (CTL) immunity. In initial studies to define a dominant CTL epitope for HIV-1 89.6 gp160, we mapped the epitope to a sequence, IGPGRAFYAR (from the V3 loop), homologous to that recognized by HIV MN loop-specific CTL and showed that HIV-1 MN-specific CTLs cross-reactively recognize the corresponding epitope from strain 89.6 presented by H-2Dd. Having defined the CTL specificity, we immunized BALB/c mice intrarectally with recombinant MVA 89.6. A single mucosal immunization with MVA 89.6 was able to elicit long-lasting antigen-specific mucosal (Peyer's patch and lamina propria) and systemic (spleen) CTL responses as effective as or more effective than those of a replication-competent vaccinia virus expressing 89.6 gp160. Immunization with MVA 89.6 led to (i) the loading of antigen-presenting cells in vivo, as measured by the ex vivo active presentation of the P18-89.6 peptide to an antigen-specific CTL line, and (ii) the significant production of the proinflammatory cytokines (interleukin-6 and tumor necrosis factor alpha) in the mucosal sites. These results indicate that nonreplicating recombinant MVA may be at least as effective for mucosal immunization as replicating recombinant vaccinia virus.

A minimal, nonamer epitope (TEMEKEGKI) from the reverse transcriptase protein of HIV-1, restricted by H-2Kk, was identified and the function of individual residues determined. Besides classical anchor residues at positions 2 and 9, methionine at position 3 was identified as an important MHC anchor and improved binding of a different (malarial) nonamer epitope to H-2Kk, albeit while also abolishing CTL recognition. Lysine at position 5 was replaceable by alanine for CTL raised against wild-type peptide but abolished recognition for CTL raised against the variant 5ALA peptide, indicating a unidirectional cross-reactivity. Interestingly, one CTL line raised against the 5ALA substituted peptide was permissive for a double substitution at positions 5 and 6, in which lysine was permissive at position 5 only if the adjacent glutamic acid was replaced by alanine. Extensive analysis revealed three distinct patterns of responses with peptides doubly substituted in this region: recognition of both single substitutions but not the double substitution, recognition of only one single substitution but also the double substitution, or recognition of both single substitutions and the double substitution. A second complementary substitution can therefore restore function lost through a first substitution. Thus, no residue acts independently of its neighbors, and pairs of substitutions may give results not predictable from the effects of each taken singly. This finding may have bearing on viral infections (such as HIV), in which the accumulation of two mutations in the epitope may lead to the reengagement of memory CTL previously silenced by the initial mutation.

Since the natural immune response to hepatitis C virus (HCV) is often unable to clear the infection, to enhance immunogenicity we studied substituted peptides from an HCV cytotoxic T lymphocyte (CTL) epitope (C7A2) from a conserved region of the HCV core protein (DLMGYIPLV) recognized by CTL lines from HLA-A2.1(+) HCV-infected patients and HLA-A2.1 transgenic mice. HLA-A2.1 binding, human and murine CTL recognition, and in vivo immunogenicity (using mice transgenic for human HLA-A2 in lieu of immunizing humans) were analyzed to define peptides with enhanced immunogenicity. Peptides substituted at position 1 showed enhanced HLA-A2 binding affinity, but paradoxically poorer immunogenicity. A peptide with Ala substituted at position 8 (8A) showed higher HLA-A2 binding affinity and CTL recognition and was a more potent in vivo immunogen in HLA-A2-transgenic mice, inducing higher CTL responses with higher avidity against native C7A2 than induced by C7A2 itself. These results suggest that peptide 8A is a more potent in vitro antigen and in vivo immunogen than C7A2 and may be useful as a vaccine component. They provide proof of principle that the strategy of epitope enhancement can enhance immunogenicity of a CTL epitope recognized by human CTL.

Point mutations in oncogene products such as ras may create neoantigenic determinants recognizable by T lymphocytes as tumor antigens, that could be marshalled to eliminate a tumor by inducing specific cytotoxic T lymphocytes (CTL) with an appropriate vaccine. Peptide-pulsed dendritic cells are a promising new approach to cancer vaccines. For such an approach to work, the determinant must be appropriately processed to the right size fragment and be presented by an appropriate HLA molecule. We have investigated both of these issues for a series of ras codon 12 and 13 point mutations that contain sequences predicted to bind to HLA-A2.1, the most common class I HLA molecule. We find that not only do the different mutations affect binding to HLA-A2.1, but also they affect extracellular antigen processing in two ways: by influencing the trimming of flanking residues from the longer sequence and by influencing the susceptibility of the optimal decamer to further proteolytic degradation. The influence of internal residues on cleavage of flanking residues downstream demonstrates the importance of distant interactions between separated amino acid side chains and/or conformational effects in determining antigen processing. These results may be important in designing an effective vaccine to induce mutant ras-specific tumor immunity.

Viral proteins are not naturally selected for high affinity major histocompatibility complex (MHC) binding sequences; indeed, if there is any selection, it is likely to be negative in nature. Thus, one should be able to increase viral peptide binding to MHC in the rational design of synthetic peptide vaccines. The T1 helper peptide from the HIV-1 envelope protein was made more immunogenic for inducing T cell proliferation to the native sequence by replacing a residue that exerts an adverse influence on peptide binding to an MHC class II molecule. Mice immunized with vaccine constructs combining the more potent Th helper (Th) epitope with a cytotoxic T lymphocyte (CTL) determinant developed greatly enhanced CTL responses. Use of class II MHC-congenic mice confirmed that the enhancement of CTL response was due to class II-restricted help. Thus, enhanced T cell help is key for optimal induction of CTL, and, by modification of the native immunogen to increase binding to MHC, it is possible to develop second generation vaccine constructs that enhance both Th cell activation and CTL induction.

Cytotoxic T lymphocytes and Th1 cells have been suggested to play a critical role in the control of HIV infection. It is therefore considered that a vaccine that induces a strong Th1 response and CTL response would be more efficacious than one that does not in providing protection against infection and progression toward AIDS. In this study we show that immunization with vaccine constructs consisting of multideterminant cluster peptides containing Th epitopes from the HIV-1IIIB envelope colinearly synthesized to peptide 18MN, is capable of inducing a Th1 response in mice and, dependent on this help, both cytotoxic T cell responses and neutralizing antibody toward the homologous strain of HIV. Moreover, the cytotoxic T cell response elicited by immunization with a mixture of cluster peptide-P18MN vaccine constructs was at least as cross-reactive against known viral variant P18 target sequences as a CTL line produced by immunization with a vaccinia construct expressing recombinant gp160 MN. Four adjuvants were compared to optimize both CTL and antibody responses. A single adjuvant formulation of peptide in ISA 51 could elicit all three: Th1 cells, CTLs, and neutralizing antibody. Thus, immunization directed toward the development of a cytotoxic T cell response does not preclude the development of neutralizing antibody and vice versa, i.e., the responses are not mutually exclusive. The immunization protocol described here should be directly applicable for study in clinical trials aimed at HIV-1 immunotherapy or prophylaxis.

To define cytotoxic T-cell (CTL) epitopes, the common approach involving the use of a series of overlapping synthetic peptides covering the whole protein sequence is impractical for large proteins. Motifs identify only a fraction of epitopes. To identify human CTL epitopes in the NS3 region of hepatitis C virus (HCV), we modified an approach using recombinant protein and the ability of short peptides to bind to class I major histocompatibility complex (MHC) molecules. Peripheral blood mononuclear cells from an HCV-infected patient were stimulated with a proteolytic digest of the recombinant NS3 protein to expand CTL to any active peptides in the digest. The digest was fractionated by reverse-phase high-performance liquid chromatography, and fractions were assessed for the ability to sensitize targets for lysis by CTL. The most active fraction was sequenced, identifying a 15-residue peptide (NS3-1J; TITTGAPVTYSTYGK). This sequence was confirmed to be the source of the activity by synthesis of the corresponding peptide. CTL lines specific for NS3-1J were established from two HCV-infected patients (both HLA-A2 and -B7 positive) by stimulation with the synthetic peptide in vitro. The CTL were HLA-A2 restricted, and the minimal epitope was mapped to a decapeptide NS3-1J (10.4). As this minimal epitope lacks the common HLA-A2-binding motif, this technique is useful for mapping CTL epitopes independent of known motifs and without the requirement for enormous numbers of overlapping peptides. Because this peptide is presented by the most common HLA class I molecule, present in almost half the population, it might be a useful component of a vaccine against HCV.

P18(IIIB) is a highly immunogenic peptide from the V3 loop of the HIV-1 gp160 envelope protein that is presented promiscuously by multiple class I MHC molecules. Understanding the molecular basis for promiscuous presentation may have many practical applications. As the highly prevalent HLA-A2.1 class I molecule is known to present P18(IIIB) for recognition by cytotoxic T lymphocytes (CTL) found in peripheral blood mononuclear cells of HIV+ donors, a P18(IIIB)-specific CTL line was generated from and HLA-A2(+), HIV- donor in order to define the molecular basis for, and ultimately improve upon the binding of, this peptide to HLA-A2.1. The minimal epitope recognized by the line was a decamer, I10, with the sequence RGPGRAFVTI. Interestingly, this decamer is identical to the minimal epitope from P18(IIIB) seen by murine CTL restricted by H-2Dd. A panel of Ala-substituted peptides was employed in MHC-binding and T cell response studies to identify MHC- and TCR-binding residues. Notably, many of the agretopic and epitopic residues identified were identical to those involved in the corresponding interactions of I10 with the H-2Dd MHC molecule and murine I10-specific CTL. The I10 peptide does not contain the described HLA-A2.1 binding motif. Instead a Pro at P3, a Phe at P7 and an Ile at P10 are utilized for MHC binding. Agretopic residue similarities with the hepatitis B nucleocapsid decamer suggest that these residues may comprise an alternative motif of anchors utilized by decamers for binding to HLA-A2.1.

We have observed and analyzed an unexpected cross-reactivity of CD8+ CTL between two nonhomologous peptides of the HIV-1 IIIB gp160 envelope protein, P18 (residues 315-329) and HP53 (834-848, also called TH4.1), in the context of four different class I MHC molecules, Dd, Dp, Dq (or Lq), and H-2u. In strains expressing Dd, the cross-reactivity between peptides was bidirectional, whereas in other strains (H-2u, H-2p, and H-2q), the cross-reactivity was unidirectional; that is, P18-specific CTLs showed no killing against targets pulsed with HP53, although HP53 stimulated CTL showed cross-reactive lysis against P18-pulsed target cells. Cross-reactivity was also shown in immunization in vivo and with target cells endogenously expressing viral protein in vitro using two different recombinant vaccinia viruses expressing only the N-terminal portion of gp160, containing P18 but not HP53. Peptide cross-contamination was excluded. Cold target inhibition and single cell cloning experiments indicated that the same CTL was responding to both peptides. Using substituted and truncated peptides, we explored amino acid residues critical for cross-reactive CTL recognition, identified fine specificity similarities among all cross-reactive CTL lines but not non-cross-reactive lines, and mapped cross-reactivity to a 10-residue core of P18 and to an eight-residue core of HP53. A comparison of these peptide sequences and recent data on residues of P18 interacting with H-2Dd provided us with clues to residues involved in the interaction of the CTL with the MHC-peptide complex.

Although several peptides have been found to bind to both class I and class II molecules, the basis for this binding of the same peptide to two classes of MHC molecules has not been compared previously. We have analyzed one such peptide, P18 from the V3 loop of HIV-1 gp160, which we have previously shown to be recognized by CD8+ CTL with the class I molecule H-2Dd, and by CD4+ Th cells with the class II molecule I-Ad. With the use of truncated and substituted peptides, we found that the minimal core peptides are very similar, that the residues required for class I binding precisely fit the recently identified consensus motif for peptides binding to Dd (XGPX[R/K/H]XXX(X) [L/I/F]), and that at least three of the same residues are involved in binding to class II I-Ad. In addition, several of the same residues are involved in TCR interaction when the peptide is presented by class I and class II molecules. Modeling shows results to be consistent with the crystal structure of a peptide-class II MHC complex. Thus, the recognition of this versatile peptide by CD4+ Th cells with class II MHC molecules and by CD8+ cytotoxic T cells with class I MHC molecules is remarkably similar in both the core peptide used and the role of different residues in the ternary complex.