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The Journal of Immunology, 2001, 166: 3589-3598.
Copyright © 2001 by The American Association of Immunologists

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

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

* Chiron Corp., Emeryville, CA 94608; and {dagger} CSL Ltd., Parkville, Victoria, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 ~1-µm particulates (as opposed to 40 nm for classical 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The hepatitis C virus (HCV)2 is now recognized as the leading cause of chronic liver disease (1). An estimated 170 million people worldwide are infected with HCV, approximately four times more than the number of people infected with HIV (2). Despite these alarming numbers, only a few therapies are available to clinicians treating patients infected with HCV. Although treatment of HCV infection with ribavirin and/or IFN has been shown to be highly effective in some patients, roughly two-thirds of patients fail to have a sustained response to treatment (3, 4). Hence, improved therapies are desperately needed, but their development is hampered by the lack of a small animal model for infection and disease.

Several studies have suggested that T cell-mediated immune responses to HCV infection can determine the outcome of HCV infection and disease (5, 6, 7). Furthermore, recent work indicated that when the early immune response to HCV is optimal, the genetic diversity of HCV quasispecies declines, and the strains become increasingly homogeneous until the final variant is cleared (8). Because response to current treatment (IFN alone or associated with ribavirin) can be predicted in part by the viral load (9), host immune factors including CD8+ CTLs, which are thought to be the major contributor to the death rate of HCV-infected cells (10, 11, 12), might be critical in determining the outcome of therapy. Using mathematical modeling, it was found that diminution of serum HCV RNA levels during the second-phase slope observed during IFN therapy was inversely correlated with baseline viral load and was positively correlated with hepatocyte death (13). Combined with the fact that there is an inverse correlation between the frequency of HCV-specific CTLs and the viral load (14) and that the presence of HCV Core-specific CTLs before IFN treatment has been associated with subsequent response of patients to IFN therapy (15), this indicated that killing of HCV-infected cells by CTLs could play an important role in determining response to therapy. Thus, a vaccine eliciting Core-specific CTLs might enhance the response rate of infected patients to therapy.

Because the HCV Core protein can modulate multiple cellular processes, such as apoptosis, lipid metabolism, and transcription, and can induce cellular transformation (16), there are potential concerns about eliciting Core-specific immune responses in patients using a genetic vaccine (naked DNA; viral, retroviral, bacterial vectors; replicons). Hence, to elicit Core-specific T cell-mediated immune responses, one must rely on the use of a subunit vaccine. Although most licensed subunit vaccines are inefficient at inducing CTLs, there has been considerable advancement in the field of adjuvant research. For example, classical ISCOM, a typically 40-nm cage-like structure composed of saponins from Quillaja saponaria Molina, cholesterol, and phospholipids, inside which the Ag is entrapped (17), have been shown to prime CD4+ and CD8+-mediated immune responses (18, 19). In this study, we evaluated and characterized the potency of a nonclassical ISCOM vaccine, in which Core was not incorporated into the ISCOMATRIX, but was adsorbed onto the cage-like structure by ionic interactions, resulting in particulates ~25 times larger than conventional ISCOM (1 µm vs 40 nm) in rhesus macaques.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Rhesus macaques (Macaca mulatta) were housed at the Southwest Foundation for Biomedical Research (San Antonio, TX). Studies were approved by the institutional animal care and use committees of Chiron and the Southwest Foundation for Biomedical Research and were performed under the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (20). Class I MHC typing of the animals was performed as previously described (21).

Female C57BL/6 (H-2b) mice were purchased from Charles River Breeding Laboratories (Wilmington, MA) and were used between 8 and 10 wk of age. Mice were housed in a pathogen-free environment and were handled according to the international guidelines for experimentation with animals. All mouse experiments were approved by Chiron’s animal care and use committee.

Immunogens and adjuvants

The full-length HCV-1a Core recombinant protein (aa 1–191) was produced in Escherichia coli. Core was purified from cells lysed and extracted with urea containing DTT. Cation exchange chromatography, hydroxyapatite chromatography, and size exclusion chromatography were used to subsequently purify the material. The resulting Core was >98% pure. The recombinant HCV-1a E1E2809 protein was produced in Chinese hamster ovary cells as described previously (22). The Core-ISCOM formulations were prepared by mixing the core protein with preformed ISCOMATRIX (empty ISCOMS) using ionic interactions to maximize the association between the Ag and the adjuvant. ISCOMATRIX was prepared essentially by previously described methods (23), except that diafiltration was used in place of dialysis. E1E2 classical ISCOM was prepared as previously described (23). The oil-in-water adjuvant MF59 has been described previously (24).

Peptides and vaccinia viruses

Peptides (15- or 20-mer overlapping by 10 aa) spanning the entire length of the Core (aa 1–191) protein of HCV-1a (25) were synthesized with free amine N termini and free acid C termini by Research Genetics (Huntsville, AL). The recombinant vaccinia virus (rVV) expressing Core and E1 (aa 1–384; rVVC/E1) and wild-type VV (VVwt) have been described previously (6).

Immunization

Rhesus macaques were immunized under anesthesia. The first study was comprised of six animals divided into two groups of three animals each. The first group (animals BB228, BB232, and DV036) was infected with 2 x 108 PFU (1 x 108 intradermally and 1 x 108 by scarification) of 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 immunized with 25 µg of Core-ISCOM by i.m. injection in the left quadriceps at 0, 1, 2, and 6 mo. For the second study, five animals (15860, 15861, 15862, 15863, and 15864) were immunized with 50 µg of Core- ISCOM by i.m. injection in the left quadriceps at 0, 1, and 2 mo. Some Core-immunized animals (see Table IGo) also received 2 x 108 PFU (1 x 108 intradermally and 1 x 108 by scarification) of rVVC/E1 9 or 11 wk after their last vaccine immunization.


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  		Table I. Summary of immunization and MHC class I type

 
Mice (10 animals/group) were immunized in the tibialis anterior muscles (50 µl/muscle) with 2 µg/dose recombinant E1E2 protein, 2 µg/dose recombinant E1E2 plus 2 µg/dose recombinant Core, 2 µg/dose recombinant E1E2 in the presence of MF59 (v/v), 2 µg/dose E1E2 classical ISCOM, or 2 µg/dose recombinant E1E2 protein plus 2 µg/dose Core- ISCOM at 0, 4, and 8 wk.

Cells and cell lines

Peripheral blood was drawn from the femoral vein while the animals were under anesthesia. PBMCs were obtained after centrifugation over a Ficoll-Hypaque gradient and were cultured in 24-well dishes at 5 x 106 cells/well. Of those cells, 1 x 106 were sensitized with 10 µM of a peptide pool (consisting of individual peptides) for 1 h at 37°C, washed and added to the remaining 4 x 106 untreated PBMCs in 2 ml of culture medium (RPMI 1640, 10% heat-inactivated FBS, and 1% antibiotics) supplemented with 10 ng/ml of IL-7 (R&D Systems, Minneapolis, MN). After 48 h, 5% (final) IL-2-containing supernatant (T-STIM without PHA; Collaborative Biomedical Products, Bedford, MA) and 50 U/ml (final) of rIL-2 (Chiron) were added to the cultures. Cultures were fed every 3–4 days. After 10 days in culture, CD8+ T cells were isolated using anti-CD8 Abs bound to magnetic beads (Dynal, Oslo, Norway) according to the manufacturer’s instructions. Purified CD8+ cells (>93% pure as determined by flow cytometry) were cultured for another 2–3 days before being assayed for cytotoxic activity. Peptide-specific CD8+ lines were obtained by periodically restimulating these CD8+ T cells with autologous B cell lymphoblastoid cell lines (B-LCLs) and peptide.

B-LCLs were derived from each animal using supernatants from the Herpesvirus papio producer cell line S394.

CTL assay

Cytotoxic activity was assayed in a standard 51Cr release assay as described previously (26). Briefly, B-LCLs were incubated with 10 µM peptides and 50 µCi of 51Cr for 1 h, washed three times, and plated at 5 x 103 cells/well in a 96-well plate. Alternatively, B-LCLs were infected at a multiplicity of infection of 10:1 with rVVC/E1 or VVwt for 1 h, washed, and cultured overnight before labeling with 51Cr. CD8+ cells were plated in duplicate at three different E:T cell ratios and incubated with target cells for 4 h in the presence of 2 x 105/well of unlabeled target cells (cold targets) that were added to minimize lysis of B-LCLs by H. papio or endogenous virus (e.g., 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 equal to the percent lysis of control targets plus 10.

Lymphoproliferation assay

This assay has been described previously (27). Briefly, freshly isolated PBMCs were plated in triplicates at 2 x 105 cells/well in 96-well round-bottom plates and cultured in the presence of 5 µg/ml recombinant Core protein or 0.05 µg/ml (the Escherichia coli-derived Core protein contains <2% impurities (>98% pure)) E. coli control. Plates were pulsed with 1 µCi/well [3H]thymidine on day 5 and harvested 6–8 h later. Results are presented as the stimulation index (SI) calculated as (mean experimental cpm)/(mean cpm in the presence of the E. coli control). An SI >=3.0 was scored positive.

FACS analysis

Freshly isolated PBMCs or PBMCs that had been restimulated in vitro with a peptide were cultured in medium alone or restimulated with 5 µg/ml Core protein, 0.05 µg/ml E. coli control, 5 µg/ml peptide, or VV-infected or peptide-sensitized autologous B-LCLs (1/1) for 12 h in culture medium containing 50 U/ml rIL-2 (Chiron) and 3 µM monensin (PharMingen, San Diego, CA). Cells were stained as previously described (28) for surface CD4 and CD8 with APC-conjugated anti-human CD4 and PerCP-conjugated anti-human CD8 and for intracellular IFN- {gamma} and TNF-{alpha} with PE-conjugated anti-human IFN-{gamma} and FITC-conjugated anti-human TNF-{alpha}. Abs were obtained from PharMingen and Becton Dickinson (San Jose, CA). Cells were analyzed on a FACSCalibur. Data files were analyzed using CellQuest software (Becton Dickinson).

Cytokine ELISA

Freshly isolated rhesus macaque PBMCs were restimulated with peptides encompassing the whole Core protein. Levels of rhesus monkey IL-2, IL-5, IL-10, and IFN-{gamma} present in 48-h cell-free culture supernatants were determined by specific ELISA (U-Cytech, Utrecht, The Netherlands) following the manufacturer’s specification.

HCV Abs

Serum levels of HCV Core and HCV E2 Abs were quantified by ELISA as previously described (29). Serum levels of Abs inhibiting the binding of E2 to the putative HCV receptor CD81 (30) were determined by immunoassay.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Priming of Core-specific CTLs in vaccinated animals

The prototype vaccine Core-ISCOM aimed at eliciting HCV-Core-specific CTLs was administered to three HCV-naive rhesus macaques (see Table IGo for animal assignment, dosage, and immunization schedule). Since it was unknown whether rhesus macaque MHC class I molecules can bind and present HCV-Core-derived peptides and whether the positively selected CD8+ T cell repertoire in these animals can recognize such MHC class I-Core-derived peptide complexes, three additional animals were inoculated with 2 x 108 PFU of rVVC/E1 to serve as positive controls (Table IGo).

None of the animals had any detectable CTLs at the time of immunization (0 wk; Table IIGo and data not shown). This confirmed that these animals had not been previously exposed to HCV-Core and that restimulation of PBMCs under the conditions described in Materials and Methods did not result in the priming of primary CTL responses in vitro. Two weeks after rVVC/E1 infection, two (BB232 and DV036) of the three animals had detectable CTLs against Core peptide pool 4 (aa 121–170) and pool 3 (aa 81–130), respectively (Table IIGo). By deconvoluting these peptide pools, it was determined that BB232’s CTLs recognized the epitopic peptide 121–135 and that DV036’s CTLs recognized peptide 86–100. The presence of 121–135- and 86–100-specific CTLs in these rVVC/E1-inoculated animals indicated that both peptides were naturally processed. No CTL responses were detectable in the other rVVC/E1-inoculated animal (BB228; data not shown). This indicated that Core-specific CTLs can be elicited in at least some rhesus monkeys. Two of the three Core-ISCOM-immunized animals (AY921 and BB231) did not mount a detectable Core-specific CTL response (data not shown). In contrast, in the other Core-ISCOM-immunized animal (DV037), CTLs recognizing pool 4 (aa 121–170) were detectable as early as 2 wk after the second immunization. This response was directed against the epitopic peptide aa 121–135 and was also detectable after the third and fourth immunizations (Table IIGo).


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

 
MHC class I alleles dictated whether a Core-ISCOM-immunized animal produced Core-specific CTLs

Only one of three animals immunized with the Core-ISCOM prototype vaccine mounted a detectable Core-specific CTL response. We hypothesized that this might be due to the fact that the MHC class I molecules of AY921 and BB231 were unable to bind and present peptides derived from this relatively small protein (191 aa). To test this hypothesis, CTL lines specific for peptides 121–135 and 86–100 were established from DV036 and DV037, respectively. As shown in Fig. 1GoA, the peptide 121–135-specific CTL line lysed peptide-sensitized B-LCLs derived from DV037, but did not kill peptide 121–135-sensitized B-LCLs from the two nonresponding animals (AY921 and BB231). Similarly, B-LCLs derived from DV036, but not AY921 or BB231, were able to present peptide 86–100 to CD8+ CTLs (Fig. 1GoB). These data indicated that AY921’s and BB231’s MHC class I molecules could not present these peptides to CD8+ T cells and suggested that MHC class I haplotypes determined whether rhesus monkeys could mount a CTL response to HCV-Core.



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  		FIGURE 1. B-LCLs from the two nonresponder (AY921 and BB231) Core-ISCOM-immunized animals cannot present Core-derived peptides 121–135 and 86–100. A, The 121–135-specific CTL line established from animal DV037 was tested in a standard 51Cr release assay for its ability to lyse DV037 B-LCL target cells sensitized with peptide 121–135 ({blacksquare}) or an irrelevant peptide ({square}), AY921 B-LCL target cells sensitized with peptide 121–135 ({blacktriangledown}) or an irrelevant peptide ({triangledown}), and BB231 B-LCL target cells sensitized with peptide 121–135 (•) or an irrelevant peptide ({circ}). B, The 86–100-specific CTL line established from animal DV036 was tested in a standard 51Cr release assay for its ability to lyse DV036 B-LCL target cells sensitized with peptide 86–100 ({blacksquare}) or an irrelevant peptide ({square}), AY921 B-LCL target cells sensitized with peptide 86–100 ({blacktriangledown}) or an irrelevant peptide ({triangledown}), and BB231 B-LCL target cells sensitized with peptide 86–100 (•) or an irrelevant peptide ({circ}).

 
Since different MHC class I alleles can bind and present different sets of peptides, these data did not rule out that the Core-ISCOM formulation was not suboptimal, i.e., it remained possible 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 x 108 PFU of rVVC/E1 11 wk after their fourth immunization with Core-ISCOM. As opposed to BB232 and DV036, which had Core-specific CTLs 2 wk after rVVC/E1 infection (Table IIGo), neither AY921 nor BB231 had any detectable CTLs after VVC/E1 inoculation (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. 2GoA). 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. 2GoB). Similarly, the 86–100-specific CTLs primed in DV036 by rVVC/E1 vaccination became undetectable 14 wk postvaccination (data not shown).



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  		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 CD8+ enrichment, cells were tested for cytotoxic activity against autologous B-LCLs sensitized with the epitopic peptide 121–135 (•) or an irrelevant peptide ({circ}). 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-{gamma} and TNF-{alpha}. Lymphocytes were gated by side vs forward scatter light and then for CD8-PerCP. Plots show log fluorescence intensity for TNF-{alpha}-FITC and IFN-{gamma}-PE.

 
In an effort to quantify the number of peptide 121–135-specific CTLs present in DV037 1 year after it had received its last boost, the animal’s PBMCs were restimulated ex vivo with 121–135, and the percentage of specific CD8+ T cells was assessed by intracellular staining for IFN-{gamma} and TNF-{alpha}. As illustrated in Fig. 2GoC (left panels), 121–135-specific CTLs represented 0.49% of the peripheral CD8+ T cells (or 490 cells/105 CD8+ T cells) that secreted IFN-{gamma} and/or TNF-{alpha} after ex vivo peptide stimulation for 12 h. In contrast, after in vitro restimulation with peptide 121–135, 71% of these CD8+ T cells were specific for this peptide, as determined by their abilities to secrete IFN-{gamma} and/or TNF-{alpha} (Fig. 2GoC, right panels).

Characterization of cellular and humoral immune responses in rhesus monkeys immunized with Core-ISCOM

Although only one of three Core-ISCOM-immunized animals had detectable CTLs, the fact that in the responding animal Core-specific CTLs were detected after only two immunizations (Table IIGo) and were long-lived (Fig. 2GoA) formed the basis to immunize five more animals (15860–4) with Core-ISCOM (see Table IGo for dosage and immunization schedule). All animals were naive at the time of vaccination. In this study we monitored the priming not only of Core-specific CTLs, but also of Core-specific CD4+ T cells and Abs.

None of the animals had any detectable Core-specific CD4+ or CD8+ T cells at the time of immunization (0 wk, Table IIIGo). Core-specific CD4+ T cells, as determined by lymphoproliferation assay, were detected in all animals except 15861 after the second immunization, but this animal had a detectable CD4+ response after the third immunization (Table IIIGo). For animals 15862 and 15863, it is unlikely that the low SI observed after the third immunization (Table IIIGo) was due to the absence of a CD4+ T cell response, as a strong proliferation was observed in these animals and at this particular time for the E. coli control (see below). None of the animals had Abs against Core before immunization. However, all animals had seroconverted to Core after two immunizations, and the level of Abs against Core was boosted by a third immunization (Fig. 3Go). Notably, the mean Core Ab titer among these animals was comparable after two immunizations (1931) and was higher after three immunizations (4566; Fig. 3Go) compared with that present in the serum of chronically infected patients with an unusually high anti-Core Ab titer (2358; data not shown) run in the same assay.


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  		Table III. Priming of Core-specific CD8+ and CD4+ T cells in rhesus macaques immunized with Core-ISCOMS

 


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  		FIGURE 3. Titer of Abs against Core in the serum of immunized animals. {square}, Preimmunization; , 2 wk after the second immunization; {blacksquare}, 2 wk after the third immunization.

 
To investigate whether Core-ISCOM elicited a Th1- or Th2-type response in these monkeys, freshly isolated PBMCs before vaccination as well as 2 wk after the second and third immunizations were tested for their capacity to produce cytokines at 48 h in response to stimulation with Core peptides spanning the entire length of the Core protein. As shown in Fig. 4Go, A and B, a significant increase in Th1 cytokines (IFN-{gamma} and IL-2) was observed postimmunization in all animals. Similarly, an increase in Th2-type cytokines (IL-5 and IL-10) was observed in all animals following vaccination (Fig. 4Go, C and D). Of note, the magnitude of the Th1-like response in animals 15860, 15861, and 15862 was lower than that in animals 15863 and 15864, but the levels of Th2-type cytokines were almost comparable in all animals. Nevertheless, these data indicated that Core-ISCOM induced a Th0-like type response in rhesus monkeys.



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  		FIGURE 4. Th-1- and Th2-type cytokines in Core-ISCOMS-immunized animals. The levels of IFN-{gamma} (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 Materials and Methods. {square}, Preimmunization; , 2 wk after the second immunization; {blacksquare}, 2 wk after the third immunization. NT, Not tested.

 
After two and three immunizations, three animals (15862, 15863, and 15864) had detectable CTL responses directed against pool B (aa 60–140), while two others (15860 and 15861) did not (Table IIIGo). This CTL response was directed against peptide 86–100 for animal 15864 and against peptide 121–135 for animals 15862 and 15863. Since 86–100- and 121–135-specific CTLs were also primed in DV036 (86–100), DV037 (121–135) and BB232 (121–135; Table IIGo), it was of importance to determine whether all 86–100- and 121–135-specific CTLs were respectively restricted by a single MHC class I allele. The 86–100-specific CTL line derived from animal 15864 efficiently lysed peptide 86–100-sensitized B-LCLs derived from 15864, but not peptide-sensitized B-LCLs derived from animal DV036, indicating that a different (unidentified) MHC class I allele presented this peptide to CTL (Fig. 5GoA and Table IGo). In contrast, the CTLs specific for peptide 121–135 from animals 15862, 15863, BB232, and DV037 were restricted by a single, as yet unidentified, MHC class I allele shared by all these animals (Fig. 5GoB and Table IGo). These data also indicated that the MHC class I molecules of 15860 and 15861 could not present either peptide (86–100 and 121–135) to specific CTLs (Fig. 5Go). As observed in the first study, rVVC/E1 infection of the two nonresponding animals (15860 and 15861) 9 wk after the third immunization with Core-ISCOM did not lead to the priming of Core-specific CTLs in these animals (data not shown). Taken together, this suggested, once again, that the MHC class I haplotype of the animals dictated whether they could mount Core-specific CTLs.



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  		FIGURE 5. MHC class I restriction of peptides 121–135 and 86–100 CTLs. A, The peptide 86–100-specific CTL line derived from animal 15864 was tested in a standard 51Cr release assay for its ability to lyse peptide 86–100-sensitized B-LCL target cells derived from animals DV036 ({circ}), 15864 (•), 15860 ({blacktriangledown}), and 15861 ({blacksquare}). B, The peptide 121–135-specific CTL line derived from animal 15862 was tested in a standard 51Cr release assay for its ability to lyse peptide 121–135-sensitized B-LCL target cells derived from animals DV037 (•), BB232 ({blacktriangleup}), 15862 ({blacktriangledown}), 15863 ({blacksquare}), 15861 ({triangledown}), and 15860 ({square}).

 
Quantification of the Core-specific cellular immune responses primed by Core-ISCOM

In an effort to quantitate the number of Core-specific CD8+ and CD4+ T cells primed in these animals, freshly isolated PBMCs were stained for intracellular IFN-{gamma} and TNF-{alpha} after ex vivo restimulation. The CD8+ T cell responses to naturally processed peptides were quantified after ex vivo restimulation with autologous B-LCLs infected with rVVC/E1 or VVwt, as a control. The CD4+ T cell responses to naturally processed peptides were quantified after ex vivo restimulation with the recombinant Core protein or an E. coli control. Intracellular staining responses revealed that while none of the animals had detectable Core-specific CD8+ T cells at the time of immunization, between 0.30 and 0.71% of peripheral CD8+ T cells in 15862, 15863, and 15864 were specific for naturally processed Core-derived peptide(s) after two immunizations (Fig. 6GoA). The number of specific CTLs was, however, not increased after the third immunization, as judged by intracellular staining responses. Notably, no CD8+ T cells secreting IFN-{gamma} and/or TNF-{alpha} 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. 6GoA and Table IIIGo). Quantification of Core-specific CD4+ T cells confirmed the data obtained by lymphoproliferation assay (Table IIIGo), in that between 0.32 and 2.21% of CD4+ T cells from all five animals were specific for naturally processed Core peptides (Fig. 6GoB). Furthermore, the fact that 0.53 and 0.28% of CD4+ T cells from animals 15862 and 15863 were positive for cytokines after the third immunization (Fig. 6GoB), strongly suggested that the negative SI observed for this time point (Table IIIGo) 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-{gamma} and TNF-{alpha} is a more sensitive assay than lymphoproliferation to assess Ag-specific CD4+ T cell responses. Indeed, for animal 15861, no CD4+ T cells were detected by lymphoproliferation 2 wk after the second immunization (Table IIIGo). In contrast, Core-specific CD4+ T cells were detected in this animal (2 wk after the second immunization) by intracellular staining for IFN-{gamma} and TNF-{alpha} (Fig. 6GoB).



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  		FIGURE 6. Quantification of the CD8+ and CD4+ T cell responses in Core-ISCOM-immunized animals. Freshly isolated PBMCs were restimulated 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-{gamma} and TNF-{alpha} as described in Materials and Methods. Lymphocytes were gated by side vs forward scatter light and then for CD8-PerCP (A) or CD4-APC (B). A, The corrected percentage of CD8+ T cells with detectable IFN-{gamma} and/or TNF-{alpha} was calculated as (percent CD8+ T cells restimulated with rVVC/E1 that were IFN-{gamma}+ and/or TNF-{alpha}+) - (percent CD8+ T cells restimulated with VVwt that were IFN-{gamma}+ and/or TNF-{alpha}+). B, The corrected percentage of CD4+ T cells with detectable IFN-{gamma} and/or TNF-{alpha} was calculated as (percent CD4+ T cells restimulated with Core that were IFN-{gamma}+ and/or TNF-{alpha}+) - (percent CD4+ T cells restimulated with the E. coli that were IFN-{gamma}+ and/or TNF-{alpha}+). {square}, Preimmunization; , 2 wk after the second immunization; {blacksquare}, 2 wk after the third immunization.

 
Core- ISCOM can serve as an adjuvant for soluble E1E2

Because vaccination with recombinant HCV envelope proteins and adjuvant can, at least in some instances, influence the outcome of infection and disease (31), we investigated whether Core-ISCOM could serve as an adjuvant for the heterodimeric envelope protein E1E2. To that end, the geometric mean E2 Ab titer of mice (10 animals/group) immunized with 2 µg/dose of recombinant E1E2 in the presence of MF59 (v/v) was compared with that of mice immunized with 2 µg/dose recombinant E1E2 protein plus 2 µg/dose Core-ISCOM or 2 µg/dose E1E2 classical ISCOM. As shown in Fig. 7Go, mice immunized with E1E2 plus Core-ISCOM had a significant anti-E2 Ab titer after three immunizations (31,099 ± 8,217), and these titers were comparable to those observed in mice immunized with E1E2 plus MF59 (24,178 ± 3,432) or E1E2 classical ISCOM (27,093 ± 2,621). The quality of Ab elicited in these mice appeared to be similar, inasmuch as the titer of Ab inhibiting the binding of HCV-1a E2 to the HCV putative receptor CD81 were comparable in these three groups of mice (Fig. 7Go). Moreover, mice immunized with 2 µg/dose of recombinant E1E2 protein plus 2 µg/dose of Core-ISCOM all seroconverted to Core (geometric mean Ab titer, 1,600 ± 835; Fig. 7Go). Further studies will be needed to decipher the mechanisms by which Core-ISCOM can adjuvant E1E2 and to determine whether this adjuvant effect is also observed in higher species.



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  		FIGURE 7. Core-ISCOM can serve as an adjuvant for E1E2. Mice (10 animals/group) were immunized with 2 µg/dose recombinant E1E2 protein, 2 µg/dose recombinant E1E2 plus 2 µg/dose recombinant Core, 2 µg/dose recombinant E1E2 in the presence of MF59 (v/v), 2 µg/dose recombinant E1E2 protein plus 2 µg/dose Core-ISCOM, or 2 µg/dose E1E2 classical ISCOM. Mice were bled 2 wk after the third immunization. Anti-E2 ({blacksquare}), anti-CD81 titers (), and anti-Core ({square}) are presented as the geometric mean of the titers obtained from individual mice from each group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The development of a vaccine against HCV and its associated diseases has been the focus of research since the virus was discovered (32). Adjuvanted subunit E1E2 vaccines can confer protection against a homologous challenge (31). There is evidence, however, that priming of CD4+ and CD8+ T cells specific for HCV gene products other than E1and E2 might increase the efficacy of an E1E2 vaccine, as such cells have been implicated in conferring immunity to HCV (5, 6, 7, 33).

One of the challenges facing the development of an HCV vaccine 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 generated in the cytosol following cleavage of cytosolic polypeptide precursors by proteases (41). Thus, the induction of CTL responses usually requires that the Ag be endogenously expressed and processed. Genetic vaccines such as naked DNA have been shown to be potent inducers of CTLs against viral proteins (42), presumably because Ag synthesis occurs in the host. Because of the concerns about using a genetic vaccine to prime Core-specific CTLs, one must rely on a subunit-based vaccine to prime CTLs against Core. Immunization with subunit protein vaccines adjuvanted in alum or oil/water emulsions can usually elicit CD4+ T cells and Abs, but are generally inefficient at priming MHC class I-restricted CTLs, as proteins in the extracellular fluid are generally processed through the exogenous processing pathway and are degraded into peptides that bind MHC class II molecules (43).

The Core-ISCOM prototype vaccine primed strong Core-specific CD4+ and CD8+ T cell-mediated immune responses in rhesus macaques (Tables IIGo and IIIGo). The fact that these immune responses were of the Th0 type might not be surprising, as both Th1- and Th2-type cytokines are secreted by spleen and draining lymph node cells from mice immunized with classical-ISCOM (44). Using intracellular staining for IFN-{gamma} and TNF-{alpha}, we determined that following immunization with Core-ISCOM, the frequency of core-specific CD4+ and CD8+ T cells ranged between 0.3 and 2.2% and between 0.3 and 0.7%, respectively (Fig. 6Go). In patients with chronic HCV infection, the frequency of CTLs was reported to be low to undetectable (7, 45), potentially a result of clonal exhaustion or anergy. Yet, using enzyme-linked immunospot, it was estimated that about 3% of the circulating CD4+ T cells and about 6% of the circulating CD8+ T cells were specific for HCV at the time of maximum responses in a patient with acute HCV infection who subsequently resolved the infection (7). Because these frequencies encompassed cells specific for multiple HCV proteins (7), this suggested that the frequency of HCV-specific T cells primed by Core-ISCOM vaccination was comparable to that present in this patient.

One year after its last immunization with Core-ISCOM, DV037 still had detectable core-specific CTLs in its periphery at a relatively high frequency (0.49%; Fig. 2Go). The nature of the mechanisms involved in the maintenance of memory CTLs remains unclear. Some experiments suggest that Ag is needed for the maintenance of memory, while other studies indicate that it is not. Similarly, whether CD8 memory requires persistence of Ag-specific CD4+ T cells is controversial (46). Whatever the mechanisms responsible for T cell memory, HCV-specific CTLs have been shown to persist for a long time following immunization (this study) and in patients who have resolved acute infection (47).

Classical ISCOM formulations are typically particulates ~40 nm in diameter in which the Ag is bound by hydrophobic interactions to the saponin, cholesterol, and phospholipid that form the cage-like pentagonal dodecahedral structure (17). Association between the Ag and the adjuvant is thought to be important for induction of CTL responses (48). With classical ISCOM formulations this association is achieved by incorporation of the hydrophobic Ag into the particle. Other methods of association include electrostatic interactions, which take advantage of the negative charge on the ISCOMATRIX and its ability to associate with positively charged proteins. The Core protein is positively charged and as such is adsorbed onto the ISCOMATRIX by electrostatic interactions to produce the Core-ISCOM formulation. A number of classical ISCOM vaccines have been shown to induce both humoral and cellular immune responses (18, 19, 23, 44, 49, 50, 51, 52, 53). In contrast to classical ISCOM, Core-ISCOM are ~1 µm in diameter (as determined by standard light scattering methodology; data not shown), and the Core Ag is adsorbed onto the cage-like structure, not trapped inside. Phagocytosis of particulate by professional APCs is much more efficient for 1-µm particles than for 100-nm or smaller particles (54). The size of the Core-ISCOM particulate might therefore directly contribute to its ability to prime potent cellular immune responses, since efficient uptake by APCs of particulate by phagocytosis may be important for the rapid and effective delivery of internalized particles for presentation by MHC molecules. However, internalization of 1-µm particulates by phagocytosis is usually directly delivered to lysosomes (55), which leads to presentation of the Ag through the MHC class II pathway (43). Yet, recent studies have documented pathways (such as the phagosome to cytosol pathway and cross-priming) that allow for peptide presentation on MHC class I molecules after phagocytosis of particulate Ag by professional APCs such as macrophages and dendritic cells (56, 57, 58). Since vaccination of rhesus macaques with Core-ISCOM-primed Core-specific CD4+ and CD8+ T cells as well as Abs against Core, mechanisms leading to presentation of exogenous Ag on both MHC class I and II molecules after phagocytosis of particulate by professional APCs must have been used. However, since ISCOM contains saponin, which can intercalate into cholesterol membranes (59), one cannot rule out that some Core-ISCOM particulates were able to pass directly through cell membranes and thus directly enter the classical pathway for MHC class I presentation. Finally, the saponin may permit endosomal escape by interfering with endosomal membrane structure (48).

There is a growing body of evidence indicating that a vaccine eliciting both Core-specific CD4+ and CD8+ T cells could have a therapeutic value. First, Core-specific CTLs have been associated in HLA-B44+ patients with a lower viral titer (60). Second, CD4+ T cell responses to Core, although they did not appear to coincide with virus clearance, have been associated with a benign course of infection (61). Such core-specific CD4+ T cells may therefore help to maintain humoral and cellular responses protective against the disease. In this context, it is conceivable that priming and maintenance of Core-specific CTLs by vaccination with Core-ISCOM were dependent and controlled, at least to some extent, by Core-specific CD4+ T cells, as observed for some tumor-specific CTLs (62, 63). This might explain why Core-specific CTLs primed by rVVC/E1 were not maintained as long as CTLs primed by Core-ISCOM (Fig. 2Go). Third, and as mentioned above, the response to IFN therapy was shown to be directly related to the rate of T cell-mediated clearance of infected cells (13) and the presence of Core-specific CTLs before initiation of treatment (15).

Overall, this study indicated that vaccination with Core-ISCOM primes strong Core-specific CD8+ and CD4+ T cells as well as anti-Core Abs. Furthermore we demonstrated that this formulation could serve as an adjuvant to elicit Abs against E1E2. Further studies in chimpanzees, the only reliable animal species for infection, or in humans, are needed to determine whether a Core-ISCOM (associated, or not, with E1E2) vaccine can confer sterilizing immunity to HCV, prevent the establishment of chronicity, and/or increase the response rate to anti-viral therapy.


   Acknowledgments
 
We thank Yiu-Lian Fong and Kevin Crawford for providing the purified recombinant proteins, Dr. Gary Ott for the MF59 adjuvant, and Diana Atchley for immunizing the mice. We are also grateful for the assistance of Jim Malliaros, Bengt Rönnberg, Megan Meinhardt, Michael McNamara, and Denise Fletcher with the development and preparation of the Core ISCOM formulations, and to David I. Watkins for MHC class I typing of the rhesus monkeys.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Xavier Paliard, Chiron Corp., 4560 Horton Street, Room 4.3132, Emeryville, CA 94608-2916. Back

2 Abbreviations used in this paper: HCV, hepatitis C virus; rVV, recombinant vaccinia virus; rVVC/E1, rVV expressing Core and E1; VVwt, wild-type VV; SI, stimulation index; B-LPCLs, B-cell lymphoblastoid cell lines. Back

Received for publication August 18, 2000. Accepted for publication December 20, 2000.


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