MHC class II invariant chain-adjuvanted viral vectored vaccines enhances T cell responses in humans

Abstract

Strategies to enhance the induction of high magnitude T cell responses through vaccination are urgently needed. Major histocompatibility complex (MHC) class II-associated invariant chain (Ii) plays a critical role in antigen presentation, forming MHC class II peptide complexes for the generation of CD4⁺ T cell responses. Preclinical studies evaluating the fusion of Ii to antigens encoded in vector delivery systems have shown that this strategy may enhance T cell immune responses to the encoded antigen. We now assess this strategy in humans, using chimpanzee adenovirus 3 and modified vaccinia Ankara vectors encoding human Ii fused to the nonstructural (NS) antigens of hepatitis C virus (HCV) in a heterologous prime/boost regimen. Vaccination was well tolerated and enhanced the peak magnitude, breadth, and proliferative capacity of anti-HCV T cell responses compared to non-Ii vaccines in humans. Very high frequencies of HCV-specific T cells were elicited in humans. Polyfunctional HCV-specific CD8⁺ and CD4⁺ responses were induced with up to 30% of CD3⁺CD8⁺ cells targeting single HCV epitopes; these were mostly effector memory cells with a high proportion expressing T cell activation and cytolytic markers. No volunteers developed anti-Ii T cell or antibody responses. Using a mouse model and in vitro experiments, we show that Ii fused to NS increases HCV immune responses through enhanced ubiquitination and proteasomal degradation. This strategy could be used to develop more potent HCV vaccines that may contribute to the HCV elimination targets and paves the way for developing class II Ii vaccines against cancer and other infections.

Figure 1. Solicited adverse events within 7 days from vaccination

Frequency of local and systemic adverse events recorded by volunteers on diary cards. The proportion of volunteers reporting symptoms at any time during 72 hours following (A) Low dose: ChAd3-hIiNSmut (5 x10^9 vp) (n=6); (B) Low dose: MVA-hIiNSmut (5 x10^7 pfu) (n=6), (C) Standard dose: ChAd3-hIiNSmut (2.5 x10^10 vp) (n=11); (D) Standard dose: MVA-hIiNSmut (2 x10^8 pfu) (n=10); (E) Standard dose: ChAd3-NSmut (2.5 x10^10 vp) and (F) Standard dose: MVA-NSmut (2 x10^8 pfu). Color code indicates maximum severity of the reaction reported: black– Grade 1 (mild); red– Grade 2 (moderate); blue-Grade 3 (severe).

Figure 2. Kinetics and breadth of T cell response to hIiNS after prime/boost vaccine regimen.

(A) The kinetics of ex vivo IFN-γ ELISpot response to the NS region of HCV is shown for 10 volunteers who received ChAd3/MVA-hIiNSmut vaccination and 1 volunteer (014) who received only ChAd3-hIiNSmut over time calculated by summing the responses of positive pools corrected for background. (B) Comparisons of IFN-γ ELISpot response to HCV NS in volunteers receiving ChAd3/MVA-hIiNSmut (black; n=10) or ChAd3/MVA-NSmut (grey; n=17). The median and IQR is shown. Arrows above graph indicate vaccination timepoints. (Two-tailed Mann-Whitney test at d14, d28, d56, d63, d84, d98) (C) Area Under the Curve (AUC) analysis up to d238 timepoint for total IFN-γ ELISpot response (Two-tailed Mann-Whitney test). Bars represent median. (D) Magnitude of T cell response to each NS region at d63. (E) The number of positive peptide pools for each volunteer is shown at peak post prime (d14 or d28), peak post boost (d63 or d84), d98 and EOS. (Two-tailed Mann-Whitney test post prime and at d98). Bars represent median. (F) Magnitude of T cell response to six peptide pools at D63, 1-week post MVA vaccination (Two-tailed Mann-Whitney test for NS3p and NS3h). Bars represent median. Only statistically significant differences are shown. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001

Figure 3. T cell and antibody response against Ii.

(A) IFN-γ ELISpot response against human Ii peptide pool at different time points: screening, d14, d28, (n= 11) and d63 and d238 (n= 10) for volunteers in group 2. Bars represent median. Each symbol represents T cell response to Ii in a specific subject at different time points of the prime/boost regimen. (B) Antibody response to human CD74 p35 ectodomain in sera from individual volunteers collected at key time-points (screen, d28, d63) as detected by ELISA (n=17). Italics denotes group 1 volunteers, bars represent the mean absorbance (OD, optical density) of triplicate wells plus standard deviation of 1:20 diluted sera or assay positive and negative controls. The dotted line set at 0.08 OD represents the assay positivity cut point, calculated as described in materials and methods.

Figure 4. Functionality of vaccine-induced CD4+ and CD8+ T cells

(A-B) Example FACS plots showing TNF-α/IFN-γ and IL-2/ IFN-γ after ICS of CD4+ and CD8+ T cells stimulated with DMSO as negative control and NS3-4 peptide pool one week after MVA-hIiNSmut boost vaccination (A) and at the end of study (EOS) (B). (C) Percentage of total CD4+ and CD8+ T cells producing IFN-γ, IL-2 or TNF-α after stimulation with NS3-5 peptide pools is shown at peak post-boost vaccination (n=10) and at EOS (n=9). Bars represent the median. (D) Percentage of CD4+ expressing CD154 and CD8+ expressing CD107a after stimulation with NS3-5 peptide pools at peak post-boost (n=10) and at EOS (n=9). Bars represent the median.

Figure 5. Sustained and enhanced proliferative T cell capacity after ChAd3/MVA-hIiNS vaccination regimen.

(A) Example FACS plots of proliferated CD8+ T cells after stimulation with pool 1 (first highest pool at boost), pool 2 (second highest pool at boost) and pool 3 (lowest at EOS but positive at boost) using cell trace violet reagent in T cell proliferation assays. (B) Percentage of proliferated CD4+ and CD8+ T cells of subjects vaccinated with ChAd3/MVA-NSmut (grey, n=7) and ChAd3/MVA-hIiNSmut (black, n=9) at EOS after stimulation with peptide pools (Two-tailed Mann-Whitney test for CD8+ pool 1, pool 2 and pool 3). Only statistically significant differences are shown. Bars represent median. *P ≤ 0.05; **P ≤ 0.01;

Figure 6. Phenotyping of pentamer+ CD8+ HCV-specific vaccine-induced T cells

(A) Example FACS plots of ex vivo staining of PBMC with using MHC class I pentamer A2-HCV NS3 1406-1415 in volunteers 001 (vaccinated with ChAd3/MVA-NSmut) and 002 (vaccinated with ChAd3/MVA-hIiNSmut) at baseline (TW0) and at peak post-boost (TW9). Values indicate percentage of CD8+ T cells binding pentamer. (B) The percentage of CD8+ T cells binding pentamer (HLA-A1-HCV 1435-1443 and HLA-A2-HCV 1406-1415) is shown over time for individual volunteers receiving ChAd3- /MVA-hIiNSmut vaccination (based on the peak of IFN-γ ELISpot response, % of pent+CD8+ are shown at TW2 or TW4). Based on HLA, a single pentamer is used for each volunteer. (C) The percentage of the pentamer+ cells expressing phenotypic markers CD38, HLA-DR, CD127, PD-1 and Granzyme A (GzA) of volunteers vaccinated with ChAd3/MVA-NSmut (gray symbols) or ChAd3/MVA-hIiNSmut (black symbols) at peak post-prime (green bars; gray, n=2 and black, n=8), at peak post-boost (yellow bars; gray, n=5 and black, n=8) and at EOS (light blue bars; gray, n=5 and black, n=8). Only statistically significant differences are shown (Two-tailed Mann-Whitney test CD38; Two-tailed Mann-Whitney test HLA-DR at prime and boost; Two-tailed Unpaired Student’s t test CD127 gray symbols boost vs EOS; black symbols boost vs EOS; Two-tailed Unpaired Student’s t test PD-1 at prime and EOS; Two-tailed Mann-Whitney test GzA). Mean with SEM is shown. (D) Pie charts display the proportion of pentamer+ CD8+ Tn, TemRA, Tcm, and Tem subsets for the two vaccination regimens as specified by staining for CD45RA and CCR7 at peak post-prime (NSmut, n=2; hIiNSmut n=8) at peak post-boost and EOS (NSmut, n=5; hIiNSmut n=8). Pie base, Median. *P ≤ 0.05; **P ≤ 0.01

Figure 7. Phenotyping of tetramer+ CD4+ HCV-specific vaccine-induced T cells.

(A) Example FACS plots of ex vivo staining with MHC class II tetramer DRB01*01 NS31806-1818 in volunteers 003 (vaccinated with ChAd3/MVA-NSmut) and 004 (vaccinated with ChAd3/MVA-hIiNSmut) over the study time course. (B) The percentage of specific CD4+ T cells stained with a single tetramer (for PEA03-01113,17, 18, 22) or a combination of them (for PEA03-01119, 26) is shown over time for individual volunteer receiving ChAd3-hIiNSmut/MVA-hIiNSmut vaccination (n=6). (C) The percentage of the tetramer+ cells expressing CD127 from volunteers vaccinated with ChAd3/MVA-NSmut (grey symbols) or ChAd3/MVA-hIiNSmut (black symbols) at peak post-prime (green bars; grey n=3, black n=4), at peak post-boost (yellow bars; grey n=4, black n=5) and EOS (light blue bars; grey n=4, black n=5). Only statistically significant differences are shown. (D) Pie charts display the proportion of tetramer+ CD4 Tn, TemRA, Tcm, and Tem subsets over the course of the study for the two vaccination regimens, as specified by staining for CD45RA and CCR7 at peak post-prime (NSmut, n=3; hIiNSmut n=4) at peak post-boost and EOS (NSmut, n=4; hIiNSmut n=5). Pie base, Median.

Figure 8. Ii modulates CD8+ T cell immune responses by targeting fused antigens for K48-specific ubiquitination and proteasome-mediated degradation.

(A) Schematic representation of full-length murine p31 Invariant chain (mIi) and deletion mutants. Functional domains are indicated: ESS, endolysosomal sorting signal; TM, transmembrane domain; KEY, key motif; CLIP, class II-associated Ii chain peptide; Trimerization, trimerization domain. (B) C57BL/6 mice were vaccinated with 3x10^6 viral particles of Ad5 encoding OVA either alone or fused to the indicated mIi deletion mutants. T cell responses were evaluated 2 weeks later by IFN-γ ELISpot assay. Data are expressed as number of T cells producing IFN-γ per million splenocytes (Ordinary one-way ANOVA test for FL, D-17, 1-105,1-80, 1-75 mIi constructs versus OVA) The experiment were repeated four times. (C) The percentage of Ad5 infected CD11c+ BMDC cells expressing SIINFEKL peptide bound to H-2Kb MHC class I left untreated or treated with MG132 (proteasome inhibitor) or Pepstatin A/E64 (lysosomal proteases inhibitor) was evaluated. Results are expressed as fold difference relative to Ad5-OVA infected cells (Kruskal-Wallis test untreated OVA vs mIi-OVA; OVA vs mIi1-75; Pepstatin+E64d treatment OVA vs mIi-OVA) The experiments were repeated five times in duplicate. (D) HeLa cells transiently transfected with ubiquitin plasmid were infected with Ad5-mIi-OVA, mIi1-75 OVA and mIi1-50 OVA and treated with MG132. Cells extracts were immunoprecipitated with anti-Lys48 antibody and analysed by WB with an anti-HA antibody detecting OVA. (E) Schematic representation of mIi and 55-75 region. (F) Immunogenicity in C57BL/6 mice of Ad5-mIi55-75 was evaluated after 2 weeks later by IFN-γ ELISpot assay (Ordinary one-way ANOVA mIi OVA versus OVA; mIi55-75 OVA versus OVA). The experiments were performed three times. (G) HeLa cells were transfected with ubiquitin plasmid, infected with Ad5 -mIi55-75 and treated with MG132. The cells lysate was immunoprecipitated using an anti-Lys48 antibody and tested by WB using anti HA antibody detecting OVA. The experiments were performed two times. (H) HeLa transiently transfected with ubiquitin plasmid were infected with ChAd3-NSmut and with ChAd3-hIiNSmut and treated or not with MG132. Cells extracts were immunoprecipitated with anti-Lys48 antibody and analysed by WB with an anti-NS3 Ab detecting NS3 protein. Mock is negative control for uninfected cells. The experiments were performed three times. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001