Potential conflict of interest: Nothing to report.
Broad T cell and B cell responses to multiple HCV antigens are observed early in individuals who control or clear HCV infection. The prevailing hypothesis has been that similar immune responses induced by prophylactic immunization would reduce acute virus replication and protect exposed individuals from chronic infection. Here, we demonstrate that immunization of naïve chimpanzees with a multicomponent HCV vaccine induced robust HCV-specific immune responses, and that all vaccinees exposed to heterologous chimpanzee-adapted HCV 1b J4 significantly reduced viral RNA in serum by 84%, and in liver by 99% as compared to controls (P = 0.024 and 0.028, respectively). However, despite control of HCV in plasma and liver in the acute period, in the chronic phase, 3 of 4 vaccinated animals developed persistent infection. Analysis of expression levels of proinflammatory cytokines in serial hepatic biopsies failed to reveal an association with vaccine outcome. However, expression of IDO, CTLA-4 (1) and PD-1 levels in liver correlated with clearance or chronicity. Conclusion: Despite early control of virus load, a virus-associated tolerogenic-like state can develop in certain individuals independent of vaccination history. (HEPATOLOGY 2007;45:602–613.)
Hepatitis C virus is the major cause of chronic liver infection leading to liver cirrhosis, with an increased risk of hepatocellular carcinoma.1 Vaccines for the prevention of new HCV infections, or therapeutic immunization to facilitate viral clearance in the more than 170 million chronic carriers worldwide, would be of tremendous public health benefit.2 Following a period of acute viremia and elevated serum ALT activity, approximately 70% of infected individuals develop persistent HCV infection leading to a slowly progressive chronic hepatitis with the subsequent sequelae.3 The specific events leading to persistent HCV infection remain unclear, and it is apparent that there is a complex interaction between virus and host, in which both adaptive4–10 and innate11 responses12 have been implicated, both influenced by the potentially tolerogenic liver environment.3, 13–15 Analysis of peripheral blood samples and, where possible, of liver biopsies from both humans and chimpanzees has revealed immune correlates of viral clearance implicating an important role for nonstructural protein 3 (NS3)-specific cellular immune responses in the control and clearance of HCV infection.5, 7, 13, 16, 17 As prospective vaccine targets in addition to NS3, the more conserved core antigen, and the envelope glycoproteins E1 and E2 are potentially important, not only for protective T cell immunity induced by the former,17–19 but also antibody responses against the envelope antigens,19–21 which may have neutralizing capacities able to control HCV viral loads as demonstrated recently in chimpanzees.10
Here we test the hypothesis that vaccine-induced rigorous and multispecific B cell, and CD4+ and CD8+ T cell responses would induce the control and consequent clearance of HCV infection after exposure. As potential immune targets, we used NS3 in combination with these 3 additional structural antigens in an HCV DNA prime, pox-virus boost vaccine strategy aimed at maximizing the breadth of adaptive B cell and T cell responses.
Six naïve mature chimpanzees (Pan troglodytes) were housed under conditions for optimal social and health care.22 All vaccine trial procedures were critically assessed for ethical care and use in accordance with international guidelines. Blood sampling and liver biopsies were performed as described.17 Body weight, temperature, hematology and biochemistry clinical values were monitored at regular monthly to bimonthly intervals.
HCV Vaccine Vectors.
Two DNA plasmids expressing the core-E1-E2 and NS3 of the HCV genotype 1b, J4 strain23 were used for priming immunizations. The NS3 encoding plasmid was described previously.25 The CoreE1-E2 (aa 1-746) gene sequence was inserted into the gWiz plasmid (Gene Therapy System Inc., San Diego, CA) to create pgWizCE1E2.24 As the immunogenicity of NS3-4 encoding DNA plasmids in primates was found to be of no additional benefit over the NS3 DNA,25 (unpublished data) the NS3 encoding DNA plasmid was selected. The empty plasmid, pgWiz, was used to immunize control animals.
Recombinant modified vaccinia Ankara (MVA) expressing core-E1-E2 and NS3 gene sequences were constructed by transient host range selection26–28 using the plasmids pCMV-C980 and pCMV-N-729-3010, encoding for HCV-1b23 structural (aa 1-830) and NS3 (aa 1028-1658) proteins. Recombinant viruses were amplified and purified by ultracentrifugation through sucrose.29 Nonrecombinant MVA was used to immunize control animals.
Peptides and Recombinant Proteins.
Fifteen-mer peptides with overlaps of 7 amino acids covering the core, E1, E2 and NS3 sequences of genotype 1b, J4 strain23 were purchased from Clonestar Biotech (Brno, Czech Republic). The peptides covering NS4 and NS5 proteins were 15-mers with 5 overlap, or 20-mer with 10 overlap for the aa positions 1875 to 2454, and were purchased from EMC microcollections GmbH (Tübingen, Germany). Core polypeptide (aa 1-120 derived from HCV genotype 1a)29 fused to a His-tag was expressed in E. coli strain BL21 (DE3), and purified under denaturing conditions on a Ni-NTA agarose column (Qiagen). Sequences of NS3 helicase (aa 1192-1457), E1 (aa 192-326), and E2 (aa 384-673) were derived from the HCV-1b strain. His-tagged NS3 was expressed in E. coli JM109 and purified on a Ni-NTA column. E1 and E2, deleted for their transmembrane domain, were cloned into pT-alpha vector. After transfection of CHO DHFR− cells, E1 or E2 were purified from supernatant on Ni-NTA agarose column. Analysis of the eluted protein fractions was performed by SDS-PAGE and Coomassie blue staining.
Immunization and HCV Exposure.
The animals were immunized at weeks 0 and 6 with DNA plasmids encoding core-E1-E2 (left arm) and NS3 (right arm) or pgWIZ (both arms) at a dose of 2 mg per inoculum, equally divided intramuscularly and intradermally. Booster immunizations were given at weeks 14 and 20 with MVA encoding core-E1-E2 and NS3 or wild-type (1 × 109 pfu per inoculum, at the corresponding locations). At week 28, eight weeks after the last immunization, all animals were challenged intravenously with 25 CID50 of in vivo titrated HCV 1b J4 (generously provided by Robert H. Purcell, NIAID, Bethesda, MD), diluted in autologous preimmune plasma. The vaccine and challenge HCV strains were both genotype 1b, differing in approximately 5% of total amino acids.
Humoral Immune Responses.
Anti-HCV antibody responses in sera were measured using microplate wells coated with HCV core (0.5 mg/ml), E1 (4 μg/ml), E2 (1 μg/ml) or NS3-helicase proteins (0.5 mg/ml). ELISA was performed as described previously (Komurian-Pradel et al., in press). The capacity of the chimpanzee sera to neutralize HCV was analyzed using HCV pseudo-particles in infection assays on HuH-7 target cells as described.31 HCV pseudoparticles were generated as described using expression vectors encoding the viral components including E1E2 glycoproteins of strain CG1b.32 Control neutralizations were performed using pseudo-particles generated with glycoproteins derived from the feline endogenous retrovirus RD114 (RD114pp).
Cellular Immune Responses.
HCV-specific lymphoproliferation was determined with peripheral blood mononuclear cells (PBMC) as described.17 Quantification of specific cytokine secreting cells was performed using IFN-γ, interleukin 2 (IL-2) and IL-4 enzyme-linked immunospot (ELISPOT) assays according to the manufacturer's instructions (U-Cytech, Utrecht, Netherlands) and as described,18 using the core, E2 and NS3 recombinant proteins, and peptide pools covering E1 and NS3. Assays were performed in triplicate. An analysis of variance was performed on the log10-transformed data for each chimpanzee at each time point. Means of antigen-stimulated wells were compared to the medium-alone wells using a Studentized range test. ICS was performed as described32 by stimulating PBMCs (5 × 106/ml) with either Con-A, peptide pools (5 μg/ml of each peptide) or medium alone, and staining with FITC labeled anti-CD3 and PerCP labeled anti-CD8, PE labeled anti-IL-2 and APC labeled anti-IFN-γ mAb (BD Pharmingen), allowing proper discrimination between CD3+ CD8+ responding cells from all other CD3+ cells The data obtained from the ICS assay were analyzed by using a test for differences between ratios, comparing the number of cytokine-positive cells in the presence of the HCV antigen to the amount of cytokine-positive cells in the medium control, with a 2-tailed test and alpha = 0.05. Only results with statistical significance are shown.
Virus Quantification and Sequencing.
For serum samples, HCV 5′ NTR sequences were detected in a nonquantitative nested real-time PCR essentially as described by Krajden et al.33 Independently, in another laboratory, all serum samples were quantified blindly for HCV RNA: RNA was extracted with the Nucleospin kit (Macherey Nagel) and eluted in RNase-free water. Sera were tested for a quantitative HCV RNA by real-time PCR of the 5′ HCV noncoding region.35, 36 In addition, several serum samples were also blindly quantified for HCV RNA in a third laboratory, using the Cobas Amplicor HCV Monitor test (Roche Molecular Diagnostics) (data not shown). For liver biopsies, frozen tissue was disrupted and RNA extracted with the High Pure RNA Tissue kit (Roche). HCV RNA-positive and -negative strand quantification in liver biopsies was carried out blindly by real-time PCR using molecular beacon detection.36 The NS3 region was targeted for amplification by nested-PCR. PCR products were directly sequenced by using the BigDye Terminator version 1.1 cycle sequencing kit (Applied Biosystems) and ABI Prism 3100 genetic analyzer (PE/Applied Biosystem). Sequence analysis was carried out with Vector NTI suite (Invitrogen). Quantification of expression of TGF-β, IFN-α, CD4, CD8, IL-10, IL-5, interferon-γ (IFN-γ), TNF-α and CCR7 from liver tissue was performed by real-time RT-PCR. RNA was extracted from the liver biopsies using the Stratagene Absolutely RNA RT-PCR miniprep kit. Real-time PCR was performed in SYBR Green PCR master mix (Applied Biosystems) containing human cDNA for positive controls, for 40 cycles using an ABI 7700 Prism. Expression of PD-1, Foxp3, CTLA-4, IDO and Cox2 mRNA in liver was quantified as described.1
To compare the serum and liver HCV RNA in the 4 vaccinated versus the 2 control animals, 2-tailed Student t tests were performed on log10-transformed data to ensure normality, with alpha = 0.05, and P values calculated by exact methods, and given to 2 significant figures. When a sample was negative, a value of 1 (serum HCV RNA) or the sensitivity of the assay (liver HCV RNA) was assigned for facilitating data analysis.
Induction of HCV-Specific Th1 and Th-2-Cytokine Responses.
DNA prime – MVA boost immunization induced strong Th1-cytokine responses against multiple antigens in all 4 vaccinated animals as determined by ELISPOT (Fig. 1A-D). As expected, DNA priming induced low to undetectable HCV antigen-specific responses, not exceeding 73 cytokine-producing cells/106 PBMCs. However, strong immune responses were induced after the first MVA boost immunization, reaching 2368 IFN-γ–producing cells/106 PBMCs (Vac2), and 1250 IL-2–producing cells/106 PBMCs (Vac3). The IFN-γ responses were 2 to 4 times higher than the IL-2 in Vac1, Vac2, and Vac3, whereas Vac4 elicited virtually no IL-2 responses. The analysis of the individual responses to each vaccine antigen after the course of immunization showed that the IFN-γ responses targeted all 4 vaccine antigens in each vaccinee, NS3 and E1 being consistently high, whereas strong IL-2 responses were observed only against NS3 (Fig. 1G).
The induction of Th-2 type T cell responses was assessed by measuring the IL-4 production in ELISPOT assays. Strong IL-4 responses were induced in Vac1, Vac2, and Vac3, ranging from 1115 (Vac1) to 2267 (Vac3) IL-4 producing cells/106 PBMCs after one MVA administration (Fig. 2A-D). In Vac4 however, the IL-4 response was lower, reaching only 473 IL-4 producing cells/106 PBMCs after the first MVA injection, and was not sustained. Similar to the IL-2 responses, the IL-4 responses targeted mainly NS3, with 58 to 568 IL-4 producing cells/106 PBMCs prior to HCV exposure (Fig. 2G).
We also assessed the proliferative capacity of the HCV-specific T cell responses (Fig. 3). All vaccinees elicited HCV-specific lymphoproliferative responses after DNA and MVA administration, Vac2 eliciting the highest response (Fig. 3A-D). However, the responses observed in Vac3 and Vac4 were transient, suggesting that the vaccine elicited poor T cell memory in these two vaccinees. The antigen targeted by the vaccine was predominantly NS3 in all 4 animals (Fig. 3G).
Induction of HCV-Specific CD8+ T Cell Responses.
The production of IFN-γ in the ELISPOT assay to specific HCV with peptide pools provided first indication of the presence of HCV-specific CD8+ T cells. Our results showed that 3 of 4 vaccinees had elicited strong NS3 peptide-specific IFN-γ responses at 2 weeks after the first MVA injection, reaching up to 1785 IFN-γ producing cells/106 PBMCs in Vac3, while the IFN-γ production in response to E1 peptides remained modest (Fig. 4A-C). Only Vac4 elicited a poor IFN-γ response to the E1 and NS3 peptides (Fig. 4D). The presence of CD8+ T cell responses was confirmed using ICS with overlapping peptide pools covering the 4 antigens encoded in the vaccine. At week 22, CD8+ IFN-γ+ responses to NS3 were confirmed in Vac1 and Vac3, reaching 0.16% of CD8+ cells in Vac1 (Fig. 4G). This represented 323 IFN-γ producing CD8+ cells/106 lymphocytes, directly suggesting that the marked IFN-γ production in the ELISPOT assay was largely generated by CD4+ T cells.
These results showed that all vaccinees elicited consistent multiantigen and multi functional specific T cell responses as indicated by high numbers of cytokine producing cells, with a cytokine bias indicative of Th0-like responses, predominantly focused on NS3.
Vaccine-Induced HCV-Specific B Cell Responses.
The B cell responses were analyzed by ELISA and by neutralization assays. The DNA prime alone was a poor inducer of antibody responses, but the prime-boost combination elicited high HCV-specific antibody titers in all 4 animals, peaking between 1300 and 64,100 after the first MVA administration, but with no further increase after the second MVA immunization (Fig. 5A). Although most of the antibody responses targeted as desired the envelope proteins E1 and E2 before (Fig. 5B) or after challenge (Fig. 5C), no neutralizing activity was detected in an HCV 1b pseudoparticle assay, either prior to or after HCV challenge (data not shown).
Vaccine-Induced Control of Early Viral Load.
Eight weeks after the last immunization, all animals were challenged with HCV 1b J4. Over the first 3 months of follow up, the 2 vector control chimpanzees (Ctrl1 and Ctrl2) developed HCV viremia with peaks of serum HCV RNA of 778,125 and 766,000 copies/ml respectively (Fig. 6A,B). Positive strand HCV RNA was detected in the liver (8990 and 3550 copies/μg of total RNA), as well as minus-strand RNA, with positive / minus strand ratios of 1.4:1 and 2.7:1.
In contrast to the control animals, all immunized animals maintained markedly lower virus loads in serum during the acute phase, as observed by the 74% reduction of peak viremia (P = 0.030, peaks of 314,500 in Vac1, 74,775 in Vac2, 307,375 in Vac3 and 242,250 copies/ml in Vac4, Fig. 7A), and the 84% reduction in total serum viral loads during the first 3 months of follow-up (P = 0.024, Fig. 7B). Quantitation of HCV loads in the liver 4 weeks after exposure provided additional evidence that HCV replication was greatly reduced in all vaccinated animals. Positive HCV RNA strand production was reduced by at least 99% in the vaccinated group as compared to the controls (P = 0.028). Moreover, HCV minus strand RNA was undetectable in all vaccinees (>99.7% reduction, P = 0.0003) (Fig. 6). These results demonstrate that HCV-specific vaccine-induced adaptive immune responses controlled and markedly limited HCV replication in the first 3 months of infection. The most striking effect was observed in Vac2, whose peak viremia never exceeded 74,775 RNA copies/ml of plasma.
Absence of Protection Against Chronic Infection.
The impact of immunization on the control of HCV virus load was observed in all 4 vaccinees during the period of primary viremia. However, these effects on viremia did not extend into the chronic phase when viral clearance in the vaccinated animals was anticipated. Ultimately, only one vaccinees (Vac1) successfully cleared HCV infection (Fig. 6C). In contrast to Vac1, the 3 other immunized chimpanzees failed to clear the virus in spite of a markedly lower peak viremia than controls. Vac3 remained HCV-positive at most time points during follow-up (Fig. 6E). Although no viral RNA was detected in the plasma of the remaining 2 immunized animals (Vac2 and Vac4) between weeks 9 and 18 after exposure using the quantitative assay (detection limit 500 copies/ml), HCV RNA could be detected at several time points (Fig. 6D,F). In addition, by week 20 after HCV exposure, viral RNA could again be quantitated at several time points in Vac2, Vac3, and Vac4, with titers ranging from 500 to 22,302 RNA copies/ml of plasma. Results were confirmed by an independent quantitative HCV RNA assay performed on other serum aliquots (COBAS, data not shown). No minus strand HCV RNA was detectable in liver biopsies from all vaccinees taken at weeks 28, 32, 36, and 42 after exposure, but positive strand RNA remained present in 3 of the 4 animals (Vac2, Vac3, and Vac4, Fig. 6A-F). Different patterns were also observed between controls in the acute versus the chronic phase. Although virus loads of the control animals during the acute viremic phase were markedly greater than those in the vaccinees, this was not the case in the chronic phase. Of the 2 control animals, Ctrl1 remained persistently infected, whereas in Ctrl2 HCV RNA levels declined and became negative by all assays (Fig. 6A,B), suggesting that mechanisms other than adaptive immune responses contributed to viral clearance. Indeed, after HCV exposure, both control animals developed weak T cell immune responses to HCV, which decreased rapidly over time (Figs. 1-3). Specific CD8+ responses could not be detected in Ctrl2 while Ctrl1 elicited a CD8+ response to NS5 pp1 (Fig. 4H).
Immune responses induced by immunization were predominantly directed to NS3 and were associated with early control of infection in all 4 vaccinees, though subsequently only transient control was achieved in 3 animals. The animal that sustained complete viral control and clearance, Vac1, did generate multifunctional T cell responses (IFN-γ and IL-2 and IL-4) to multiple HCV antigens. However, these were not necessarily of greater magnitude prior to, nor following HCV exposure compared to the other 3 vaccinees (Figs. 1-3). However, Vac1 did elicit stronger CD8+ responses to NS3 prior to challenge (0.19% of CD8+ lymphocytes). Additionally, this animal mounted a strong CD8+ response to E2, peaking at 1.36% of CD8+ cells 6 weeks after exposure (Fig. 4H). These responses eventually decreased in Vac1 over time indicative of successful clearance, while Vac2 and Vac3 subsequently developed detectable NS3-specific CD8+ responses (from 0.45 to 0.77% of IFN-γ secreting CD8+ T cells) (Fig. 4H) that were maintained up to the end of the follow up (data not shown).
To investigate underlying causes of the discordance between the vaccine-induced control in the acute phase, to the unpredicted causes of chronic persistence, we undertook an analysis of cytokines and immune regulatory molecules in hepatic tissue, Comparison between animals which cleared HCV with those which became persistently infected revealed no significant differences in the expression of TGF-β, IFN-α, CD4 and CD8, IL-10, IL-5, IFN-γ, TNF-α, CCR7, FoxP3 and Cox2 in liver biopsies before and after HCV challenge (data not shown). Strikingly, despite fluctuations in the acute phase, by week 12 after infection a consistent correlation of higher PD-1 expression in hepatic tissues was observed in animals which developed persistent infection versus lower levels in those which resolved infection (Vac1 and Ctl2, Fig. 8). Not only was this effect observed with PD-1 but also with levels of expression of IDO and CTLA-4 (data not shown),37 There is a close interaction between CTLA-4 and IDO, given CTLA-4′s ability to stimulate IDO activity which ultimately favors development of tolerogenic dendritic cells.38
With worldwide estimates of more than 170 million infected humans, chronically carrying HCV, the potential risk of transmission is great and the need for a prophylactic vaccine is apparent. Here, we report on a prophylactic HCV vaccine study aimed at inducing immune responses capable of (1) reducing acute phase viral load, and (2) subsequently preventing persistent infection, and thus chronic liver disease. For this study, we used a heterologous HCV 1b J4 isolate that has previously established chronic infection in all 7 naïve chimpanzees in which it had been used.18, 39-41 Following immunization, we demonstrated that robust vaccine-induced immune responses to multiple HCV antigens were possible. Upon exposure to the heterologous 1b strain there was a dramatic reduction of HCV replication in the early phase in all vaccinated individuals in vivo. Similar results were observed recently with a T cell–based vaccine against an HCV 1a challenge.8 However, based on the characteristics of this 1b genotype to induce a high prevalence of persistent infection, we were able to demonstrate that the transition to chronic phase HCV viremia is not directly linked to early control.
Early peak viral titers are a characteristic of this inoculum, occurring between 1 to 2.5 weeks after exposure.18, 39, 40 It has been suggested that T cell responses must be sustained for weeks or months beyond the point of apparent control of virus replication to prevent relapse and establishment of persistent infection.42, 43 In the absence of data concerning HCV-specific immune responses of vaccinated animals that were not exposed to HCV, we can only speculate that the relatively rapid decline of the HCV-specific immune responses after infection may be due to a specific immune impairment by the virus itself. Indeed, impairment of adaptive immune responses in the presence of HCV infection in humans has been reported at both the level of dendritic and T helper cells.44–47 Recently, several studies have reported that T helper dependent CD8+ T cell effector function is reduced in HCV infection (reduced proliferation, perforin and IFN-γ expression), similar to observations in HIV infected individuals.48–52 In addition, whereas strong HCV specific humoral and CD4+ T cell responses were induced in all vaccinees, vaccine-induced CD8+ T cell responses detected by intracellular cytokine staining were rather weak in 3 of the 4 vaccinees. The strongest response was observed in the vaccinee with sustained viral clearance. In an effort to understand potential local and limiting effects in the liver, we measured mRNA expression of IFNγ, IL-5, IL-10, TNFα, CCR7 and TGF-β in hepatic biopsies of all study animals. This panel of cytokines unfortunately failed to reveal any correlation with effective viral clearance or persistence.
There was little or no boost of the T cell responses in the vaccinated chimpanzees following the second MVA boost or challenge. Overstimulation or exhaustion of T cell responses by MVA booster immunization seems only likely in the case of Vac4, as in this particular animal high levels of non-HCV background IFN-γ were induced following MVA boosting which were maintained until shortly before challenge.
Following acute HCV infection, T cell responses became poorly functional particularly in individuals which became chronically infected, which is reminiscent of immune exhaustion. Longitudinal liver biopsies revealed higher PD-1 expression in relationship to actin as well as CD4 expression in the 4 animals which became chronically infected in contrast to the lower levels in the 2 animals which cleared infection. These findings are supported by recent observations of elevated PD-1 expression in HCV infected persons53 in which PD-1/PDL-1 receptor ligand blocking was reported to improve function of HCV-specific CD8 T cells. Our findings of elevated PD-1 in hepatic tissue provide mechanistic insight into HCV persistence as well as possible therapeutic and vaccine strategies. Findings of elevated levels of IDO and CTLA-4 expression from the same biopsies provided additional insights into underlying mechanisms for this impaired immune function.1 Increased IDO provokes tolerogenic-like effects when induced by pro-inflammatory cytokines such as IFN-γ and TNFα.37, 38, 54 Both lines of evidence point toward the risk of driving an over zealous IFN-γ CD8+ T cell response that in particular may be counter productive if directed to promiscuous HCV epitopes that fail to impact sufficiently on viral fitness. Such rigorous but largely ineffective T cell responses may in fact drive or compound a tolerogenic environment in the liver. Detailed follow-up studies are currently underway to characterize the conserved or promiscuous nature of epitope-specific CD4+ and CD8+ T cell responses and their MHC-restricting molecules in these animals.
The data presented here clearly demonstrate the potential of HCV vaccine candidates to elicit host immune responses capable of controlling HCV 1b viremia both in plasma as well as liver in the acute phase of infection. While these are important proof of principle findings with respect to HCV vaccine development, they also point to the challenges ahead. Identifying conserved vaccine targets for CTL based vaccines that will provide efficacy in a large outbred population is a tall order. This is underscored by a cluster of recent reports4, 8, 9 highlighting the potential for CTL escape by HCV in infected human cohorts.55 These data, together with growing evidence of HCV induced tolerance in hepatic sites provides new insight into the challenges, as well as potential strategies, for the induction of effective immunity to HCV.
The Animal Science Department provided excellent animal care and support. Alexander van den Berg, Gerrit Koopman, David Davis, Ed Remarque, Axel Ulsenheimer (University of Munich) and all participants of the European HCVacc cluster provided helpful comments and assistance. The authors have no conflicting financial interest.