Broadening CD4⁺and CD8⁺T Cell Responses against Hepatitis C Virus by Vaccination with NS3 Overlapping Peptide Panels in Cross-Priming Liposomes

Abstract

Despite the introduction of effective drugs to treat patients with chronic hepatitis C virus (HCV) infection, a vaccine would be the only means to substantially reduce the worldwide disease burden. 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 ϩ and CD8 ϩ 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 epitopespecific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 induced only CD4 ϩ T cells, whereas the NS3 pepmix induced a far more vigorous CD4 ϩ T cell response and was as potent a CD8 ϩ T cell inducer as an adenovirus-vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells, such as gamma interferonpositive (IFN-␥ ϩ ) tumor necrosis factor alpha-positive (TNF-␣ ϩ ) 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.IMPORTANCE 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 ϩ and CD8 ϩ 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-

Rational Design and In Vivo Characterization of Vaccine Adjuvants

“…This approach was used to elucidate the induction of CD4 + and CD8 + T-cell responses following immunization with recombinant NS3 protein antigen or the corresponding peptide mix both adjuvanted with CAF09. 92 The epitope mapping revealed that immunization with the peptide mix resulted in recognition of more CD4 epitopes compared with the recombinant protein, whereas 2 CD8 epitopes were induced by the peptide mix, while none were observed with the NS3 protein. 92 Pentamer/tetramer/dextramer-conjugated CD8 epitopeloaded MHC-I molecules are used to assess the level of antigenspecific CD8 + T cells in the relevant organs using flow cytometry.…”

“…92 The epitope mapping revealed that immunization with the peptide mix resulted in recognition of more CD4 epitopes compared with the recombinant protein, whereas 2 CD8 epitopes were induced by the peptide mix, while none were observed with the NS3 protein. 92 Pentamer/tetramer/dextramer-conjugated CD8 epitopeloaded MHC-I molecules are used to assess the level of antigenspecific CD8 + T cells in the relevant organs using flow cytometry. In a DC-based vaccine pulsed with the antigen TRP2 and adjuvanted with soluble poly(I:C), the percentage of TRP2specific CD8 + T cells was assessed using a K b /TRP2 tetramer and an anti-CD8 antibody.…”

“…injection of splenocytes labeled with 3 different concentrations of CFSE and 10 μg/mL of each peptide. 92 A complex protocol involving up to 216 separately fluorescently stained splenocyte populations was developed by Quah et al, intended for detailed in vivo assessment of CD8 + T-cell avidity and concomitant evaluation of several CD8 epitopes. 101 Splenocyte populations derived from naïve mice were stained with 4-6 concentrations of the fluorescent dyes CFSE, celltrace violet, and cell proliferation dye, including a nonstained population, followed by pulsing with different concentrations of minimal CD8 epitopes prior to injection into immunized mice.…”

Innate Immune Response against Hepatitis C Virus: Targets for Vaccine Adjuvants

“…Again, the adjuvant helped to exceed the immunostimulatory activity of alum adjuvant, increasing antibody and IFN-γ levels, and polarizing the immune response to Th1 type [ 163 ]. A vaccine, based on a mixture of 62 overlapping 20-mer peptides covering HCV NS3 and CAF09 adjuvant, induced polyfunctional T-cell responses in CB6F1 mice [ 164 ]. CAF09 consists of cationic liposomes combined with the immunostimulants monomycolyl glycerol and the TLR3 ligand poly (I:C) [ 171 ].…”

Features of Effective T Cell-Inducing Vaccines against Chronic Viral Infections

“…An increased breadth of the vaccine-induced T cell response has been found beneficial against many chronic viral pathogens ( 5 , 54 , 83 – 86 ). Induction of T cells with multiple antigen-specificities correlates with advanced killing capacity for control of HCV or even complete eradication during primary infection with HCV and superior protection upon reinfection ( 80 , 86 , 87 ). Analysis of CD8 + T cell responses in untreated HIV-infected individuals showed that an increasing breadth of Gag-specific responses is associated with decreased viremia ( 88 ).…”