TRAP specific response in non-human primates 12 weeks after MVA boost.

<p>Frozen PBMCs samples taken from male rhesus macaque 12 weeks after the MVA boost (Week 20 sample) were thawed and restimulated for 16 hours with TRAP peptides prior staining for IFN-γ production and memory/effector T cell markers. a.) Graphs represent the frequency of IFN-γ⁺ CD4⁺ T cells (left panel), frequency of T cell subset⁺ CD4⁺ T cells (central panel) or the frequency of each T cell subsets as a percentage of IFN-γ⁺ CD4⁺ T cells (right panel). b.) Graphs represent the frequency of IFN-γ⁺ CD8⁺ T cells (left panel), frequency of T cell subset⁺ CD8⁺ T cells (central panel) or the frequency of each T cell subsets as a percentage of IFN-γ⁺ CD8⁺ T cells (right panel). Analysis with a two-way anova showed a significant effect of hIi on the frequency of IFN-γ⁺ T cells (p = 0.0227), frequency of CD4⁺ T cells subset (p = 0.0117) and CD8⁺ T cell subsets (p = 0.0057) T cells; post-hoc Bonferoni positive test were performed to determine the effect the hIi on either CD4 or CD8 cells, or each T cell subset, asterisk denote the level of significance (*p<0.05, **p<0.01).</p

Human invariant chain fusion in mice.

<p>a.) C57BL/6 mice were immunized i.m. with 10⁸ and 10⁶ iu of ChAd63.ME-TRAP (circles) or ChAd63.hIi-ME-TRAP (squares) with spleens harvested two weeks later to measure TRAP specific CD4⁺ (left) and CD8⁺ (right) T cell responses by ICS. b.) CD-1 (ICR) mice were vaccinated i.m. with 10⁸ iu of ChAd63.ME-TRAP (circles) or ChAd63.hIi-ME-TRAP (squares) with spleens harvested 2 weeks after vaccination to measure TRAP specific CD4⁺ (left) and CD8⁺ (right) T cell response by ICS. In each graph, individual mice are denoted by a single point after background subtraction (unstimulated) and lines represent the median response per group. Data in each experiment was analysed with a two-way analysis of variance and where a significant effect of the human Ii chain was observed, a post-hoc Bonferoni test was performed, asterisks denote the level of statistical significance when compared to the control ME-TRAP vaccinated group (*p<0.05, **p<0.01, ***p<0.001).</p

Human invariant chain in an MVA vector.

<p>a.) C57BL/6 mice were immunized i.m. with 10⁷ and 10⁶ PFU of MVA.ME-TRAP (circles) or MVA.hIi-ME-TRAP (squares) with spleens harvested 1 week later to measure TRAP specific CD4⁺ (left) and CD8⁺ (right) T cell responses by ICS. b.) CD-1 (ICR) mice were vaccinated i.m. with 10⁷ PFU of MVA.ME-TRAP (circles) or MVA.hIi-ME-TRAP (squares) and spleens harvested 1 week later to measure TRAP specific CD4⁺ (left) and CD8⁺ T cell response by ICS. In each graph, individual mice are denoted by a single point after background subtraction (unstimulated) and lines represent the median response per group. Data in each experiment was analysed with a two-way analysis of variance and where a significant effect of the human Ii chain was observed, a post-hoc Bonferoni test was performed, asterisks denote the level of statistical significance when compared to the control ME-TRAP vaccinated group (*p<0.05).</p

ChAd63 MVA prime-boost regimens in mice.

<p>a.) C57BL/6 mice were immunized i.m. with 10⁶ iu of ChAd63.ME-TRAP and 8 weeks later boosted with either 10⁶ PFU of MVA.ME-TRAP (circles) or MVA.hIi-ME-TRAP (squares). b.) C57BL/6 mice were immunized i.m. with 10⁶ iu of ChAd63.ME-TRAP (circles) or ChAd63.hIi-ME-TRAP (squares) and boosted 8 weeks later with the 10⁶ PFU of MVA vector containing the same insert. c.) CD-1 (ICR) mice were immunized i.m. with 10⁸ iu of ChAd63.ME-TRAP (circles) or ChAd63.hIi-ME-TRAP (squares) and boosted 8 weeks later with the 10⁶ PFU of MVA vector containing the same insert. In all experiments, spleens and sera were harvested 1 week after the MVA vaccination, individual mice are denoted with a single point and lines represent the median response per group. TRAP specific CD4⁺ (left) and CD8⁺ (middle) T cells responses were measured by ICS with the data from each experiment analysed with a 2-way analysis of variance and a post-hoc Bonferoni positive test; asterisk denotes the level of statistical significance compared to the control ME-TRAP vaccinated group (***p<0.001). Abs against TRAP were measured by LIPS assay (right), the dotted line in each graph denotes the luminescence of a naïve mouse. Data from each experiment was analysed with a Mann-Whitney test, a significant effect of hIi was only observed in outbred mice (*p = 0.0152).</p

Effect of fusion of mIi to ME-TRAP in mice immunized with ChAd63 vectors.

<p>a. & b.) C57BL/6 mice were vaccinated i.m. with 10⁷ or 5×10⁵ iu of ChAd63.ME-TRAP (circles) or ChAd63.mIi-ME-TRAP (triangles) and spleens were harvested two weeks later to measure the response to the dominant CD4⁺ (a.) and CD8⁺ (b.) T cell epitopes by IFN-γ ELISpot. c. & d.) Outbred CD-1 (ICR) mice were vaccinated i.m. with 10⁷ iu of ChAd63.ME-TRAP (circles) or ChAd63.mIi-ME-TRAP (triangles) and spleens harvested two weeks later to measure IFN-γ producing TRAP specific CD4⁺ (c.) and CD8⁺ (d.) T cell responses by ICS. Points represent individual mice after subtraction of background responses and lines represent the median. ****p<0.0001, ***p<0.01, *p<0.05 compared to ChAd63.ME-TRAP (2-way ANOVA with Bonferroni post-test).</p

ChAd63-MVA prime boost regimen in non-human primates.

<p>Male rhesus macaque were vaccinated i.m. with either ChAd63.ME-TRAP followed 8 weeks later by MVA.ME-TRAP (open symbols), or ChAd63.hIi-ME-TRAP followed 8 weeks later by MVA.hIi-ME-TRAP (closed symbols). Blood samples were taken on the day of vaccination, weeks 2, 4, 6, 8 following ChAd63 vaccination and 1 (9), 3 (11) and 12 (20) weeks following MVA vaccination. a.) The graphs represent the summed response to TRAP peptides over the course of the experiment for animals immunised with ME-TRAP (left) or hIi-ME-TRAP (right) vectors. The data was analysed with a 2-way anova (repeated measures) demonstrating a significant effect of the human Ii chain (p = 0.0182) and a post-hoc Bonferoni positive test to compare the two groups at each timepoint. b.) Graphs represent the response at week 2 to ME, TRAP and human Ii chain peptides measured by ELISpot at (left) and the IFN-γ TRAP specific response of CD4⁺ (centre) or CD8⁺ (right) T cells as measured by ICS at this same time. Each data set (ELISpot or ICS) was analysed with a 2-way analysis of variance and where a significant effect of human Ii was observed (ELISpot p = 0.0080), a post-hoc Bonferoni positive was performed (asterisks denote the level of significance) (****p<0.0001). c.) Graphs represent the response at week 9 to ME, TRAP and human Ii chain peptides measured by ELISpot at (left) and the IFN-γ TRAP specific response of CD4⁺ (centre) or CD8⁺ (right) T cells as measured by ICS at this same time. Each data set (ELISpot or ICS) was analysed with a 2-way analysis of variance and where a significant effect of human Ii was observed (ELISpot p = 0.0094, ICS p = 0.0274), a post-hoc Bonferoni positive was performed (asterisks denote the level of significance) (***p<0.01, *p<0.05). d.) Graphs represent the antibody response to TRAP over the course of the vaccination (left panel) or as a fold increase from the pre-vaccination timepoint. Data was analysed with a 2-way analysis of variance (repeated measure) with a significant effect of the human Ii chain observed (timecourse p = 0.0443, fold change p = 0.0341) with a post-hoc Bonferoni positive test to determine the significance at each timepoint, asterisks denote the level of significance (*p<0.05).</p

DataSheet_1_Adenovirus Encoded Adjuvant (AdEnA) anti-CTLA-4, a novel strategy to improve Adenovirus based vaccines against infectious diseases and cancer.docx

IntroductionVirus vectored genetic vaccines (Vvgv) represent a promising approach for eliciting immune protection against infectious diseases and cancer. However, at variance with classical vaccines to date, no adjuvant has been combined with clinically approved genetic vaccines, possibly due to the detrimental effect of the adjuvant-induced innate response on the expression driven by the genetic vaccine vector. We reasoned that a potential novel approach to develop adjuvants for genetic vaccines would be to “synchronize” in time and space the activity of the adjuvant with that of the vaccine.MethodsTo this aim, we generated an Adenovirus vector encoding a murine anti-CTLA-4 monoclonal antibody (Ad-9D9) as a genetic adjuvant for Adenovirus based vaccines.ResultsThe co-delivery of Ad-9D9 with an Adeno-based COVID-19 vaccine encoding the Spike protein resulted in stronger cellular and humoral immune responses. In contrast, only a modest adjuvant effect was achieved when combining the vaccine with the same anti-CTLA-4 in its proteinaceous form. Importantly, the administration of the adjuvant vector at different sites of the vaccine vector abrogates the immunostimulatory effect. We showed that the adjuvant activity of Ad-α-CTLA-4 is independent from the vaccine antigen as it improved the immune response and efficacy of an Adenovirus based polyepitope vaccine encoding tumor neoantigens.DiscussionOur study demonstrated that the combination of Adenovirus Encoded Adjuvant (AdEnA) with an Adeno-encoded antigen vaccine enhances immune responses to viral and tumor antigens, representing a potent approach to develop more effective genetic vaccines.</p

Effect of hIi or mIi fusion to ME-TRAP in mice immunized with ChAd63 vectors.

<p>C57BL/6 mice were vaccinated i.m. with 10⁸ iu of ChAd63.ME-TRAP (circles), ChAd63.hIi-ME-TRAP (squares) or ChAd63.mIi-ME-TRAP (triangles) with spleens and sera harvested two weeks after vaccination. T cell responses to TRAP (a. & b.) or hIi (c. & d.) peptides were measured by ICS. Points represent individual mice after subtraction of background responses and lines represent the median. ****p<0.0001 compared to ChAd63.ME-TRAP (2-way ANOVA with Bonferroni post-test).</p

Local and systemic adverse events (AEs) possibly, probably or definitely related to vaccination shown as percentage of volunteers affected.

<p>(a) Post ChAd63 CS 5×10⁹ vp; (b) Post ChAd63 CS 5×10¹⁰ vp; (c) Post MVA CS 2×10⁸ pfu.</p

CONSORT diagram of study progress.

<p>39 volunteers were screened. Reasons for not meeting the inclusion criteria in 7 excluded volunteers were: psychiatric morbidity (2), history of malignancy (2), one each of: history of headaches, Carbohydrate Deficient Transferrin (CDT) >3% and neutropenia.</p