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IntroductionTherapeutic vaccination based on synthetic long peptides (SLP®) containing both CD4+ and CD8+ T cell epitopes is a promising treatment strategy for chronic hepatitis B infection (cHBV).MethodsWe designed SLPs for three HBV proteins, HBcAg and the non-secreted proteins polymerase and X, and investigated their ability to induce T cell responses ex vivo . A set of 17 SLPs was constructed based on viral protein conservation, functionality, predicted and validated binders for prevalent human leukocyte antigen (HLA) supertypes, validated HLA I epitopes, and chemical producibility.ResultsAll 17 SLPs were capable of inducing interferon gamma (IFNɣ) production in samples from four or more donors that had resolved an HBV infection in the past (resolver). Further analysis of the best performing SLPs demonstrated activation of both CD8+ and CD4+ multi-functional T cells in one or more resolver and patient sample(s). When investigating which SLP could activate HBV-specific T cells, the responses could be traced back to different peptides for each patient or resolver.DiscussionThis indicates that a large population of subjects with different HLA types can be covered by selecting a suitable mix of SLPs for therapeutic vaccine design. In conclusion, we designed a set of SLPs capable of inducing multifunctional CD8+ and CD4+ T cells ex vivo that create important components for a novel therapeutic vaccine to cure cHBV.

Synthetic long peptides (SLPs) are a promising vaccine modality that exploit dendritic cells (DC) to treat chronic infections or cancer. Currently, the design of SLPs relies on in silico prediction and multifactorial T cells assays to determine which SLPs are best cross-presented on DC human leukocyte antigen class I (HLA-I). Furthermore, it is unknown how TLR ligand-based adjuvants affect DC cross-presentation. Here, we generated a unique, high-quality immunopeptidome dataset of human DCs pulsed with 12 hepatitis B virus (HBV)-based SLPs combined with either a TLR1/2 (Amplivant®) or TLR3 (PolyI:C) ligand. The obtained immunopeptidome reflected adjuvant-induced differences, but no differences in cross-presentation of SLPs. We uncovered dominant (cross-)presentation on B-alleles, and identified 33 unique SLP-derived HLA-I peptides, several of which were not in silico predicted and some were consistently found across donors. Our work puts forward DC immunopeptidomics as a valuable tool for therapeutic vaccine design.

Immunopeptidomics is used to identify novel epitopes for (therapeutic) vaccination strategies in cancer and infectious disease. Various false discovery rates (FDRs) are applied in the field when converting liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra to peptides. Subsequently, large efforts have recently been made to rescue peptides of lower confidence. However, it remains unclear what the overall relation is between the FDR threshold and the percentage of obtained HLA-binders. We here directly evaluated the effect of varying FDR thresholds on the resulting immunopeptidomes of HLA-eluates from human cancer cell lines and primary hepatocyte isolates using HLA-binding algorithms. Additional peptides obtained using less stringent FDR-thresholds, although generally derived from poorer spectra, still contained a high amount of HLA-binders and confirmed recently developed tools that tap into this pool of otherwise ignored peptides. Most of these peptides were identified with improved confidence when cell input was increased, supporting the validity and potential of these identifications. Altogether, our data suggest that increasing the FDR threshold for peptide identification in conjunction with data filtering by HLA-binding prediction, is a valid and highly potent method to more efficient exhaustion of immunopeptidome datasets for epitope discovery and reveals the extent of peptides to be rescued by recently developed algorithms.

BackgroundIn melanoma, cancer germline antigen (CGA)-directed vaccination has shown to induce objective clinical responses accompanied by strong anti-tumor immune responses.1 As CGAs are immunogenic and highly expressed by hepatocellular carcinoma (HCC) tumor cells, these have demonstrated to be attractive targets to be implemented in therapeutic anti-liver cancer vaccination as well.2Synthetic long peptide (SLP) vaccination has proven to elicit efficient anti-tumor CD4+ and CD8+ T cell responses and to have promising clinical effects.3 We aimed to develop an SLP vaccine targeting HCC-restricted CGA-epitopes covering at least five different HLA super types that are highly prevalent globally.MethodsWe applied an integrative pre-clinical approach of in silico epitope prediction, immunopeptidomics, and in vitro tools to select GSAs and validate CGA-SLPs in HCC patient-derived tumor infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs).ResultsOut of a set of 13 CGAs, previously shown to be expressed in primary human HCC tissues, two CGAs (i.e., CGA-A and -B) demonstrated no healthy tissue expression and covered >75% of HCC patients collectively (N = 55). Immunopeptidome analysis of human HCC-derived hepatocytes (N = 12), together with in silico CGA-related epitope predictions according to epitope immunogenicity, enabled identification of 196 and 220 potential epitopes for CGA-A and -B, respectively. HLA-A*02:01 binding of these epitopes was validated in vitro using a HLA-A2 stabilization assay and ranked accordingly. Six SLPs were designed incorporating 54 HLA-A*02:01, 25 HLA-A*01:01, 24 HLA-A*03:01, 27 HLA-A*24:01, and 15 HLA-B*07:02 predicted and/or validated CGA-A- and -B-related epitopes. Top three-ranked epitopes were selected to validate ex vivo intra-tumor immune reactivity using corresponding peptide-HLA-A*02:01 dextramers in human HCC-derived TILs. Tumors of 8/11 patients contained CGA-A- and CGA-B-specific TILs that were characterized by a tumor reactive phenotype. Upon in vitro enrichment, SLP immunogenicity was demonstrated through Interferon gamma ELISPOT in 2/3 of human HCC-derived PBMCs using an in vitro co-culture system with autologous antigen presenting cells.ConclusionsHere, we describe the intelligent design of a set of immunogenic SLPs comprising CGA-related epitopes for the global population that can be further exploited for the development of an off-the shelf anti-cancer vaccine to treat HCC.ReferencesAn RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature 2020;585(7823):107–112.Expression of cancer testis antigens in tumor-adjacent normal liver is associated with post-resection recurrence of hepatocellular carcinoma. Cancers (Basel) 2021;13(10):2499.Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med. 2009;361(19):1838.Ethics ApprovalAll study procedures were approved by the local ethics committee (Medische Ethische Toetsings Commissie Erasmus MC Rotterdam; NL58534,078.16). Patients had given informed consent for tissue and blood donation as well as usage of personal data.ConsentPatients had given informed consent for tissue and blood donation as well as usage of personal data.