Fiber-modified recombinant adenoviral constructsencoding hepatitis C virus proteins induce potentHCV-specific T cell responseDuangtawan Thammanichanonda,1, Sarah Moneera, Patricia Yotndab,Campbell Aitkenc, Linda Earnest-Silveirad, David Jacksona,Margaret Hellard c, James McCluskeya,Joseph Torresi d, Mandvi Bharadwaja,⁎,1
a Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australiab Baylor College of Medicine, Texas, USAc Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria 3004, Australiad Department of Medicine, RMH, University of Melbourne, Victoria 3050, Australia
Received 10 January 2008; accepted with revision 7 April 2008Available online 3 June 2008
⁎ Corresponding author. Department oFax: +61 3 9347 1540.
E-mail address: [email protected] MB and JT are joint senior authors.
Cellular immunity, comprised of TCD8 and TCD4 responses, isessential for spontaneous resolution of acute hepatitis C andlong-term protection from persistent infection [1,2]. Studies ofCTL responses in HCV-infected individuals have been sub-stantially impeded by the low frequency of HCV-specific CTLprecursors in the peripheral blood. To overcome this problem,various approaches have been adopted to stimulate and expandthe resting memory T cells. Most studies have so far utilisedsynthetic peptides to stimulate PBMCs in an antigen-specificmanner [1,3]. However, reactivation of memory T cells usingsynthetic peptides selectively expands T cells, is limited toknown epitopes or epitopes predicted by algorithms and pre-cludes any information on the hierarchy of T cell responsetowards the different CTL determinants in natural infection.HCV peptides corresponding to subdominant CTL-epitopes caninduce strong responses when memory T cells are repeatedlystimulated in vitro. Hence, in vitro studies based on syntheticpeptide stimulationmay not precisely reflect the responses thatoccur during natural infection and that are associated withclearance of HCV. This requires an efficient strategy that wouldmimic the natural cytosolic generation of viral peptides, thusenabling an accurate and comprehensive analysis of HCV-spe-cific T cell responses against the multitude naturally processedpeptides afterHCV infection. Theuseof recombinant adenoviralsystem to stimulate and expand HCV-specific CTL responses istherefore an attractive strategy. This systemwould result in theintracellular expression of recombinant HCV proteins, allowingendogenously synthesized antigens to be processed, bound toMHC class I molecules and presented on the cell surface, as innatural infection.
So far, most human studies based on recombinant adeno-virus-HCV constructs have utilised the adenovirus serotype-5(Ad5). Although these constructs were able to expand HCV-specific CTL responses and stimulate maturation of dendriticcells (DCs) [4,5], Ad5 is known to be relatively inefficient ininfecting PBMC and dendritic cells [6]. In this study,we utilised achimeric virus containing anAd5backbonewith the fiber proteinsubstituted fromadenovirus serotype-35 (referred to as Ad5F35)[7]. This chimeric virus is known to be efficient in transducinghematopoietic stem cells and DCs [8] and has been used forin vitro human studies before [9]. Here, we have successfullyused recombinant Ad5F35-HCV to induce HCV-specific CTL re-sponses from HCV-infected individuals. rAd5F35-HCV thus pro-vides a significant advance in studies of CTL responses againstHCV and is a potential candidate for immunotherapy/vaccinestrategies based on reactivation of anti-viral cellular immuneresponse.
Materials and methods
Construction of Ad5F35-HCV vector
Recombinant Ad5F35 viruses expressing individual HCV proteins(core and NS3) were generated using a ligation-based method[10]. The HCV-Core and NS3 genes were amplified by PCR fromthe plasmid containing H77 sequences using sequence-specificprimers containing enzyme restriction sites for subsequent clon-ing. The amplified DNAwas gel purified using the UltraClean™15DNA purification kit (Mo Bio Laboratories Inc.) and ligated intopGEM T-easy vector (Promega, Madison, WI, USA) according to
themanufacturer's instructions. The NS3 amplicon was digestedwith XbaI and AflII (New England Biolabs) and the core ampliconwas digestedwith NotI and AflII (New England Biolabs). The pre-digested amplicons were cloned into pShuttle expression vector(BD biosciences). The expression cassette from pShuttle wasexcised and ligated into an Ad5F35 expression vector [7]. Theintegrity of rAd5F35-HCV constructs was confirmed by perform-ing both restriction analysis and DNA sequencing.
Production of rAd5F35-HCV viruses
The rAd5F35-HCV virions were produced by transfection ofrAd5F35-HCV DNA into human embryonic kidney (HEK) 293cells and amplified by serial passaging in culture. The HEK 293line was maintained in Dulbecco's minimal essential medium(DMEM) containing 10% fetal calf serum (FCS) at 37 °C and 5%CO2. For the first passage of rAd5F35-HCVvirions,HEK 293 cellswere transfected with PacI-linearized rAd5F35-HCV DNA usingLipofectamine™ 2000 (Invitrogen) according to the manufac-turer's instruction. Cells were collected at day 3 post-trans-fection and lysed by freeze-thawing. Lysates were centrifugedat 3000 g for 15 min and supernatants containing rAd5F35-HCVvirions were filtered with a 0.22 μm filter (Millipore) andstored at −80 °C. High titres of the final virus stocks wereprepared by serial passaging in HEK 293 cells (Fig. 1A). Thetitre of the final virus stock was determined using Adeno-X™Rapid Titer Kit (BD Biosciences, Palo Alto, CA). RecombinantAd5F35 containing preproinsulin (rAd5F35-PPI) was generatedin a similar manner to serve as a negative control virus.
Western blot analysis
HeLa cells were transduced with rAd5F35-NS3/Ad5F35-Corevirus at various multiplicity of infection (MOI) and protein ex-pression was examined by Western blot analysis after a 72-hourincubation at 37 °C. Representative data for the expression ofthe Core protein is shown in Figure 1B, a 22 kDa protein bandcorresponding to the HCV-Core confirmed the expression of theCore protein in the transduced cells. This was further verified bythe observation of an equal sized band in the pcDNA-coretransfected Huh7 cell lysate used as a positive control. Incontrast, no specific bands were observed in both uninfectedcell lysate and rAd-PPI infected cell lysate.
Donors
Peripheral blood was collected from HCV-infected donorsrecruited from the Royal Melbourne Hospital, Australia andcommunity-based intravenous drug users (IDUs) wererecruited through a social network study [11]. This researchwas approved by the Human Research and Ethics Committee(HREC) of Melbourne health and the Victorian Department ofHuman Services, and informed consent was obtained from allsubjects. Peripheral blood mononuclear cells (PBMCs) wereisolated from whole blood by Ficoll–Hypaque density centri-fugation. Class I HLA typing (Table 1) was performed at theVictorian Transplantation and Immunogenetics Service (VTIS).
In vitro expansion of HCV-specific T cells
PBMCs transduced with 50 or 100 MOI of Ad5F35-HCV were co-culturedwith autologous PBMCs at stimulator:responder ratio of
Figure 1 Quantification and expression of HCV-Core in cells infected with rAd5F35-Core. HEK 293 cells were infected with differentdilutions of rAd5F35-HCV, after 48hrs of incubation the cells were fixed and stained with an anti-hexon antibody specific to the adenovirushexon protein. A dark brown staining indicated cells infected with rAd5F35-HCV (A). There was no staining in the uninfected cells. Acytopathic effect, identified by the detached dead cells was evident at a high titre (10−1). Serial 10-fold dilution of the virus stockproduced a gradual decline in the numbers of infected cells. Expression of the core protein (B) was verified by immunoblotting cell lysateswith an anti-coremonoclonal antibody. No specific bandwas observed in cell lysates from uninfected HeLa cells (lane 1) and the rAd5F35-PPI infected HeLa cells (lane 2: negative control). The HCV-Core protein was detected in lysates from rAd5F35-Core infected HeLa cells atMOI 100:1 (rAd-Core: lane 3) and 200:1 (lane 4). Lysate from pcDNA-core transfected Huh 7 cells was used as a positive control (lane 5).
331rAd5F35-HCV stimulates potent HCV specific CTLs
1:2. To generate peptide-specific T cells, PBMCs were co-cul-tured with autologous PBMCs coated with peptide at 10 μg/mLfor 1 h at 37 °C and later washed twice before adding back toculture. The growth medium (RPMI+10% FCS) in all the cultureswas supplemented with recombinant interleukin 7 (rIL-7)(Chemicon International, Inc) and recombinant IL-2 (Cetus,Emeryville, USA). Stimulated T cells were tested for cytotoxiccapacity in a 51Cr release assay and for IFN-γ and TNF-αsecretion by intracellular cytokine staining (ICS) on day 10.
Intracellular cytokine staining
ICS for IFN-γ and TNF-α was performed as previously described[12]. Briefly, autologous phytohemagglutinin antigen (PHA)blasts were pre-sensitized with 5 μg/mL of synthetic peptidesderived fromHCV-NS3/Core for 1 h. After 4washes, these targetcells were incubated with in vitro expanded HCV-specific Tcellcultures at effector:target ratio of 2:1. After incubation,Brefeldin A (BFA) (Sigma) was added to a final concentrationof 10 μg/mL and incubated for a further 5 h at 37 °C. Cells werewashed and stained with Tricolor-conjugated anti-CD8 (Caltag),phycoerythrin (PE)-conjugated anti-CD3 (eBiosciences). Thecells were then fixed for 20 min at room temperature with 1%
paraformaldehyde, permeabilizedwith 0.3% saponin and stainedwith FITC-conjugated anti-human IFN-γ (BD Biosciences) or anti-TNF-α-FITC (eBiosciences). The cells were washed and resus-pended in staining buffer before analysis by flow cytometry.
Cytotoxicity assay
Standard chromium (51Cr) release assaywas performed to assessthe HCV-specific cytolytic activity of in vitro expanded T cellsused as effectors, as previously described [9]. Autologous PHAblasts were used as targets after being pre-sensitized withsynthetic peptides and incubated with 51Cr for 60 min at 37 °C.All assays were done in triplicates and the mean of the threeobservations for each parameter was utilised to calculate thepercentage of specific cytotoxicity according to the followingformula: %lysis=[(experimental release−spontaneous release)/(maximum release−spontaneous release)]×100.
IFN-γ ELISPOT assay
Interferon-γ (IFN-γ) ELISPOT assays were performed by anovernight incubation of 2×105 PBMCs/well with appropriatepeptides (Table 2) at a final concentration of 10 μg/mL in
Table 1 Donor details
Donors Age (years) HLA typing Anti-HCV HCV RNA HCV genotype
ninety-six well, ELISPOT plates (Multiscreen, Millipore, MA)precoated with anti-human IFN-γ monoclonal antibody(MabTech, Sweden). Peptide was not added to 3–5 negativecontrol wells for calculating background IFN-γ productionand cells were stimulated with phytohemagglutinin (PHA) at10 μg/mL to confirm the presence of functional Tcells. After24 h, plates were washed and stained with biotinylated anti-human IFN-γ as the secondary antibody (MabTech), followedby streptavidin-alkaline phosphatase (Sigma) and freshlyprepared BCIP/NBT substrate (100 μL/well, Sigma) todevelop the colorimetric reaction. The plates were washed,dried and colored spots were counted using the AID EliSpotReader System (Autoimmun Diagnostika, Germany). Resultsrepresent IFN-γ spot forming cells (SFC)/106 PBMCs.
Results
HCV-specific T cells expanded by rAd5F35-Core andrAd5F35-NS3 are antigen-specific and possessmultipleanti-viral activities
To investigate the efficiency of rAd5F35-HCV in expandinganti-HCV TCD8 in vitro, PBMCs from HCV-infected donors werestimulated with autologous PBMCs transduced with rAd5F35-Core. After 10 days, the expanded T cells were assessed forHCV-specific activity by measuring TCD8 responses in an IFN-γand TNF-α ICS assay against autologous PHA blasts pre-sen-sitized with a known HLA-B7 restricted CTL determinant
Table 2 List of HLA class I-restricted HCV peptides used in the s
Peptide sequence Code Antigen Localiz
GPRLGVRAT GPR Core aa 41–HSKKKCDEL HSK NS3 aa 1395HSKKKCDEI HSK NS3 aa 1395LIRLKPTL LIR NS3 aa 1611LVRLKPTL LVR NS3 aa 1611HPNIEEVAL HPN NS3 aa 1359CINGVCWTV CIN NS3 aa 1073ALYDVVTKL ALY NS5 aa 2594ILAGYGAGV ILA NS4 aa 1851DLMGYIPLV DLM Core aa 132–GLQDCTMLV GLQ NS5 aa 2727KLVALGINAV KLV NS3 aa 1406LLFNILGGWV LLF NS4 aa 1807YLVAYQATV YLV NS3 aa 1585GLCTLVAML GLC EBV BMLF1
GPRLGVRAT (Core 41–49, denoted as GPR) [13]. Non-specificcytokine production was determined for each culture bytesting against target cells without peptide pre-sensitization.Figure 2A shows a high proportion of cytokine-producing TCD8observed against GPR, with 35.76% IFN-γ and 37.17% TNF-αproduction, in an HCV-infected donor (H#1). Thus indicating,that stimulation with rAd5F35-Core resulted in the expansionof HCV-specific Tcells that were potent producers of anti-viralcytokines. To verify that TCD8 responses were specific to theHCV insert and not the adenovirus, rAd5F35 containing pre-proinsulin (rAd5F35-PPI) was generated. Preproinsulin is aprecursor protein of proinsulin, containing an additional poly-peptide sequence at the N-terminus and normal individuals donot mount a CTL response to this protein. Tcells derived fromcultures infected with rAd5F35-preproinsulin did not show anyGPR-specific activity, with insignificant amounts of cytokine(0.12% of IFN-γ and 0.02% of TNFα) producing TCD8 (Fig. 2B)detected, as compared to the negative controls.
These observationswere extended by utilising rAd5F35-NS3to expand HCV-specific T cells from a donor (H#2) chronicallyinfected with HCV-G1. These T cells were tested for cytokineproduction against the HLA-B8 restricted peptide HSKKKCDEL(NS3 1395–1403, denoted as HSK) as well the specificity of thisresponse was also confirmed by using an irrelevant peptideGLCTLVAML (GLC) [14] derived from the lytic protein (BMLF1)of Epstein–Barr virus (EBV). IFN-γ and TNF-α secretion wasdemonstrated specifically against HSK (NS3 1395–1403) butnot against the control peptide GLC.
Figure 2 rAd5F35-Core and rAd5F35-NS3 can expand HCV-specific TCD8 in vitro. Representative ICS data from an HCV-infected donor(H#1) show that high amounts of IFN-γ and TNF-α production were observed against GPR (Core 41–49) by TCD8 expanded in rAd5F35-Corestimulated (A) but not from rAd5F35-preproinsulin (rAd5F35-PPI) stimulated culture (B). Similarly, TCD8 expanded in rAd5F35-NS3stimulated culture fromadonor (H#2) chronically infectedwithHCV-G1produced IFN-γ andTNF-α specifically against HSK (NS3 1395–1403)but not against an irrelevant peptide GLC, derived from the Epstein–Barr virus.
333rAd5F35-HCV stimulates potent HCV specific CTLs
These observations were further confirmed in another fourHCV-infected donors (H#3–H#6). As shown in Figure 3, HCV-specific IFN-γ and TNF-α secreting HCV-specific TCD8 weredetected in cultures from all four donors when analysed byutilising theHLA-B8 restricted peptide HSK. Further, Tcells fromcultures stimulated with rAd5F35-NS3 (NS3 derived from HCV-G1) were cross-reactive when tested against the HCV peptidesHSKKKCDEL and HSKKKCDEI originating from HCV-G1 and HCV-G3 respectively. Comparable numbers of IFN-γ (Fig. 3A) andTNF-α (Fig. 3B) secreting TCD8were observed in donors H#4, H#5and H#6 against both peptides. It is also noteworthy that donorH#3 was known to be infected with HCV-G2b (HSRKKCDEL) and
significant numbers of HSK-specific TCD8 were observed afterin vitro stimulation with rAd5F35-NS3(G1).
Since themeasurement of IFN-γ and TNF-α production onlypartially assessed the anti-viral function of the HCV-specificTCD8 cells, Tcells from cultures stimulated with rAd5F35-NS3,Core and PPI were also tested in a 51Cr release assay againsttargets pre-sensitized either with HSK or GPR. Significantly,high cytotoxic activities were detected with the HSK-specificlysis ranging between 27.9 and 76% in cultures from thedifferent donors (Fig. 4A). In contrast, TCD8 from culturesstimulated with rAd5F35-PPI had no detectable HSK-specificcytolytic activity (Fig. 4B). These results clearly demonstrated
334 D. Thammanichanond et al.
Figure 4 rAd5F35-HCV stimulated Tcells possess potent cytotoxic activity. Tcells stimulated with either rAd5F35-NS3 (A), a negativecontrol rAd5F35-PPI (B) or rAd5F35-Core (C) were used as effectors against HSK (NS3 1395–1403) or GPR (Core 41–49)-pulsed targets (■).Strong cytotoxic activity was observed in rAd5F35-NS3 stimulated cultures of 4 different donors (H#3, #5, #7 and #8) when tested at an E:Tratio of 20:1, against targets pulsedwith HSK as compared to targetswithout peptide (▤). In contrast, Tcells from rAd5F35-PPI stimulatedcultures had no cytotoxic activity. Similarly, TCD8 expanded by rAd5F35-Core stimulation but not in rAd5F35-PPI stimulated culture,possessed strong cytotoxic activity against GPR pulse targets (C). Tcells from donor H#9 were assessed at an E:Tratio of 20:1 (lower leftpanel) and donor H#1 (lower right panel) at different E:Tratios (20:1, 10:1 and 5:1). Targets without peptide are depicted as (E) and GPRpulsed targets depicted as (■).
335rAd5F35-HCV stimulates potent HCV specific CTLs
that HCV-specific TCD8 induced by rAd5F35-NS3 possessedsignificant cytolytic activity against HCV. Similar observationswere also made when the rAd5F35-Core was utilised. As shownin Figure 4C, T cells expanded in rAd5F35-Core, but not inrAd5F35-PPI stimulated culture from donor H#9 possessedsignificant cytolytic activity against GPR pulsed targets. Tofurther assess the potency of this CTL response, T cells fromrAd5F35-Core stimulated culture of another donor H#1, wereassessed at different E:Tratios. GPR-specific cytotoxic activity(Fig. 4C) was detected at all the different E:T ratios assessedwhile there was no cytotoxic activity in the rAd5F35-PPI sti-mulated Tcells, thus indicating the expansion of highly specificTCD8 with potent anti-HCV activity.
Figure 3 rAd5F35-NS3 stimulates HCV-specific TCD8 that are potedonors were stimulated with rAd5F35-NS3 and tested for NS3 peptirAd5F35-NS3 stimulation displayed a remarkable ability to produce IFHSK. A comparable level of cytokine production was observed againsHCV-G2b prior to spontaneous clearance of virus and was only teste
CTL from HCV-infected donors can be expanded in vitro bypeptide stimulation, however these are selective expansionsand may not always reflect the hierarchy of CTL response afternatural infection. Therefore, rAd5F35-NS3 stimulated culturesfrom two different donors were assessed for a CTL responseagainst peptides restricted by the different HLA alleles specificfor each donor. TCD8 from donor H#5 (HLA A2, B8, B41) weretested against a known HLA-A2 restricted peptide, CINGVCWTV(NS3 1073–1081, denoted CIN) and HSK. Peptide-specific cyto-kine-producing TCD8 were detected against both the peptides
nt producers of anti-viral cytokines. PBMCs from HCV-infectedde-specific cytokine production by ICS assay. TCD8 expanded byN-γ (A) and TNF-α (B) against autologous PHA blasts coated witht HSK derived from HCV G1 and G3. Donor H#3 was infected withd on APCs pulsed with HSK-G1.
336 D. Thammanichanond et al.
(Fig. 5A) however the HSK-specific TCD8 response was strongerthan the CIN-specific response, with 1.45% of TCD8 producing aCIN-specific IFN-γ versus 8.9% in response to HSK. T cells fromcultures derived fromdonor H#6 (HLA-A1, 3 B8, 35) were testedagainst target cells pulsed with an HLA-B35 restricted peptide,HPNIEEVAL (NS3 1359–1367, denotedHPN) andHSK. Again, TCD8responses against both peptides were detected, with 10.49%
Figure 5 rAd5F35-HCV stimulates TCD8 directed towards multiple Hfrom three donors (H#5, H#6, H#7) were stimulated with rAd5F35-NS3(A) for peptide-specific cytokine secretion and a 51Cr release assay (Balleles. TCD8 from,H#5 (HLAA2, 2 B8, 41)were tested against targets purestricted peptide HSK, H#6 (HLA-A1, 3 B8, 35) were tested against HLAtested against with two HLA-B8 restricted peptides, HSK and LVRLKPpotential (B) was detected against the different HCV peptides with thewithin each donor. This determinant hierarchy was similar to that oresponse against a panel of HCV peptides is shown from donor H#5described in Table 2. Selective peptide stimulation with LVR of PBMCs fas demonstrated by the significant proportion of IFN-γ secreting TCD8
TCD8 producing IFN-γ in response to HSK compared to 0.85% inresponse to HPN (Fig. 5A). Thus, the TCD8 response to HSK wasconsistently greater than that observed against either CIN orHPN. This trend of determinant hierarchy was also observed inthe cytotoxic potential of TCD8 generated in these cultures withthe % specific lysis against HSK-pulsed targets being greatertheneither CIN (in H#5) or HPN (in H#6) pulsed targets (Fig. 5B).
CV peptides while preserving the determinant hierarchy. PBMCsand TCD8 were assessed on day 10 post-stimulation, in an ICS assay) against different HCV peptides based on the donor's class I HLAlsedwithHLA-A2 restricted peptide, CINGVCWTV (CIN) andHLA-B8-B35 restricted peptide, HPNIEEVAL (HPN) and HSK, and H#7 were
TL (LVR). A variable degree of IFN-γ production (A) and cytotoxicHSK-specific response dominating the response to other peptides
bserved in an ex vivo response. Representative data of the TCD8when tested in an ex vivo ELISPOT assay (C). Peptide details arerom donor H#7 yielded considerable expansion of LVR-specific TCD8(D).
337rAd5F35-HCV stimulates potent HCV specific CTLs
As depicted in representative data (Fig. 5C) from an ex vivoELISPOTassay utilised to screen the PBMCs fromdonor H#5 on apanel of HCV peptides, we observed that the frequency of TCD8response in an ex vivo ELISPOTassay, was greater against HSKthen other TCD8 determinants derived fromHCV irrespective ofthe restricting HLA allotype. Furthermore, when rAd5F35-NS3stimulated culture from donor H#7 was tested individuallyagainst two HLA-B8-restricted peptides within the NS3 pro-tein, HSK (NS3 1395–1403) and LVRLKPTL (NS3 1611–1618,denoted LVR), only 0.59% of LVR-specific IFN-γ producing TCD8were observed, as compared to, 5.52% of IFN-γ producing TCD8against HSK (Fig. 5A). In contrast, single peptide stimulationwith the HCV-peptide LVR resulted in a significant expansion(12.2%) of LVR-specific IFN-γ producing TCD8 (Fig. 5D). Theseresults demonstrate that rAd5F35-NS3, not only stimulates aCTL response to the different peptides encoded within the NS3protein, but also preserves the determinant hierarchies ob-served in natural infection.
Discussion
HCV-specific CTL responses have been studied extensively,however previous reports have mostly focused on HCV-specificCTLs from PBMCs stimulated in an antigen-specific mannerusing synthetic HCV peptides. Peptide-specific stimulation hasvarious limitations mentioned above, as well, it precludesinformation on CTL determinants presented in the context ofthe various autologous HLA class I alleles simultaneously.Several studies have described the use of recombinant virusesencoding transgenic HCV proteins to study HCV-specific CTLresponses. Wong et al utilised a recombinant vaccinia-HCVvirus to express the HCV proteins in autologous B-lymphoblas-toid cells [15] for detecting HCV-specific CTL responsesfrom PBMCs of patients with chronic HCV infection. However,very high non-specific responses were observed against thevaccinia infectedB-LCLs,withmore than 20% lysis in 8 of the 12subjects. This high background possibly arose from pre-existing immunity to the vaccinia virus antigens due tovaccination or due to the fact that most healthy individualsare EBV seropositive and generate a high T cell responseagainst B-LCLs. Another study where rAd5 encoding the HCVgenes was utilised to induce HCV-specific responses in vitro[16] reported a low T cell response against HCV. This is notsurprising, in view of reports suggesting that PBMCs and DCsare refractory to Ad5 transduction [17,18]. We therefore uti-lised a modified Ad5 construct with the fiber protein substi-tuted from adenovirus serotype-35 known for its transductionefficiency into hematopoietic stem cells and dendritic cells[8].
We found that significant HCV-specific TCD8 responses wereinduced independent of MHC class I alleles following rAd5F35-HCV stimulation of PBMCs from HCV-infected individuals. Asshown in Figure 5, TCD8 expanded in rAd5F35-NS3 stimulatedcultures from the different donors simultaneously respondedto peptides restricted by the different class I alleles, thusunderlining the potential of utilising these constructs to ex-pandHCV-specific CTL in anHLA-diverse population.Moreover,stimulation with rAd5F35-NS3 demonstrated a definite hier-archy of CTL responses with the TCD8 response against HLA-B8restricted peptide HSK being stronger than responses to otherCTL determinants restricted by either HLA-B8 or other HLAalleles (CIN restricted by HLA-A2 and HLA-B35 restricted HPN).
In this context, several peptides that are adequately pre-sented on APCs are classified as non-dominant because theyelicit Tcell responses when they are presented alone, but areneglected when they are presented in conjunction with otherpeptides [19]. In our study, we found that when rAd5F35-NS3was used for stimulation and potentially all the HLA-B8 res-tricted peptideswere expressed simultaneously on the antigenpresenting cells, the LVR-specific response was much lowerthan the HSK-specific response. In contrast, when the LVR-peptide was used for stimulation, there was a significantexpansion of LVR-specific TCD8. These results suggest thatwhile peptide stimulation can selectively expand the CTLresponse, the adenoviral constructs permit both the subdomi-nant and immunodominant peptides to be presented on thesameantigenpresenting cells (APCs), thusmaking it feasible tostudy the hierarchy of CTL stimulation in the context of naturalinfection. The specificity of the TCD8 responses induced bythe rAd5F35-HCV constructs was confirmed by utilising therAd5F35-PPI. Indeed, TCD8 expanded from rAd5F35-PPI stimu-lated cultures had no cytotoxic activity against target cellspre-sensitized with HCV peptides.
Interestingly, similar to that observed with syntheticpeptide stimulation, utilisation of the rAd5F35-HCV constructsalso demonstrated the cross-genotypic recognition of peptideanalogues derived fromdifferentHCV genotypes. rAd5F35-NS3from genotype 1a could be used to expand HSK-specific TCD8response from donors infected with HCV genotype 2b and 3a.Although a substitution in the anchor residue at position 3of HSK occurs in genotype 2b (HSRKKCDEL), this did not abro-gate the MHC class-I binding and T cell receptor recognition.This phenomenonmay be explained by the similar biochemicalproperties of positively charged lysine (K) and arginine I.Similarly, a substitution of leucine (L) with isoleucine (I) isfound at position 9 of the HSK peptide (HSKKKCDEI) derivedfrom genotype 3a. These two amino acid residues are bothnon-polar hence the MHCbinding and TcR recognition were notcompromised. This is particularly important since HCVG1a and3a are the two most prevalent viral genotypes in HCV-infectedindividuals worldwide.
Recombinant Ad5 vectors are known to be important vac-cine delivery modalities since they induce potent transgeneproduct-specific antibody and T cell responses in other infec-tious disease models such as Ebola virus [20] and HIV [21].Although our study was limited to the characterisation of TCD8induced by the rAd5F35-HCV constructs, it is highly likely thatthe HCV-specific TCD4 were also stimulated by these constructssince the vectors contained the entire proteins that alsoencode thedifferentTCD4 determinants. Furthermore,wehaveshown that HCV-specific TCD8 responses were induced inde-pendent of MHC class I alleles following rAd5F35-HCV stimula-tion. Considering the high HLA polymorphisms in the humanpopulations, this advantage highlights the potential of utilisingthe rAd5F35-HCV for the development of HCV vaccine andimmunotherapy. A Phase II clinical trial with autologous den-dritic cells transduced with rAd5F35 expressing the latentmembrane protein-1 and 2 genes from the Epstein–Barr virus,is already underway in patients with metastatic Nasophar-yngeal Carcinoma [22] and rAd5F35 based vectors have beenrecommended as promising tools for malignantmelanoma [23]and glioblastoma treatment [24]. While rAd5F35 may be anexcellent candidate vector for ex vivo vaccination strategiesor adoptive immunotherapynevertheless, in vivo vaccine studies
338 D. Thammanichanond et al.
in nonhuman primates have suggested that pre-existing Ad5immunity diminishes the cellular response to vaccine antigens[25] and that higher doses of rAd5 are required to overcomethe inhibiting effects of pre-existing Ad5 immunity. Conver-sely, rAd vectors based on other rare human serotypes testedso far have been found to be less potent than rAd5-basedvectors in the absence of anti-Ad5 immunity [26]. The mostpromising approaches recently proposed to circumvent thecomplications of pre-existing anti-Ad5 immunity and increasethe efficacy of in vivo rAd5F35 vaccines are prime-boost stra-tegies based on either a combination of a rare serotype rAdvector such as rAd26 followed by rAd5 [27] or recombinant DNAfollowed by rAd5F35 [28].
In conclusion, this study provides a highly efficient strategy,mimicking natural processing and presentation of antigen, toinduce an HCV-specific CTL response, thereby permitting ananalysis of the Tcell response in the context of the hierarchy ofTCD8 determinants within HCV. Future studies of HCV-specificTCD4 will further enhance its potential as a study tool. Wepropose that, this system has future implication for an immu-notherapeutic vaccine against HCV.
Acknowledgments
This work was supported by project grants funded by theNHMRC (#331312) and the Australian centre for HIV and HCVresearch. We would like to acknowledge the participants inthe Network 2 study.
Appendix A. Supplementary data
Supplementary data associatedwith this article can be found,in the online version, at doi:10.1016/j.clim.2008.04.002.
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