Modulation of the Immune Response Induced by Gene Electrotransfer ...

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Lisa Kierstead,† Janine Burns,† Lionello Ruggeri,* Mirko Arcuri,* Manuela Cappelletti,* ...... O'Hagan, D. T., M. Singh, C. Dong, M. Ugozzoli, K. Berger, E. Glazer,.

The Journal of Immunology

Modulation of the Immune Response Induced by Gene Electrotransfer of a Hepatitis C Virus DNA Vaccine in Nonhuman Primates Stefania Capone,1* Immacolata Zampaglione,1* Alessandra Vitelli,* Monica Pezzanera,* Lisa Kierstead,† Janine Burns,† Lionello Ruggeri,* Mirko Arcuri,* Manuela Cappelletti,* Annalisa Meola,* Bruno Bruni Ercole,* Rosalba Tafi,* Claudia Santini,* Alessandra Luzzago,* Tong-Ming Fu,† Stefano Colloca,* Gennaro Ciliberto,* Riccardo Cortese,* Alfredo Nicosia,* Elena Fattori,* and Antonella Folgori2* Induction of multispecific, functional CD4ⴙ and CD8ⴙ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4ⴙ and CD8ⴙ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-␥ ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4ⴙ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-␥ⴙCD8ⴙ and CD4ⴙ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV. The Journal of Immunology, 2006, 177: 7462–7471.


fficient vaccination against viral pathogens requires potent and sustained T cell-mediated immune responses. DNA vaccination has emerged as an effective and safe strategy for inducing protective T cell immunity in preclinical models of infectious diseases (1). However, experiments in nonhuman primates and human trials suggested that DNA vaccines are not nearly as immunogenic in these species as they are in rodents (2– 6). Several strategies to improve the immunogenicity of DNA vaccines have been described, but the level of improvement has been modest and not applicable to all Ags (7). Recently, two physical strategies were described that could overcome the limitation of naked DNA injection: cationic microparticles and electroporation. The mechanism is unclear, but in both cases, likely to include increased transfection rates together with increased inflammatory responses, possibly including attraction of APC to the injection site. Cationic microparticles have been evaluated as a delivery system for a hepatitis C virus (HCV)3 and HIV vaccine candidates in

*Istituto di Ricerche di Biologia Molecolare, “P. Angeletti,” Rome, Italy; and †Merck Research Laboratories, West Point, PA 19486 Received for publication June 20, 2006. Accepted for publication August 25, 2006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

S.C. and I.Z. contributed equally to this work.


Address correspondence and reprint requests to Dr. Antonella Folgori, Technology Department, Istituto di Ricerche di Biologia Molecolare, via Pontina Kilometer 30600, 00040 Rome, Italy. E-mail address: [email protected] 3 Abbreviations used in this paper: HCV, hepatitis C virus; B-LCL, B lymphoblastoid cell line; CMI, cellular-mediated immunity; GET, gene electrotransfer; ICS, intracellular staining; SFC, spot-forming cell; SI, stimulation index; T, time.

Copyright © 2006 by The American Association of Immunologists, Inc.

rhesus macaques (8, 9). This strategy was particularly effective at enhancing Ab responses but had less or no effect on T cells. We and others (10 –13) have shown that uptake and expression of various reporter genes upon i.m. plasmid DNA delivery in vivo can be dramatically increased by applying an electric field to the muscle fibers immediately after the injection (gene electrotransfer (GET)). This approach results in an improved genetic immunization strategy for eliciting B and T cell immune responses specific for the HCV E2 envelope glycoprotein in mice and rabbits (14). To define an electrical treatment suitable for human application, we have recently made an extensive comparative analysis of different electrical conditions (15). We were able to design a treatment that lasts only 3 s, which has comparable efficiency to the longer treatment previously used in terms of muscle transduction in mice, rabbits, and monkeys. In the present work, we extended these studies showing that DNA immunization by GET results in a substantially improved strategy for eliciting broad and long-lasting T cell responses against a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (pV1JnsNSOPTmut) in mice and rhesus macaques. A crucial factor to be considered in the design of T cell-based vaccines is the ability to induce a complete repertoire of Ag-specific T lymphocytes with the correct balance between CD4⫹ and CD8⫹. GET immunization could induce both CD4⫹ and CD8⫹ T cells against multiple viral determinants with robust production of IFN-␥ in mice, macaques, and chimpanzees and proved to be superior at inducing Th1 CD4 responses than a potent adenovirus 6-based viral vector. We also showed that GET of the pV1JnsNSOPTmut vaccine elicited antiviral T cell responses in nonhuman primates quantitatively and qualitatively 0022-1767/06/$02.00

The Journal of Immunology equivalent to that observed in five acute/resolving infected humans.

Materials and Methods Animals BALB/c mice were purchased by Charles River Laboratories. Rhesus macaques (Macaca Mulatta) were housed at Biomedical Primates Research Center and at the New Iberia Research Center (NIRC). Chimpanzees (Pan troglodytes) were housed at NIRC. All animal care and treatment was in accordance with standards approved by the Institutional Animal Care and Use Committee in conformity with national and international laws and policies (European Economic Community Council Directive 86/609, Official Journal L 358, 1, 1987; Italian Legislative Decree 116/92, Gazzetta Ufficiale della Repubblica Italiana 40, 1992). The animals involved in the studies all met the following criteria: in good health, free of known infectious or immunological disease, and no previous contact with a pathogen related to HCV.

Vectors A DNA fragment encoding the HCV NS3-NS5A region from a BK isolate was derived from plasmid pcD3-5a (16) by HindIII digestion, filling in with Klenow and subsequent restriction with XbaI. The fragment was cloned into pV1JnsApoly (a derivative of the pV1Jns vector modified by insertion of a polylinker), which was linearized by BglII digestion, bluntended with Klenow, and subsequently digested with XbaI to generate the plasmid pV1JnsNS. From this construct, the plasmid pV1JNS3-5Akozak was obtained by homologous recombination into the bacterial strain BJ5183, cotransforming a AflII-digested pV1JNS3-5A and a PCR fragment containing the proximal part of intron A, the restriction site BglII and an optimal translation initiation (Kozak) sequence fused to NS3 coding sequence. A mutated version of the NS5B gene was obtained by replacing the Gly-Asp-Asp sequence corresponding to amino acid positions 1711–1713 of the complete polyprotein with Ala-Ala-Gly. This polymerase motif is conserved among all positive-stranded RNA viruses, and mutating these three residues inhibits or abolishes the RNA-dependent RNA polymerase activity of purified HCV NS5B and the infectivity of HCV RNA in chimpanzees. The mutated NS5B gene was inserted into pV1JNS3-5Akozak by homologous recombination as follows. The plasmid pV1JNS3-5Akozak was linearized by XbaI digestion and cotransformed into the bacterial strain BJ5183 with a PCR fragment, containing ⬃200 bp from the 3⬘-end of NS5A, the NS5B mutated sequence, the strong translation termination TAAA, and ⬃60 bp homologous to the 5⬘-end of the bovine growth hormone polyadenylation signal. The resulting plasmid pV1JnsNSmut was verified by restriction endonuclease and DNA sequence analysis. A synthetic DNA fragment encoding a fully codon-optimized NS3NS5B HCV sequence with the already described NS5B mutation to abrogate RNA-dependent RNA polymerase activity, an optimal translation initiation (Kozak) sequence, and a strong translation termination signal (NSOPTmut) was cloned into the pV1Jns plasmid as follows. The NSOPTmut DNA was digested with BamHI and SalI restriction endonucleases and then ligated into the BglII and SalI restriction sites present in the polylinker of pV1JnsA plasmid generating the plasmid pV1JnsNSOPTmut. The structure of pV1JnsNSOPTmut was verified by restriction endonuclease and sequence analysis. MRKAd6NSmut vector construction and rescue has been described elsewhere (17).

In vitro expression of HCV NS proteins in mammalian cells Expression of HCV NS proteins was tested by transfection of HEK 293 cells grown in 10% FCS/DMEM supplemented by L-glutamine (all media from Invitrogen Life Technologies). Twenty-four hours before transfection, cells were plated in 6-well plates to reach 90 –95% confluence on the day of transfection. Forty nanograms of plasmid DNA were cotransfected with 100 ng of pRSV-Luc plasmid containing the luciferase reporter gene under the control of Rous sarcoma virus promoter, using the LipofectAMINE 2000 reagent (catalog no. 11668019; Invitrogen Life Technologies). Cells were kept in a CO2 incubator for 48 h at 37°C. Cell extracts were prepared in 1% Triton/TEN buffer. The extracts were normalized for luciferase activity and run in serial dilution on 10% SDS-acrylamide gel. Proteins were transferred on nitrocellulose and assayed with Abs directed against NS3 (mAb 10E5/24) (18) and NS5A (rabbit polyclonal anti-serum) (16) to assess expression efficiency and correct proteolytic cleavage. Mocktransfected cells were used as a negative control. Expression of HCV NS proteins by MRKAd6NSmut has been described elsewhere (17).

7463 Vaccination Six-week-old female BALB/c mice were immunized with 50 ␮g of either pV1JnsNS or pV1JnsNSOPTmut plasmid DNA in 50 ␮l of sterile PBS. Two injections were performed 3 wk apart in the quadriceps muscle of each hind limb and were immediately followed by electrical treatment. Devices and electrical condition for GET in mice have been described elsewhere (14 –15). Two weeks after the boosting injection, mice were euthanized, and spleens were harvested and processed for detection of IFN-␥. Rhesus macaques and chimpanzees were vaccinated as detailed in Table I. Animals were injected with 5 mg of DNA into the quadriceps (rhesus macaques: groups A and B) or deltoid (chimpanzees: group D) muscle using 1 ml of solution (split over two sites, one each leg or arm, with 0.5 ml/site). The apparatus and the needle/electrodes used for GET in primates have been already described (15). The electrical field comprised two trains of 100 square bipolar pulses delivered every other second with each pulse lasting 2 ms/phase. The electrical field strength was 150 V (constant voltage mode) with current limitation at 100 mA. Macaques from group C (Table I) were immunized with 1010 viral particles of MRKAd6NSmut in 1 ml of buffer A105 (17) injected in the deltoid muscle split over two sites (0.5 ml/site).

Peptides and vaccinia virus for T cell assays The peptide sequence, spanning the NS3-NS5B region, reproduced the amino acid sequence of the HCV BK strain (19). Peptides were purchased from Biosynth International. All peptides were synthesized with free Nterminal amine and free C-terminal carboxylate and were purified by preparative HPLC. The peptides, 15 aa in length and overlapping by 11 aa (NS3-NS5B), were reconstituted in 100% DMSO at 40 mg/ml and mixed in pools so that each peptide is represented equally in the mixture. To facilitate the analysis, the 491 NS3-NS5B peptides were combined in six pools covering NS3 protease (NS3p), NS3 helicase (NS3h), NS4, NS5A, and NS5B (split into two pools NS5B-I and NS5B-II). The recombinant vaccinia virus encoding the nonstructural proteins NS2, NS3, NS4, and NS5, strain BK 1b (VacHCV-NS), was provided by J. Condra (Merck Research Laboratories, West Point, PA).

Ex vivo IFN-␥ ELISPOT IFN-␥ ELISPOT assay with mouse splenocytes has been described in details elsewhere (14). IFN-␥ ELISPOT assay with freshly isolated Rhesus PBMC was performed as described, with minor modifications (20). PBMC from EDTA-treated peripheral blood were isolated by lymphocyte separation medium density gradient centrifugation (lymphocyte separation medium, Organon, Teknika). PBMC were plated in duplicate at two different cell concentration/well (400,000 and 200,000). HCV peptide pools (5 ␮g/ml of each peptide/well), DMSO, and Con A (C 2010; Sigma-Aldrich) were used, respectively, as Ag, negative, and positive control. Biotinylated rabbit anti-monkey IFN-␥ mAb (U-Cytech) was diluted 1/100. Spots were quantified by an automated ELISPOT reader system (ELR03 AID ELISPOT Scientific). Since stimulation of PBMC with an unrelated peptide pool resulted in IFN-␥ spot counts not different from those obtained with DMSO only (data not shown), DMSO was used as negative control in all assays. The ELISPOT response was considered positive when all of the

Table I. Immunization schedule for rhesus monkeys (A, B, C) and chimpanzees (D)







21G pVIJnsNSOPTmut 99C161 99C166 C035 pVIJnsNSOPTmut C145 C034 C010 00D065 MRKAd6NSmut 00D116 00D159 00D088 Ch152 pVIJnsNSOPTmut Ch208



Schedule (wk)


5 mg

0, 4, 8


5 mg

0, 4, 8, 24



0, 4


5 mg

0, 4, 8, 16

7464 following conditions were met: IFN-␥ production present in Con A-stimulated wells; the number of specific spots/million PBMC to at least one HCV peptide pool was ⬎55; the number of spots seen in positive wells was three times the number detected in the mock control wells (DMSO); and responses decreased with dilutions of PBMC.

T-CELL BASED HCV DNA VACCINE Statistical analysis Immune responses against individual peptide pools for each animal within a group were counted as individual values for statistical analysis. Student’s t test was used to assess differences between distributions. A value of p ⬍0.05 was considered significant.

IFN-␥ intracellular staining (ICS)


One to 2 million freshly isolated rhesus PBMC were incubated with peptide pools (5 ␮g/ml of each peptide) and anti-CD28 and anti-CD49d mAbs (1 ␮g/ml each; catalog nos. 550630 and 340976, respectively; BD Biosciences) at 37°C and 5% CO2 for 1 h before the addition of brefeldin A (1 ␮g/ml, catalog no. B-7651; Sigma-Aldrich). Staphylococcal enterotoxin B (3 ␮g/ml, catalog no. S-4881; Sigma-Aldrich) and DMSO were used as positive and negative controls, respectively. The cells were incubated for an additional 12 h at 37°C in 5% CO2. PBMC were then washed and stained with surface Abs, CD3-allophycocyanin (rhesus clone FN-18, custom conjugated), CD4-PE (clone L-200, catalog no. 550630; BD Pharmingen), and CD8-PerCP (catalog no. 345774; BD Biosciences) for 30 min at room temperature. After the washing, PBMC were fixed and permeabilized (FACS Perm 2; BD Biosciences), and the IFN-␥-FITC (catalog no. AHC4338; BioSource International) Ab was added. Cells were then washed and analyzed on a FACSCalibur flow cytometer, using CellQuest software (BD Biosciences); at least 30,000 CD8⫹,CD3⫹ gated events were acquired for each sample. A threshold for positive response was set by calculating the average and SD of IFN-␥⫹CD8⫹ and IFN-␥⫹CD4⫹ T cells obtained after mock stimulation of PBMC from different animals at multiple time points. A cutoff was set at a value corresponding to the average plus 4 SDs (200 IFN-␥⫹CD8⫹ and 180 IFN-␥⫹CD4⫹ T cells) and at least three times the number of IFN-␥⫹ T cells detected in the mock control (DMSO) for each sample. IFN-␥ ICS with chimpanzee and human’s PBMC was performed exactly as described above using the following Abs: CD4-PerCP-cy5.5 (catalog no. 347324; BD Biosciences), CD8␤-PE (catalog no. 2217; Beckman Coulter), CD3-allophycocyanin (catalog no. 555335; BD Pharmingen), and IFN-␥FITC (Mabtech mAb 1-D1K-FITC F/P: 2.1). A cutoff was calculated as described above and was set at a value corresponding to the average plus 4 SDs (100 IFN-␥⫹CD8⫹ and 120 IFN-␥⫹CD4⫹ T cells) and at least three times the number of IFN-␥⫹ T cells detected in the mock control (DMSO) for each sample.

A novel codon-optimized DNA vaccine encoding for the HCV nonstructural region

Cytotoxicity assay Bulk CTL cultures set up and 51Cr CTL assay were performed as described previously (21). Generally, effector cells were prepared from a 2-wk incubation with vaccine PBMC infected previously with recombinant vaccinia virus VacHCV-NS. The harvested effector cells were tested against autologous B lymphoblastoid cell line (B-LCL) sensitized for 2 h with peptide pools (at 4 ␮g/ml for each peptide). Then, 30 ␮l of supernatants/well was transferred into a Lumaplate-96 (catalog no. 4096; Packard Instrument), and radioactivity was determined using a Top-Count microplate scintillation counter (Packard Instrument). The percent-specific lysis was calculated using the formula: (E ⫺ S)/(M ⫺ S), where E represents the average counts per minute released from target cells in the presence of effector cells, S is the spontaneous counts per minute released in the presence of medium only, and M is the maximum counts per minute released in the presence of 1% Triton X-100. CTL responses were scored positive when percent-specific lysis at the two highest E:T ratios was greater or equal to the percent lysis of control target wells plus 10.

A synthetic DNA fragment encoding a fully codon-optimized NS3-NS5B HCV sequence from a 1b genotype (BK strain) was designed and subcloned into the pV1Jns plasmid. Nucleotides 1– 6 provided for an optimized ribosome entry site and nt 7–5961 encoded for a HCV Met-NS3-NS4A-NS4B-NS5A-NS5B polypeptide. To improve the safety profile of this candidate vaccine, the original amino acid sequence GlyAspAsp at positions 1711–1713 in the catalytic site of the NS5B was substituted with AlaAlaGly to abrogate RNA-dependent RNA polymerase activity (Materials and Methods). Expression levels and poly-protein processing were assayed by transient transfection of the resulting plasmid (pV1JnsNSOPTmut) into 293 cells and Western blotting on whole cell extracts. As controls, similar plasmids encoding for the wildtype sequence with and without the NS5B-inactivating mutation were constructed (pV1JnsNSmut and pV1JnsNS, respectively) and tested in parallel. As shown in Fig. 1, mature NS3 and NS5A products were detected with specific Abs in extracts from 293 cells transfected with each of the three plasmids, confirming that the NS poly-protein was processed correctly. Comparable levels of expression were displayed by the two plasmids encoding for the nonoptimized NS sequence, indicating that mutation of the NS5B does not affect expression and proteolytic cleavage by the cis-encoded NS3 viral protease. In contrast, codon optimization led to ⬃4-fold increase in the expression levels while maintaining the expected pattern of poly-protein processing. GET of the pV1JnsNSOPTmut DNA vaccine induces potent and broad T cell responses in mice We previously reported that GET improves B and T cell immune responses to a DNA plasmid (pV1JnsE2), encoding for a secreted version of the HCV E2 envelope glycoprotein upon injection into the muscle of mice and rabbits (14). To test the potential of GET in enhancing cellular immune responses against the HCV nonstructural proteins, we performed extensive immunization studies

BrdU proliferation assay PBMC from immunized chimpanzee were plated in 24-well plates (2 million/well in 1 ml of medium) with peptide pool (5 ␮g/ml final concentration of each peptide) or equivalent volume of DMSO as negative control and anti-CD28 at 10 ␮g/ml final concentration. Plates were incubated for 4 days at 37°C and 5% CO2. BrdU (10 ␮M final, FITC BrdU Flow kit, catalog no. 559619; BD Biosciences) was added for the last 14 h of incubation. Cells were then harvested, washed, and stained for surface Abs (CD3, CD4, and CD8␤, same Abs used in IFN-␥-ICS) for 30 min at room temperature. After washing, cells were fixed and permeabilized as described in the assay kit. DNase I treatment and BrdU staining were also performed according to kit instructions using the kit reagents. Cells were washed and analyzed on a FACSCalibur flow cytometer, using CellQuest software (BD Biosciences). Results are expressed as stimulation index (SI): percentage of BrdU⫹/CD8⫹ or CD4⫹ cells divided by percentage in mock stimulations (DMSO). A SI ⬎ 4 was considered positive.

FIGURE 1. Expression and processing of NS proteins by transfection of 293 cells with HCV-NS DNA plasmids. Western blot on whole cell extracts from 293 cells transfected with plasmid DNA expressing the different HCV NS cassettes. Whole cell extracts were normalized for transfection efficiency by measuring the luciferase activity from a cotransfected vector. Two-fold dilutions of each extract were tested (indicated at the top of each lane). Mature NS3 and NS5A proteins (indicated by arrows) were detected with specific Abs. Extract from mock-transfected cells was used as a negative control (mock). Molecular weight markers are indicated on the left side of each panel.

The Journal of Immunology


in mice with the pV1JnsNS plasmid with or without electrical stimulation. T cell responses were measured by ex vivo IFN-␥ ELISPOT assays on splenocytes from immunized animals using a panel of six peptide pools (NS3p, NS3h, NS4, NS5A, NS5B-I, and NS5B-II) spanning the HCV NS3-NS5B region. The results of this experiment indicated that GET induced broad (Fig. 2A) and more potent (⬃5-fold, p ⫽ 0.02; Fig. 2B) T cell responses than DNA injection alone. We then wanted to compare immunogenicity of pV1JnsNS and pV1JnsNSOPTmut to verify if the increase in expression levels of the HCV proteins by the optimized version of the construct observed in vitro resulted in a stronger induction of immune response in vivo. To this end, we immunized groups of 10 BALB/c mice with two injections of each construct (50 ␮g/injection), followed by GET, and monitored IFN-␥ production by ELISPOT. As shown in Fig. 2C, a significant improvement of the cellular immune response was observed when using pV1JnsNSOPTmut as vaccine ( p ⫽ 0.004). GET of the pV1JnsNSOPTmut DNA vaccine induces potent and broad T cell responses in rhesus macaques A crucial question we wished to address is whether the GET protocol would improve T cell responses induced by the pV1JnsNSOPTmut plasmid in nonhuman primates. To this end, we used an improved GET protocol lasting only 3 s (15). Two groups of rhesus macaques were immunized with 5 mg of pV1JnsNSOPTmut with or without electrical stimulation (n ⫽ 4 and 3, respectively) at weeks 0, 4, and 8 (Table I). Another group of rhesus macaques (n ⫽ 4) received 1010 viral particle of MRKAd6NSmut at weeks 0 and 4. This schedule and dosage proved to be very effective at inducing a strong cellular immune response in rhesus macaques (17). Serial PBMC samples were collected at periodic intervals after vaccination and tested for the induction of HCV-specific T cells with effector function by IFN-␥ ELISPOT assay. When compared with naked DNA, GET treatment led to a faster kinetics of response with three animals showing anti-HCV cellular-mediated immunity (CMI) already after the first injection with an average of 463 spot-forming cells (SFC) per 106 PBMC. All macaques in this immunization group eventually developed a response after the second dose, and the response peaked (average of 3,557 SFC per 106 PBMC) after the third dose (see Fig. 4A and data not shown). In contrast, only two of three animals in the DNA immunization group without GET responded to the vaccine after two doses (average of 408 SFC per 106 PBMC), and the response did not increase after an additional injection. The third animal did not respond even after the third injection. As expected, all four macaques immunized with MRKAd6NSmut responded already after the first administration of the vector, and the second dose did not improve the overall response strength, presumably due to anti-vector Abs reducing infectivity of the second dose. Fig. 3A shows peak ELISPOT responses against HCV peptide pools in individual macaques (postdose 3 for groups A and B; postdose 1 for group C). A strong difference in the potency ( p ⫽ 0.002) and breadth of response was observed between the two DNA immunization groups. In fact, GET treatment induced numbers of IFN-␥-positive T cells against individual peptide pools ranging from 83 to ⬎3,000 SFC/106 PBMC. Moreover, all animals developed a T cell response targeting all six different HCV Ags. In contrast, naked DNA immunization induced a weaker T cell response, which was directed against one or two Ags. The immune response in GET-treated macaques was even stronger than that induced by MRKAd6NSmut vaccination ( p ⫽ 0.05). In this last group, responses against individual peptide pools ranged between 82 and 1,329 SFC/106 PBMC, and two to five Ags per macaque were recognized.

FIGURE 2. Immunogenicity of HCV-NS DNA plasmids in mice. A, GET of pV1JnsNS induces a broad response in BALB/c mice. IFN-␥ ELISPOT responses to NS peptide pools 3 wk after boosting in two groups of four mice immunized with or without GET treatment. Results are expressed as IFN-␥ SFC per 106 splenocytes and are reported on the vertical axis. Each bar represents the average response from four mice per group to individual NS peptide pools and to DMSO (negative control) ⫾ SD. B, GET treatment enhances immunogenicity of pV1JnsNS. IFN-␥ ELISPOT responses to NS peptide pools 3 wk after boosting. The total anti-NS response, calculated by adding up to the responses to the six NS peptide pools and subtracting six times the response to DMSO, is shown for each single mouse (Œ). Average number of spots in DMSO control samples was 2 per 106 splenocytes. The black horizontal bars represent the average response for each group. Similar results were obtained in five independent experiments. C, Codon optimization of the NS3-NS5B sequence enhances immunogenicity. Two groups of 10 mice were vaccinated with pV1JnsNS or pV1JnsNSOPTmut plus GET. Immune responses were measured by IFN-␥ ELISPOT against NS peptide pools 3 wk after boosting. Œ represents the total anti-NS responses for each single mouse. Similar results were obtained in three independent experiments.



FIGURE 3. Comparison of T cell responses induced by pV1JnsNSOPTmut vaccine with or without GET and by MRKAd6NSmut in rhesus macaques. A, IFN-␥ ELISPOT responses to NS peptide pools in rhesus macaques immunized with pV1JnsNSOPTmut plasmid DNA without any treatment, pV1JnsNSOPTmut with GET, and with MRKAd6NSmut. Peak ELISPOT responses after priming (week 12 for groups A and B; week 4 for group C) are shown for individual macaques. Results are expressed as IFN-␥ SFC per 106 PBMC. Each bar represents the response to the individual NS peptide pools and to DMSO (negative control). Average number of SFC/106 PBMC in DMSO-stimulated wells was 11. B, IFN-␥ ICS responses to NS peptide pools in rhesus macaques immunized with pV1JnsNSOPTmut plasmid DNA without any treatment, pV1JnsNSOPTmut with GET, and with MRKAd6NSmut. Each bar represents the total anti-NS response for each subset (CD8 or CD4), calculated by adding up the responses to individual NS peptide pools and subtracting six times the response to DMSO. Data are expressed as number of IFN-␥⫹/CD3⫹/CD8⫹ (f) or CD4⫹ (䡺) per 106 lymphocytes. Average numbers of IFN-␥⫹/CD3⫹/CD8⫹ or CD4⫹ per 106 lymphocytes in DMSO stimulated wells were 58 and 39, respectively.

GET of the pV1JnsNSOPTmut DNA vaccine induces more potent CD4⫹ responses than naked DNA or Adenovirus immunization A second crucial question we wished to address is whether the GET protocol would induce a complete repertoire of Ag-specific T lymphocytes consisting of both helper (CD4⫹) and cytotoxic (CD8⫹) T cells. The relative contribution of the two T cell subsets to the overall anti-HCV NS T cell response was evaluated by IFN-␥ ICS of PBMC collected at time (T) ⫽ 12 wk for DNA and at T ⫽ 8 for MRKAd6NSmut-immunized macaques. All animals immunized with GET showed a strong response mediated by both CD4⫹ and CD8⫹ T cells. Fig. 3B shows the total HCV Ag-specific response for each T cell subset, calculated as the sum of the reactivities against each individual positive peptide pool (from 2,827 to 21,862 IFN-␥⫹CD8⫹/106 lymphocytes and from 2,357 to 5,478 IFN-␥⫹CD4⫹/106 lymphocytes). A weaker CD8⫹ T cell response was detected in two of three naked DNA-vaccinated animals ( p ⫽ 0.02). Remarkably, no or weak HCV-specific CD4⫹ T cell response could be measured in the naked DNA and MRKAd6NSmut immunization groups. These data showed that GET strategy was significantly more potent in inducing CD4⫹ Th1 response to HCV than naked DNA or adenoviral vector ( p ⫽ 0.0001 and p ⫽ 0.0003, respectively).

Strenght, breadth, and longevity of GET induced immune responses after boost in rhesus macaques Animals in the GET immunization group were given a booster injection at week 24 (see Table I) after a slow decline in immune responses, and HCV-specific T cell responses were followed prospectively by IFN-␥ ELISPOT and ICS. After boosting, T cell responses peaked at week 28 (average of the total reactivity in the four animals: 2,188 SFC/106 PBMC; Fig. 4A) with values of circulating Ag-specific IFN-␥⫹ T cells up to 4,417/106 lymphocytes (macaque C035) and 28,220/106 lymphocytes (macaque C145) for the CD4⫹ and CD8⫹ subsets, respectively (Fig. 4B). Vaccine-induced CMI was sustained for the following 5 mo with numbers of SFC/106PBMC ⬎ 1,000 (Fig. 4A). The longevity of both T cell subsets was tested by IFN-␥ ICS at week 46 and showed that all animals had high levels of both CD4⫹ and CD8⫹ effector T cells with total HCV-specific T lymphocytes up to 19,713 for CD8⫹ and to 4,802 for CD4⫹ (data not shown). HCV NS-specific T cells elicited by GET in rhesus macaques displayed cytotoxic activity, as assessed by bulk CTL assays using PBMC collected from immunized animals at several time points after priming and boosting immunizations. At week 38, all macaques exhibited strong CTL activity with levels of specific lysis ranging from 20 to 60% at an E:T ratio of 40:1 (Fig. 4C). The

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FIGURE 4. Characterization of GET-induced immune responses after boost in rhesus macaques. A, Longevity of the anti-HCV T cell response induced by GET of the pV1JnsNSOPTmut vaccine. The immune response was measured by the IFN-␥ ELISPOT assay. The average of the total anti-NS response and the SD among the four treated animals during 50 wk of follow-up is shown. The total anti-NS response was calculated for each time point and each animal by adding up responses to individuals NS peptide pools and correcting for DMSO background. Numbers on the vertical axis represent SFC per 106 PBMC and arrows indicate time points of each immunization. B, HCV-specific CD4⫹ and CD8⫹ T cell responses in GET-treated macaques after boosting (T ⫽ 28 wk). Results are expressed as numbers of IFN-␥⫹/CD3⫹/CD8⫹ or CD4⫹ per 106 lymphocytes. Each bar represents either CD8⫹ or CD4⫹ responses to the individual NS peptide pools and to DMSO (negative control). C, Bulk cytotoxicity assay in GET-treated macaques. PBMC collected at T ⫽ 38 wk were restimulated in vitro with VacHCV-NS and then tested for their ability to kill autologous B-LCL pulsed with either DMSO alone or with the individual NS peptide pools. Results are expressed as percentage of specific lysis at each E:T ratio tested.

animals displayed cytotoxic activity in response to one (C035), two (C145 and C010), and four HCV Ags (C034). Immunization of chimpanzees by GET elicited anti-HCV CMI qualitatively and quantitatively comparable to that observed in acute/resolving-infected humans To evaluate the immunological potency of the pV1JnsNSOPTmut vaccine candidate in a human-like species, we immunized two adult chimpanzees with 5 mg of pV1JnsNSOPTmut with GET at weeks 0, 4, 8, and 16. To exclude a prior HCV infection, the two chimpanzees were screened for the presence of anti-HCV Abs using commercially available ELISA and all scored negative. Moreover, HCV-specific T cell response was tested at two time points before vaccination with peptide pools covering core and NS pro-

teins. Similar background responses were detected with all tested HCV peptide pools and controls (DMSO and an unrelated peptide pool). After vaccination, we collected serial PBMC samples and tested them by IFN-␥ ICS. The vaccine induced a strong and polyspecific CD8⫹ T cell response in chimp Ch152 targeting NS3p, NS3h, NS4, NS5A, and NS5B Ags with values of circulating Agspecific CD8⫹IFN-␥⫹ T cells/106 lymphocytes ranging from 154 to 1,823 (Fig. 5A). Weaker but significant CD4⫹ responses were also measured against NS3p, NS3h, NS4, and NS5A. In the second chimpanzee (Ch208), CD8⫹ and CD4⫹ T cell responses targeted mostly NS5A. The effector function of HCV-specific CD8⫹ in vaccinated chimpanzees was confirmed by bulk CTL assay using autologous BLCL pulsed with the NS3p and NS5A peptide pools (Fig. 5B).



FIGURE 5. Immune response induced by GET in chimpanzees. A, HCV-specific CD4⫹ and CD8⫹ T cell responses in two chimpanzees vaccinated with pV1JnsNSOPTmut with GET by IFN-␥ ICS. Each bar represents the number of IFN-␥⫹/CD3⫹/CD8⫹ or CD4⫹ per 106 lymphocytes after stimulation with the individual NS peptide pools and DMSO. B, Bulk cytotoxicity assay in GET-treated chimpanzees. PBMC collected at T ⫽ 21 wk were restimulated in vitro with VacHCV-NS and then tested for their ability to kill autologous B-LCL pulsed with either DMSO alone or with selected NS peptide pools. Results are expressed as percentage of specific lysis at each E:T ratio tested. C, HCV-specific CD4⫹ and CD8⫹ T cell responses in five acutely infected human subjects that spontaneously resolved the infection (H24, H50, H25, H46, and H43). Each bar represents the number of IFN-␥⫹/CD3⫹/CD8⫹ or CD4⫹ per 106 lymphocytes after stimulation with the individual NS peptide pools.

In a recent study, we comprehensively analyzed HCV-specific T cell response in a large cohort of individuals during the natural course of acute infection (22). To compare vaccine-induced CMI with that observed in acute/resolving subjects, we performed IFN-␥ ICS assays on PBMC from five such individuals tested at peak of immune response before resolution using the same procedure and peptide pools used for the evaluation of CMI in immunized chimpanzees. The results of this comparative analysis indicated that the levels of CD8⫹ and CD4⫹ T lymphocytes with ability to secrete IFN-␥ in response to HCV peptide pools are very similar between vaccinated chimpanzees and at least a subset of infected humans progressing toward virus clearance (Fig. 5C). One of the immunological hallmarks of a resolving infection in humans is the ability of virus-specific T cells to proliferate in vitro in response to Ag stimulation (22–25). Therefore, we performed proliferative assays on PBMC from immunized chimpanzees at T ⫽ 34 wk. To this end, the HCV peptide pools were used to stimulate the PBMC in an ex vivo cytofluorometric proliferation assay based on BrdU incorporation. This approach allowed the evaluation of the individual contribution of CD4⫹ and CD8⫹ T cells to the proliferative response. Proliferation of both subsets of HCV-specific T lymphocytes was measured in both animals, and SI were comparable to those observed by performing the same assays with PBMC from acute/resolving HCV-infected individuals (Fig. 6). Thus, i.m. injection of the pV1JnsNSOPTmut vaccine candidate followed by GET is capable of inducing a fully functional anti-HCV CMI in a

human-like species, which is qualitatively and quantitatively comparable to that induced in humans who can resolve the infection.

Discussion Our strategy toward developing a novel DNA vaccine against HCV was based on a three step approach: 1) the generation of a plasmid DNA encoding for a more immunogenic version of the HCV nonstructural region based on codon optimization (pV1JnsNSoptmut); 2) the development of a highly efficient in vivo DNA delivery system based on muscle GET and capable of inducing a balanced CD4 and CD8 immunity in different species, including nonhuman primates; and 3) a comparative analysis of the immune responses induced by such vaccine candidate in chimpanzees with that observed in acute/resolving individuals using the same reagents and functional T cell assays. Several studies have demonstrated that protection against chronic HCV infection requires the early generation of a potent T cell response directed against multiple viral determinants (for review, see Ref. 26). Patients with self-limited acute hepatitis C were shown to mount an early and vigorous Th response that was most frequently directed toward epitopes located within the NS3 and NS4 proteins (27). Remarkably, all mapped epitopes in these two Ags were highly conserved among HCV genotypes and bound promiscuously with a high affinity to a large panel of different HLA class II molecules. CTL epitopes have been identified in all viral proteins, but recent data collected in chronically and acutely

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FIGURE 6. HCV-specific T cell proliferation in GET-treated chimpanzees and in acutely infected human subjects that spontaneously resolved the infection. Proliferation was measured in the two chimpanzees (Ch152 and Ch208) and in human subjects (H24, H50, H25, H46, and H43) by a FACS-based BrdU incorporation assay. Results are reported as the SI. The SI was calculated as the ratio between the percentage of BrdU⫹/CD8⫹ or CD4⫹ cells measured after NS peptide pools stimulation and the percentage of BrdU⫹/CD8⫹ or CD4⫹ cells measured after mock stimulation. Responses were considered positive when the SI was ⬎4.

infected patients indicated that responses against the nonstructural region are more prevalent in the latter group (28). On the basis of these observations, we designed a novel HCV DNA vaccine vector encoding the region spanning from NS3 to NS5B (NS) of a 1b (BK strain) viral isolate. This choice was based on a number of considerations. First, the NS region is well conserved between the six HCV genotypes and several of the major subtypes (between 74 and 79% sequence identity at the amino acids level). Second, this HCV consists of ⬃2000 aa and thus contains a large amount of antigenic information. In fact, the majority of the HCV T cell epitopes described to date is contained in this part of the viral polyprotein. Third, genotype 1 is one of the most prevalent in the United States, Europe, and Japan and accounts for ⬎80% of HCV infections worldwide (29). In addition, by a comparative sequence analysis, the BK strain sequence resulted to be a very good representative of the observed variability between different genotypes and subtypes (data not shown), supporting the hypothesis that a vaccine based on this sequence may have the potential for protection against different HCV strains. Finally, by engineering a methionine at the N terminus of the NS3 sequence, we obtained a polyprotein fragment fully competent for posttranslational processing. We believe that the ability of the NS DNA vaccine to mirror the HCV maturation process upon expression in vivo represents a better way to induce an effective immunological imprinting against the real virus. Since DNA-vectored vaccines encoding sequence-modified high-level expression cassettes have been shown to improve immunization efficiency of HIV genes in mice and monkeys (30 –31), we synthesized a fully codon-optimized NS sequence and showed that the optimized vector resulted in increased in vitro expression and higher immune responses in mice as compared with the corresponding wild-type construct. Intramuscular delivery of DNA vaccines is the preferred route for eliciting T cell responses, but this approach has met with limited success in large species, including nonhuman primates, chimpanzees, and humans. A few laboratories, including our own, have shown that increasing in vivo transduction of muscle cells by GET of plasmid DNA significantly enhanced humoral and cellular immunity in mice, rabbits, guinea pigs, and pigs (14, 32–35). Very recently, the GET technology was shown to be effective in rhesus macaques for enhancing humoral and Th immune responses

7469 against HIV Ags (36). In line with these data, in the present study, we showed that GET of plasmid pV1JnsNSOPTmut in rhesus macaques greatly improved the kinetics and frequency of anti-NS CMI with respect to DNA injection without treatment. In fact, macaques immunized with plasmid DNA in combination with GET developed a T cell response already after a single injection of the HCV plasmid, whereas at least two doses were required to induce a measurable response in the naked DNA immunization group. Peak responses were seen 4 wk after the third injection. These data are in agreement with previous studies in primates showing that the kinetics of T cell responses may depend on several different parameters, including number of doses and regimen, type of vector, and adjuvants (9, 36, 38, 39). One of the most remarkable aspects of the anti-HCV CMI elicited in GET-treated animals was the large number of Ags recognized, with T cells from all immunized animals being capable of secreting IFN-␥ in response to all five NS Ags. Given the high degree of viral variability and the heterogeneity of the human MHC alleles, such a broad response induced in GET-treated animals represents a highly encouraging finding in view of potential clinical development of the present DNA vaccine candidate. A crucial factor to be considered in the design of T cell-based HCV vaccines is the ability to induce a complete repertoire of Ag-specific T lymphocytes with a balance between CD4⫹ and CD8⫹ T cell responses (37). While CD8⫹ T cells have been clearly shown to mediate HCV clearance in both humans and chimpanzees (25–26, 40), several human studies have also shown that a strong HCV-specific CD4⫹ T cell response is associated with resolution of acute infection or a benign carrier state (41– 42). The important role of CD4⫹ T cell response in viral control is also suggested by HCV recurrence after loss of virus-specific CD4⫹ T cells during acute hepatitis C (43). Moreover, recent data point to the role of CD4⫹ T cells in promoting the expansion of primary CD8⫹ T cell responses, but also in regulating the quality and longevity of CD8⫹ T memory cells (44 – 45). In the present study, we show that GET of the NS DNA vaccine in rhesus macaques could induce both CD4⫹ and CD8⫹ T cells against multiple viral determinants with robust production of IFN-␥. Notably, injection of the pV1JnsNSOPTmut plasmid without GET treatment or by a potent adenoviral vector failed to elicit a potent CD4⫹ response, further underlining the importance of GET for DNA immunization of primates. This is not a species-specific phenomenon because we obtained similar results in mice (data not shown). The extent of the induced response in GET-immunized rhesus macaques was extremely high especially after a boosting injection, as witnessed by the frequency of HCV-specific CD8⫹ T cells reaching over 11% of the total number of circulating CD8⫹ T lymphocytes. Most importantly, GET-immunized macaques maintained high levels of IFN-␥-producing anti-HCV T cells (⬎500 SFC/106PBMC on average) throughout the whole observation period, lasting more than 6 mo after the boosting immunization, thus indicating that a considerable memory T cell pool was induced by the vaccine. Induction of CD8⫹ T cells with effector function in rhesus macaques was confirmed by bulk cytotoxicity assay. It must be emphasized that the induced response effectively targeted epitopes resulting from physiological processing, since CD8⫹ killer cells were amplified using autologous PBMC infected with a vaccinia virus expressing NS before their testing. Chimpanzees are the only other species susceptible to HCV infection besides humans and currently provide the only means to evaluate safety and efficacy of prophylactic or therapeutic vaccines against HCV (46 –50). However, a head-to-head comparison of T cell responses induced in vaccinated chimpanzees and in acute/

7470 resolving humans was still missing. We immunized two chimpanzees by GET of the pV1JnsNSOPTmut vaccine vector and compared the induced HCV-specific T cell response to that of five infected humans. Potent and broad CD4⫹ and CD8⫹ T cells with ability to secrete IFN-␥ in response to HCV Ags were elicited in both chimpanzees. The functional phenotype of CD8⫹ T cells was confirmed by their being able to kill target cells in bulk cytotoxicity assays. Finally, by IFN-␥ ICS, we could show that the frequencies of Ag-specific T cells elicited by GET treatment in both chimpanzees were much higher than that measured in chronic HCV-infected humans (22, 28), but most importantly, this response had the same magnitude and breadth of that observed in five acutely infected humans experiencing a self-limiting infection. In a recently performed prospective study on a cohort of 31 acutely infected HCV individuals, we could show that the ability of HCV-specific CD4⫹ and CD8⫹ T lymphocytes to proliferate in response to Ag stimulation in vitro represents one of the immunological hallmarks of a protective CMI (22). Conversely, acutely infected individuals who eventually progress toward chronic disease only transiently develop a T cell response, which is rapidly exhausted due to an impaired ability of the HCV-specific lymphocytes to proliferate. Given this scenario, it was essential to demonstrate that GET of HCV DNA in chimpanzees could induce CD4⫹ and CD8⫹ T lymphocytes with vigorous ability to proliferate. In fact, since the pV1JnsNSOPTmut vaccine described in the present work would rely solely on T cell-dependent function to achieve virus control, the ability of vaccine-induced T cells to self-renew is expected to play a crucial role in tilting the balance between HCV replication and antiviral T cell activity toward the latter being capable of achieving disease resolution. The level of anti-HCV CMI induced in chimpanzees was somewhat lower than that observed in rhesus macaques by the same immunization strategy. However, it must be noted that in the former case ⬃10-fold lower dose/kg of the vaccine was administered. In any case, the GET strategy appears to be fairly scalable from mice to large primates, holding promise for its successful application in humans. Noteworthy, all immunizations in nonhuman primates reported in this work were performed with a novel GET protocol lasting only 3 s, which was developed to improve tolerability of the treatment (15). Relevant to this point is our recent finding from a preliminary study in humans that confirmed tolerability of the GET treatment (data not shown). The data obtained in the current studies were sufficiently encouraging to move the GET/DNA approach forward into a chimpanzee challenge model with HCV (50). The results from this study showed that a T cell-based genetic vaccine was effective in protecting the animals against challenge with a heterologous viral strain.

Acknowledgments We acknowledge Dr. B. Rosenwirth, H. Oostermeijer, and the staff at the Biomedical Primate Research Centre (Rijswijk, The Netherlands) for the excellent care of macaques and samples and M. Emili for artwork.

Disclosures The authors have no financial conflict of interest.

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