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An incomplete understanding of how HCV interacts with its human host and evades immune surveillance has hampered vaccine development. It is generally accepted that in infected individuals, a narrow repertoire of exhausted T cells is a hallmark of persistent infection, whereas broad, vigorous CD4<jats:sup>+<\/jats:sup>and CD8<jats:sup>+<\/jats:sup>T cell responses are associated with control of acute hepatitis C. We employed a vaccine approach based on a mixture of peptides (pepmix) spanning the entire sequence of HCV nonstructural protein 3 (NS3) in cross-priming cationic liposomes (CAF09) to facilitate a versatile presentation of all possible T cell epitopes, regardless of the HLA background of the vaccine recipient. Here, we demonstrate that vaccination of mice with NS3 pepmix broadens the repertoire of epitope-specific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 induced only CD4<jats:sup>+<\/jats:sup>T cells, whereas the NS3 pepmix induced a far more vigorous CD4<jats:sup>+<\/jats:sup>T cell response and was as potent a CD8<jats:sup>+<\/jats:sup>T cell inducer as an adenovirus-vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells, such as gamma interferon-positive (IFN-\u03b3<jats:sup>+<\/jats:sup>) tumor necrosis factor alpha-positive (TNF-\u03b1<jats:sup>+<\/jats:sup>) coproducers, and displayed cytotoxic capacity in mice. In conclusion, we present a novel vaccine approach against HCV, inducing a broadened T cell response targeting both immunodominant and potential subdominant epitopes, which may be key elements to counter T cell exhaustion and prevent chronicity.<\/jats:p><jats:p><jats:bold>IMPORTANCE<\/jats:bold>With at least 700,000 annual deaths, development of a vaccine against hepatitis C virus (HCV) has high priority, but the tremendous ability of the virus to dodge the human immune system poses great challenges. Furthermore, many chronic infections, including HCV infection, have a remarkable ability to drive initially strong CD4<jats:sup>+<\/jats:sup>and CD8<jats:sup>+<\/jats:sup>T cell responses against dominant epitopes toward an exhausted, dysfunctional state. Thus, new and innovative vaccine approaches to control HCV should be evaluated. Here, we report on a novel peptide-based nanoparticle vaccine strategy (NS3 pepmix) aimed at generating T cell immunity against potential subdominant T cell epitopes that are not efficiently targeted by vaccination with full-length recombinant protein (rNS3) or infection with HCV. As proof of concept, we found that NS3 pepmix excels in broadening the repertoire of epitope-specific, multifunctional, and cytotoxic CD4<jats:sup>+<\/jats:sup>and CD8<jats:sup>+<\/jats:sup>T cells compared to vaccination with rNS3, which generated only CD4<jats:sup>+<\/jats:sup>T cell responses.<\/jats:p>","DOI":"10.1128\/jvi.00130-17","type":"journal-article","created":{"date-parts":[[2017,4,27]],"date-time":"2017-04-27T00:40:24Z","timestamp":1493253624000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":21,"title":["Broadening CD4<sup>+<\/sup>and CD8<sup>+<\/sup>T Cell Responses against Hepatitis C Virus by Vaccination with NS3 Overlapping Peptide Panels in Cross-Priming Liposomes"],"prefix":"10.1128","volume":"91","author":[{"given":"Jonathan","family":"Filskov","sequence":"first","affiliation":[{"name":"Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, 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