Immunization with Hepatitis C Virus-Like Particles Induces Humoral and Cellular Immune Responses in Nonhuman Primates

E1 and E2 proteins were expressed in vvHCV.S-infected BSC-1 cells and purified by using a lectin column (4). HCV core protein (amino acids [aa] 1 to 115) was purchased from Mikrogen (Munich, Germany). Synthetic peptides spanning the whole HCV structural genome of HCV 1b genotype J strain were synthesized by Chiron Mimotopes (Clayton, Victoria, Australia). Peptides were 15 aa in length with a 10-aa overlap. The overlapping peptide 1 (OLP1) consisted of a pool of 38 peptides spanning the HCV core sequence, OLP2 consisted of 38 peptides spanning the HCV E1 sequence, OLP3 consisted of 35 peptides spanning the N-terminal half of the HCV E2 sequence, and OLP4 consisted of 36 peptides spanning the C-terminal half of the HCV E2 sequence. Peptide stock solutions were prepared in 5% dimethyl sulfoxide in phosphate-buffered saline (PBS) at the concentration of 1 mg/ml and stored at −20°C.

Procedures for production and purification of HCV-LP were as described previously (3), with some modifications. Briefly, Sf9 cells (2 × 10⁹) were infected with recombinant baculovirus (bvHCV.Sp7⁻) at a multiplicity of infection of 5 to 10 and incubated at 27°C for 3 days in Sf-900 serum-free medium (Gibco-Invitrogen Corporation, Carlsbad, Calif.). Cells were harvested by centrifugation at 2,500 × g for 5 min at room temperature, and the cell pellet was washed once with PBS. The cell pellet was resuspended in 18 ml of prewarmed PBS and 3 ml of 90% glycerol containing 10 mM HEPES buffer, 1 mM phenylmethylsulfonyl fluoride [PMSF]; Sigma-Aldrich, St. Louis, Mo.) and a cocktail of EDTA-free protease inhibitors (PI; one tablet per 50 ml; Roche, Indianapolis, Ind.). The cell suspension was mixed and incubated at 37°C for 5 min. The process was repeated twice so that the final glycerol concentration in the cell suspension reached 30% (32). The cells were then chilled on ice for 5 min and centrifuged at 2,500 × g for 10 min at 4°C. The following purification steps were performed at 4°C unless specified. The cell pellet was resuspended with 50 ml of lysis buffer (10 mM Tris-HCl [pH 7.4], 1 mM MgCl₂, 1 mM CaCl₂, 1 mM PMSF, PI [TNC-PI buffer]) containing 0.5% digitonin and allowed to sit on ice with gentle agitation. When the percentage of cell lysis reached satisfactory levels after periodic monitoring for cell lysis by trypan blue exclusion, the cell lysate was centrifuged at 26,000 × g in an SW 28 rotor (Beckman) for 30 min to remove cell debris. To maximize the HCV-LP yield, the lysis process may be repeated once more by resuspending the cell pellet in fresh lysis buffer containing 0.5% digitonin. The supernatant was loaded onto 3 ml of a 20% sucrose cushion in TNC-PI buffer and centrifuged at 160,000 × g for 3 h by using an SW41 rotor (Beckman). The pellet was then redissolved in a small volume of TNC-PI buffer and then loaded onto 9 ml of a 20 to 60% sucrose gradient and centrifuged at 120,000 × g for 16 h in an SW41 rotor. One-milliliter fractions were collected from the top of the tube, and these were tested for core, E1, and E2 proteins by enzyme-linked immunosorbent assay (ELISA) (37) and Western blotting. Total protein was quantitated by using Coomassie Blue Plus protein assay reagent (Pierce, Rockford, Ill.), and ultrastructural study of HCV-LP was performed by electron microscopy.

Recombinant vaccinia virus (wild type, Western reserve) expressing HCV structural proteins core, E1, and E2 (vvHCV.S containing the cDNA for HCV 1b genotype J strain) was purified from the BSC-1 cells as previously described (4).

Two different adjuvants were used in this study. ASO1B (MPL and QS21) and CpG 10105 were generously provided by GlaxoSmithKline (Rixensart, Belgium) and Coley Pharmaceutical Group (Wellesley, Mass.), respectively. These adjuvants (AS01B at 1× and CpG 10105 at 500 μg per animal as recommended by the providers) were mixed with HCV-LP just before injection.

Baboons (Papio cynocephalus) were housed in a large outdoor cage at Southwest Foundation for Biomedical Research (San Antonio, Tex.), which is an AALAC (American Association of Laboratory Animal Care)-accredited animal facility. Fourteen baboons, all males with an average age of 8.3 years and weighing between 12 and 17 kg, were randomly selected and divided into four groups for this study. Three groups of four baboons were immunized four times at 0, 4, 8, and 34 weeks with 100 μg of HCV-LP in a volume of 200 μl injected into each gluteal muscle. Group 1 received HCV-LP, group 2 received HCV-LP + ASO1B, and group 3 received HCV-LP + ASO1B/CpG 10105. Two remaining baboons were immunized twice at a 4-week interval with 5 × 10⁸ PFU of the purified recombinant vaccinia virus expressing HCV structural proteins (vvHCV.S) as positive controls for HCV-specific immune response. All animal experiments were conducted according to the criteria published by the National Institutes of Health (NIH publication 86-23, revised in 1985).

Baboon sera collected before vaccination (week 0) and 4 weeks after each vaccination (weeks 4, 8, 12, and 35) were tested for antienvelope and anticore antibodies by ELISA. Briefly, 96-well microtiter plates (Nunc Immunoplates; Nunc Corp., Naperville, Ill.) were coated with purified E1 and E2 protein solution (5 μg/ml) or core protein (2 μg/ml; Mikrogen, Munich, Germany) diluted in 0.05 M bicarbonate buffer (pH 9.6) at 4°C overnight. The wells were washed three times with PBS to remove unbound proteins and then blocked 2 h at 37°C with 5% skim milk in PBS. After the plate was washed three times with PBS containing 0.05% Tween 20 (PBST), 100 μl of sera diluted 1:100 in PBST containing 5% skim milk was added to the wells and incubated for 1 h at 37°C. The wells were washed six times with PBST, and 100 μl of peroxidase-conjugated goat anti-human IgG (Sigma-Aldrich) diluted 1:2,000 in PBST with 5% skim milk was added and incubated for 45 min at room temperature. The wells were washed six times and incubated with a peroxidase substrate (ABTS, Microwell Peroxidase Substrate System; KPL Laboratories, Gaithersburg, Md.). The optical density at 405 nm (OD₄₀₅) of the plate was read. All assays were run in triplicates. Antibody titers were determined by the serial end point dilution method. End point titers were defined as the highest serum dilution that gave a positive reading, and the cutoff for positivity was defined as two times the OD value of the preimmune sera (signal-to-noise ratio of ≥2).

Detection and titration of vaccinia virus neutralizing antibodies was performed as previously described by using BSC-1 cells (9). Briefly, virus inoculum of wild-type vv-WR (Western Reserve) was mixed with fivefold serial dilutions of pre- and postvaccination sera (week 8) for 2 h at 4°C after heat inactivation of the sera. The virus-serum mixture was added to near-confluent BSC-1 cell monolayers in six-well plate and incubated for 1 h at 37°C. After being washed with 2× PBS, cells were incubated for 2 days in 37°C with 5% CO₂. Cells were then stained with 0.1% crystal violet for 5 min, and the plaques were counted. The dilution of serum capable of reducing the number of virus plaques by 50% compared to the virus control was set as the 50% inhibitory concentration.

Baboon peripheral blood mononuclear cells (PBMC) prior to immunization (week 0) and 4 weeks after each immunization (weeks 4, 8, 12, and 38) were used to measure the number of antigen-specific gamma interferon (IFN-γ)-producing cells by using an ELISPOT kit (U-CyTech, Utrecht, The Netherlands). Briefly, microtiter plates were coated with monoclonal antibody specific for monkey IFN-γ at 4°C overnight. After being washed five times with 0.05% PBST, the wells were blocked with 1% bovine serum albumin for 2 h at 37°C. PBMC (3 × 10⁵) were added to the wells in duplicates and stimulated with soluble protein antigens (1 μg/ml of HCV core protein or 2 μg of HCV E1/E2 proteins/ml), or OLP pools of core, E1, and E2 (1 μg of each peptide/ml), respectively. As a negative control, media (without any antigen) was added and as a positive control, Concanavalin A (Sigma-Aldrich) was added at a concentration of 5 μg/ml. Cells were incubated for 24 h at 37°C and then lysed with distilled water for 10 min. After being washed 10 times with PBST, biotinylated detecting antibody was incubated for 1 h at 37°C. After being washed 10 times with PBST, antibiotin antibody was incubated for 1 h at 37°C followed by the addition of chromogenic substrate according to the manufacturer's instructions. Tap water was added to the wells to stop the reaction, and the dried plate was read by use of the automated ELISPOT Reader System with KS software (Carl Zeiss, Thornwood, N.Y.). IFN-γ spot-forming units (SFU) representing single cells were counted and expressed as SFU per 3 × 10⁵ PBMC. The numbers of antigen-specific SFU per well were calculated by subtracting background values (wells without antigen, typically less than 5). The sample was considered positive when the SFU (after subtracting the background) was greater than 10 and more than or equal to twice the mean SFU of the sample prior to immunization in the same animal. The PBMC were also tested against an irrelevant peptide (HBV core peptide, aa 18 to 27) as an additional control, and in all cases, the SFU were less than 10 (without subtracting the background).

PBMC (10⁶ cells) in 0.5 ml of RPMI medium in 5-ml polystyrene tubes (Falcon; Becton Dickinson, Franklin Lakes, N.J.) were stimulated with OLP pools of core, E1, or E2 (1 μg of each peptide/ml) and costimulated with 1 μg of anti-human CD28 (clone CD28.2) and anti-human CD49d (9F10) (BD Biosciences)/ml for 1 h at 37°C and then for an additional 5 h in the presence of monesin (Golgi Stop; BD PharMingen). As controls, cells were incubated without peptide (as a negative control) or stimulated with phorbol myristate acetate (PMA) (50 ng/ml) and ionomycin (1 μM) instead of peptides (as a positive control), respectively. After being washed twice with PBS-1% fetal calf serum, cells were incubated with fluorescein isothiocyanate-conjugated anti-CD8 (mouse anti-human monoclonal antibody [clone RPA-T8, 5 μl/10⁶ PBMC]) for 30 min at 4°C. The cells were then washed with permeabilization buffer using Cytofix/Cytoperm kit (BD PharMingen) and stained with 10 μl of phycoerythrin-conjugated mouse anti-human IFN-γ (clone 4S.B3; BD Biosciences) for 30 min at 4°C. The stained cells were washed with PBS-1% fetal calf serum, fixed with 2% paraformaldehyde, and analyzed by flow cytometry by using CellQuest software (BD Biosciences). At least 100,000 events and in some cases, up to 300,000 events were acquired from each sample. The percentage of IFN-γ⁺CD8⁺ T cells was defined as the difference between the percentage detected in peptide-stimulated cells and the percentage detected in unstimulated cells in the PBMC of the same animal. The cutoff for positive percentage of IFN-γ⁺CD8⁺ T cells was set at ≥50 events in the IFN-γ⁺ gate and greater than twice the mean percentage of sample prior to immunization in the same animal. As a negative control, the PBMC were also tested against an irrelevant peptide (HBV core peptide, aa 18 to 27), which typically gave <0.01% of IFN-γ⁺CD8⁺ T cells.

Comparisons between groups of baboons were analyzed by the Kruskal-Wallis test using software from the SAS Institute Inc (Cary, N.C.), and differences were considered significant when P values were ≤0.05.

The 12 baboons subjected to HCV-LP immunization were housed together, and the remaining 2 baboons subjected to vvHCV.S immunization were housed separately. All baboons were healthy throughout the study period, and there were no signs of systemic or local reactions to HCV-LP, adjuvants, or vvHCV.S. Blood chemistry and complete blood counts performed before immunization and after each immunization were normal.

Anti-HCV-E1/E2- and core-specific humoral responses were analyzed by ELISA (20), and the results are shown in Fig. 1. Blood samples collected before immunization and 4 weeks after the third and fourth immunization were tested in parallel and in duplicate. Seroconversion was defined as an increase of twofold or more of the OD₄₀₅ of postimmunization sera over that of the preimmune sera at a 1:100 dilution.

All baboons immunized with HCV-LP developed an anti-E response (100% seroconversion rate) after the fourth immunization (week 38) (Fig. 1A). However, the seroconversion rate after the third immunization (week 12) was increased by the use of adjuvant(s) from 0% in the HCV-LP group to 50% in the HCV-LP + ASO1B group and 100% in the HCV-LP + AS01B/CpG 10105 group. The combination of ASO1B and CpG 10105 seemed to be superior to no adjuvant or to single adjuvant ASO1B in promoting a faster and stronger anti-E1 or anti-E2 response. However, the overall anti-E titers after the fourth immunization were not significantly different in baboons immunized with HCV-LP or HCV-LP plus adjuvant(s) (Table 1).

In contrast to the anti-E1 and anti-E2 response, the anticore response was slow to develop in all animals (Fig. 1B and D) and no anticore antibodies were detected in any of the three groups of animals after three immunizations (week 12). However, all animals (with or without adjuvant) developed anticore antibodies after the fourth immunization and the antibody titers of the animals immunized with HCV-LP plus adjuvant(s) were only slightly higher than those of animals immunized with HCV-LP (P value not significant). End point titers of anti-E1 or -E2 and anticore among the three groups after the fourth immunization are summarized in Table 1. The anti-HCV antibodies induced in all three groups persisted (less than twofold reduction in titer) 8 months after the last immunization (Fig. 1C and D).

As immunization controls, two baboons were immunized twice with 5 × 10⁸ PFU of purified recombinant vaccinia virus expressing HCV structural proteins (vvHCV.S) at weeks 0 and 4. ELISA results showed that these baboons developed low levels of anti-E response (titers of 50 and 100) after the second immunization while anticore response was not detected at all in either animal. We detected vaccinia virus-neutralizing antibodies in both animals (titers, 200 and 400) by virus neutralization assay.

To quantitatively examine the T-helper response against the HCV structure proteins in baboons immunized with HCV-LP alone or HCV-LP combined with adjuvant(s), we performed ELISPOT assays for IFN-γ. PBMC collected before immunization (week 0) and after the third and fourth immunization (weeks 8, 12, 30, and 38) were tested in parallel and in duplicate. These cells were stimulated with recombinant core or E1 and E2 proteins or with four pools of OLP (core, E1, and E2) as described in Materials and Methods. The number of HCV-specific SFU was calculated by subtracting the number of spots in the absence of stimulant from those in the presence of antigen or OLP. Figure 2 displays the IFN-γ ELISPOT results of all 12 animals after immunization with HCV-LP, HCV-LP + ASO1B, or HCV-LP + ASO1B/CpG 10105.

Immunization with HCV-LP (Fig. 2, far left column) resulted in IFN-γ secretion after stimulation of PBMC with HCV proteins or OLPs. The addition of ASO1B or ASO1B + CpG 10105 seemed to further enhance the number of IFN-γ producing T cells (Fig. 2, middle and far right columns), although the difference did not reach statistical significance. The IFN-γ responses of these animals suggested that HCV-LP immunization induced a Th1 response against HCV structural proteins and OLPs. The number of SFU was markedly increased after the last booster immunization in all the baboons, indicating that the fourth immunization at month 8 (6 months after the previous immunization) is important in generating high-level HCV-specific cellular immune responses. There was no obvious trend in the three groups of animals in their responses to different HCV-specific stimulants (HCV proteins or OLPs).

The HCV-specific CD4⁺ response was maintained for an extended period of time after the fourth immunization. Blood samples obtained 8 months after the fourth immunization showed IFN-γ⁺ SFU results by ELISPOT similar to those obtained with the samples taken 1 month after the fourth immunization (Fig. 2).

To quantitatively determine the CD8⁺ responses in HCV-LP immunized animals, we performed ex vivo intracellular cytokine staining (ICS) for IFN-γ-producing CD8⁺ cells (Fig. 3). PBMC collected before immunization (week 0) and after the second, third, and fourth immunizations were stimulated with OLPs of HCV core protein and E1 and E2 proteins, respectively. As shown in Fig. 3B, all the immunized baboons demonstrated significant production by CD8⁺ cells of IFN-γ to one or more of the OLPs (greater than twofold over the baseline value) after the fourth immunization. The fluorescence-activated cell sorter histograms of representative immunized baboons after the fourth immunization are shown in Fig. 3A. Some of the animals also exhibited a positive response at earlier time points, either after the second or the third immunizations. However, many animals did not demonstrate any detectable IFN-γ-producing CD8⁺ response until after the fourth immunization, again supporting the importance of the final boost after an extended period of time from the third immunization. Combining all the OLP responses, the total frequency of the IFN-γ-producing CD8⁺ cells after the fourth immunization reached up to 0.45% in some animals. HCV-LP combined with adjuvants ASO1 and CpG (Fig. 3B) appeared to induce higher frequencies of IFN-γ-producing CD8⁺ T-cell response, but statistical significance was not observed among the three groups of baboons (Table 1).

As an immunization control for cellular immune response, we immunized two baboons with vvHCV.S. PBMC were collected and tested in the same way as those of HCV-LP-immunized animals. HCV-specific SFU with E protein stimulation was approximately 40 to 50 SFU/3 × 10⁵ cells after two immunizations. Similarly, PBMC stimulated with OLPs of HCV core and E proteins yielded detectable SFU (Table 1). Intracellular cytokine staining of HCV OLP-stimulated PBMC did not show detectable induction of IFN-γ-producing cells among CD8⁺ cells after two immunizations with vvHCV.S (Table 1).