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A human vaccine strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts, and sustains functional HCV-specific T cell memory
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"Although the diversity of the HCV genome represents a major challenge to vaccine development, a proportion of people infected with HCV are able to eradicate the virus spontaneously and effective T cell immunity appears to play a crucial role in this. Overall, we have generated a potent T cell vaccine that we believe may recapitulate and accelerate these events in vivo to prevent the development of chronic disease, thus paving the way for the first human efficacy studies. Suitable cohorts of IVDU populations have now been identified (50, 51), and the first efficacy study of ChAd3-NSmut/MVA-NSmut in IVDUs has recently started in the United States (NCT01436357). This study will enable the assessment of vaccine immunogenicity, efficacy, and safety in a larger cohort of volunteers with a broad range of HLA types, exposed to different viral subtypes."
"We describe the development of a highly immunogenic T cell vaccine for HCV, using replication-defective chimpanzee Ad and MVA encoding the HCV NS proteins in a prime-boost strategy. This approach generates very high numbers of both CD4+ and CD8+ T cells, targeting multiple HCV antigens irrespective of host HLA background. Using established technologies and single-cell mass spectrometry (CyTOF), we show that T cells induced by vaccination are polyfunctional, that functionality increases over time, and that heterologous prime-boost with ChAd3 and MVA induced T cells with phenotypic and functional profiles distinct from those elicited by heterologous Ad vaccination. Furthermore, the strategy is simple, safe, and well tolerated in this phase 1 human study."
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A human vaccine strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts, and sustains functional HCV-specific T cell memory
Sci Transl Med 5 November 2014
A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies, and assessment of host immunity during acute infection highlight the critical role that effective T cell immunity plays in viral control. In this first-in-man study, we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in natural infection, and improved upon a vaccine based on adenoviral vectors alone. We assessed a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A, and NS5B proteins of HCV genotype 1b. Analysis used single-cell mass cytometry and human leukocyte antigen class I peptide tetramer technology in healthy human volunteers. We show that HCV-specific T cells induced by ChAd3 are optimally boosted with MVA, and generate very high levels of both CD8+ and CD4+ HCV-specific T cells targeting multiple HCV antigens. Sustained memory and effector T cell populations are generated, and T cell memory evolved over time with improvement of quality (proliferation and polyfunctionality) after heterologous MVA boost. We have developed an HCV vaccine strategy, with durable, broad, sustained, and balanced T cell responses, characteristic of those associated with viral control, paving the way for the first efficacy studies of a prophylactic HCV vaccine.
INTRODUCTION
Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and hepatocellular cancer, with millions of people afflicted worldwide (1). Although new oral antivirals are available [reviewed in (2, 3)], representing a real advance in the field, these are unaffordable and unavailable to most people, are least effective in patients with advanced liver disease, are associated with the development of viral resistance, and do not provide protection from reinfection (4). For these reasons, an effective vaccine to prevent chronic infection remains of clinical importance.
After primary infection, a proportion of those infected spontaneously resolve infection, leading to viral eradication and effectively representing long-term clinical cure (1, 5). Therefore, an effective vaccine against HCV would not need to provide sterilizing immunity, but would aim to recapitulate or accelerate the immune pathway followed in natural infection to prevent disease chronicity.
HCV may be particularly susceptible to a T cell vaccination strategy (6). Although the correlates of protection in HCV are imperfectly defined, studies of host genetic and antiviral immune responses have shown that T cells play a critical role in viral control during primary infection. This is evidenced by associations of class I human leukocyte antigens [for example, HLA-A3, HLA-B27, and HLA-B57 (7-9)] and class II antigens [for example, HLA-DR1101 and HLA-DQ0301 (10)] with clearance, the temporal association of HCV-specific interferon-γ (IFN-γ)-secreting T cells with resolution of infection (11), and the generation of polyfunctional, durable CD4+ and CD8+ T cell subsets directed against multiple HCV antigens in spontaneous control (5, 12). Additionally, the depletion of CD4+ and CD8+ T cell subsets in chimpanzees is associated with viral persistence after challenge (13, 14), whereas secondary exposure to HCV in intravenous drug users (IVDUs) is associated with the generation of robust T cell immunity (15), which correlates with protection from chronic infection upon subsequent exposure to HCV. Together, these data suggest that an HCV T cell vaccine could prevent persistent HCV infection.
Although broadly neutralizing antibodies have been identified (16) and may contribute to viral control both in natural infection (15, 17) and in chimpanzee challenge models after vaccination with HCV envelope proteins (18), it is not yet possible to generate these in most people by vaccination (19). In contrast, the development of a potent T cell vaccine is a practical and attainable goal. Proof of principle that a prophylactic T cell vaccine for HCV may be an effective strategy was first obtained in a chimpanzee model. High levels of anti-HCV-specific T cell immunity were induced in animals vaccinated with adenoviral (Ad) and DNA vectors encoding the nonstructural (NS) HCV proteins (20); after heterologous viral challenge, four of five vaccinated animals developed low-level viremia and minimal hepatitis, followed by accelerated viral clearance.
To overcome the issue of preexisting anti-Ad immunity in humans, which may limit vaccine efficacy (21), we developed a large panel of replication-defective chimpanzee and human adenoviruses found at low seroprevalence that could be used as vaccine vectors (22, 23). We trialed the first HCV prophylactic T cell vaccine using heterologous Ad vectors derived from human (Ad6) and chimpanzees (ChAd3) encoding the HCV NS3-NS5B polyprotein, with a genetically inactivated NS5B polymerase (24) in healthy volunteers. The magnitude and breadth of polyfunctional HCV-specific T cells induced after a single priming vaccination with either vector were the most potent described to date in human studies (25).
However, there were two limitations: first, heterologous Ad boosting failed to increase T cell responses to the level observed after priming. Subsequent analysis showed that this was most likely due to the induction of cross-reactive anti-Ad antibodies. This was unexpected because heterologous Ad vaccination using the same vectors in macaques had generated substantial responses after boosting, suggesting that the vectors were serologically distinct. Second, CD8+ T cells were the dominant subset induced by vaccination, whereas natural history, genetic, and T cell depletion studies show that both CD4+ and CD8+ T cell subsets are critical for viral control (5, 11-15, 26-29).
Here, we have overcome these limitations and developed an HCV prophylactic T cell vaccination strategy based on heterologous viral vectors [ChAd3 and modified vaccinia Ankara (MVA)] that is highly immunogenic after both priming and boosting vaccination, inducing both CD4+ and CD8+ T cells targeting multiple HCV antigenic targets, and associated with an acceptable safety profile.
We elected to use ChAd3 rather than Ad6 because the seroprevalence of ChAd3 is markedly lower than Ad6 (3% versus 28%), and in a heterologous Ad vectored vaccine regime, boosting after ChAd3 prime was optimal (25). Additionally, we have performed a comprehensive characterization of vaccine-induced T cell phenotype and functionality using both traditional cytometry and (for the first time in a clinical trial) cytometry by time of flight (CyTOF), revealing that this vaccine induced a functional T cell memory profile.
DISCUSSION
We describe the development of a highly immunogenic T cell vaccine for HCV, using replication-defective chimpanzee Ad and MVA encoding the HCV NS proteins in a prime-boost strategy. This approach generates very high numbers of both CD4+ and CD8+ T cells, targeting multiple HCV antigens irrespective of host HLA background. Using established technologies and single-cell mass spectrometry (CyTOF), we show that T cells induced by vaccination are polyfunctional, that functionality increases over time, and that heterologous prime-boost with ChAd3 and MVA induced T cells with phenotypic and functional profiles distinct from those elicited by heterologous Ad vaccination. Furthermore, the strategy is simple, safe, and well tolerated in this phase 1 human study.
Whereas the correlates of protection in HCV are not precisely defined, studies of T cell immunity in natural infection suggest that a number of key parameters will be required. These include the targeting of multiple HCV antigens (12), the generation of CD4+ and CD8+ T cell subsets (11, 12, 14, 15, 26, 28, 29), the maintenance of a memory pool over time with the capacity to proliferate (31), and a population of circulating T cells with immediate effector function (26, 27). A critical threshold for the magnitude of the T cell response required has not been established, although it is likely that in the context of a prophylactic vaccine, "more is better." The T cell vaccine regimen described here meets each of these criteria.
The magnitude of the HCV-specific T cell response generated by heterologous Ad-NSmut/MVA-NSmut vaccination is unprecedented. Priming with ChAd3-NSmut typically induced more than 1000 SFCs/106 PBMCs, whereas boosting with MVA-NSmut typically doubles this, with responses up to 7000 SFCs/106 PBMCs in some volunteers. The beneficial effect of MVA boosting is sustained overtime, with a clear elevation in the T cell "set point" and the maintenance of a sustained memory pool long term. Furthermore, all vaccinees respond to multiple HCV epitopes spanning the NS protein. The association of T cell breath with spontaneous viral resolution is controversial; some studies suggest that a T cell response against multiple NS antigens as measured by IFN-γ ELISpot is a predictor of viral clearance (5), whereas others have failed to demonstrate a clear association (32). Nevertheless, in the context of a prophylactic HCV vaccine, the generation of a broad antiviral response is likely to be important because HCV exists as quasispecies within an infected host and as distinct strains between individuals; targeting multiple HCV antigens increases the likelihood of vaccine-induced T cells recognizing incoming viral strains and escape variants.
Here, we could show that ChAd3/MVA vaccination induced T cell responses against all six NS antigenic pools in most individuals, and further mapping revealed as many as 31 different epitopes in a single vaccinee.
The ChAd3/MVA vaccination regimen is a significant improvement on the ChAd3/Ad6 regimen previously tested (25); a higher magnitude of T cell response is seen at all time points after boost vaccination, and both the breadth and proliferative capacity of the T cell response are significantly enhanced when measured immediately or long term after boost vaccination. The T cells induced by MVA boost vaccination have comparable cytolytic potential and polyfunctionality to those induced by heterologous Ad/Ad vaccination, but a distinct combination of T cell memory phenotypes is induced.
Because HCV exists as distinct genotypes that are broadly segregated geographically, we assessed the capacity of T cells generated by MVA boost encoding a subtype 1b immunogen to target genotypes 1a, 3a, and 4a. Although we have previously shown that T cell targets in genotypes 1 and 3 infection are distinct in the setting of natural infection (33), we find that in the context of a highly immunogenic vaccine, cross-reactive T cell responses between heterologous viral genotypes are readily generated but at a reduced magnitude. Whether these responses are sufficient to provide protection will require efficacy studies in mixed genotype populations.
Little is known about the ability of potent virally vectored vaccines to induce Treg subsets, which may influence vaccine efficacy. The induction of Tregs has been associated with persistent infection in humans (34) and with repeated HCV antigen exposure in chimpanzees (35). Treg expansion in these studies may reflect priming of T cells in the tolerogenic liver environment, because we found no induction of Tregs during vaccination with viral vectors encoding HCV antigens in the periphery.
CD4+ T cell responses are known to play a central role in the generation of effective CD8+ T cell immunity (36) and have been reproducibly associated with HCV viral control both in natural infection (28, 29) and in chimpanzee challenge studies (14). Whereas heterologous boosting with MVA-NSmut markedly increased the magnitude of the CD8+ T cell responses compared to heterologous Ad vaccination, the increase in the CD4+ T cell response, producing IL-2, TNFα, and IFN-γ, was particularly striking. Furthermore, we show that the ability of this important T cell subset to secrete multiple cytokines is enhanced, and the capacity of CD4+ T cells to proliferate increases over time after MVA-NSmut vaccination.
To increase the resolution of the functional and phenotypic assessment of vaccine-induced T cells, we used CyTOF. We show that there is a progressive increase in CD8 T cell polyfunctionality after MVA vaccination, and identified a clear hierarchy of cytokine production (MIP-1ß > IFN-γ > IL-2). Evidence of a hierarchy in cytokine production has been previously described, whereby MIP-1ß and IFN-γ are most readily released by T cells after limited stimulation, whereas IL-2 production is only triggered when a T cell has been exposed to high levels of antigen and costimulation (30, 37, 38).
Next, using both CyTOF and conventional flow cytometry, we characterized in detail the phenotype of HCV-specific T cells after Ad and MVA vaccination. Significant differences might be expected because the vector for immunogen delivery exerts a profound influence on the type of T cell response elicited due to differences in innate signaling pathways and the persistence and quantity of antigen expressed (39, 40). Low levels of transcriptionally active Ad have been shown to persist long term in both mice (41, 42) and primates (42), whereas transgene expression by MVA becomes undetectable within a few days (43).
We find that the expression of markers of activation and cytolytic capacity are significantly greater after MVA boost. In addition, distinct combinations of T cell memory phenotypes are seen; after ChAd3 vaccination, CD45RA is strongly down-regulated, inducing dominant populations of Tcm and Tem cells. After heterologous Ad boost, the response is dominated by T cells with a "naïve"-like phenotype (CD45RA+CCR7+), even though these are functional antigen-experienced T cells. In contrast, after MVA-NSmut boost, there is a marked expansion of Tem cells that increase further over time, so that by the end of the study, ~50% of cells are Tem, ~30% display a Temra phenotype, and ~10 to 15% appear to be Tcm or naïve-like in phenotype. Through 3D-PCA analysis of the CyTOF data, we confirmed the previous observation that antigen-experienced cells exists along a continuum that extends from naïve toward memory populations, associated with a progressive loss of markers associated with naïve/early differentiated cells (CD28/CD27/CCR7) and a gain in expression of markers associated with senescence (CD57, CD45RA) (30). We show that vaccine-induced HCV-specific T cells cluster in restricted niches; after Ad priming, HCV-specific T cells cluster near the area occupied by influenza-specific and Tcm CD8+ T cells. In contrast, after MVA boost, these become more heterogeneous, occupying areas further along the differentiation pathway with a phenotype that is more typical of a CMV-specific T cell population.
This evolution of CD8+ T cell memory is striking because a contraction toward a Tcm phenotype might have been expected. Overall, the phenotype of T cells after Ad/MVA vaccination is remarkably similar to that induced by the highly efficacious yellow fever and smallpox (Dryvax) vaccines (that is, Temra/Tem phenotype and PD-1+ expression), which are associated with lifelong protection (44). Furthermore, Tem and Temra cell subsets have been associated with protection against HIV (45) and influenza (46) in natural history studies, and against simian immunodeficiency virus (47) and malaria (48) in vaccine studies. It is plausible that low-level persistence of Ad-derived transcripts contributes to the molding and maintenance of long-lived Tem populations; recent data from studies of recombinant Ad vaccines in mice reveal long-term evolution of sustained Tem populations, with a close resemblance to expanded CD8+ T cell memory induced after CMV infection, and akin to those described here (41, 49).
Although the diversity of the HCV genome represents a major challenge to vaccine development, a proportion of people infected with HCV are able to eradicate the virus spontaneously and effective T cell immunity appears to play a crucial role in this. Overall, we have generated a potent T cell vaccine that we believe may recapitulate and accelerate these events in vivo to prevent the development of chronic disease, thus paving the way for the first human efficacy studies. Suitable cohorts of IVDU populations have now been identified (50, 51), and the first efficacy study of ChAd3-NSmut/MVA-NSmut in IVDUs has recently started in the United States (NCT01436357). This study will enable the assessment of vaccine immunogenicity, efficacy, and safety in a larger cohort of volunteers with a broad range of HLA types, exposed to different viral subtypes.
RESULTS
Vaccination with ChAd3-NSmut and MVA-NSmut is well tolerated
Vaccines were administered intramuscularly, and the volunteer group protocols are described in table S1. Most local and systemic adverse events (AEs) were mild or moderate (92%) and resolved within 48 hours. Systemic AEs were more common after MVA-NSmut (mean number/volunteer; 4.1 MVA-NSmut versus 1.9 ChAd3-NSmut; P = 0.032). The proportion of volunteers experiencing one or more moderate/severe AE was not significantly different between the two vaccines (P = 0.689). Severe AEs were observed in five volunteers after MVA-NSmut and two volunteers after ChAd3-NSmut (local pain/swelling, fatigue, migraine, and feverishness), but these resolved within 24 to 48 hours. Overall, both ChAd3-NSmut and MVA-NSmut were very well tolerated with no serious adverse reactions (fig. S1).
MVA-NSmut optimally and specifically boosts HCV-specific T cell responses after ChAd3-NSmut priming without the induction of regulatory T cells
We previously showed that boosting with heterologous Ad6-NSmut did not enhance anti-HCV immune responses above magnitudes observed with ChAd3-NSmut prime vaccination (25). We therefore evaluated the immunogenicity of a heterologous MVA-NSmut boost [2 x 108 plaque-forming units (pfu)] 8 weeks after ChAd3-NSmut [2.5 x 1010 viral particles (vp)] prime in nine healthy volunteers (arm A2, table S1) using IFN-γ enzyme-linked immunospot (ELISpot). All volunteers responded to ChAd3-NSmut prime, peaking 2 to 4 weeks after vaccination [median, 1140; range, 87 to 4427 spot-forming cells (SFCs)/106 peripheral blood mononuclear cells (PBMCs); Fig. 1A]. HCV-specific T cell responses were significantly enhanced by MVA-NSmut boost in all volunteers, peaking 1 week after vaccination (median, 2355; range, 1490 to 6117 SFCs/106 PBMCs; peak after ChAd3-NSmut prime versus peak after MVA-NSmut boost; P = 0.0039; Fig. 1, A and B). Furthermore, in comparison to heterologous Ad vaccination, ChAd3-NSmut/MVA-NSmut prime-boost generated responses that were more sustained over time, and significantly greater at the end of the study both to the HCV NS region overall (Fig. 1, B and C; median, 443; range, 138 to 1783 versus median, 98; range, 10 to 1092 SFCs/106 PBMCs; P = 0.0109) and to all six individual peptide pools covering HCV NS (Fig. 1D). The peak response after MVA-NSmut boost correlated significantly with the durability of the T cell response [trial week 9 (TW9) versus TW34; linear regression R2 = 0.784; P = 0.0034; Fig. 1E) and between TW8 (before MVA-NS boost vaccination; R2 = 0.68; P = 0.0429). T cell responses remained detectable by IFN-γ ELISpot in four of five patients tested at weeks 70 to 73 (median, 302; range, 10 to 732 SFCs/106 PBMCs; Fig. 1A).
No HCV-specific T cell response was detected in the four volunteers vaccinated with 2 x 108 pfu MVA-NSmut prime alone (arm A1; table S3).
To assess a possible nonspecific "bystander" expansion of non-HCV antigen-specific T cells after vaccination, we monitored the T cell response to HLA class I influenza A (FLU), Epstein-Barr virus (EBV), and cytomegalovirus (CMV) epitopes and to CMV lysate; no change in the magnitude of the responses to these antigens was observed (fig. S2). The induction of regulatory T cells (Tregs) was also assessed in five volunteers before vaccination, at the peak of the T cell response to ChAd3-NSmut prime and MVA-NS boost vaccination (TW2-4 and TW9, respectively), and long-term (TW47-72) after vaccination. We found no significant change in the magnitude of Treg subsets, and levels of Tregs in vaccinated volunteers were comparable to those seen in PBMCs from eight healthy unvaccinated volunteers (fig. S3).
MVA-NSmut boost increases the breadth of ChAd3-NSmut-primed T cell responses
We compared the breadth of T cell responses induced by ChAd3-NSmut prime/MVA-NSmut boost to that induced by heterologous Ad boosting using peptides corresponding to the entire immunogen in six pools and defined further using 8 to 11 peptides in minipools (Table 1). At the peak response after MVA-NSmut boost, most individuals responded to all six peptide pools (range, 4 to 6; Fig. 2, A and B), and the breadth was significantly higher than that observed after ChAd3-NSmut prime or after heterologous Ad boost (Fig. 2, A and C; P = 0.0156 and P = 0.0010, respectively). The breadth of response was also significantly greater at the end of the study after MVA vaccination when compared to heterologous Ad boost vaccination (P = 0.0355; Fig. 2A). Further dissection of responses to minipool level revealed that the number of HCV epitopes targeted after MVA boost was as high as 31 in a single individual (subject 310; Table 1); because the minipool analysis was performed at TW12, after contraction of the T cell response, the true number of epitopes targeted in each subject may be underestimated. Although all peptide pools were targeted in patients from diverse HLA backgrounds, responses to NS3 dominated after both ChAd3 prime and MVA boost [Friedman analysis of variance (ANOVA), P = 0.0033; Fig. 2, B and C]. MVA boost increased the magnitude but did not affect the overall hierarchy of HCV antigen recognition (Fig. 2C). Responses to two epitopes located in NS3h restricted by HLA-A1 and HLA-A2 (marked bold in Table 1) were particularly prevalent and selected for subsequent pentamer analysis.
All individuals showed a major increase in T cell response with MVA boost compared to Ad prime, but three individuals showed a particularly strong response to MVA boost vaccination (Fig. 1A); however, there were no known differences in the three "super-responders" when compared to the other volunteers: In particular, no one HLA type was overrepresented in these three volunteers, and there was no evidence that these volunteers had previous exposure to HCV. After removing the super-responders from the analysis, the difference between Ad6-NSmut and MVA-NSmut boost vaccination remained statistically significant at multiple time points, including the end of the study TW34/36 (P = 0.0287).
T cell responses after MVA-NSmut boosting vaccination recognize multiple HCV genotypes
We determined the capacity of the T cells induced by ChAd3-NSmut/MVA-NSmut encoding the genotype 1b immunogen to target other globally prevalent HCV subtypes using peptides derived from genotypes 1a, 3a, and 4a sequences in IFN-γ ELISpot assays. Although subtypes 1a, 3a, and 4a diverge significantly from genotype 1b at the amino acid level (86, 77, and 78% sequence homology, respectively), responses to these subtypes were generated, albeit at a lower magnitude (Fig. 2D). Responses to genotype 1a were about 60%, and to genotype 3a/4a were 30% of those generated against genotype 1b, whereas breadth is maintained (fig. S4). We observed a direct correlation between the response to the genotype 1b immunogen and subtypes 1a and 3a but not to 4a (1b versus 1a: R2 = 0.974, P = 0.0018; 1b versus 3a: R2 = 0.975, P = 0.0017; 1b versus 4a: R2 = 0.045, P = 0.734).
MVA-NSmut boost induces polyfunctional CD4 and CD8+ T cell subsets
Next, we assessed the relative contribution and functionality of CD4+ and CD8+ T cell subsets to the total response. We found that MVA-NSmut boosting vaccination induced higher numbers of both T cell subsets compared to those seen after ChAd3-NSmut prime, and also in comparison to heterologous Ad6-NSmut boost (Fig. 3). Using intracellular cytokine staining (ICS) and SPICE analysis, we showed that vaccine-induced HCV-specific CD4+ and CD8+ T cells were polyfunctional with an equal proportion of CD4+ T cells producing one [interleukin-2 (IL-2) or IFN-γ], two [IL-2 and IFN-γ or IFN-γ and tumor necrosis factor-α (TNFα)], or three (IL-2, IFN-γ, and TNFα) cytokines, whereas CD8+ T cells predominantly produced IFN-γ early after vaccination (TW4/TW9) and produced IFN-γ in conjunction with TNFα or TNFα and IL-2 10 to 14 weeks after boost (fig. S5). The polyfunctionality of CD4+ and CD8+ T cells increased after MVA vaccination, peaking at weeks 18 and 22, respectively (fig. S5). We also assessed the per-cell production of cytokine in polyfunctional compared to single cytokine-producing T cells (fig. S6). The geometric mean fluorescent intensity (GeoMFI) of each cytokine (with the exception of IL-2 production by CD8+ cells) was significantly higher in CD4+ and CD8+ T cells that produced three cytokines versus one cytokine. The biggest differences were seen in per-cell production of IFN-γ [median GeoMFI of triple- versus single-producing CD4+ T cells was 4131 versus 1322 (P < 0.0001), and 10,329 versus 1543 for CD8+ T cells (P < 0.0001)]. Polyfunctional CD4+ and CD8+ T cells were readily detectable by ICS 74 weeks after prime vaccination (Fig. 3B).
The proliferative capacity of PBMCs was assessed in [3H]thymidine incorporation assays. HCV recombinant protein antigens were used in this assay, which detects predominantly CD4+ T cell proliferation. Strong proliferative responses to multiple HCV antigens could be detected 6 weeks after MVA-NSmut boost, and these increased further when assessed 26 weeks after MVA-NSmut boost (Wilcoxon, P = 0.0391, NS3; Fig. 3D), consistent with the generation of a population of memory T cells capable of rapid proliferation on reexposure to antigen. Proliferative responses after MVA-NSmut boost were significantly greater than those seen after heterologous Ad boost (Fig. 3D).
A detailed characterization of vaccine-induced CD8+ T cells was performed using HLA class I multimers
After fine mapping of vaccine-induced T cell responses (Table 1), we used HLA class I pentamers (HLA-A*0201 HCV NS31406-1415 KLSALGINAV; HLA-A*0101 HCV NS31435-1443 ATDALMTGY) to track the characteristics of vaccine-induced T cells over time (Fig. 4, A and B; example FACS plots in fig. S7). After boosting with MVA-NSmut, HCV-specific T cells were highly activated, expressing CD38 in 80 to 100% pentamer+ cells, with about 60% coexpressing HLA-DR (Fig. 4C). In contrast, after heterologous Ad boosting, 25% expressed CD38 with minimal HLA-DR coexpression. PD-1 expression (a molecule that has been associated with both T cell activation and exhaustion) was also high after MVA-NSmut, declining over the duration of the study (Fig. 4C). HCV-specific T cells after MVA-NSmut also expressed granzyme A, granzyme B, and variable levels of perforin (Fig. 4D).
After ChAd3-NSmut priming vaccination, a mixed pool of memory populations was detected [central memory T cells (Tcm; CD45RA-CCR7+), effector memory T cells (Tem; CD45RA-CCR7-), naïve or naïve-like memory (CD45RA+CCR7+), and "terminally differentiated" effector memory T cells (Temra; CD45RA+CCR7-)] that remained after heterologous Ad boosting. In contrast, the large expansion of HCV-specific T cells after MVA-NSmut boost was dominated by CD45RA- populations. The long-term memory population at the end of the study after heterologous Ad vaccination was predominantly lymph node homing (CCR7+) T cells and that had reexpressed CD45RA; in contrast, after Ad/MVA regime, the dominant population were peripheral organ-homing (CCR7-) Tem with low expression of CD45RA (Fig. 5).
An analysis of T cell functionality using CyTOF technology reveals the evolution of memory T cells over time
Single-cell mass cytometry (CyTOF) was used to produce an in-depth analysis of vaccine-induced HCV-specific T cells in two volunteers (319 and 322) at multiple time points. Antibodies labeled with heavy metal isotopes (n = 35; table S2) that bind to surface and intracellular proteins allowed T cell phenotypes to be quantified. HCV-specific T cells were observed using metal-labeled peptide-MHC tetramers (gating strategy for CyTOF shown in fig. S8). Validating this approach, the expression of surface and intracellular markers measured by CyTOF correlated with those previously assessed using FACS (Spearman's rank r = 0.8449, P < 0.0001; fig. S9).
We assessed the overall potential for cytokine production of HCV-specific CD8+ T cells after a 3-hour stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin, so that the cytokine production by HCV-specific T cells could be compared to that of bulk CD8+ T cells and avoid the rapid down-regulation of T cell receptor and subsequent lack of tetramer staining that accompanies peptide stimulation. Newell et al. (30) have previously shown that PMA/ionomycin stimulation induced comparable cytokine production by T cells to CD3/CD28 bead activation and that it allowed accurate multimer staining.
In keeping with the ICS data by FACS (fig. S5), we showed a progressive increase in HCV-specific CD8+ T cells producing multiple cytokines over time (a feature not seen in the bulk CD8+ population), with about 80% of cells having three or more functions by TW22 (14 weeks after MVA-NSmut; fig. S10). Evidence of a hierarchy of cytokine production by vaccine-induced HCV-specific T cells was observed, with single cytokine-producing CD8+ T cells making MIP-1ß (macrophage inflammatory protein 1ß) and dual cytokine-producing T cells making MIP-1ß and IFN-γ or TNFα, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-2 were only produced in combination with MIP-1ß, IFN-γ, and TNFα by the most polyfunctional T cells (fig. S10).
To further analyze the CyTOF data, we used principal components analysis (PCA). The PCA was loaded with expression data from patient 319 at TW22, because this patient had the largest populations of "memory" CD8+ T cells and a relatively large HCV-specific pentamer cloud (observed by FACS). Although this analysis is unsupervised, the apparent meaning of each component can be deduced on the basis of previously defined CD8+ T cell subsets by looking at the markers that most influence the PCs (PC1: naïve versus memory, PC2: effector function, PC3: T cell differentiation status; Fig. 6, A to C). The first three PCs accounted for >50% of the variation within the data set (Fig. 6D), and therefore, these alone were plotted in PyMOL [three-dimensional PCA (3D-PCA)] to visualize CD8+ T cell complexity. In theory, CD8+ T cells could occupy any space within these plots; however, CD8+ T cells clustered in defined, continuous regions, resulting in an "L-shaped" plot along the PC1-PC3 axis (Fig. 7). This pattern was observed in both individuals at all time points and in the two vaccine-naïve control patients.
We identified the location of classical and viral specific T cell subsets on this continuum before defining the location of the vaccine-induced HCV-specific T cells. Analysis of the relative expression of single markers (for example, IFN-γ, CD57, CD28, CD45RA, and CD27) showed that classical T cell subsets cluster in discrete niches (Fig. 7A and fig. S11). For example, the niche occupied by the naïve T cell population is easily identified by its high expression of CD45RA, CD27, and CD28 and low expression of IFN-γ, whereas most non-naïve (memory) T cells are positioned within an "L-shaped arm" that extends from the naïve population (Fig. 7A). The memory populations were further dissected through the analysis of CD45RA and CCR7, which showed that Tem, Tcm, and Temra occupied niches along the L-shaped arm (fig. S11). HCV, influenza, and CMV-specific tetramer+ T cells were superimposed on the 3D-PCA plot of bulk CD8, showing that they also cluster in tightly restricted niches (Fig. 7B).
After ChAd3-NSmut prime (TW2) vaccination, HCV-specific T cells appear to occupy a niche close to the naïve population, in the area occupied by Tcm (CD45RA-CCR7+) and FLU-specific T cells (Fig. 7B). After MVA-NSmut boost vaccination (TW9), the cells appear more heterogeneous and occupy a broader area across the continuum, with most HCV-specific T cells matching the position of Tem and CMV+ T cells in the 3D-PCA plots (Fig. 7B). At the latest time point studied, TW22, most HCV-specific T cells sit in a niche at the end of the L-shaped arm, in a similar location to Temra cells (Fig. 7B).
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