A human vaccine strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts, and sustains functional HCV-specific T cell memory

Abstract

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.

Trial registration: ClinicalTrials.gov NCT01296451.

Figure 1. Magnitude of T-cell responses to HCV NS after vaccination

a-d) The total ex-vivo IFN-γ ELISpot response to the NS region of HCV is shown over time during the vaccine trial (calculated by summing the responses to positive peptide pools corrected for background; see methods). a) The kinetics of the response is shown for the nine volunteers who received ChAd3-NSmut/MVA-NSmut vaccination. 4 volunteers were assessed at an extra time point a year and half after initial vaccination (TW70-73). b) The median ex-vivo IFN-γ ELISpot response to HCV NS is shown for volunteers receiving ChAd3-NSmut/MVA-NSmut (black; n=9) or ChAd3-NSmut/Ad6-NSmut (grey n=9) vaccine regimens. Arrows indicate vaccinations and trial week indicates weeks since prime vaccination. (ChAd3/MVA TW12 vs ChAd3/Ad6 TW12 p= 0.0012; ChAd3/MVA TW9 vs ChAd3/Ad6 TW10 p=0.0009) c-d) A comparison of the total ex-vivo response to HCV NS at TW34-36 in volunteers receiving ChAd3-NSmut/MVA-NSmut (black) or ChAd3-NSmut/Ad6-NSmut (grey) vaccinations (p = 0.0109) by IFN-γ ELISpot; c) magnitude of summed total T-cell response to HCV NS. d) magnitude of T-cell response to six peptide pools covering HCV NS. e) The magnitude of response to HCV NS at the peak after MVA-NSmut vaccination (TW9) vs. the end of the study (TW34) for volunteers receiving ChAd3-NSmut/MVA-NSmut vaccinations with linear regression (no TW34 data available for volunteer 320).

Figure 2. Breadth, specificity and cross-reactivity of T-cell responses to HCV NS after vaccination

a) The breadth of the T-cell response to HCV NS (the number of positive pools, F-M) assessed ex-vivo at peak magnitude after ChAd3-NSmut prime vaccination (TW2-4; open circles), after MVA-NSmut boost (TW9; black triangles) or Ad6-NSmut boost (TW12-16; grey triangles), and at the end of the study (EOS; TW34-36) after MVA-NSmut boost (black squares) or Ad6-NSmut boost (grey squares) measured by IFN-γ ELISpot. Bars, median. (ChAd3 prime vs MVA boost p=0.0156; Ad6 boost vs MVA boost p =0.0010; Ad6 EOS vs MVA EOS p=0.0355) b) The magnitude of the T-cell response to individual peptide pools 1 week after boost (TW9) is shown for volunteers who received ChAd3-NSmut/MVA-NSmut vaccination (IFN-γ ELISpot) c) The peak magnitude of the T-cell response to each individual pool is shown for volunteers vaccinated with ChAd3-NSmut/MVA-NSmut (n=9) after ChAd3-NSmut prime vaccination (TW2-4; open circles) and after MVA-NSmut boost vaccination (TW9; black triangles; IFN-γ ELISpot; ChAd3/Ad6 vs ChAd3/MVA pool F p=0.0106, pool G p=0.0106). d) The cross-reactivity of T-cell response: The total magnitude of T-cell responses using peptide pools covering the NS region of HCV genotype 1b (vaccine strain) compared to (i) genotypes 1a, 3a and (ii) genotypes 4a in volunteers by IFN-γ ELISpot assay at TW9. Bars at median with values shown (SFC/10⁶ PBMC).

Figure 3. Functionality of vaccine-induced CD4+ and CD8+ T-cells

a-b) Example FACS plots showing TNFα/IFN-γ and IL-2/IFN-γ after intracellular cytokine staining are shown for CD4+ and CD8+ T-cells stimulated with NS3-4 or DMSO control, in volunteer 310 one week (a) and 62 weeks (b) after MVA-NSmut boost vaccination (TW9 and TW70 respectively). c) A comparison of cytokine production by T-cells at peak response post vaccination. The percentage of total CD4+ or CD8+ T-cells producing IFN-γ, TNFα, or IL-2 after stimulation with NS3-5 is shown at the peak after ChAd3-NSmut prime vaccination (open circles; TW4, n=16) and at the peak after boost vaccination with either heterologous Ad6-NSmut (grey triangles; TW12, n=8) or MVA-NSmut (filled circles: TW9, n=9). PBMC were stimulated with pools F+G+H (NS3-4) or I+L+M (NS5) overnight and the percentage of cytokine secreting cells for these two stimulations were summed after background subtraction (DMSO stimulation) to get the total NS response. Values ≤0.01 are assigned 0.01. Bars, Median. d) Proliferative capacity of T-cells after boost vaccination: The proliferative response to stimulation with HCV proteins, plotted as box and whisker plots (max-min, IQR and median), is shown 6-8 weeks (TW14/16) and 26-28 weeks (TW34/36) after boost vaccination for ChAd3-NSmut/Ad6-NSmut (checkered bars; n=9) and ChAd3-NSmut/MVA-NSmut (solid bars; n=9) groups. Data are expressed as Stimulation index (SI). A dashed line at SI=3 indicate positivity cut off (Data shown in table S4).

Figure 4. Phenotyping of vaccine-induced HCV-specific T-cells

a) Example FACS plots of staining with tetramer A2-HCV-NS3_1406-1415 in volunteers 319 and 322 (vaccinated with ChAd3-NSmut/MVA-NSmut) over the study time course. Gating is on live CD3+ cells. Values indicate Percentage of CD8+ cells binding pentamer. b) Magnitude of pentamer cloud: The percentage of CD8+ T-cells binding pentamer (HLA-A2-HCV_1406-1415 or HLA-A1-HCV_1435-1443) is shown for individual volunteers over the study time course. c-d) Phenotype of vaccine induced T-cells: The percentage of the pentamer+ cells expressing phenotypic markers CD38, HLA-DR and PD-1 c), or granzyme A, granzyme B, and perforin d), are shown 4 weeks after ChAd3-NSmut prime (open circles; peak prime-PP), after Ad6-NSmut boost (PB = post boost, TW12; EOS = end of study, TW36 grey, n=5) and after MVA-NSmut boost (PB, TW9; EOS, TW34 respectively; black, n=7). Comparisons are made between vaccine regimens and between time points within a single regimen (ChAd3-NSmut/Ad6-NSmut vs ChAd3-NSmut/MVA-NSmut). Only statistically significant differences are shown. All pentamer staining and phenotyping performed ex vivo without culture. Example FACS plots shown in fig. S7.

Figure 5. Memory phenotype of vaccine-induced CD8 T-cells

PBMC were costained with pentamers to immunodominant epitopes in NS3 and with antibodies to human CD45RA and CCR7 ex-vivo. Data is shown at the following timepoints: Post ChAd3-NSmut prime (TW4, n=8), peak post Ad6-NSmut boost (TW12, n=5), at the end of the study (EOS) after ChAd3-NSmut/Ad6-NSmut (TW36, n=5; EOS ChAd3/Ad6), peak post MVA-NSmut boost (TW9, n=7) and at the end of the study after ChAd3-NSmut/MVA-NSmut (TW22-74, n=7; EOS ChAd3/MVA). a) Pie charts show the proportion of the Pentamer+ cells which display the Naïve, Tem, Temra, Tcm phenotype at the time points listed above. Pie base, Median. b) The bar graph shows the percentage of Pentamer+ cells displaying a particular phenotype at the time points listed above. Median and upper quartile are shown.

Figure 6. Principal component analysis (PCA) of T-cell immunity in human vaccinees

The PCA was loaded with the relative expression of the markers listed on the x-axis of the bar graphs above (a-c) for all CD8+ T-cells from volunteer 319 at TW22. PCA produces summary variables/principal components that summaries as much variation in the expression of these markers across CD8 T-cells as possible. a-c) The three components (PC1, PC2, PC3) summarising the most variation are shown with the weighting coefficients/component loading for each marker. d) The percentage of the overall variation in the markers assayed on CD8 T-cells explained by each principal component is plotted individually (bars) and cumulatively (line). The first three components, shown in a-c) and used in the 3D-PCA plots in Fig. 7 are shown by filled bars/dots.

Figure 7. 3D Principle Component Analysis of vaccine-induced CD8+ T-cells

The first three principal components were plotted using the protein imaging program PyMOL (PC1 axis in red, PC2 axis in green and PC3 axis in blue). a) Bulk CD8 T-cells. Each dot represents a single CD8 T-cell and in the images above the cells are coloured (blue = low, red = high) according to their relative expression within the CD8 population of IFN-γ, CD57, CD45RA, CD28, and CD27. A plot showing all CD8 T-cells as grey dots shows the shape of the CD8 T-cells in space according to the first 3 PCs with the relative locations of Naïve, Tcm and Temra populations highlighted by arrows. Data from volunteers 319 and 322. b) 3D-PCA of vaccine-induced HCV-specific T-cells: Each pink dot represents a single CD8+ T-cell (bulk CD8+ T-cells from 319 and 322) and plotted on the same axis are the tetramer+ (NS3 _1406-1415 HLA-A2) T-cells at TW2 (peak after ChAd3-NSmut prime, green) at TW9 (peak after MVA-NSmut boost, red) and at TW22 (blue). The location of CMV (black) and FLU (purple) specific T-cells, stained using tetramers on PBMC from healthy unvaccinated volunteers (LC037 and LC046), are also shown. The bulk CD8+ T-cells are shown in the bottom left image, coloured for their relative expression of IFN-γ with the locations of Naïve, Tcm and Temra populations highlighted by arrows for reference.