Circulating Proteins as Diagnostic Markers in Gastric Cancer.

Gastric cancer (GC) is a highly malignant disease affecting humans worldwide and has a poor prognosis. Most GC cases are detected at advanced stages due to the cancer lacking early detectable symptoms. Therefore, there is great interest in improving early diagnosis by implementing targeted prevention strategies. Markers are necessary for early detection and to guide clinicians to the best personalized treatment. The current semi-invasive endoscopic methods to detect GC are invasive, costly, and time-consuming. Recent advances in proteomics technologies have enabled the screening of many samples and the detection of novel biomarkers and disease-related signature signaling networks. These biomarkers include circulating proteins from different fluids (e.g., plasma, serum, urine, and saliva) and extracellular vesicles. We review relevant published studies on circulating protein biomarkers in GC and detail their application as potential biomarkers for GC diagnosis. Identifying highly sensitive and highly specific diagnostic markers for GC may improve patient survival rates and contribute to advancing precision/personalized medicine.

A DSC Test for the Early Detection of Neoplastic Gastric Lesions in a Medium-Risk Gastric Cancer Area.

In this study, we aimed to assess the accuracy of the proposed novel, noninvasive serum DSC test in predicting the risk of gastric cancer before the use of upper endoscopy. To validate the DSC test, we enrolled two series of individuals living in Veneto and Friuli-Venezia Giulia, Italy (n = 53 and n = 113, respectively), who were referred for an endoscopy. The classification used for the DSC test to predict gastric cancer risk combines the coefficient of the patient's age and sex and serum pepsinogen I and II, gastrin 17, and anti-Helicobacter pylori immunoglobulin G concentrations in two equations: Y1 and Y2. The coefficient of variables and the Y1 and Y2 cutoff points (>0.385 and >0.294, respectively) were extrapolated using regression analysis and an ROC curve analysis of two retrospective datasets (300 cases for the Y1 equation and 200 cases for the Y2 equation). The first dataset included individuals with autoimmune atrophic gastritis and first-degree relatives with gastric cancer; the second dataset included blood donors. Demographic data were collected; serum pepsinogen, gastrin G17, and anti-Helicobacter pylori IgG concentrations were assayed using an automatic Maglumi system. Gastroscopies were performed by gastroenterologists using an Olympus video endoscope with detailed photographic documentation during examinations. Biopsies were taken at five standardized mucosa sites and were assessed by a pathologist for diagnosis. The accuracy of the DSC test in predicting neoplastic gastric lesions was estimated to be 74.657% (65%CI; 67.333% to 81.079%). The DSC test was found to be a useful, noninvasive, and simple approach to predicting gastric cancer risk in a population with a medium risk of developing gastric cancer.

Quantitative Plasma Proteomics to Identify Candidate Biomarkers of Relapse in Pediatric/Adolescent Hodgkin Lymphoma.

Classical pediatric Hodgkin Lymphoma (HL) is a rare malignancy. Therapeutic regimens for its management may be optimized by establishing treatment response early on. The aim of this study was to identify plasma protein biomarkers enabling the prediction of relapse in pediatric/adolescent HL patients treated under the pediatric EuroNet-PHL-C2 trial. We used untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics at the time of diagnosis­before any therapy­as semiquantitative method to profile plasma proteins specifically associated with relapse in 42 children with nodular sclerosing HL. In both the exploratory and the validation cohorts, six proteins (apolipoprotein E, C4b-binding protein α chain, clusterin, fibrinogen γ chain, prothrombin, and vitronectin) were more abundant in the plasma of patients whose HL relapsed (|fold change| ≥ 1.2, p < 0.05, Student's t-test). Predicting protein function with the Gene Ontology classification model, the proteins were included in four biological processes (p < 0.01). Using immunoblotting and Luminex assays, we validated two of these candidate biomarkers­C4b-binding protein α chain and clusterin­linked to innate immune response function (GO:0045087). This study identified C4b-binding protein α chain and clusterin as candidate early plasma biomarkers of HL relapse, and important for the purpose of shedding light on the molecular scenario associated with immune response in patients treated under the EuroNet-PHL-C2 trial.

PDCD1 and IFNL4 genetic variants and risk of developing hepatitis C virus-related diseases.

BACKGROUND: Genetic variants of IFNL4 and PDCD1 genes have been shown to influence the spontaneous clearance of hepatitis C virus (HCV) infection. We investigated the IFNL4 rs12979860 and the PDCD1 polymorphisms in 734 HCV-positive patients, including 461 cases with liver disease of varying severity and 273 patients with lymphoproliferative disorders to determine the association of these genes with patient's outcome. METHODS: Expression levels of PDCD1 mRNA encoded by haplotypes were investigated by quantitative PCR in hepatocellular carcinoma (HCC) tissue and peripheral blood mononuclear cells. Flow cytometry was used to detect PD-1 and its ligand PD-L1. RESULTS: The frequency of IFNL4 rs12979860 C/T or T/T genotypes was significantly higher in patients with HCV-related diseases than blood donors (P < .0001). Patients expressing the IFNλ4 variant with one amino acid change that reduces IFNλ4 secretion was found increased in frequency in HCV-related diseases compared to HCC PDCD1 mRNA levels in HCC tissue were significantly higher in cases carrying the PD-1.3 A or the PD-1.7 G allele (P = .0025 and P = .0167). Linkage disequilibrium (LD) between PD-1.3 and IFNL4 was found in patients with mixed cryoglobulinaemia (MC) only (LD = 0 in HCC; LD = 72 in MC). PBMCs of MC patients expressed low levels of PD-L1 in CD19+IgM+B cells and of PD-1 in CD4+T cells suggesting the involvement of regulatory B cell-T cell interaction to the pathogenesis of MC. CONCLUSION: Collectively, our data indicate an important contribution of IFNλ4 expression to the development of HCV-related HCC and an epistatic contribution of IFNL4 and PDCD1 in MC. LAY SUMMARY: Studies of IFNL4 and PDCD1 genes are helpful to better understand the role of host genetic factors and immune antigens influencing the outcome of HCV-related diseases. Our data support an association between the expression of IFNλ4, which prevents the expression of IFNλ3, with all the different HCV-related diseases studied, and besides, evidence that a higher IFNλ4 expression is associated with hepatocellular at a younger age. The expression pattern of low PD-L1 on B cells and high PD-1 on CD4+T-cells in patients with HCV-positive cryoglobulinaemia suggests a critical role of the PD-1/PD-L1 signaling in modulating B cell-T cell interaction in this lymphoproliferative disease.

Proteomics signature of autoimmune atrophic gastritis: towards a link with gastric cancer.

BACKGROUND: Autoimmune atrophic gastritis (AAG) is a chronic disease that can progress to gastric cancer (GC). To better understand AAG pathology, this proteomics study investigated gastric proteins whose expression levels are altered in this disease and also in GC. METHODS: Using two-dimensional difference gel electrophoresis (2D-DIGE), we compared protein maps of gastric corpus biopsies from AAG patients and controls. Differentially abundant spots (|fold change|≥ 1.5, P < 0.01) were selected and identified by LC-MS/MS. The spots were further assessed in gastric antrum biopsies from AAG patients (without and with Helicobacter pylori infection) and from GC patients and unaffected first-degree relatives of GC patients. RESULTS: 2D-DIGE identified 67 differentially abundant spots, with 28 more and 39 less abundant in AAG-corpus than controls. LC-MS/MS identified these as 53 distinct proteins. The most significant (adjusted P < 0.01) biological process associated with the less abundant proteins was "tricarboxylic acid cycle". Of the 67 spots, 57 were similarly differentially abundant in AAG-antrum biopsies irrespective of H. pylori infection status. The differential abundance was also observed in GC biopsies for 14 of 28 more abundant and 35 of 39 less abundant spots, and in normal gastric biopsies of relatives of GC patients for 6 and 25 spots, respectively. Immunoblotting confirmed the different expression levels of two more abundant proteins (PDIA3, GSTP gene products) and four less abundant proteins (ATP5F1A, PGA3, SDHB, PGC). CONCLUSION: This study identified a proteomics signature of AAG. Many differential proteins were shared by GC and may be involved in the progression of AAG to GC.

Family's History Based on the CDH1 Germline Variant (c.360delG) and a Suspected Hereditary Gastric Cancer Form.

Hereditary diffuse gastric cancer (HDGC) is a cancer susceptibility syndrome caused by germline pathogenic variant in CDH1, the gene encoding E-cadherin. The germline loss-of-function variants are the only proven cause of the cancer syndrome HDGC, occurring in approximately 10-18% of cases and representing a helpful tool in genetic counseling. The current case reports the family history based on a CDH1 gene variant, c.360delG, p.His121Thr in a suspected family for hereditary gastric cancer form. This frameshift deletion generates a premature stop codon at the amino acid 214, which leads to a truncated E-cadherin protein detecting it as a deleterious variant. The present study expands the mutational spectra of the family with the CDH1 variant. Our results highlight the clinical impact of the reported CDH1 variant running in gastric cancer families.

Proteomic Profiles and Biological Processes of Relapsed vs. Non-Relapsed Pediatric Hodgkin Lymphoma.

The identification of circulating proteins associated with relapse in pediatric Hodgkin lymphoma (HL) may help develop predictive biomarkers. We previously identified a set of predictive biomarkers by difference gel electrophoresis. Here we used label-free quantitative liquid chromatography-mass spectrometry (LC-MS/MS) on plasma collected at diagnosis from 12 children (age 12-16 years) with nodular sclerosis HL, including six in whom the disease relapsed within 5 years of treatment in the LH2004 trial. Plasma proteins were pooled in groups of three, separately for non-relapsing and relapsing HL, and differentially abundant proteins between the two disease states were identified by LC-MS/MS in an explorative and validation design. Proteins with a fold change in abundance >1.2 or ≤0.8 were considered "differentially abundant". LC-MS/MS identified 60 and 32 proteins that were more abundant in non-relapsing and relapsing HL plasma, respectively, in the explorative phase; these numbers were 39 and 34 in the validation phase. In both analyses, 11 proteins were more abundant in non-relapsing HL (e.g., angiotensinogen, serum paraoxonase/arylesterase 1, transthyretin), including two previously identified by difference gel electrophoresis (antithrombin III and α-1-antitrypsin); seven proteins were more abundant in relapsing HL (e.g., fibronectin and thrombospondin-1), including two previously identified proteins (fibrinogen ß and γ chains). The differentially abundant proteins participated in numerous biological processes, which were manually grouped into 10 biological classes and 11 biological regulatory subclasses. The biological class Lipid metabolism, and its regulatory subclass, included angiotensinogen and serum paraoxonase/arylesterase 1 (more abundant in non-relapsing HL). The biological classes Immune system and Cell and extracellular matrix architecture included fibronectin and thrombospondin-1 (more abundant in relapsing HL). These findings deepen our understanding of the molecular scenario underlying responses to therapy and provide new evidence about these proteins as possible biomarkers of relapse in pediatric HL.

Molecular Features Distinguish Gastric Cancer Subtypes.

Gastric cancer (GC) is a leading cause of cancer deaths [...].

Immunotherapy for Gastric Cancer: Time for a Personalized Approach?

Over the last decade, our understanding of the mechanisms underlying immune modulation has greatly improved, allowing for the development of multiple therapeutic approaches that are revolutionizing the treatment of cancer. Immunotherapy for gastric cancer (GC) is still in the early phases but is rapidly evolving. Recently, multi-platform molecular analyses of GC have proposed a new classification of this heterogeneous group of tumors, highlighting subset-specific features that may more reliably inform therapeutic choices, including the use of new immunotherapeutic drugs. The clinical benefit and improved survival observed in GC patients treated with immunotherapeutic strategies and their combination with conventional therapies highlighted the importance of the immune environment surrounding the tumor. A thorough investigation of the tumor microenvironment and the complex and dynamic interaction between immune cells and tumor cells is a fundamental requirement for the rational design of novel and more effective immunotherapeutic approaches. This review summarizes the pre-clinical and clinical results obtained so far with immunomodulatory and immunotherapeutic treatments for GC and discusses the novel combination strategies that are being investigated to improve the personalization and efficacy of GC immunotherapy.

Proposed Molecular and miRNA Classification of Gastric Cancer.

Gastric cancer (GC) is a common malignant neoplasm worldwide and one of the main cause of cancer-related deaths. Despite some advances in therapies, long-term survival of patients with advanced disease remains poor. Different types of classification have been used to stratify patients with GC for shaping prognosis and treatment planning. Based on new knowledge of molecular pathways associated with different aspect of GC, new pathogenetic classifications for GC have been and continue to be proposed. These novel classifications create a new paradigm in the definition of cancer biology and allow the identification of relevant GC genomic subsets by using different techniques such as genomic screenings, functional studies and molecular or epigenetic characterization. An improved prognostic classification for GC is essential for the development of a proper therapy for a proper patient population. The aim of this review is to discuss the state-of-the-art on combining histological and molecular classifications of GC to give an overview of the emerging therapeutic possibilities connected to the latest discoveries regarding GC.

Use of Metabolomics as a Complementary Omic Approach to Implement Risk Criteria for First-Degree Relatives of Gastric Cancer Patients.

A positive family history is a strong and consistently reported risk factor for gastric cancer (GC). So far, it has been demonstrated that serum pepsinogens (PGs), and gastrin 17 (G17) are useful for screening individuals at elevated risk to develop atrophic gastritis but they are suboptimal biomarkers to screen individuals for GC. The main purpose of this study was to investigate serum metabolomic profiles to find additional biomarkers that could be integrated with serum PGs and G17 to improve the diagnosis of GC and the selection of first-degree relatives (FDR) at higher risk of GC development. Serum metabolomic profiles included 188 serum metabolites, covering amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, sphingomyelins and hexoses. Serum metabolomic profiles were performed with tandem mass spectrometry using the Biocrates AbsoluteIDQ p180 kit. The initial cohort (training set) consisted of n = 49 GC patients and n = 37 FDR. Differential metabolomic signatures among the two groups were investigated by univariate and multivariate partial least square differential analysis. The most significant metabolites were further selected and validated in an independent group of n = 22 GC patients and n = 17 FDR (validation set). Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic power and the optimal cut-off for each of the discriminant markers. Multivariate analysis was applied to associate the selected serum metabolites, PGs, G17 and risk factors such as age, gender and Helicobacter pylori (H. pylori) infection with the GC and FDR has been performed and an integrative risk prediction algorithm was developed. In the training set, 40 metabolites mainly belonging to phospholipids and acylcarnitines classes were differentially expressed between GC and FDR. Out of these 40 metabolites, 9 were further confirmed in the validation set. Compared with FDR, GC patients were characterized by lower levels of hydroxylated sphingomyelins (SM(OH)22:1, SM(OH)22:2, SM(OH)24:1) and phosphatidylcholines (PC ae 40:1, PC ae 42:2, PC ae 42:3) and by higher levels of acylcarnitines derivatives (C2, C16, C18:1). The specificity and sensitivity of the integrative risk prediction analysis of metabolites for GC was 73.47% and 83.78% respectively with an area under the curve of the ROC curve of 0.811 that improves to 0.90 when metabolites were integrated with the serum PGs. The predictive risk algorithm composed of the C16, SM(OH)22:1 and PG-II serum levels according to the age of individuals, could be used to stratify FDR at high risk of GC development, and then this can be addressed with diagnostic gastroscopy.

Quantitative Proteomic Approach Targeted to Fibrinogen ß Chain in Tissue Gastric Carcinoma.

Elevated plasma fibrinogen levels and tumor progression in patients with gastric cancer (GC) have been largely reported. However, distinct fibrinogen chains and domains have different effects on coagulation, inflammation, and angiogenesis. The aim of this study was to characterize fibrinogen ß chain (FGB) in GC tissues. Retrospectively we analyzed the data of matched pairs of normal (N) and malignant tissues (T) of 28 consecutive patients with GC at diagnosis by combining one- and two-dimensional electrophoresis (1DE and 2DE) with immunoblotting and mass spectrometry together with two-dimensional difference in gel electrophoresis (2D-DIGE). 1DE showed bands of the intact FGB at 50 kDa and the cleaved forms containing the fragment D at ~37-40 kDa, which corresponded to 19 spots in 2DE. In particular, spot 402 at ~50 kDa and spots 526 and 548 at ~37 kDa were of interest by showing an increased expression in tumor tissues. A higher content of spot 402 was associated with stomach antrum, while spots 526 and 548 amounts correlated with corpus and high platelet count (>208 × 108/L). The quantification of FGB and cleaved products may help to further characterize the interconnections between GC and platelet/coagulation pathways.

Molecular and Pathological Features of Gastric Cancer in Lynch Syndrome and Familial Adenomatous Polyposis.

Lynch syndrome (LS) and familial adenomatous polyposis (FAP) are autosomal dominant hereditary diseases caused by germline mutations leading to the development of colorectal cancer. Moreover, these mutations result in the development of a spectrum of different tumors, including gastric cancers (GCs). Since the clinical characteristics of GCs associated with LS and FAP are not well known, we investigated clinical and molecular features of GCs occurring in patients with LS and FAP attending our Institution. The Hereditary Tumor Registry was established in 1994 at the Department of Oncologic Gastroenterology, CRO Aviano National Cancer Institute, Italy. It includes 139 patients with LS and 86 patients with FAP. Patients were recruited locally for prospective surveillance. Out of 139 LS patients, 4 developed GC­3 in the presence of helicobacter pylori infection and 1 on the background of autoimmune diseases. All GCs displayed a high microsatellite instability (MSI-H) and loss of related mismatch repair (MMR) protein. One of the FAP patients developed a flat adenoma, displaying low-grade dysplasia at the gastric body, and another poorly differentiated adenocarcinoma with signet ring cells like Krukenberg without HP infection. LS carriers displayed a risk of GC. The recognition of HP infection and autoimmune diseases would indicate those at higher risk for an endoscopic surveillance. Regarding FAP, the data suggested the need of suitable endoscopic surveillance in long survivals with diffuse fundic gland polyps.

Dendritic cells accumulate in the bone marrow of myeloma patients where they protect tumor plasma cells from CD8+ T-cell killing.

Many researchers have speculated that the clinical progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is driven by defects in dendritic cell (DC) function. However, evidence supporting this assumption is controversial, and no mechanism for the putative DC dysfunction has been demonstrated thus far. We studied DC subsets from the bone marrow of MM patients compared with those of MGUS patients and control subjects. We found that myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) accumulate in the bone marrow during the MGUS-to-MM progression. After engulfment of apoptotic tumor plasma cells via CD91, bone marrow mDCs and pDCs mature and are able to activate tumor-specific CD8(+) T cells. However, by interacting directly with CD28 on live (nonapoptotic) tumor plasma cells, bone marrow mDCs downregulate the expression of proteasome subunits in these cells, thus enabling their evasion from human leukocyte antigen (HLA) class I-restricted CD8(+) T-cell killing. These results suggest that DCs play a dual, but opposing, role in MM: for one, DCs activate CD8(+) T cells against tumor plasma cells and, for the other, DCs protect tumor plasma cells from CD8(+) T-cell killing. This information should be taken into account in designing immunotherapy approaches to enhance immune surveillance in MGUS and to break down immune tolerance in MM.

Characterizing Metastatic HER2-Positive Gastric Cancer at the CDH1 Haplotype.

The CDH1 gene, coding for the E-cadherin protein, is linked to gastric cancer (GC) susceptibility and tumor invasion. The human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in a portion of GC. HER2 is an established therapeutic target in metastatic GC (mGC). Trastuzumab, in combination with various chemotherapeutic agents, is a standard treatment for these tumors leading to outcome improvement. Unfortunately, the survival benefit is limited to a fraction of patients. The aim of this study was to improve knowledge of the HER2 and the E-cadherin alterations in the context of GC to characterize subtypes of patients that could better benefit from targeted therapy. An association between the P7-CDH1 haplotype, including two polymorphisms (rs16260A-rs1801552T) and a subset of HER2-positive mGC with better prognosis was observed. Results indicated the potential evaluation of CDH1 haplotypes in mGC to stratify patients that will benefit from trastuzumab-based treatments. Moreover, data may have implications to understanding the HER2 and the E-cadherin interactions in vivo and in response to treatments.

Differential proteomics of Helicobacter pylori associated with autoimmune atrophic gastritis.

Atrophic autoimmune gastritis (AAG) is a condition of chronic inflammation and atrophy of stomach mucosa, for which development can be partially triggered by the bacterial pathogen Helicobacter pylori (HP). HP can cause a variety of gastric diseases, such as duodenal ulcer (DU) or gastric cancer (GC). In this study, a comparative proteomic approach was used by two-dimensional fluorescence difference gel electrophoresis (DIGE) to identify differentially expressed proteins of HP strains isolated from patients with AAG, to identify markers of HP strain associated with AAG. Proteome profiles of HP isolated from GC or DU were used as a reference to compare proteomic levels. Proteomics analyses revealed 27 differentially expressed spots in AAG-associated HP in comparison with GC, whereas only 9 differential spots were found in AAG-associated HP profiles compared with DU. Proteins were identified after matrix-assisted laser desorption ionization (MALDI)-TOF and peptide mass fingerprinting. Some AAG-HP differential proteins were common between DU- and GC-HP (peroxiredoxin, heat shock protein 70 [HSP70], adenosine 5'-triphosphate [ATP] synthase subunit α, flagellin A). Our results presented here may suggest that comparative proteomes of HP isolated from AAG and DU share more common protein expression than GC and provide subsets of putative AAG-specific upregulated or downregulated proteins that could be proposed as putative markers of AAG-associated HP. Other comparative studies by two-dimensional maps integrated with functional genomics of candidate proteins will undoubtedly contribute to better decipher the biology of AAG-associated HP strains.

The frequency of CD127(+) hepatitis C virus (HCV)-specific T cells but not the expression of exhaustion markers predicts the outcome of acute HCV infection.

T cells are exhausted and overexpress inhibitory molecules in chronic hepatitis C virus (HCV) infection. It is unclear whether this is the cause or consequence of HCV persistence. By studying serial blood and liver samples of chimpanzees during acute infection, we demonstrate that the early expression of the memory precursor marker CD127 on HCV-specific T cells, but not the expression of inhibitory molecules on those T cells or their ligands in the liver, predicts the outcome of acute infection.

Immune escape of multiple myeloma cells results from low miR29b and the ensuing epigenetic silencing of proteasome genes.

BACKGROUND: Activation of CD28 on multiple myeloma (MM) plasma cells, by binding to CD80 and CD86 on dendritic cells, decreases proteasome subunit expression in the tumor cells and thereby helps them evade being killed by CD8+ T cells. Understanding how CD28 activation leads to proteasome subunit downregulation is needed to design new MM therapies. METHODS: This study investigates the molecular pathway downstream of CD28 activation, using an in vitro model consisting of myeloma cell lines stimulated with anti-CD28-coated beads. RESULTS: We show that CD28 engagement on U266 and RPMI 8226 cells activates the PI3K/AKT pathway, reduces miR29b expression, increases the expression of DNA methyltransferase 3B (DNMT3B, a target of miR29b), and decreases immunoproteasome subunit expression. In vitro transfection of U266 and RPMI 8226 cells with a miR29b mimic downregulates the PI3K/AKT pathway and DNMT3B expression, restores proteasome subunit levels, and promotes myeloma cell killing by bone marrow CD8+ T cells from MM patients. Freshly purified bone marrow plasma cells (CD138+) from MM patients have lower miR29b and higher DNMT3B (mRNA and protein) than do cells from patients with monoclonal gammopathy of undetermined significance. Finally, in MM patients, high DNMT3B levels associate with shorter overall survival. CONCLUSIONS: Altogether, this study describes a novel molecular pathway in MM. This pathway starts from CD28 expressed on tumor plasma cells and, through the PI3K-miR29b-DNMT3B axis, leads to epigenetic silencing of immunoproteasome subunits, allowing MM plasma cells to elude immunosurveillance. This discovery has implications for the design of innovative miR29b-based therapies for MM.

Successful vaccination induces multifunctional memory T-cell precursors associated with early control of hepatitis C virus.

BACKGROUND & AIMS: T cells are an important component for development of a vaccine against hepatitis C virus (HCV), but little is known about the features of successful vaccine-induced T cells. METHODS: We compared the phenotype, function, and kinetics of vaccine-induced and infection-induced T cells in chimpanzees with HCV infection using multicolor flow cytometry and real-time polymerase chain reaction. RESULTS: In chimpanzees successfully vaccinated with recombinant adenovirus and DNA against HCV NS3-5, HCV-specific T cells appeared earlier, maintained better functionality, and persisted at higher frequencies for a longer time after HCV challenge, than those of mock-vaccinated chimpanzees. Vaccine-induced T cells displayed higher levels of CD127, a marker of memory precursors, and lower levels of programmed death-1 (PD-1) than infection-induced T cells. Vaccine-induced, but not infection-induced, T cells were multifunctional; their ability to secrete interferon gamma and tumor necrosis factor α correlated with early expression of CD127 but not PD-1. Based on a comparison of vaccine-induced and infection-induced T cells from the same chimpanzee, the CD127(+) memory precursor phenotype was induced by the vaccine itself rather than by low viremia. In contrast, induction of PD-1 correlated with viremia, and levels of intrahepatic PD-1, PD-L1, and 2,5-OAS-1 messenger RNAs correlated with peak titers of HCV. CONCLUSIONS: Compared with infection, vaccination-induced HCV-specific CD127(+) T cells with high functionality that persisted at higher levels for a longer time. Control of viremia prevented up-regulation of PD-1 on T cells and induction of PD-1, PD-L1, and 2,5-OAS-1 in the liver. Early development of a memory T-cell phenotype and, via control of viremia, attenuation of the inhibitory PD1-PD-L1 pathway might be necessary components of successful vaccine-induced protection against HCV.