Vaccine Rhesus Macaques by a Nonclassical ISCOM Core-Specific Immune Responses Primed in Characterization of Hepatitis C Virus

Characterization of Hepatitis C Virus Core-Specific Immune Responses Primed in Rhesus Macaques by a Nonclassical ISCOM Vaccine This information is current as of October 4, 2021. Noelle K. Polakos, Debbie Drane, John Cox, Philip Ng, Mark J. Selby, David Chien, Derek T. O'Hagan, Michael Houghton and Xavier Paliard J Immunol 2001; 166:3589-3598; ;

doi: 10.4049/jimmunol.166.5.3589 Downloaded from http://www.jimmunol.org/content/166/5/3589

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Characterization of Hepatitis C Virus Core-Speciﬁc Immune Responses Primed in Rhesus Macaques by a Nonclassical ISCOM Vaccine

Noelle K. Polakos,* Debbie Drane,† John Cox,† Philip Ng,* Mark J. Selby,* David Chien,* Derek T. O’Hagan,* Michael Houghton,* and Xavier Paliard1*

Current therapies for the treatment of hepatitis C virus (HCV) infection are only effective in a restricted number of patients. Cellular immune responses, particularly those mediated by CD8؉ CTLs, are thought to play a role in the control of infection and the response to antiviral therapies. Because the Core protein is the most conserved HCV protein among genotypes, we evaluated the ability of a Core prototype vaccine to prime cellular immune responses in rhesus macaques. Since there are serious concerns about using a genetic vaccine encoding for Core, this vaccine was a nonclassical ISCOM formulation in which the Core protein was adsorbed onto (not entrapped within) the ISCOMATRIX, resulting in ϳ1-␮m particulates (as opposed to 40 nm for classical Downloaded from .ISCOM formulations). We report that this Core-ISCOM prototype vaccine primed strong CD4؉ and CD8؉ T cell responses Using intracellular staining for cytokines, we show that in immunized animals 0.30–0.71 and 0.32–2.21% of the circulating CD8؉ and CD4؉ T cells, respectively, were speciﬁc for naturally processed HCV Core peptides. Furthermore, this vaccine elicited a Th0-type response and induced a high titer of Abs against Core and long-lived cellular immune responses. Finally, we provide evidence that Core-ISCOM could serve as an adjuvant for the HCV envelope protein E1E2. Thus, these data provide evidence that Core-ISCOM is effective at inducing cellular and humoral immune responses in nonhuman primates. The Journal of Immunol- http://www.jimmunol.org/ ogy, 2001, 166: 3589–3598.

he hepatitis C virus (HCV)2 is now recognized as the infected cells (10–12), might be critical in determining the out- leading cause of chronic liver disease (1). An estimated come of therapy. Using mathematical modeling, it was found T 170 million people worldwide are infected with HCV, that diminution of serum HCV RNA levels during the second- approximately four times more than the number of people infected phase slope observed during IFN therapy was inversely corre- with HIV (2). Despite these alarming numbers, only a few thera- lated with baseline viral load and was positively correlated with

pies are available to clinicians treating patients infected with HCV. hepatocyte death (13). Combined with the fact that there is an by guest on October 4, 2021 Although treatment of HCV infection with ribavirin and/or IFN inverse correlation between the frequency of HCV-specific has been shown to be highly effective in some patients, roughly CTLs and the viral load (14) and that the presence of HCV two-thirds of patients fail to have a sustained response to treatment Core-specific CTLs before IFN treatment has been associated (3, 4). Hence, improved therapies are desperately needed, but their with subsequent response of patients to IFN therapy (15), this development is hampered by the lack of a small animal model for indicated that killing of HCV-infected cells by CTLs could play infection and disease. an important role in determining response to therapy. Thus, a Several studies have suggested that T cell-mediated immune vaccine eliciting Core-specific CTLs might enhance the re- responses to HCV infection can determine the outcome of HCV sponse rate of infected patients to therapy. infection and disease (5–7). Furthermore, recent work indicated Because the HCV Core protein can modulate multiple cellu- that when the early immune response to HCV is optimal, the lar processes, such as apoptosis, lipid metabolism, and tran- genetic diversity of HCV quasispecies declines, and the strains scription, and can induce cellular transformation (16), there are become increasingly homogeneous until the final variant is potential concerns about eliciting Core-specific immune re- cleared (8). Because response to current treatment (IFN alone or sponses in patients using a genetic vaccine (naked DNA; viral, associated with ribavirin) can be predicted in part by the viral ϩ retroviral, bacterial vectors; replicons). Hence, to elicit Core- load (9), host immune factors including CD8 CTLs, which are specific T cell-mediated immune responses, one must rely on thought to be the major contributor to the death rate of HCV- the use of a subunit vaccine. Although most licensed subunit vaccines are inefficient at inducing CTLs, there has been con- *Chiron Corp., Emeryville, CA 94608; and †CSL Ltd., Parkville, Victoria, Australia siderable advancement in the field of adjuvant research. For Received for publication August 18, 2000. Accepted for publication December example, classical ISCOM, a typically 40-nm cage-like structure 20, 2000. composed of saponins from Quillaja saponaria Molina, cholesterol, The costs of publication of this article were defrayed in part by the payment of page and phospholipids, inside which the Ag is entrapped (17), have been charges. This article must therefore be hereby marked advertisement in accordance ϩ ϩ with 18 U.S.C. Section 1734 solely to indicate this fact. shown to prime CD4 and CD8 -mediated immune responses (18, 1 Address correspondence and reprint requests to Dr. Xavier Paliard, Chiron Corp., 19). In this study, we evaluated and characterized the potency of a 4560 Horton Street, Room 4.3132, Emeryville, CA 94608-2916. E-mail address: nonclassical ISCOM vaccine, in which Core was not incorporated [email protected] into the ISCOMATRIX, but was adsorbed onto the cage-like structure 2 Abbreviations used in this paper: HCV, hepatitis C virus; rVV, recombinant vac- ϳ cinia virus; rVVC/E1, rVV expressing Core and E1; VVwt, wild-type VV; SI, stim- by ionic interactions, resulting in particulates 25 times larger than ulation index; B-LPCLs, B-cell lymphoblastoid cell lines. conventional ISCOM (1 ␮m vs 40 nm) in rhesus macaques.

totoxic activity. Peptide-specific CD8ϩ lines were obtained by periodically Materials and Methods ϩ Animals restimulating these CD8 T cells with autologous B cell lymphoblastoid cell lines (B-LCLs) and peptide. Rhesus macaques (Macaca mulatta) were housed at the Southwest Foun- B-LCLs were derived from each animal using supernatants from the dation for Biomedical Research (San Antonio, TX). Studies were approved Herpesvirus papio producer cell line S394. by the institutional animal care and use committees of Chiron and the Southwest Foundation for Biomedical Research and were performed under CTL assay the National Institutes of Health Guidelines for the Care and Use of Lab- Cytotoxic activity was assayed in a standard 51Cr release assay as described oratory Animals (20). Class I MHC typing of the animals was performed previously (26). Briefly, B-LCLs were incubated with 10 ␮M peptides and as previously described (21). ␮ 51 ϫ 3 b 50 Ci of Cr for 1 h, washed three times, and plated at 5 10 cells/well Female C57BL/6 (H-2 ) mice were purchased from Charles River in a 96-well plate. Alternatively, B-LCLs were infected at a multiplicity of Breeding Laboratories (Wilmington, MA) and were used between 8 and 10 infection of 10:1 with rVVC/E1 or VVwt for 1 h, washed, and cultured wk of age. Mice were housed in a pathogen-free environment and were overnight before labeling with 51Cr. CD8ϩ cells were plated in duplicate at handled according to the international guidelines for experimentation with three different E:T cell ratios and incubated with target cells for4hinthe animals. All mouse experiments were approved by Chiron’s animal care presence of 2 ϫ 105/well of unlabeled target cells (cold targets) that were and use committee. added to minimize lysis of B-LCLs by H. papio or endogenous virus (e.g., Immunogens and adjuvants foamy virus)-specific CTLs. CTL responses were scored positive when percent specific lysis at the two highest E:T cell ratios was greater than or The full-length HCV-1a Core recombinant protein (aa 1–191) was pro- equal to the percent lysis of control targets plus 10. duced in Escherichia coli. Core was purified from cells lysed and extracted with urea containing DTT. Cation exchange chromatography, hydroxyap- Lymphoproliferation assay atite chromatography, and size exclusion chromatography were used to This assay has been described previously (27). Briefly, freshly isolated

Ͼ Downloaded from subsequently purify the material. The resulting Core was 98% pure. The PBMCs were plated in triplicates at 2 ϫ 105 cells/well in 96-well round- recombinant HCV-1a E1E2809 protein was produced in Chinese hamster ovary bottom plates and cultured in the presence of 5 ␮g/ml recombinant Core cells as described previously (22). The Core-ISCOM formulations were pre- protein or 0.05 ␮g/ml (the Escherichia coli-derived Core protein contains pared by mixing the core protein with preformed ISCOMATRIX (empty Ͻ2% impurities (Ͼ98% pure)) E. coli control. Plates were pulsed with 1 ISCOMS) using ionic interactions to maximize the association between the Ag ␮Ci/well [3H]thymidine on day 5 and harvested 6–8 h later. Results are and the adjuvant. ISCOMATRIX was prepared essentially by previously de- presented as the stimulation index (SI) calculated as (mean experimental scribed methods (23), except that diaﬁltration was used in place of dialysis. cpm)/(mean cpm in the presence of the E. coli control). An SI Ն3.0 was E1E2 classical ISCOM was prepared as previously described (23). The oil-

scored positive. http://www.jimmunol.org/ in-water adjuvant MF59 has been described previously (24). FACS analysis Peptides and vaccinia viruses Freshly isolated PBMCs or PBMCs that had been restimulated in vitro with Peptides (15- or 20-mer overlapping by 10 aa) spanning the entire length a peptide were cultured in medium alone or restimulated with 5 ␮g/ml Core of the Core (aa 1–191) protein of HCV-1a (25) were synthesized with free amine protein, 0.05 ␮g/ml E. coli control, 5 ␮g/ml peptide, or VV-infected or N termini and free acid C termini by Research Genetics (Huntsville, AL). The peptide-sensitized autologous B-LCLs (1/1) for 12 h in culture medium recombinant vaccinia virus (rVV) expressing Core and E1 (aa 1–384; rVVC/E1) containing 50 U/ml rIL-2 (Chiron) and 3 ␮M monensin (PharMingen, San and wild-type VV (VVwt) have been described previously (6). Diego, CA). Cells were stained as previously described (28) for surface Immunization CD4 and CD8 with APC-conjugated anti-human CD4 and PerCP-conjugated anti-human CD8 and for intracellular IFN- ␥ and TNF-␣ with PE- by guest on October 4, 2021 Rhesus macaques were immunized under anesthesia. The first study was conjugated anti-human IFN-␥ and FITC-conjugated anti-human TNF-␣. comprised of six animals divided into two groups of three animals each. Abs were obtained from PharMingen and Becton Dickinson (San Jose, The first group (animals BB228, BB232, and DV036) was infected with CA). Cells were analyzed on a FACSCalibur. Data files were analyzed 2 ϫ 108 PFU (1 ϫ 108 intradermally and 1 ϫ 108 by scarification) of using CellQuest software (Becton Dickinson). rVVC/E1 at 0 mo. This group served as a positive control for CTL priming. Animals from the second group (AY921, BB231, and DV037) were im- Cytokine ELISA ␮ munized with 25 g of Core-ISCOM by i.m. injection in the left quadri- Freshly isolated rhesus macaque PBMCs were restimulated with peptides ceps at 0, 1, 2, and 6 mo. For the second study, five animals (15860, 15861, ␮ encompassing the whole Core protein. Levels of rhesus monkey IL-2, IL-5, 15862, 15863, and 15864) were immunized with 50 g of Core- ISCOM IL-10, and IFN-␥ present in 48-h cell-free culture supernatants were de- by i.m. injection in the left quadriceps at 0, 1, and 2 mo. Some Core- ϫ 8 ϫ 8 termined by specific ELISA (U-Cytech, Utrecht, The Netherlands) follow- immunized animals (see Table I) also received 2 10 PFU (1 10 ing the manufacturer’s specification. intradermally and 1 ϫ 108 by scarification) of rVVC/E1 9 or 11 wk after their last vaccine immunization. HCV Abs Mice (10 animals/group) were immunized in the tibialis anterior mus- cles (50 ␮l/muscle) with 2 ␮g/dose recombinant E1E2 protein, 2 ␮g/dose Serum levels of HCV Core and HCV E2 Abs were quantified by ELISA as recombinant E1E2 plus 2 ␮g/dose recombinant Core, 2 ␮g/dose recombi- previously described (29). Serum levels of Abs inhibiting the binding of E2 nant E1E2 in the presence of MF59 (v/v), 2 ␮g/dose E1E2 classical to the putative HCV receptor CD81 (30) were determined by ISCOM, or 2 ␮g/dose recombinant E1E2 protein plus 2 ␮g/dose Core- immunoassay. ISCOM at 0, 4, and 8 wk. Results Cells and cell lines Priming of Core-specific CTLs in vaccinated animals Peripheral blood was drawn from the femoral vein while the animals were under anesthesia. PBMCs were obtained after centrifugation over a Ficoll- The prototype vaccine Core-ISCOM aimed at eliciting HCV-Core- Hypaque gradient and were cultured in 24-well dishes at 5 ϫ 106 cells/ specific CTLs was administered to three HCV-naive rhesus ma- well. Of those cells, 1 ϫ 106 were sensitized with 10 ␮M of a peptide pool caques (see Table I for animal assignment, dosage, and immuni- (consisting of individual peptides) for1hat37°C, washed and added to the zation schedule). Since it was unknown whether rhesus macaque ϫ 6 remaining 4 10 untreated PBMCs in 2 ml of culture medium (RPMI MHC class I molecules can bind and present HCV-Core-derived 1640, 10% heat-inactivated FBS, and 1% antibiotics) supplemented with ϩ 10 ng/ml of IL-7 (R&D Systems, Minneapolis, MN). After 48 h, 5% (final) peptides and whether the positively selected CD8 T cell reper- IL-2-containing supernatant (T-STIM without PHA; Collaborative Bio- toire in these animals can recognize such MHC class I-Core-de- medical Products, Bedford, MA) and 50 U/ml (final) of rIL-2 (Chiron) rived peptide complexes, three additional animals were inoculated were added to the cultures. Cultures were fed every 3–4 days. After 10 with 2 ϫ 108 PFU of rVVC/E1 to serve as positive controls days in culture, CD8ϩ T cells were isolated using anti-CD8 Abs bound to magnetic beads (Dynal, Oslo, Norway) according to the manufacturer’s (Table I). instructions. Purified CD8ϩ cells (Ͼ93% pure as determined by flow cy- None of the animals had any detectable CTLs at the time of tometry) were cultured for another 2–3 days before being assayed for cy- immunization (0 wk; Table II and data not shown). This confirmed The Journal of Immunology 3591

Table I. Summary of immunization and MHC class I type

MHC Class I Typing Immunization Study Animal (Mamu A* and B*)a Immunogen Dose (route) Schedule (wk)

First BB228 A*08; B*17 BB232 B*03 rVVC/E1 1 ϫ 108 PFU (scariﬁcation) 0 DV036 B*03 1 ϫ 108 PFU (i.d.)b AY921c A*08; B*03 BB231c A*01; A*02 HCV Core-ISCOM 25 mg (i.m.) 0, 4, 8, 27 DV037 B*03; B*04 Second 15860c B*01; B*03 15861c B*03 HCV Core-ISCOM 50 mg (i.m.) 0, 4, 8 15862 B*03 15863 – 15864 B*01

a Mamu alleles tested for A*01, *02, *08, and *11; B*01, *03, *04, and *17. b i.d., Intradermal; i.m., intramuscular. c These animals also received 2 ϫ 108 PFU of rVVC/E1 9–11 wk after the last immunization. that these animals had not been previously exposed to HCV-Core The presence of 121–135- and 86–100-specific CTLs in these and that restimulation of PBMCs under the conditions described in rVVC/E1-inoculated animals indicated that both peptides were Downloaded from Materials and Methods did not result in the priming of primary naturally processed. No CTL responses were detectable in the CTL responses in vitro. Two weeks after rVVC/E1 infection, two other rVVC/E1-inoculated animal (BB228; data not shown). This (BB232 and DV036) of the three animals had detectable CTLs indicated that Core-specific CTLs can be elicited in at least some against Core peptide pool 4 (aa 121–170) and pool 3 (aa 81–130), rhesus monkeys. Two of the three Core-ISCOM-immunized ani- respectively (Table II). By deconvoluting these peptide pools, it mals (AY921 and BB231) did not mount a detectable Core-spe- was determined that BB232’s CTLs recognized the epitopic pep- cific CTL response (data not shown). In contrast, in the other Core- http://www.jimmunol.org/ tide 121–135 and that DV036’s CTLs recognized peptide 86–100. ISCOM-immunized animal (DV037), CTLs recognizing pool 4 (aa

Table II. Priming of Core-speciﬁc CTLs in rhesus macaques

% Speciﬁc Lysisa by guest on October 4, 2021 DV037 BB232 DV036 (Core-ISCOMS) (rVVC/E1) (rVVC/E1)

Core pool 4 Core pool 4 Core pool 3 (aa 121–170) (aa 121–170) (aa 81–130) E:T Week Ratio Hmb Htb Hmb Htb Hmb Htb

0 40:1 Ͻ1 Ͻ19Ͻ18 12 (pre) 13:1 10 Ͻ17Ͻ1 Ͻ14 4:1 2 Ͻ17Ͻ1 Ͻ1 Ͻ1 2 40:1 NTc NT 44d Ͻ124e 5 (2 wk post-1st) 13:1 NT NT 25 Ͻ115Ͻ1 4:1 NT NT 13 Ͻ16Ͻ1 6 40:1 20 Ͻ1 N/Ac N/A N/A N/A (2 wk post-2nd) 13:1 10 Ͻ1 N/A N/A N/A N/A 4:1 5 Ͻ1 N/A N/A N/A N/A 10 40:1 20 Ͻ1 N/A N/A N/A N/A (2 wk post-3rd) 13:1 12 Ͻ1 N/A N/A N/A N/A 4:1 Ͻ1 Ͻ1 N/A N/A N/A N/A 14 40:1 30d 5 N/A N/A N/A N/A (6 wk post-3rd) 13:1 16 Ͻ1 N/A N/A N/A N/A 4:1 12 Ͻ1 N/A N/A N/A N/A 29 40:1 50 2 N/A N/A N/A N/A (2 wk post-4th) 13:1 35 3 N/A N/A N/A N/A 4:1 28 2 N/A N/A N/A N/A

a Percent speciﬁc lysis is only shown for animals with detectable CTL activity and for the peptide pools against which such CTL activity was detected. b CTL activity of CD8ϩ T cells restimulated in vitro with a peptide pool (1–5) was tested against autologous B-LCLs sensitized with the same peptide pool (Hm) or an irrelevant peptide pool (Ht). c NT, Not tested; N/A, not applicable. d The epitopic peptide recognized is aa 121-135 (KVIDTLTCGFADLMG). e The epitopic peptide recognized is aa 86-100 (YGNEGCGWAGWLLSP). 3592 PRIMING OF HCV-SPECIFIC T CELLS IN NONHUMAN PRIMATES

121–170) were detectable as early as 2 wk after the second im- Characterization of cellular and humoral immune responses in munization. This response was directed against the epitopic pep- rhesus monkeys immunized with Core-ISCOM tide aa 121–135 and was also detectable after the third and fourth Although only one of three Core-ISCOM-immunized animals had immunizations (Table II). detectable CTLs, the fact that in the responding animal Core-specific CTLs were detected after only two immunizations (Table II) MHC class I alleles dictated whether a Core-ISCOM-immunized and were long-lived (Fig. 2A) formed the basis to immunize five animal produced Core-specific CTLs more animals (15860–4) with Core-ISCOM (see Table I for dos- Only one of three animals immunized with the Core-ISCOM pro- age and immunization schedule). All animals were naive at the totype vaccine mounted a detectable Core-specific CTL response. time of vaccination. In this study we monitored the priming not ϩ We hypothesized that this might be due to the fact that the MHC only of Core-specific CTLs, but also of Core-specific CD4 T class I molecules of AY921 and BB231 were unable to bind and cells and Abs. ϩ present peptides derived from this relatively small protein (191 aa). None of the animals had any detectable Core-specific CD4 or ϩ To test this hypothesis, CTL lines specific for peptides 121–135 CD8 T cells at the time of immunization (0 wk, Table III). Core- ϩ and 86–100 were established from DV036 and DV037, respec- specific CD4 T cells, as determined by lymphoproliferation as- tively. As shown in Fig. 1A, the peptide 121–135-specific CTL line say, were detected in all animals except 15861 after the second ϩ lysed peptide-sensitized B-LCLs derived from DV037, but did not immunization, but this animal had a detectable CD4 response kill peptide 121–135-sensitized B-LCLs from the two nonrespond- after the third immunization (Table III). For animals 15862 and ing animals (AY921 and BB231). Similarly, B-LCLs derived from 15863, it is unlikely that the low SI observed after the third im- ϩ

DV036, but not AY921 or BB231, were able to present peptide munization (Table III) was due to the absence of a CD4 T cell Downloaded from 86–100 to CD8ϩ CTLs (Fig. 1B). These data indicated that response, as a strong proliferation was observed in these animals AY921’s and BB231’s MHC class I molecules could not present and at this particular time for the E. coli control (see below). None these peptides to CD8ϩ T cells and suggested that MHC class I of the animals had Abs against Core before immunization. How- haplotypes determined whether rhesus monkeys could mount a ever, all animals had seroconverted to Core after two immuniza- CTL response to HCV-Core. tions, and the level of Abs against Core was boosted by a third immunization (Fig. 3). Notably, the mean Core Ab titer among

Since different MHC class I alleles can bind and present differ- http://www.jimmunol.org/ ent sets of peptides, these data did not rule out that the Core- these animals was comparable after two immunizations (1931) and ISCOM formulation was not suboptimal, i.e., it remained possible was higher after three immunizations (4566; Fig. 3) compared with that these animals could mount a Core-speciﬁc CTL response directed against peptides other than 86–100 and 121–135. To address this, AY921 and BB231 were challenged with 2 ϫ 108 PFU of rVVC/E1 11 wk after their fourth immunization with Core- ISCOM. As opposed to BB232 and DV036, which had Core-spe- ciﬁc CTLs 2 wk after rVVC/E1 infection (Table II), neither

AY921 nor BB231 had any detectable CTLs after VVC/E1 inoc- by guest on October 4, 2021 ulation (data not shown). This strongly suggested that the absence of detectable Core-speciﬁc CTLs after Core-ISCOM immunization of these animals was due not to a suboptimal vaccine formulation, but to an intrinsic inability of these animals to mount such a response, presumably a consequence of their MHC class I haplotype.

CTLs primed by immunization with Core-ISCOM are long-lived To investigate whether immunization with Core-ISCOM induced long-lived CTLs, we monitored DV037 for up to 51 wk (1 year) after its fourth immunization. Peptide 121–135-specific CTLs were detected 10, 15, 31, 38, 45, and 51 wk after the last immunization (Fig. 2A). In contrast, the 121–135-specific CTL response primed by rVVC/E1 in BB232 was barely detectable 14 wk postvaccination and was undetectable 18 wk postvaccination (Fig. 2B). Sim- ilarly, the 86–100-specific CTLs primed in DV036 by rVVC/E1 vaccination became undetectable 14 wk postvaccination (data not shown). FIGURE 1. B-LCLs from the two nonresponder (AY921 and BB231) In an effort to quantify the number of peptide 121–135-specific Core-ISCOM-immunized animals cannot present Core-derived peptides CTLs present in DV037 1 year after it had received its last boost, 121–135 and 86–100. A, The 121–135-specific CTL line established from the animal’s PBMCs were restimulated ex vivo with 121–135, and animal DV037 was tested in a standard 51Cr release assay for its ability to the percentage of specific CD8ϩ T cells was assessed by intracel- lyse DV037 B-LCL target cells sensitized with peptide 121–135 (f)oran lular staining for IFN-␥ and TNF-␣. As illustrated in Fig. 2C (left irrelevant peptide (Ⅺ), AY921 B-LCL target cells sensitized with peptide ƒ panels), 121–135-specific CTLs represented 0.49% of the periph- 121–135 ( ) or an irrelevant peptide ( ), and BB231 B-LCL target cells ϩ ϩ sensitized with peptide 121–135 (F) or an irrelevant peptide (E). B, The eral CD8 T cells (or 490 cells/105 CD8 T cells) that secreted ␥ ␣ 86–100-specific CTL line established from animal DV036 was tested in a IFN- and/or TNF- after ex vivo peptide stimulation for 12 h. In standard 51Cr release assay for its ability to lyse DV036 B-LCL target cells contrast, after in vitro restimulation with peptide 121–135, 71% of f Ⅺ ϩ sensitized with peptide 86–100 ( ) or an irrelevant peptide ( ), AY921 these CD8 T cells were specific for this peptide, as determined by B-LCL target cells sensitized with peptide 86–100 () or an irrelevant their abilities to secrete IFN-␥ and/or TNF-␣ (Fig. 2C, right peptide (ƒ), and BB231 B-LCL target cells sensitized with peptide 86–100 panels). (F) or an irrelevant peptide (E). The Journal of Immunology 3593 Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

FIGURE 2. Longevity of the CTL responses primed by vaccination. PBMCs from DV037 (A) and BB232 (B) were restimulated in vitro with the epitopic peptide 121–135. After CD8ϩ enrichment, cells were tested for cytotoxic activity against autologous B-LCLs sensitized with the epitopic peptide 121–135 (F) or an irrelevant peptide (E). C, Freshly isolated PBMCs from DV037 51 wk after its last immunization (two left panels) or in vitro restimulated PBMCs from the same time point (two right panels) were restimulated for 12 h with peptide 121–135 or a control peptide and stained for surface CD8 and intracellular IFN-␥ and TNF-␣. Lymphocytes were gated by side vs forward scatter light and then for CD8-PerCP. Plots show log fluorescence intensity for TNF-␣-FITC and IFN-␥-PE. that present in the serum of chronically infected patients with an 100 for animal 15864 and against peptide 121–135 for animals unusually high anti-Core Ab titer (2358; data not shown) run in the 15862 and 15863. Since 86–100- and 121–135-specific CTLs same assay. were also primed in DV036 (86–100), DV037 (121–135) and To investigate whether Core-ISCOM elicited a Th1- or Th2- BB232 (121–135; Table II), it was of importance to determine type response in these monkeys, freshly isolated PBMCs before whether all 86–100- and 121–135-specific CTLs were respectively vaccination as well as 2 wk after the second and third immuniza- restricted by a single MHC class I allele. The 86–100-specific CTL tions were tested for their capacity to produce cytokines at 48 h in line derived from animal 15864 efficiently lysed peptide 86–100- response to stimulation with Core peptides spanning the entire sensitized B-LCLs derived from 15864, but not peptide-sensitized length of the Core protein. As shown in Fig. 4, A and B, a signif- B-LCLs derived from animal DV036, indicating that a different icant increase in Th1 cytokines (IFN-␥ and IL-2) was observed (unidentified) MHC class I allele presented this peptide to CTL postimmunization in all animals. Similarly, an increase in Th2- (Fig. 5A and Table I). In contrast, the CTLs specific for peptide type cytokines (IL-5 and IL-10) was observed in all animals fol- 121–135 from animals 15862, 15863, BB232, and DV037 were lowing vaccination (Fig. 4, C and D). Of note, the magnitude of the restricted by a single, as yet unidentified, MHC class I allele shared Th1-like response in animals 15860, 15861, and 15862 was lower by all these animals (Fig. 5B and Table I). These data also indi- than that in animals 15863 and 15864, but the levels of Th2-type cated that the MHC class I molecules of 15860 and 15861 could cytokines were almost comparable in all animals. Nevertheless, not present either peptide (86–100 and 121–135) to specific CTLs these data indicated that Core-ISCOM induced a Th0-like type (Fig. 5). As observed in the first study, rVVC/E1 infection of the response in rhesus monkeys. two nonresponding animals (15860 and 15861) 9 wk after the third After two and three immunizations, three animals (15862, immunization with Core-ISCOM did not lead to the priming of 15863, and 15864) had detectable CTL responses directed against Core-specific CTLs in these animals (data not shown). Taken to- pool B (aa 60–140), while two others (15860 and 15861) did not gether, this suggested, once again, that the MHC class I haplotype of (Table III). This CTL response was directed against peptide 86– the animals dictated whether they could mount Core-specific CTLs. 3594 PRIMING OF HCV-SPECIFIC T CELLS IN NONHUMAN PRIMATES

Table III. Priming of Core-speciﬁc CD8ϩ and CD4ϩ T cells in rhesus macaques immunized with Core-ISCOMS

Animal 15860 Animal 15861 Animal 15862 Animal 15863 Animal 15864

CD8 resp. CD8 resp. CD8 resp. CD8 resp. CD8 resp. (% lysis)a (% lysis)a (% lysis)a (% lysis)a (% lysis)a

E:T Core pool B CD4ϩ Core pool B CD4ϩ Core pool B CD4ϩ Core pool B CD4ϩ Core pool B CD4ϩ Week Ratio (aa 61–140) resp. (SI)b (aa 61–140) resp. (SI)b (aa 61–140) resp. (SI)b (aa 61–140) resp. (SI)b (aa 61–140) resp. (SI)b

Hm Ht Hm Ht Hm Ht Hm Ht Hm Ht 0 40:1 8 13 4 1 14 14 11 5 13 3 (pre) 13:1 2 11 1.7 Ͻ1 Ͻ1 0.4 6 8 0.9 Ͻ1 4 1.2 Ͻ1 13 0.8 4:1 2 5 Ͻ1 Ͻ13Ͻ113Ͻ1 Ͻ1 6 40:1 8 3 7 Ͻ131c 834c 942d 18 (2 wk post-2nd) 13:1 Ͻ1 2 5.5 Ͻ1 Ͻ1 1.9 16 6 5.0 18 6 3.5 20 4 3.8 4:1 Ͻ1 Ͻ1 Ͻ1 Ͻ14Ͻ181123 10 40:1 Ͻ1 3 13 13 31 5 29 1 79 10 (2 wk post-3rd) 13:1 Ͻ1 12 7.1 5 7 4.5 14 4 2.1e 17 Ͻ1 1.1e 60 11 8.8 4:1 Ͻ1 Ͻ1 Ͻ12 6Ͻ18Ͻ13311

a CTL activity of CD8ϩ T cells restimulated in vitro with a peptide pool (A–C) was tested against autologous B-LCLs sensitized with the same peptide pool (Hm) or an irrelevant peptide pool (Ht). Percent speciﬁc lysis is only shown for pool B since no CTL activity was detected in any animals for pool A (aa 1-70) or pool C (aa 131-191). b CD4ϩ response was determined by lymphoproliferation assay as described in Materials and Methods. A SI of 3.0 or greater was scored positive. resp., Responses. Downloaded from c Epitope recognized was aa 121-130 (KVIDTLTCGFADLMG). d Epitope recognized was aa 86-100 (YGNEGCGWAGWLLSP). e High proliferation to E. coli control.

Quantification of the Core-specific cellular immune responses Core- ISCOM can serve as an adjuvant for soluble E1E2 http://www.jimmunol.org/ primed by Core-ISCOM Because vaccination with recombinant HCV envelope proteins and In an effort to quantitate the number of Core-specific CD8ϩ and adjuvant can, at least in some instances, influence the outcome of CD4ϩ T cells primed in these animals, freshly isolated PBMCs infection and disease (31), we investigated whether Core-ISCOM were stained for intracellular IFN-␥ and TNF-␣ after ex vivo re- could serve as an adjuvant for the heterodimeric envelope protein stimulation. The CD8ϩ T cell responses to naturally processed E1E2. To that end, the geometric mean E2 Ab titer of mice (10 peptides were quantified after ex vivo restimulation with autolo- animals/group) immunized with 2 ␮g/dose of recombinant E1E2 gous B-LCLs infected with rVVC/E1 or VVwt, as a control. The in the presence of MF59 (v/v) was compared with that of mice ϩ CD4 T cell responses to naturally processed peptides were quan- immunized with 2 ␮g/dose recombinant E1E2 protein plus 2 ␮g/ by guest on October 4, 2021 tified after ex vivo restimulation with the recombinant Core protein dose Core-ISCOM or 2 ␮g/dose E1E2 classical ISCOM. As shown or an E. coli control. Intracellular staining responses revealed that in Fig. 7, mice immunized with E1E2 plus Core-ISCOM had a while none of the animals had detectable Core-specific CD8ϩ T significant anti-E2 Ab titer after three immunizations (31,099 Ϯ cells at the time of immunization, between 0.30 and 0.71% of 8,217), and these titers were comparable to those observed in mice peripheral CD8ϩ T cells in 15862, 15863, and 15864 were specific immunized with E1E2 plus MF59 (24,178 Ϯ 3,432) or E1E2 clas- for naturally processed Core-derived peptide(s) after two immuni- sical ISCOM (27,093 Ϯ 2,621). The quality of Ab elicited in these zations (Fig. 6A). The number of specific CTLs was, however, not mice appeared to be similar, inasmuch as the titer of Ab inhibiting increased after the third immunization, as judged by intracellular the binding of HCV-1a E2 to the HCV putative receptor CD81 staining responses. Notably, no CD8ϩ T cells secreting IFN-␥ were comparable in these three groups of mice (Fig. 7). Moreover, and/or TNF-␣ in response to Core were detected in the two animals (15860 and 15861) in which no Core-specific CTL activity was observed by 51Cr release assay (Fig. 6A and Table III). Quan- tification of Core-specific CD4ϩ T cells confirmed the data obtained by lymphoproliferation assay (Table III), in that between 0.32 and 2.21% of CD4ϩ T cells from all five animals were specific for naturally processed Core peptides (Fig. 6B). Furthermore, the fact that 0.53 and 0.28% of CD4ϩ T cells from animals 15862 and 15863 were positive for cytokines after the third immunization (Fig. 6B), strongly suggested that the negative SI observed for this time point (Table III) was indeed a false negative, most likely due to the high proliferation observed in response to the E. coli control (data not shown). This suggested that intracellular staining for IFN-␥ and TNF-␣ is a more sensitive assay than lymphoproliferation to assess Ag-specific CD4ϩ T cell responses. Indeed, for animal 15861, no CD4ϩ T cells were detected by lymphoproliferation 2 wk after the second immunization (Table III). In contrast, ϩ Core-specific CD4 T cells were detected in this animal (2 wk FIGURE 3. Titer of Abs against Core in the serum of immunized ani- after the second immunization) by intracellular staining for IFN-␥ mals. Ⅺ, Preimmunization; o, 2 wk after the second immunization; f,2 and TNF-␣ (Fig. 6B). wk after the third immunization. The Journal of Immunology 3595

FIGURE 4. Th-1- and Th2-type cytokines in Core-ISCOMS-immunized animals. The levels of IFN-␥ (A), IL-2 (B), IL-5 (C), and IL-10 (D) were measured by speciﬁc ELISA in cell-free supernatant of freshly isolated PBMCs stimulated for 48 h as described in Ma- terials and Methods. Ⅺ, Preimmunization; o,2 wk after the second immunization; f, 2 wk after the third immunization. NT, Not tested. Downloaded from

mice immunized with 2 ␮g/dose of recombinant E1E2 protein plus Discussion http://www.jimmunol.org/ 2 ␮g/dose of Core-ISCOM all seroconverted to Core (geometric The development of a vaccine against HCV and its associated mean Ab titer, 1,600 Ϯ 835; Fig. 7). Further studies will be needed diseases has been the focus of research since the virus was dis- to decipher the mechanisms by which Core-ISCOM can adjuvant covered (32). Adjuvanted subunit E1E2 vaccines can confer pro- E1E2 and to determine whether this adjuvant effect is also ob- tection against a homologous challenge (31). There is evidence, served in higher species. however, that priming of CD4ϩ and CD8ϩ T cells speciﬁc for HCV gene products other than E1and E2 might increase the efﬁ- cacy of an E1E2 vaccine, as such cells have been implicated in conferring immunity to HCV (5–7, 33).

One of the challenges facing the development of an HCV vac- by guest on October 4, 2021 cine is that HCV exhibits extensive genetic variation, resulting in multiple distinct genotypes (34) and that HCV exists as a population of related, yet heterogeneous, sequences (35). In that regard, the use of Core in a vaccine is attractive, in that Core is the most conserved HCV protein among genotypes (36), and Core-specific CTLs can recognize and lyse target cells expressing Core derived from most, if not all, genotypes (14). Furthermore, in HCV-infected patients, no mutations could be detected within Core despite the presence of CTLs specific for these epitopes (37), suggesting that immune responses to Core did not lead to the appearance of escape variants. This is in sharp contrast to the fact that CTL escape mutations have been described for other HCV proteins, such as NS3 (38) and E2 (39). Whether the appearance of such mutations in E2 and NS3 resulted from true immune selection remains unclear, but nonetheless this has important implications for vaccine development. Hence, the inclusion of Core in an HCV vaccine might broaden its effectiveness, as the immune responses elicited should be relevant for most, if not all, HCV genotypes and quasispecies. CTLs recognize peptide fragments of 8–10 aa in length bound to MHC class I molecules (40). Such peptides are usually gener- FIGURE 5. MHC class I restriction of peptides 121–135 and 86–100 ated in the cytosol following cleavage of cytosolic polypeptide CTLs. A, The peptide 86–100-specific CTL line derived from animal precursors by proteases (41). Thus, the induction of CTL responses 51 15864 was tested in a standard Cr release assay for its ability to lyse usually requires that the Ag be endogenously expressed and pro- peptide 86–100-sensitized B-LCL target cells derived from animals cessed. Genetic vaccines such as naked DNA have been shown to DV036 (E), 15864 (F), 15860 (), and 15861 (f). B, The peptide 121– 135-specific CTL line derived from animal 15862 was tested in a standard be potent inducers of CTLs against viral proteins (42), presumably 51Cr release assay for its ability to lyse peptide 121–135-sensitized B-LCL because Ag synthesis occurs in the host. Because of the concerns target cells derived from animals DV037 (F), BB232 (Œ), 15862 (), about using a genetic vaccine to prime Core-specific CTLs, one 15863 (f), 15861 (ƒ), and 15860 (Ⅺ). must rely on a subunit-based vaccine to prime CTLs against Core. 3596 PRIMING OF HCV-SPECIFIC T CELLS IN NONHUMAN PRIMATES

FIGURE 7. Core-ISCOM can serve as an adjuvant for E1E2. Mice (10 ␮

animals/group) were immunized with 2 g/dose recombinant E1E2 pro- Downloaded from tein, 2 ␮g/dose recombinant E1E2 plus 2 ␮g/dose recombinant Core, 2 ␮g/dose recombinant E1E2 in the presence of MF59 (v/v), 2 ␮g/dose recombinant E1E2 protein plus 2 ␮g/dose Core-ISCOM, or 2 ␮g/dose E1E2 classical ISCOM. Mice were bled 2 wk after the third immunization. An- ti-E2 (f), anti-CD81 titers (o), and anti-Core (Ⅺ) are presented as the FIGURE 6. Quantiﬁcation of the CD8ϩ and CD4ϩ T cell responses in geometric mean of the titers obtained from individual mice from each

Core-ISCOM-immunized animals. Freshly isolated PBMCs were restimu- group. http://www.jimmunol.org/ lated ex vivo with rVVC/E1- or VVwt-infected autologous B-LCLs (A)or with the recombinant Core protein of an E. coli control (B). Cells were then stained for surface CD8 or CD4, and intracellular IFN-␥ and TNF-␣ as described in Materials and Methods. Lymphocytes were gated by side vs HCV proteins (7), this suggested that the frequency of HCV-spe- forward scatter light and then for CD8-PerCP (A) or CD4-APC (B). A, The cific T cells primed by Core-ISCOM vaccination was comparable corrected percentage of CD8ϩ T cells with detectable IFN-␥ and/or TNF-␣ to that present in this patient. was calculated as (percent CD8ϩ T cells restimulated with rVVC/E1 that One year after its last immunization with Core-ISCOM, DV037 were IFN-␥ϩ and/or TNF-␣ϩ) Ϫ (percent CD8ϩ T cells restimulated with still had detectable core-specific CTLs in its periphery at a rela- ϩ ϩ VVwt that were IFN-␥ and/or TNF-␣ ). B, The corrected percentage of tively high frequency (0.49%; Fig. 2). The nature of the mecha- ϩ by guest on October 4, 2021 CD4 T cells with detectable IFN-␥ and/or TNF-␣ was calculated as (per- nisms involved in the maintenance of memory CTLs remains un- ϩ ␥ϩ cent CD4 T cells restimulated with Core that were IFN- and/or TNF- clear. Some experiments suggest that Ag is needed for the ␣ϩ Ϫ ϩ ) (percent CD4 T cells restimulated with the E. coli that were maintenance of memory, while other studies indicate that it is not. IFN-␥ϩ and/or TNF-␣ϩ). Ⅺ, Preimmunization; o, 2 wk after the second Similarly, whether CD8 memory requires persistence of Ag-spe- immunization; f, 2 wk after the third immunization. cific CD4ϩ T cells is controversial (46). Whatever the mechanisms responsible for T cell memory, HCV-specific CTLs have been Immunization with subunit protein vaccines adjuvanted in alum or shown to persist for a long time following immunization (this oil/water emulsions can usually elicit CD4ϩ T cells and Abs, but study) and in patients who have resolved acute infection (47). are generally inefficient at priming MHC class I-restricted CTLs, Classical ISCOM formulations are typically particulates ϳ40 as proteins in the extracellular fluid are generally processed nm in diameter in which the Ag is bound by hydrophobic inter- through the exogenous processing pathway and are degraded into actions to the saponin, cholesterol, and phospholipid that form the peptides that bind MHC class II molecules (43). cage-like pentagonal dodecahedral structure (17). Association be- The Core-ISCOM prototype vaccine primed strong Core-spe- tween the Ag and the adjuvant is thought to be important for in- cific CD4ϩ and CD8ϩ T cell-mediated immune responses in rhe- duction of CTL responses (48). With classical ISCOM formula- sus macaques (Tables II and III). The fact that these immune tions this association is achieved by incorporation of the responses were of the Th0 type might not be surprising, as both hydrophobic Ag into the particle. Other methods of association Th1- and Th2-type cytokines are secreted by spleen and draining include electrostatic interactions, which take advantage of the neg- lymph node cells from mice immunized with classical-ISCOM ative charge on the ISCOMATRIX and its ability to associate with (44). Using intracellular staining for IFN-␥ and TNF-␣, we deter- positively charged proteins. The Core protein is positively charged mined that following immunization with Core-ISCOM, the fre- and as such is adsorbed onto the ISCOMATRIX by electrostatic quency of core-specific CD4ϩ and CD8ϩ T cells ranged between interactions to produce the Core-ISCOM formulation. A number of 0.3 and 2.2% and between 0.3 and 0.7%, respectively (Fig. 6). In classical ISCOM vaccines have been shown to induce both hu- patients with chronic HCV infection, the frequency of CTLs was moral and cellular immune responses (18, 19, 23, 44, 49–53). In reported to be low to undetectable (7, 45), potentially a result of contrast to classical ISCOM, Core-ISCOM are ϳ1 ␮m in diameter clonal exhaustion or anergy. Yet, using enzyme-linked immuno- (as determined by standard light scattering methodology; data not spot, it was estimated that about 3% of the circulating CD4ϩ T shown), and the Core Ag is adsorbed onto the cage-like structure, cells and about 6% of the circulating CD8ϩ T cells were specific not trapped inside. Phagocytosis of particulate by professional for HCV at the time of maximum responses in a patient with acute APCs is much more efficient for 1-␮m particles than for 100-nm or HCV infection who subsequently resolved the infection (7). Be- smaller particles (54). The size of the Core-ISCOM particulate cause these frequencies encompassed cells specific for multiple might therefore directly contribute to its ability to prime potent The Journal of Immunology 3597 cellular immune responses, since efficient uptake by APCs of par- for treatment of chronic infection with hepatitis C virus: International Hepatitis ticulate by phagocytosis may be important for the rapid and effec- Interventional Therapy Group (IHIT). Lancet 352:1426. 4. McHutchison, J. G., S. C. Gordon, E. R. Schiff, M. L. Shiffman, W. M. Lee, tive delivery of internalized particles for presentation by MHC V. K. Rustgi, Z. D. Goodman, M. H. Ling, S. Cort, and J. K. Albrecht. 1998. molecules. However, internalization of 1-␮m particulates by Interferon ␣-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C: Hepatitis Interventional Therapy Group. N. Engl. J. Med. phagocytosis is usually directly delivered to lysosomes (55), which 339:1485. leads to presentation of the Ag through the MHC class II pathway 5. Missale, G., R. Bertoni, V. Lamonaca, A. Valli, M. Massari, C. Mori, (43). Yet, recent studies have documented pathways (such as the M. G. Rumi, M. Houghton, F. Fiaccadori, and C. Ferrari. 1996. Different clinical behaviors of acute hepatitis C virus infection are associated with different vigor phagosome to cytosol pathway and cross-priming) that allow for of the anti-viral cell-mediated immune response. J. Clin. Invest. 98:706. peptide presentation on MHC class I molecules after phagocytosis 6. Cooper, S. L., A. L. Erickson, E. J. Adams, J. Kansopon, A. J. Weiner, of particulate Ag by professional APCs such as macrophages and D. Y. Chien, M. Houghton, P. Parham, and C. M. Walker. 1999. Analysis of a dendritic cells (56–58). Since vaccination of rhesus macaques with successful immune response against hepatitis C virus. Immunity 10:439. ϩ ϩ 7. Lechner, F., D. K. Wong, P. R. Dunbar, R. Chapman, R. T. Chung, Core-ISCOM-primed Core-specific CD4 and CD8 T cells as P. Dohrenwend, G. Robbins, R. Phillips, P. Klenerman, and B. D. Walker. 2000. well as Abs against Core, mechanisms leading to presentation of Analysis of successful immune responses in persons infected with hepatitis C exogenous Ag on both MHC class I and II molecules after phago- virus. J. Exp. Med. 191:1499. 8. Farci, P., A. Shimoda, A. Coiana, G. Diaz, G. Peddis, J. C. Melpolder, cytosis of particulate by professional APCs must have been used. A. Strazzera, D. Y. Chien, S. J. Munoz, A. Balestrieri, et al. 2000. The outcome However, since ISCOM contains saponin, which can intercalate of acute hepatitis C predicted by the evolution of the viral quasispecies. Science into cholesterol membranes (59), one cannot rule out that some 288:339. 9. Boyer, N., and P. Marcellin. 2000. Pathogenesis, diagnosis and management of Core-ISCOM particulates were able to pass directly through cell hepatitis C. J. Hepatol. 32:98. membranes and thus directly enter the classical pathway for MHC 10. Ballardini, G., P. Groff, P. Pontisso, F. Giostra, R. Francesconi, M. Lenzi, class I presentation. Finally, the saponin may permit endosomal D. Zauli, A. Alberti, and F. B. Bianchi. 1995. Hepatitis C virus (HCV) genotype, Downloaded from tissue HCV antigens, hepatocellular expression of HLA-A,B,C, and intercellular escape by interfering with endosomal membrane structure (48). adhesion-1 molecules: clues to pathogenesis of hepatocellular damage and re- There is a growing body of evidence indicating that a vaccine sponse to interferon treatment in patients with chronic hepatitis C. J. Clin. Invest. eliciting both Core-specific CD4ϩ and CD8ϩ T cells could have a 95:2067. 11. Liaw, Y. F., C. S. Lee, S. L. Tsai, B. W. Liaw, T. C. Chen, I. S. Sheen, and therapeutic value. First, Core-specific CTLs have been associated C. M. Chu. 1995. T-cell-mediated autologous hepatocytotoxicity in patients with in HLA-B44ϩ patients with a lower viral titer (60). Second, CD4ϩ chronic hepatitis C virus infection. Hepatology 22:1368. T cell responses to Core, although they did not appear to coincide 12. Calabrese, F., P. Pontisso, E. Pettenazzo, L. Benvegnu, A. Vario, L. Chemello, http://www.jimmunol.org/ A. Alberti, and M. Valente. 2000. Liver cell apoptosis in chronic hepatitis C with virus clearance, have been associated with a benign course of correlates with histological but not biochemical activity or serum HCV-RNA infection (61). Such core-specific CD4ϩ T cells may therefore help levels. Hepatology 31:1153. to maintain humoral and cellular responses protective against the 13. Neumann, A. U., N. P. Lam, H. Dahari, D. R. Gretch, T. E. Wiley, T. J. Layden, and A. S. Perelson. 1998. Hepatitis C viral dynamics in vivo and the antiviral disease. In this context, it is conceivable that priming and main- efficacy of interferon-␣ therapy. Science 282:103. tenance of Core-specific CTLs by vaccination with Core-ISCOM 14. Nelson, D., C. Marousis, G. Davis, C. Rice, J. Wong, M. Houghton, and J. Lau. were dependent and controlled, at least to some extent, by Core- 1997. The role of hepatitis C virus-specific cytotoxic T lymphocytes in chronic ϩ hepatitis C. J. Immunol. 158:1473. specific CD4 T cells, as observed for some tumor-specific CTLs 15. Nelson, D. R., C. G. Marousis, T. Ohno, G. L. Davis, and J. Y. Lau. 1998. (62, 63). This might explain why Core-specific CTLs primed by Intrahepatic hepatitis C virus-specific cytotoxic T lymphocyte activity and re- ␣ by guest on October 4, 2021 rVVC/E1 were not maintained as long as CTLs primed by Core- sponse to interferon therapy in chronic hepatitis C. Hepatology 28:225. 16. McLauchlan, J. 2000. Properties of the hepatitis C virus core protein: a structural ISCOM (Fig. 2). Third, and as mentioned above, the response to protein that modulates cellular processes. J. Viral Hepat. 7:2. IFN therapy was shown to be directly related to the rate of T 17. Morein, B., B. Sundquist, S. Hoglund, K. Dalsgaard, and A. Osterhaus. 1984. cell-mediated clearance of infected cells (13) and the presence of Iscom, a novel structure for antigenic presentation of membrane proteins from enveloped viruses. Nature 308:457. Core-specific CTLs before initiation of treatment (15). 18. Takahashi, H., T. Takeshita, B. Morein, S. Putney, R. Germain, and J. Berzofsky. Overall, this study indicated that vaccination with Core-ISCOM 1990. Induction of CD8ϩ cytotoxic T cells by immunization with purified HIV-1 primes strong Core-specific CD8ϩ and CD4ϩ T cells as well as envelope protein in ISCOMs. Nature 344:873. 19. Maloy, K. J., A. M. Donachie, and A. M. Mowat. 1995. Induction of Th1 and Th2 anti-Core Abs. Furthermore we demonstrated that this formulation CD4ϩ T cell responses by oral or parenteral immunization with ISCOMS. Eur. could serve as an adjuvant to elicit Abs against E1E2. Further J. Immunol. 25:2835. studies in chimpanzees, the only reliable animal species for infec- 20. National Institutes of Health. 1985. Guide for the Care and Use of Laboratory Animals. U.S. Department of Health and Human Services Publication 82-23. tion, or in humans, are needed to determine whether a Core- National Institutes of Health, Bethesda. ISCOM (associated, or not, with E1E2) vaccine can confer steril- 21. Urvater, J. A., N. Otting, J. H. Loehrke, R. Rudersdorf, I. I.Slukvin, izing immunity to HCV, prevent the establishment of chronicity, M. S. Piekarczyk, T. G. Golos, A. L. Hughes, R. E. Bontrop, and D. I. Watkins. 2000. Mamu-I: a novel primate MHC class I B-related locus with unusually low and/or increase the response rate to anti-viral therapy. variability. J. Immunol. 164:1386. 22. Heile, J. 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