Induction of Broad CD4+ and CD8+ T-Cell Responses and Cross- Neutralizing Antibodies against...

JOURNAL OF VIROLOGY, Aug. 2008, p. 7492–7503 Vol. 82, No. 150022-538X/08/$08.00�0 doi:10.1128/JVI.02743-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Induction of Broad CD4� and CD8� T-Cell Responses and Cross-Neutralizing Antibodies against Hepatitis C Virus by Vaccination

with Th1-Adjuvanted Polypeptides Followed by Defective AlphaviralParticles Expressing Envelope Glycoproteins gpE1 and gpE2

and Nonstructural Proteins 3, 4, and 5�†Yinling Lin,* Taewoo Kwon, John Polo, Yi-Fei Zhu, Stephen Coates, Kevin Crawford, Christine Dong,

Mark Wininger, John Hall, Mark Selby, Doris Coit, Angelica Medina-Selby, Colin McCoin, Philip Ng,Debbie Drane, David Chien, Jang Han, Michael Vajdy, and Michael Houghton‡

Novartis Vaccine and Diagnostic, Inc., Emeryville, California 94608

Received 26 December 2007/Accepted 19 May 2008

Broad, multispecific CD4� and CD8� T-cell responses to the hepatitis C virus (HCV), as well as virus-cross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated inchronic HCV patients with a favorable response to antiviral treatment. In order to recapitulate all of theseresponses in an ideal vaccine regimen, we have explored the use of recombinant HCV polypeptides combinedwith various Th1-type adjuvants and replication-defective alphaviral particles encoding HCV proteins invarious prime/boost modalities in BALB/c mice. Defective chimeric alphaviral particles derived from theSindbis and Venezuelan equine encephalitis viruses encoding either the HCV envelope glycoprotein gpE1/gpE2heterodimer (E1E2) or nonstructural proteins 3, 4, and 5 (NS345) elicited strong CD8� T-cell responses butlow CD4� T helper responses to these HCV gene products. In contrast, recombinant E1E2 glycoproteinsadjuvanted with MF59 containing a CpG oligonucleotide elicited strong CD4� T helper responses but no CD8�

T-cell responses. A recombinant NS345 polyprotein also stimulated strong CD4� T helper responses but noCD8� T-cell responses when adjuvanted with Iscomatrix containing CpG. Optimal elicitation of broad CD4�

and CD8� T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteinsand then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologousHCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protectionagainst this highly heterogeneous global pathogen.

The hepatitis C virus (HCV) is responsible for essentially allparentally transmitted non-A, non-B hepatitis cases. An esti-mated 170 million humans, or 3% of the world’s population,are infected with HCV, with an even higher prevalence in thedeveloping parts of the world (27). There is no vaccine avail-able, and the standard combination treatment with pegylatedinterferon (IFN) and ribavirin is curative in less than one-halfof all HCV patients (16). There is therefore an urgent need foralternative therapies and effective prophylactic vaccines.

A key feature of most vaccines is the induction of neutral-izing antibodies. In many cases, infusion of neutralizing anti-bodies is also used for passive postexposure prophylaxis. Pre-clinical studies with chimpanzees have indicated the ability ofpolyclonal antibodies derived from plasma of HCV-infectedpatients to prevent or delay HCV infection. The antibodieswere shown to prevent or delay the onset of acute hepatitis C

when given before or soon after inoculation of chimpanzeeswith the virus (13, 14, 22, 63). In addition, vaccination ofchimpanzees with recombinant HCV envelope glycoproteinsgpE1 and gpE2 induced strong antibody responses that pre-vented infection from a homologous viral (HCV-1) challenge(8). The HCV 1a strain predominates in the United States.Subsequent studies in which animals were vaccinated with ad-juvanted, clade 1a-derived gpE1/gpE2 and then challengedwith a heterologous 1a viral strain demonstrated a substantialand statistically significant reduction in the carrier rate of thevaccinees versus a control, unimmunized group of chimpan-zees (9, 20). Recently, it was also demonstrated that a sus-tained anti-E2 antibody response correlates with reduced peakviremia after HCV infection in the chimpanzee (62). Recentstudies have also correlated the early induction of HCV cross-neutralizing antibody with recovery from acute infection inhumans (28, 39).

Other studies have emphasized the role of the cellular im-mune response in protection against HCV by showing thatbroad, multispecific CD4� and CD8� T-cell responses to thevirus are associated with naturally resolving infection (10, 11,12, 15, 17, 29). Furthermore, a series of rechallenge studieswith chimpanzees that recovered spontaneously, in which theCD4� or CD8� T-cell compartments were first depleted, have

* Corresponding author. Mailing address: 4 Captain Dr. 411, Em-eryville, CA 94608-2916. Phone: (510) 923-8170. Fax: (510) 923-2586.E-mail: [email protected].

† Supplemental material for this article may be found at http://jvi.asm.org/.

‡ Present address: Epiphany Biosciences Inc., 1 California St., Suite2800, San Francisco, CA 94111.

� Published ahead of print on 28 May 2008.

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demonstrated the crucial role of both of these cell types inprotective immunity against HCV infection (17, 52). This alsohas been successfully adopted in a vaccine approach using aprime/boost immunization regimen utilizing adenovirus andplasmid DNA expressing HCV nonstructural genes 3, 4, and 5.Most of the naı̈ve chimpanzees vaccinated in this way wereprotected against the onset of chronic hepatitis and viremiafollowing an experimental challenge with a highly heterologousHCV strain (5). Thus, HCV immunogens able to elicit strongand broad cell-mediated immunity, as well as cross-neutraliz-ing antibodies, may represent the optimal approach to HCVvaccination (20).

Replication-defective alphaviral vectors have been shown toinduce robust cellular, humoral, and mucosal immune re-sponses specific to the replicon-expressed antigen in severalanimal models (6, 18, 21, 38, 42). A number of features makealphavirus replicon vectors attractive for gene-based vaccines,including high-level expression of the heterologous gene, vec-tor amplification through double-stranded RNA intermediates(which stimulates aspects of innate immunity such as activationof the IFN cascade), induction of apoptosis in some cell types(which may enhance immunogenicity via antigen cross-prim-ing), and the overall lack of preexisting immunity in the humanpopulation (42). In addition, alphavirus replicon particles canbe used for the delivery of antigen to antigen-presenting cellssuch as dendritic cells (42). It is also possible to repeat immu-nize with defective alphavirus particles (42).

Alphaviruses such as Semliki Forest virus, Venezuelanequine encephalitis virus (VEE), and Sindbis virus (SIN) haveall been developed as potential vaccine and gene therapy vec-tors. Each alphavirus has unique properties in terms of func-tion, potency, and safety when applied to recombinant vectorsystems. VEE replicon particles have been shown to inducepotent and protective immune responses in primates. How-ever, production of VEE replicon particles must be conductedat biosafety level III (38). In contrast, since SIN is not associ-ated with serious human disease, the use of SIN-derived rep-licon particles largely obviates safety concerns. In addition,particular engineered SIN variants target lymphoid cells, andstable replicon-packaging cell lines have been developed (41),which may simplify large-scale production for human testing ofvaccine candidates. A novel VEE/SIN defective-particle chi-mera that comprises SIN structural outer membrane proteinsencapsidating VEE replicon RNA (38) was used in this studyto express the HCV E1E2 envelope or nonstructural NS345protein.

MF59, CpG, and Iscomatrix have been used as adjuvants forvarious recombinant proteins. The oil-in-water adjuvant MF59(34) has been shown to be a potent stimulator of cellular andhumoral responses to subunit antigens in both animal modelsand clinical studies (34) and is licensed for human use in theEuropean Union as part of an influenza vaccine. RecombinantHCV gpE1/gpE2 glycoproteins combined with MF59-like ad-juvant were shown to be protective against both homologousand heterologous viral challenges (8, 9, 20). CpG 7909 is animmunomodulating synthetic oligonucleotide designed to spe-cifically target Toll-like receptor 9 in dendritic antigen-present-ing cells and augments the adaptive immune response to vac-cine antigens (23). It also activates memory B cells (24) andthus is a promising adjuvant for vaccines against cancer and

infectious diseases (2). Iscomatrix (36, 37, 40) comprises phos-pholipid-cholesterol particles containing purified saponins.When it was combined with a recombinant HCV nucleocapsidprotein (C) and used to immunize rhesus macaques, verystrong C-specific Th1-type CD4� T helper cells were elicited,as well as long-lived CD8� cytotoxic T cells (40).

In the present study, we have explored the use of adjuvantedrecombinant HCV proteins and defective alphaviral vectors toelicit cross-neutralizing antibodies and broad cellular immuneresponses to HCV in BALB/c mice. We show that a prime/boost regimen can elicit all of the humoral and cellular im-mune responses known to be associated with protectionagainst this common and global pathogen.

MATERIALS AND METHODS

Replicon vectors and defective helper constructs. E1E2 (amino acids [aa] 192to 746) was generated by PCR amplification of pCMVtpaE1E2p7, which con-tains the upstream tissue plasminogen activator (TPA) signal sequence at the 5�end and E1E2p7 (aa 192 to 809) from HCV strain 1a (32, 44, 55), and the cDNAwas inserted into the VCR-Chim2.1 replicon vectors (38), resulting in the con-struct VCRChim-E1E2 (aa 192 to 746). NS345 generated by PCR amplificationof HCV strain 1a cDNA (7) and the cDNA (aa 1027 to 3012) were inserted intothe VCR-Chim2.1 replicon vectors, resulting in construct VCRChim-NS345.Sequences encoding either capsid or envelope glycoproteins from SIN wereinserted into the VEE-based defective helper backbone (VCR-DH) and namedVCR-DH-Scap and VCR-DH-Sgly (38).

Production of alphavirus replicon particles. Replicon particles were gener-ated by coelectroporation of in vitro-transcribed RNAs corresponding to a rep-licon vector (VCRChim-E1E2or VCRChim-NS345) and two defective helpers(VCR-DH-Scap and VCR-DH-Sgly) (38). Replicon particles (VEE/SIN-E1E2and VEE/SIN-NS345) expressing HCV E1E2 and NS345 were harvested asculture supernatants at 24 h postelectroporation, clarified by filtration, andpurified by cation-exchange chromatography. Replicon particle titers, reportedin infectious units (IU) per milliliter, were determined by intracellular staining(ICS) of expressed HCV proteins (E1 and E2 for VEE/SIN-E1E2; NS3, NS4,NS5A, and NS5B for VEE/SIN-NS345) following overnight infection of BHK-21cells (38). Infected cells were permeabilized and fixed by using a Cytofix/Cyto-perm kit (Pharmingen) and then stained with fluorescein isothiocyanate-conju-gated antibodies to HCV antigen. By flow cytometry analysis, the percentage ofHCV protein-positive cells was determined and used to calculate titers. Theabsence of contaminating replication-competent virus was established by fiveconsecutive infections of naive BHK-21 cells and determination of titers. Finally,endotoxin levels were measured for all replicon particle samples and shown to be�0.03 U/ml.

HCV recombinant proteins. HCV strain 1a E1E2 contains E1E2p7 (aa 192 to809) with the TPA at the 5� end. E1E2 was expressed from recombinant CHOcells and extracted with Triton X-100 detergent. The expression and purificationmethods used have been described previously (32). HCV polyprotein NS345core(HCV strain 1a NS345core, aa 1018 to 3012 and 1 to 191) (59) contains HCVstrain 1a NS3 to NS5 and has the core protein at the C terminus (59). Themethods used for plasmid construction, protein expression, and purification fromyeast cells have been described in detail previously (59).

Adjuvants. MF59 was used at a 1:1 dilution with the immunogens immediatelybefore immunization. CpG oligodeoxynucleotides (CpG) 7909 (10 �g per dose;Coley Pharmaceutical Group, Wellesley, MA) (35) were mixed with the proteinplus MF59 or Iscomatrix (5 �g per mouse) immediately before immunization.Iscomatrix was diluted with isotonic buffer (sodium chloride at 145 mM, diso-dium hydrogen phosphate anhydrate at 2.5 mM and sodium dihydrogen phos-phate dihydrate at 7.7 mM; pH 6.2) to 100 �g/ml and used at a 1:1 dilution withisotonic-buffer-diluted polyprotein immediately before immunization.

Immunizations. Ten female BALB/c mice per group were injected intramus-cularly in the tibialis anterior muscle with a total volume of 100 �l (i.e., 50 �l perthigh) of the indicated vaccine formulations at weeks 0, 3, and 6, and serumsamples were collected at week 8. For the prime-boost studies, the mice wereprimed at weeks 0 and 3 and boosted at week 6. We used 4 � 106 IU ofVEE/SIN-E1E2 and 2 �g of HCV strain 1a E1E2 protein (E1E2P7 derived fromE1E2-809) (55) mixed with MF59 in the presence or absence of CpG for injec-tion. The mice were sacrificed at week 8, and the spleens were harvested forfurther assays. For nonstructural protein, 5 � 106 replication particles of VEE/

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SIN-NS345 and 50 �g of polyprotein were mixed with 5 �g of Iscomatrix (fromCSL) (36, 37, 40) for injection. The mice were injected and sacrificed at week 8to detect T-cell responses in the spleen and antibody responses in serum.

ICS. Spleen cells (1 � 106) were stimulated with the indicated peptides orproteins at 10 �g/ml (Table 1) for 6 h at 37°C in the presence of anti-CD28antibody (1 �g/ml) (BD Biosciences, San Jose, CA) and brefeldin A (BD Bio-sciences, San Jose, CA) and then stained with antibodies against CD4 (anti-CD4PerCP-Cy5.5 conjugate, clone RM4-5; BD Pharmingen) and CD8 (anti-CD8�fluorescein isothiocyanate conjugate, clone 53-6.7; BD Pharmingen), perme-abilized with Cytofix/Cytoperm (Pharmingen), and IFN-� (clone XMG1.2, allo-phycocyanin conjugate; BD Pharmingen). Stained cells were analyzed with aFACScalibur flow cytometer (BD Bioscience). The mean frequencies of cyto-kine-positive cells were calculated for each pair of duplicates. The antigen-specific frequency was determined by comparing the unstimulated mean fre-quency (no peptide) with the stimulated mean frequency (with HCV peptides,Table 1), and P � 0.05 is considered statistically significant by t test (Table 1).

CD81 inhibition assay. E1E2 protein at 5 �g/ml was mixed with 5E5/H7*Eu3�

(anti-E2 specific monoclonal antibody labeled with europium) at 0.33 �g/ml andthen incubated with serial diluted serum samples. The mixture was transferred toCD81 recombinant receptor-coated 96-well plates. After washing, the plateswere treated with Enhancement Solution (Wallac) and then placed in a Wallac1420 Multilabel Counter for reading by the europium protocol. Fifty percentinhibition multiplied by the dilution factor is used to estimate the CD81 titer.The preimmunized blood of a given subject at the assay dilution of 1:10 is usedas the negative control (1, 19, 47).

Luminex multiplex cytokine assay. Spleen cells (1 � 106) were stimulated withHCV peptides or proteins at 10 �g/ml (Table 1) for 24 h at 37°C in the presenceof anti-CD28 antibody (1 �g/ml) (BD Biosciences, San Jose, CA), and thesupernatant was harvested and stored at �20°C for cytokine assay. The BeadlyteMouse Multi-Cytokine Flex Kit Beadlyte (Upstate, Charlottesville, VA) wasused to detect interleukin-5 (IL-5), IL-10, tumor necrosis factor alpha (TNF-�),and IFN-� levels in cell culture supernatants according to the manufacturer’sprotocol. Cytokine concentrations from the mean fluorescence values obtainedwere calculated from standard curves of each cytokine tested with MiraibioMaster Plex QT software (Miraibio, Alameda, CA).

HCVcc neutralization assay. pRlucF2Aubi-JFH1 is a full-length JFH1 genome(60) carrying Renilla luciferase (Rluc) as a reporter in a monocistronic construct(T. W. Kwon et al., unpublished data). The RlucF2Aubi-JFH1 virus (RJ1) wasgenerated by electroporation of IFN-cured Huh-7 cells with in vitro-transcribedRluc-JFH1 RNA. The conditioned medium that was harvested at 10 to 14 daysposttransfection was used as the viral stock. For the neutralization assay, Huh-7cells were seeded at 5 � 104 per well in a 24-well plate the day before infection.Mouse serum was mixed with viral medium (900 tissue culture infective doses/ml) at 1:100 and incubated at 37°C for 1 h. A 200-�l volume of a virus-serummixture per well was applied to Huh-7 cells. After a 4-h infection at 37°C, theviral medium was removed and replaced with fresh complete cell medium (Dul-becco modified Eagle medium–10% fetal bovine serum). As an indication ofinfection, Renilla luciferase activity was measured 3 days postinfection by usingthe Renilla luciferase assay system (Promega).

Statistical analysis. For antibody responses, each individual animal immuneresponse was counted as an individual value for statistical analysis. The signifi-cance of the responses was calculated by Student’s t test (two tailed, type 2) with

Prism (version 3.0 for Windows; GraphPad, San Diego, CA). A P value of �0.05was considered statistically significant.

RESULTS

Immunization with VEE/SIN particles is optimal for elicit-ing E1E2-specific CD8� T cells. Defective alphavirus particlesexpressing HCV E1E2 (aa 192 to 746) and NS345 (aa 1027 to3012) were generated and designated VEE/SIN-E1E2 andVEE/SIN-NS345. The expression of glycoproteins E1 and E2,as well as NS3, NS4, NS5A, and NS5B, from the replicons wasconfirmed by Western blotting (see Fig. S1 in the supplementalmaterial). The particular antigen-adjuvant combinations usedin this study were based on individual formulations currentlybeing tested in clinical trials. Anticipated prophylactic formu-lations comprising E1E2 with MF59 have been used to vacci-nate humans in the presence or absence of CpG with a view toeliciting broadly cross-neutralizing antibodies, whereas poten-tial therapeutic vaccine formulations have focused on the useof the Iscomatrix adjuvant to boost optimal cellular immuneresponses in HCV-infected patients (unpublished data).

To compare the immune responses elicited by defectiveVEE/SIN vectors encoding E1E2 with adjuvanted E1E2 pro-tein, mice were immunized three times either with 4 � 106 IUVEE/SIN-E1E2 particles or with recombinant E1E2 (aa 192 to809) protein adjuvanted with MF59 (with or without CpG)(35). Spleen cells were assessed 14 days after the final immu-nization with each vaccine. The cells were stimulated eitherwith HCV strain 1a-specific peptide pools encompassing E1 orE2 or with single peptides known to represent CD4� or CD8�

T-cell epitopes for E2 in BALB/c mice (Table 1). Negativecontrols consisted of cells treated with medium alone or cellsfrom phosphate-buffered saline (PBS)-injected mice stimu-lated in vitro with HCV peptides. In contrast to MF59-adju-vanted recombinant E1E2 immunizations, VEE/SIN-E1E2 im-munizations stimulated readily detectable HCV-specific CD8�

T-cell responses to the E1 peptide pool (64) (Fig. 1A and B;see Fig. S3 in the supplemental material). MF59-adjuvantedE1E2, in either the presence or the absence of CpG, did notstimulate significant HCV-specific CD8� T-cell responses (Fig.1A and B; see Fig. S3 in the supplemental material). Twoimmunizations with E1E2/MF59/CpG followed by a boost withVEE/SIN-E1E2 produced strong CD8� responses to an E2

TABLE 1. Peptides and proteins used in this study

Name Peptide or protein information Reference(s)

E1 pool 20-mer overlapping peptides covering E1 region 64E2 pool 20-mer overlapping peptides covering E2 region 64CD4 E2 pep QTHTTGGQAGHQAHSLTGLFSPGAKQN 43, 64CD8 E2 pep DATYSRCGSGPWITPRCLVD 64NS3 pool 20-mer overlapping peptides covering NS3 53, 61NS4 pool 20-mer overlapping peptides covering NS4 53, 61NS5A pool 20-mer overlapping peptides covering NS5A 53, 61NS5B pool 20-mer overlapping peptides covering NS5B 53, 61NS3-1 pep LVALGINAVAYYRGL 54NS3-2 pep TTVRLRAYMNTPGLP 54NS3-3 pep SSPPVVPQSF 3, 4NS5B pep MSYSWTGALVTPCAAE 51, 58SOD-C100 (NS34) Recombinant NS34 protein purified from yeast (HCV strain 1a aa 1569–1931) 25, 30SOD-NS5 Recombinant NS5A/B protein purified from yeast (HCV strain 1a aa 2054–2995) 48

7494 LIN ET AL. J. VIROL.

epitope (Fig. 1C; see Fig. S2B and S3 in the supplementalmaterial). This could also be accomplished by administeringtwo immunizations with E1E2/MF59 followed by a third im-munization with VEE/SIN-E1E2 (Fig. 1B and C). This re-sponse was greater than that obtained with VEE/SIN-E1E2alone, although surprisingly, CD8� responses to the E1 poolwere reduced by the vaccine combination regimen.

A prime/boost regimen with E1E2/MF59/CpG followed byVEE/SIN-E1E2 elicits strong CD4� responses. To analyzeHCV-specific CD4� T-cell responses after immunization,spleen cells were stained for CD4 and IFN-� after in vitrostimulation with HCV-specific peptides (Table 1). Spleen cellsobtained from PBS-immunized mice and from vaccinated micewithout in vitro peptide stimulation (medium) were both usedas negative controls. Immunization with E1E2/MF59/CpGclearly elicited the strongest CD4� responses to the E1 pool,with weaker responses to the E2 pool (P � 0.05 compared withE1E2/MF59 or VEE/SIN-E1E2 immunization; Fig. 1D; seeFig. S2A in the supplemental material). These responses werealso obtained in mice that were primed by two immunizationswith E1E2/MF59/CpG followed by boosting with VEE/SIN-E1E2 (Fig. 1D). Interestingly, lower responses were obtained ifVEE/SIN-E1E2 was used to prime CD4� responses prior toboosting with E1E2/MF59/CpG (Fig. 1D). Therefore, an op-timal prime/boost regimen for eliciting E1E2-specific CD4� Tcells (priming with recombinant E1E2 adjuvanted with MF59-CpG followed by boosting with VEE/SIN-E1E2) was also

nearly optimal at eliciting E2-specific CD8� T cells (Fig. 1C;see Fig. S2B and S3 in the supplemental material). It should benoted that VEE/SIN-E1E2 induced a high, peptide-indepen-dent background of IFN-� production in CD4� T cells ingeneral, but no HCV-specific CD4� T-cell responses weredetected (Fig. 1D).

CD4� Th1 cells assist in priming antigen-specific CTLs byactivating DCs and secreting IL-2. They are also required forthe maintenance of CD8� T-cell number and function (33).Clinical data indicate that patients who spontaneously clearviremia have stronger multiclonal Th1-based T-cell responsesto HCV antigens than those who do not (45, 49). Therefore, weanalyzed the Th cell status of the immunized mice by measur-ing the cytokines released from the spleen cells after in vitrostimulation with HCV-specific peptides. E1E2/MF59 stimu-lated high IL-5 production but not IFN-� or TNF-�, indicatingthe induction of a Th2 response (Fig. 2). On the other hand,E1E2/MF59/CpG induced high levels of IFN-� and TNF-�,but not IL-5 or IL-10, indicating a Th1 response. VEE/SINalphavirus particles (VEE/SIN-E1E2, VEE/SIN-E1E2p7, andVEE/SIN-NS345) induced nonspecific cytokine productionfrom CD4� T cells in both Th1 (IFN-� and TNF-�) and Th2(IL-5 and IL-10) cytokines, as no difference in cytokine pro-duction was observed after in vitro stimulation with eithercontrol medium or HCV peptides (Fig. 2). However, theprime/boost regimen using E1E2/MF59/CpG to prime fol-lowed by a boost with VEE/SIN-E1E2 induced the strongest

FIG. 1. Priming with E1E2/MF59/CpG followed by boosting with VEE/SIN-E1E2 stimulates strong, good CD4� and CD8� T-cell responses.BALB/c mice (n 10) received three intramuscular injections (1, 2, 3) at 3-week intervals with the immunogens indicated. The spleen cells wereharvested at 2 weeks after the last immunization and stimulated with 10 �g/ml HCV-specific peptides for ICS and fluorescence-activated cell sorteranalysis as described in Materials and Methods. Twenty-mer overlapping peptide pools for the HCV strain 1a E1 region (E1 pool) and E2 region(E2 pool) and single E2 peptides specific for CD4� T cells (CD4 E2 pep) and CD8� T cells (CD8 E2 pep) were used for stimulation. The dataare presented as the mean total percentage of CD8� IFN-�� (A, B, and C) and CD4� IFN-�� (D) cells in two pools (five mice per pool). Datafrom one representative experiment of two performed are shown. *, P � 0.05 compared with the medium control and the PBS-immunized group.


and broadest HCV-specific Th1 cytokine responses (Fig. 2).On the other hand, the prime/boost regimen with E1E2/MF59and VEE/SIN-E1E2 induced both HCV-specific Th1 and Th2cytokines (Fig. 2).

There is strong stimulation of IFN-� and TNF-� productionby the E2 peptide pool using E1E2/MF59/CpG or E1E2/MF59(priming) followed by VEE/SIN-E1E2 (boosting) in the bead-array assay (Fig. 2), but this regimen did not strongly stimulateIFN-� from CD8� (Fig. 1C) or CD4� T cells (Fig. 1D) by theE2 peptide pool. The cytokines detected by the bead arrayassay might therefore be derived from other spleen cell pop-ulations in addition to CD4� and CD8� T cells.

Elicitation of optimal cellular immune responses againstHCV nonstructural proteins. BALB/c mice were immunizedwith an HCV NS345core polyprotein or with VEE/SIN particlesexpressing NS345. T-cell responses were detected by ICS ofIFN-� on CD4� or CD8� T cells after in vitro stimulation withoverlapping HCV strain 1a peptide pools for either NS3, NS4,NS5A, or NS5B, as well as recombinant HCV NS34 protein

[SOD-C100 (NS34); aa 1569 to 1931 (25, 30)] and recombinantHCV NS5 protein (SOD-NS5A/B; aa 2054 to 2995 [48]) (Table1). Iscomatrix was used as an adjuvant to the HCV NS345corepolyprotein, with or without CpG, since previous studies haveshown Iscomatrix to be a very effective adjuvant for primingTh1-type CD4� and CD8� CTL responses to the HCV nucleo-capsid protein in nonhuman primates (40). HCV NS345 polypro-tein adjuvanted with Iscomatrix plus CpG elicited strong CD4�

T-cell responses against the NS4 and NS5b peptide pools, as wellas against HCV NS34protein (SOD-C100) and recombinantHCV NS5 protein (SOD-NS5) (48) (Fig. 3A). In the absence ofCpG, Iscomatrix-adjuvanted NS345 polyprotein still primedCD4� T cells but at an approximately fivefold lower level (no. 2in Fig. 3A). VEE/SIN particles expressing HCV NS345 could alsoprime HCV-specific CD4� T cells (no. 4 in Fig. 3A) but at a muchlower level than the Iscomatrix-CpG-adjuvanted NS345 polypro-tein. Again, however, a prime/boost regimen comprising primingwith the latter followed by boosting with VEE/SIN-NS345 reca-pitulated the optimal CD4� T-cell responses (no. 7 in Fig. 3A).

FIG. 2. Prime/boost regimen with E1E2/MF59/CpG and VEE/SIN-E1E2 stimulated strong HCV-specific Th1 cytokine responses. Spleen cellsfrom the immunized mice (immunogens as indicated) were stimulated with HCV-specific peptides for 24 h, and the supernatants were collectedfor detection of the cytokines IFN-�, TNF-�, IL-5, and IL-10 with the Beadlyte Luminex mouse multicytokine detection system (Upstate,Charlottesville, VA). The data are presented as the means of pooled spleens from the same group (two pools per vaccine regimen, five spleensper pool). Data from one representative experiment of two performed are shown.


This same prime/boost regimen was also clearly optimal forpriming HCV-specific CD8� T cells (no. 7 in Fig. 3B). Adju-vanted HCV NS345core polyprotein did not elicit CD8� T cells,and while VEE/SIN-NS345 was effective, maximal levels wereachieved via the prime/boost regimen.

The cytokine profile of spleen cells from immunized mice

was also analyzed after stimulation with HCV-specific pep-tide pools. Poly/Iscomatrix/CpG induced strong Th1 cyto-kines (IFN-� and TNF-�) and low or undetectable Th2 cyto-kines (IL-10 and IL-5) (Fig. 4). Interestingly, a prime/boostregimen using Poly/Iscomatrix/CpG for the first two immuni-zations followed by a third immunization with VEE/SIN-

FIG. 3. A prime/boost regimen using NS345Poly/IMX/CpG and VEE/SIN-NS345 is optimal for stimulating HCV-specific CD4� (A) and CD8�

(B) T-cell responses. BALB/c mice in each test group (n 10) received three injections (1, 2, 3) at 3-week intervals with immunogens as indicated.Intracellular cytokine staining was performed 2 weeks after the last injection, and the spleen cells were stimulated with 10 �g/ml HCV-specificpeptides for ICS and fluorescence-activated cell sorter analysis as described in Materials and Methods. Twenty-mer overlapping peptide pools forthe HCV strain 1a NS3 (NS3 pool), NS4 (NS4 pool), NS5A (NS5A pool), and NS5B (NS5B pool) regions; single peptides from the NS3 region(NS3-1 pep, NS3-2 pep, and NS3-3 pep) and NS5 (NS5B pep); and recombinant proteins SOD-C100 (NS34) and SOD-NS5 were used forstimulation. The data are presented as the means of pooled spleens from the same group (two pools per vaccine regimen, five spleens per pool).Data from one representative experiment of two performed are shown. *, P � 0.05 compared with the medium control and the PBS-immunizedgroup. IMX, Iscomatrix.


NS345 stimulated the highest IFN-� and TNF-� production,indicating a strong Th1 response (Fig. 4).

Also of note are the high but nonspecific background levelsof IL-10 and IL-5 elicited by three immunizations with VEE/SIN-NS345, which are similar to those found following immu-nization with VEE/SIN-E1E2 (Fig. 2). The nonspecific induc-tion of Th1 and Th2 cytokines may be due to the ability of SINand VEE to infect dendritic cells and induce proinflammatorycytokine production (31, 57).

A prime/boost regimen using MF59-CpG-adjuvanted E1E2followed by VEE/SIN-E1E2 elicits virus-cross-neutralizing an-tibodies. Previous chimpanzee vaccination studies using adju-vanted recombinant E1E2 vaccines generated sterilizing im-munity against a homologous viral challenge. This sterilizationcorrelated directly with both total serum anti-E1E2 antibodytiters (enzyme immunoassay) and titers of antibodies whichblock the binding of gpE2 to the essential HCV receptor com-ponent CD81 (8, 20, 46). To explore if the immunogens wetested could stimulate HCV-neutralizing antibodies, we firstanalyzed serum samples obtained from mice 2 weeks after the

final immunization for antibodies that could block the bindingof gpE2 to CD81. Immunizations with E1E2/MF59 were bestat eliciting these antibodies, whereas immunization with defec-tive VEE/SIN particles stimulated poor antibody responses(Fig. 5A and B). However, a prime/boost regimen using E1E2/MF59/CpG followed by VEE/SIN-E1E2 was similar to E1E2/MF59/CpG in stimulating antibodies that block the binding ofHCVgpE2 to CD81, although their titers are lower than thoseobtained by E1E2/MF59 immunization (Fig. 5A). To deter-mine if these immunizations could stimulate antibodies capa-ble of neutralizing the infectivity of HCV derived from infectedcell cultures (HCVcc), we generated HCVcc derived fromHCV JFH-1 cDNA fused to a luciferase reporter (JFH1-Rluc)as described by Wakita et al. (60). This HCV strain is part ofthe 2a subtype, whereas the vaccine is derived from a 1a sub-type. Mice immunized with PBS were used as controls. Thedata are expressed as the percent inhibition of luciferase ac-tivity relative to that obtained from control mice. Mice immu-nized with E1E2/MF59 elicited substantial cross-neutralizingantibody titers against this highly heterologous HCV strain,

FIG. 4. Prime/boost regimen with NS345Poly/IMX/CpG and VEE/SIN-NS345 stimulates strong HCV-specific Th1 cytokine responses. Spleencells from immunized mice (immunogens as indicated) were stimulated with HCV-specific peptides for 24 h, and the supernatants were collectedfor the detection of the cytokines IFN-�, TNF-�, IL-5, and IL-10 with the Beadlyte Luminex mouse multicytokine detection system (Upstate,Charlottesville, VA). The data are presented as the means of pooled spleens from the same group (two pools per vaccine regimen, five spleensper pool). Data from one representative experiment of two performed are shown. IMX, Iscomatrix.


indicating the induction of B-cell responses to a conservedneutralizing epitope(s) within E1E2 (Fig. 5B).

In addition, the immunization regimen optimal for primingHCV-specific CD4� and CD8� T cells (priming with E1E2/MF59/CpG followed by boosting with VEE/SIN-E1E2) elic-ited somewhat lower (about 20% less relative to the E1E2/MF59 group) but still significant cross-neutralizing antibodytiters against this diverse 2a genotype (Fig. 5B and C).

Eliciting humoral and cellular immune responses to HCVstructural and nonstructural proteins in the same immuniza-tion regimen. A combination immunization regimen was thentested in order to determine if all of the above immune responsescould be elicited successfully. BALB/c mice were immunizedtwice with adjuvanted recombinant E1E2 and NS345 polyproteinprior to boosting with defective VEE/SIN alphaviral particlesexpressing these genes. Figure 6 shows that, compared with an

individual regimen for either structural or nonstructural proteins,a combination regimen elicited comparable CD4� (Fig. 6A andC) and CD8� (Fig. 6B and D) T-cell responses. While this com-bination vaccine elicited somewhat lower CD4� T-cell responsesto E1 (Fig. 6A), it increased CD8� T-cell responses to NS34 (Fig.6D). In addition, cross-neutralizing antibody titers were elicitedthat were equivalent to those obtained with the single-immuniza-tion regimen for E1E2 (Fig. 6E).

DISCUSSION

Our first-generation HCV vaccine was based on the use ofrecombinant, native strain 1a E1E2 glycoproteins derived frommammalian cell lines that were adjuvanted with oil-wateremulsions. Such vaccines have been shown to be highly effec-tive at preventing the development of chronic infection in the

FIG. 5. Induction of cross-neutralizing antibodies in vaccinated mice. (A) A prime/boost regimen with E1E2/MF59/CpG and VEE/SIN-E1E2stimulates antibodies that block the binding of HCV gpE2 to CD81. BALB/c mice were immunized with immunogens as indicated. Mouse serumwas collected 2 weeks after the last injection. The antibody titers are expressed as reciprocal values of serum dilutions which inhibit 50% of E2binding to CD81. Serum samples from each individual mouse were measured. (B) Induction of antibodies that cross-neutralize the infectivity ofJFH-1 2a HCVcc for Huh-7 cell lines. Serum samples collected as described for panel A were mixed at a 1:100 dilution with JFH-1 2a HCVcc andpreincubated at 37°C for 1 h. The mixture was then applied to Huh-7 cells in triplicate, and the cell lysate was measured for luciferase activity atday 3 postinfection. The luciferase activity from mouse serum with PBS immunization was used as a control. The data are shown as percentinhibition of the luciferase activity from serum with different immunizations relative to the control. (C) A prime/boost regimen with E1E2/MF59/CpG and VEE/SIN-E1E2 stimulates cross-neutralizing antibodies against JFH-1 2a HCVcc infection of Huh-7 cells. Serum samples fromimmunized mice (antigens as indicated) were diluted 1:100 and mixed with JFH-1 2a HCVcc. The experiment was performed as described for panelB. The luciferase activity detectable with mouse serum following PBS immunization was used as a control. The data are shown as the percentinhibition of the luciferase activity from serum with different immunizations relative to the control. �, P � 0.05 relative to PBS immunization.


large majority (80%) of vaccinated chimpanzees that weresubsequently challenged with either homologous 1a virus (8)or heterologous 1a virus (9, 20). Recently, vaccination ofguinea pigs, chimpanzees, and humans with this formulationhas been shown to elicit antibodies that cross-neutralize HCV/HIV pseudoparticles displaying HCV E1E2 envelope glyco-proteins derived from multiple global HCV clades. SubstantialT helper responses to E1E2 were also elicited in vaccinatedindividuals (56; M. Houghton et al., unpublished data). Whilethis vaccine represents a highly promising candidate, furtherresearch is aimed at developing a second-generation vaccine ofeven higher potency.

The present work aimed to identify a vaccine regimen capa-ble of eliciting all of the immune responses known to be asso-

ciated with protection from HCV infection, including virus-cross-neutralizing antibodies (8, 28, 39, 46) and broad cellularimmune responses against HCV. The latter includes broadTh1-type CD4� T helper responses (12, 17), as well as broadCD8� T-cell responses (10, 52). Since the Hepacivirus genus isvery heterogeneous, a global HCV vaccine needs to elicit pro-tective, broadly cross-neutralizing antibodies, as well as widelycross-reactive cellular immune responses.

The present study explored the use of defective alphavirusparticles expressing HCV genes and various adjuvanted recom-binant proteins in order to achieve these immunogenicity ob-jectives. The data presented allow the following conclusions.Firstly, it is clear that optimal Th1-type CD4� T helper re-sponses were obtained through the use of adjuvanted recom-

FIG. 6. A combination prime/boost immunization regimen elicits broad cellular responses to HCV structural and nonstructural proteins, as wellas cross-neutralizing antibodies. Groups of 10 BALB/c mice were immunized as indicated. For the combination immunization regimen, primingwith E1E2/MF59/CpG and NS345Poly/IMX vaccines and boosting with VEE/SIN-E1E2 and VEE/SIN-NS345 were done individually to separatemuscles for two different antigens at weeks 0, 3, and 6. At week 8, two pools of spleen cells were prepared (five spleens per pool) and stimulatedwith HCV-specific peptides prior to staining for intracellular IFN-� and fluorescence-activated cell sorter analysis. The mean values obtained fromthe two pools are shown. Panels: A, CD4� cells stimulated by E1 and E2 peptides; B, CD8� cells stimulated by E1 and E2 peptides; C, CD4� cellsstimulated by NS3, -4, and -5 peptides; D, CD8� cells stimulated by NS3, -4, and -5 peptides; E, cross-neutralizing antibody activity elicited againstJFH-1 2a HCVcc. Serum obtained at week 8 was diluted 1:100 and preincubated with virus for 1 h at 37°C prior to infection of Huh-7 cells. Threedays later, luciferase activity in cell lysates was determined. Data are expressed as percent inhibition (based on means from triplicate assays) relativeto control mice immunized with PBS. *, P � 0.05 compared with the medium control and the PBS-immunized group. IMX, Iscomatrix.


binant E1E2. The inclusion of CpG with MF59 substantiallyincreases Th1-type CD4� T-cell responses to E1E2 (Fig. 1D).While defective chimeric alphaviral particles derived fromboth SIN and VEE (38) could not elicit such good CD4� T-cellresponses (Fig. 1D), they were effective at eliciting E1E2-specific CD8� T-cell responses (Fig. 1A and B; see Fig. S2Band S3 in the supplemental material). Priming with MF59-CpG-adjuvanted E1E2 followed by boosting with VEE/SINexpressing E1E2 was found to be optimal for eliciting bothCD4� and CD8� T-cell responses (Fig. 1C and D; see Fig. S2Band S3 in the supplemental material). This regimen also elic-ited substantial titers of virus-cross-neutralizing antibody (Fig.5). Although these titers were somewhat (�20%) lower thanthose obtained by the use of recombinant E1E2 adjuvantedwith MF59 alone (Fig. 5), the prime/boost regimen allowed theinduction of all protective components of the immune re-sponse against E1E2 (Fig. 1 and 5; see Fig. S2 and S3 in thesupplemental material). The addition of at least 125 �g CpG toMF59-adjuvanted E1E2 has been associated with some sys-temic side effects in a recent phase 1 clinical trial (S. E. Frey etal., unpublished data), and so it will be important in futuretrials to explore a lower dose range in order to establish awell-tolerated dose for these HCV antigen formulations.

It should be noted that some variation in the T-cell re-sponses (as measured by ICS assays) was observed betweenexperiments, but a positive result was confirmed in at leastthree independent experiments (Fig. 1; see Fig. S3 in the sup-plemental material).

In order to recapitulate cellular immune responses to non-structural proteins, which have also been associated with pro-tection against HCV infection (12), we also explored the use ofan adjuvanted recombinant NS345core polyprotein derivedfrom yeast, as well as replication-defective chimeric VEE/SINparticles expressing NS345 genes. Again, we found that CD8�

T-cell responses were induced efficiently by the alphaviral par-ticles and poorly by adjuvanted NS345core polyprotein (Fig.3B). However, NS345core polyprotein adjuvanted with Isco-matrix plus CpG was optimal in mice for the elicitation ofTh1-type CD4� T-cell responses (Fig. 3A). As with E1E2-specific immune responses, optimal levels of both CD4� andCD8� T-cell responses to the NS3, -4, and -5 proteins could beinduced via a regimen comprising priming with adjuvantedNS345core polyprotein followed by boosting with VEE/SINparticles expressing NS345 (Fig. 3A and B). It is known thatmany T-cell epitopes are conserved between different HCVclades (50) and also that eliciting broad cellular immune re-sponses to the HCV NS3, -4, and -5 gene products is associatedwith the protection of chimpanzees against the development ofchronic, persistent infection by highly diverse HCV clades (15,26). It is to be expected, therefore, that the broad cellularimmune responses elicited here by this prime/boost immuni-zation regimen utilizing multiple HCV gene products will bebroadly cross-protective against multiple HCV clades.

Importantly, a single immunization regimen was shown to becapable of eliciting these broad T-cell responses, as well ascross-neutralizing antibodies (Fig. 6). We conclude, therefore,that the prime/boost regimen identified here involving primingwith adjuvanted envelope glycoproteins and nonstructural pro-teins of HCV, followed by boosting with defective VEE/SINparticles expressing these genes, is optimal for eliciting the

array of known protective immune responses against HCVinfection. It should be noted that this vaccine may have poten-tial therapeutic use, as well as prophylactic efficacy, especiallywhen combined with antiviral drugs.

ACKNOWLEDGMENTS

We thank CSL Ltd. for providing Iscomatrix and Heather Davisfrom Coley Pharmaceuticals Inc. for the gift of CpG. We also thankCatherine E. Greer and Harold S. Legg for preparing alphavirus par-ticles.

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