Chimpanzees and HCV Challenge
Chimpanzees were intravenously (IV) inoculated with 100 chimpanzee infectious dose 50 HCV (H77 strain) and studied under standard conditions for humane care, in compliance with National Institutes of Health guidelines, at an Association for Assessment and Accreditation of Animal Care accredited facility under a protocol approved by the New Iberia Research Center Animal Care and Use Committee. The clinical and virologic course of HCV infection were described previously.20,21
Liver biopsies and plasma were cryopre-served for the present study.
HCV Infection and Polyinosinic/Polycytidylic Acid Transfection of PHH
The purity of PHH (Invitrogen, Carlsbad, CA and Celsis, Chicago, IL) was confirmed by intracellular staining with anti-albumin (clone 2F11; Sigma-Aldrich, St. Louis, MO), followed by staining with anti-mouse immunoglobulin G-phycoerythrin (Invitrogen), treatment with 10% mouse serum (Innovative Research, Southfield, MI), staining with ethidium monoazide (EMA; Sigma-Aldrich), anti-CD14-Pacific Blue (clone M5E2), anti-CD45RA-fluorescein isothio-cyanate (FITC) (clone HI100), anti-CD45RO-FITC (clone UCHL1), anti-CD11c-allophycocyanin (clone B-ly6), and anti-CD16-PE-cyanine 5 (clone 3G8; all from BD Biosciences, Bedford, MA) and analysis on an LSRII flow cytometer (BD Biosciences). Hepatocytes constituted >95% and EMA-CD16-CD45+ hematopoietic cells <0.4% of the cells. Of the latter, 85% were CD14+ and 18.6% were CD11c+ .
PHHs shipped in plates were incubated with InVitroGRO HI medium (Celsis) for 4 hours at 37° C (5% CO2), followed by polyinosinic/polycytidylic acid (polyI:C) transfection or HCV infection, as described below.
PHHs shipped in suspension were centrifuged at 50×g
for 5 minutes, incubated in InVitro
GRO CP medium (Celsis) in 12- (7 × 105
/well) or 24-well (3.5 × 105
/well) plates overnight, and transfected with 5 μg of polyI:C (InvivoGen, San Diego, CA) and 6.4 μL of Lipofectamine 2000 in Opti-MEM medium (Invitrogen), or infected with HCV (Japanese fulminant hepatitis type I [JFH-1] strain)22
at a multiplicity of infection (MOI) of 0.4–2.7. After 3–6 hours, culture medium was replaced with InVitro
GRO HI including Torpedo
antibiotic mix (Celsis).
In some experiments, PHHs were incubated 30 minutes before HCV infection with neutralizing antibodies (Abs) against type I IFNs (10 μg/mL each of anti-IFN-α [clone MMHA-2; PBL Interferon Source, Piscataway, NJ] and polyclonal anti-IFN-β [R&D Systems, Minneapolis, MN]) or type III IFNs (10 μg/ mL each of polyclonal anti-IL-29, polyclonal anti-IL-28A, and anti-IL-29/IL-28B [clone 247801;, R&D Systems]).
Extraction of RNA, Complementary DNA synthesis, and Quantitative Polymerase Chain Reaction
Total RNA was isolated from snap-frozen, mechanically homogenized liver biopsies or from PHH using the RNeasy Mini Kit (Qiagen, Valencia, CA) with on-column DNase digestion.
A complementary DNA (cDNA) equivalent to 20–80 ng of total RNA, generated with the Monster-Script 1st-Strand cDNA Synthesis Kit (EPICENTRE Biotechnologies, Madison, WI), was used to determine IFN-induced protein with tetratricopeptide repeats 1 (IFIT1), myxovirus resistance 1 (MX1), chemokine (C-X-C motif) ligand (CXCL)10, CXCL11, IFN-α2, IFN-β, IL-29, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and proteasome subunit, beta type, 4 (PSMB4) messenger RNA (mRNA) levels with pre-designed human TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA). Because IL28A and IL28B share 98% of their nucleotide sequence, their mRNA levels were quantitated with shared forward primer 5′-TGAGGCAGCTGCAGGTGA-3′, reverse primer 5′-CTCCAGAACCTTCAGCGTCAG-3′, and probe 5′-FAM-TGGCTTTGGAGGCTGA-MGB-3′ designed with Primer Express (Applied Biosystems). The specific mRNA amount was quantitated using comparative cycle threshold values and 1–107 copies/well standard curves, and normalized to mean GAPDH and PSMB4 mRNA levels. Relative mRNA levels represent fold-increase over pre-infection or pre-transfection samples.
For HCV RNA quantitation, we used TaqMan EZ RTPCR Core Reagents (Applied Biosystems) and forward primer 5′-CGGGAGAGCCATAGTGG-3′, reverse primer 5′AGTACCACAAGGCCTTTCG-3′, and probe 5′-FAM-CTGCGGAACCGGTGAGTA CAC -TAMRA-3′ (Sigma-Aldrich). RT-PCR conditions are 2 minutes at 50° C, 30 minutes at 60° C, 5 minutes at 95° C, followed by 50 cycles at 95° C for 20 seconds and at 60° C for 1 minute. HCV RNA levels were normalized to microgram of total input RNA.
Enzyme-Linked Immunosorbent Assay
IFN-α and IFN-β (PBL Interferon Source, Piscataway, NJ), IL-28A, CXCL10, and CXCL11 (R&D Systems), and IL-29 (eBioscience, San Diego, CA) enzyme-linked immunosorbent assays (ELISAs) were used to quantitate cytokines in chimpanzee plasma or PHH culture supernatants. IL-28A exhibited 30% cross-reactivity with IL-28B.
Antiviral Activity Assay
Huh7-Lunet cells harboring a subgenomic, luciferase-expressing HCV JFH-1 replicon (Luc-ubi-neo/JFH-1 cells,23
here designated Huh7-HCV-Luc cells) were treated for 30 hours with the indicated amounts of recombinant IL-29, IL-28A, IL-28B (R&D Systems), IFN-αA2, and IFN-β (PBL Interferon Source) or with 72-hour supernatants from HCV-infected PHH. Luciferase activity was measured with the Luciferase Assay System (Promega, Madison, WI) using a POLARstar Omega instrument (BMG Labtech, Cary, NC). For some experiments, the neutralizing Abs to type I or III IFNs described above were added to either (1) supernatants from HCV-infected PHH that had been treated with the same neutralizing Abs or (2) recombinant cytokines, 30 minutes before adding the mixture to Huh7-HCV-Luc cells.
Isolation of pDCs and Coculture With PHH
Peripheral blood mononuclear cells were isolated from buffy coats, using density gradient centrifugation, and pDCs were selected with BDCA-4 magnetic beads (Miltenyi Biotec, Auburn, CA). Purity of CD123+ BDCA2+ pDCs was 86%–95% by flow cytometry. PHHs (3 × 105/well) were infected with HCV JFH-1 at an MOI of 2 in a 24-well plate for 30 hours, followed by removal of culture supernatants and the addition of 3 × 104 pDCs/200 μL directly or into a transwell. The total coculture volume was 500 μL/ well. Released IFN-α, IL-29, CXCL10, and CXCL11 were quantitated after 24 hours by ELISA.
DNA samples of 90 chimpanzees, including the 6 of this study, were genotyped by Sanger sequencing with primers, designed by Primer Express software to flank human SNPs associated with HCV-related traits12–15
(). Cleaned polymerase chain reaction (PCR) products were sequenced on a 3730 Automatic Sequencer (Applied Biosystems). Genetic variants were analyzed in relation to human sequences using Sequencher 4.1 software.
Analysis of Human SNPs Associated With HCV-Related Traits