Ex vivo IFN-γ ELISpot responses to the vaccine insert.

<p>(A) Median and individual ex vivo IFN-γ ELISpot responses from vaccinated volunteers at baseline (week 0), and weeks 1, 3, 8, 12, 24, and 52. Significant differences between the pre- and post-vaccination time points were detected using the Wilcoxon signed rank test: week 1 (p = 0·0001), week 3 (p = 0·0001), week 8 (p = 0·0001), and week 12 (p = 0·001). (B) Median <i>ex vivo</i> IFN-γ ELISpot responses to the NP+M1 insert stratified according to age: black bars  =  group 1 (50–59 years), white bars  =  group 2 (60–69 years), and grey bars  =  group 3 (70+ years). Error bars indicate interquartile ranges. Significant differences between the pre- and post-vaccination time points were detected using the Wilcoxon signed rank test as follows. Group 1: week 1 (p = 0·002), week 3 (p = 0·002), week 8 (p = 0·002), week 12 (p = 0·039), week 24 (p = 0·002), and week 52 (p = 0·0039). Group 2: week 1 (p = 0·002), week 3 (p = 0·002), week 8 (p = 0·002), and week 12 (p = 0·0371). Group 3: week 1 (p = 0·0039) and week 3 (p = 0·0195). Significant differences were also detected between groups using the Mann-Whitney U-test, with responses in group 1 being higher than those in group 3 at week 3 (p = 0·043) and week 8 (p = 0·023). (C) Median and individual <i>ex vivo</i> IFN-γ ELISpot responses at week 1 and week 3 stratified according to age, and including a vaccinated cohort of younger (18–45 years) volunteers.</p

Clonality of HTLV-2 in Natural Infection

<div><p>Human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) both cause lifelong persistent infections, but differ in their clinical outcomes. HTLV-1 infection causes a chronic or acute T-lymphocytic malignancy in up to 5% of infected individuals whereas HTLV-2 has not been unequivocally linked to a T-cell malignancy. Virus-driven clonal proliferation of infected cells both in vitro and in vivo has been demonstrated in HTLV-1 infection. However, T-cell clonality in HTLV-2 infection has not been rigorously characterized. In this study we used a high-throughput approach in conjunction with flow cytometric sorting to identify and quantify HTLV-2-infected T-cell clones in 28 individuals with natural infection. We show that while genome-wide integration site preferences in vivo were similar to those found in HTLV-1 infection, expansion of HTLV-2-infected clones did not demonstrate the same significant association with the genomic environment of the integrated provirus. The proviral load in HTLV-2 is almost confined to CD8⁺ T-cells and is composed of a small number of often highly expanded clones. The HTLV-2 load correlated significantly with the degree of dispersion of the clone frequency distribution, which was highly stable over ∼8 years. These results suggest that there are significant differences in the selection forces that control the clonal expansion of virus-infected cells in HTLV-1 and HTLV-2 infection. In addition, our data demonstrate that strong virus-driven proliferation <i>per se</i> does not predispose to malignant transformation in oncoretroviral infections.</p></div

IFN-γ, IL-2, TNF and CD107a responses to the vaccine insert measured by flow cytometry.

<p>Production of IFN-γ (A), IL-2 (B) and TNF (C), and mobilization of CD107a (D), after background subtraction in CD3⁺CD4⁺ (black circles) and CD3⁺CD8⁺ (white circles) cell populations stimulated with a single pool of peptides spanning the complete NP+M1 vaccine insert. Volunteers in group 3 were tested at weeks 0, 1, and 3. Significant differences between pre- and post-vaccination time points were detected using the Wilcoxon signed rank test as follows: IFN-γ CD4⁺, week 1 (p = 0·0001) and week 3 (p = 0·0001); IFN-γ CD8⁺, week 1 (p = 0·001) and week 3 (p = 0·0005); IL-2 CD4⁺, week 1 (p = 0.001) and week 3 (p = 0·0001); IL-2 CD8⁺, week 1 (p = 0·006) and week 3 (p = 0·03); TNF CD4⁺, week 1 (p = 0·002) and week 3 (p = 0·0003); TNF CD8⁺, week 1 (p = 0·0003) and week 3 (p = 0·002); CD107a CD8⁺, week 3 (p = 0·004).</p

Patterns of clonotype usage in M1-specific CD8⁺ T cell populations before and after vaccination with MVA-NP+M1.

<p>TRBV and TRBJ usage, CDR3 amino acid sequence and relative frequency are shown for GILGFVFTL-specific CD8⁺ T cell clonotypes on day 0 (pre-vaccination) and day 7 (post-vaccination). Public clonotypes within the present dataset are colour-coded. Non-public clonotypes present at both time points within an individual are highlighted in bold type.</p

Functional profile of T cell responses to the vaccine insert measured by flow cytometry.

<p>Mobilization of CD107a and production of IFN-γ, IL-2 and TNF after background subtraction in CD3⁺CD4⁺ (A) and CD3⁺CD8⁺ (B) cell populations stimulated with a single pool of peptides spanning the complete NP+M1 vaccine insert. Median percentages of quadruple (black), triple (dark grey), double (light grey) and single (white) functional cells are shown.</p

Phenotypic and clonotypic properties of M1-specific CD8⁺ T cells elicited by MVA-NP+M1.

<p>(A) Phenotype of vaccine-elicited CD8⁺ T cells specific for the HLA A*0201-restricted M1-derived epitope GILGFVFTL (residues 58–66). Antigen-specific CD3⁺CD8⁺tetramer⁺ cells are shown as coloured dots superimposed on bivariate plots showing the phenotypic distribution of the total CD8⁺ T cell population (grey density plots). Response sizes were 1·48% (left panels) and 0·75% (right panels) with respect to the total CD8⁺ T cell population. (B) TRBV and TRBJ usage, CDR3 amino acid sequence and relative frequency of the GILGFVFTL-specific CD8⁺ T cell clonotypes contained within the antigen-specific populations depicted in (A). Public clonotypes within the present dataset are colour-coded. Representative analyses are shown for volunteers in group 3 (70+ years).</p

HTLV-2 infection is found almost exclusively in CD8⁺ T-cells.

<p>Cryopreserved PBMCs from 28 HTLV-2-infected individuals were sorted by flow-cytometry into separate CD3⁺CD4⁺CD8⁻ and CD3⁺CD4⁻CD8⁺ cell populations. Integration site content was determined by high-throughput sequencing for both sorted populations and unsorted PBMCs. In the unsorted cells, integration sites were positively assigned to CD4⁺ or CD8⁺ cells based on the sorted fraction in which the same sites were found. The proportion of the load was calculated as the sum of the relative frequencies of the clones. Unknown – proportion of the load made up by clones that were not resampled in either the CD4⁺ or CD8⁺ fraction. Since the redetection of clones is most unlikely if proviral load is very low, only individuals in whom >100 proviruses were found are shown here.</p

Demographic characteristics of volunteers vaccinated in each cohort.

<p>Demographic characteristics of volunteers vaccinated in each cohort.</p

Frequency of local and systemic adverse events that were possibly, probably or definitely related to vaccination.

<p>(A) Volunteers aged 50+ (n = 30). (B) Volunteers aged 18–45 (n = 15). For both age groups pain was the most frequently recorded local adverse event followed by erythema. A similar pattern of systemic adverse events was observed in both age groups with the majority of solicited adverse events occurring in 20–60% of individuals. For volunteers aged 18–45, 85% of adverse events were mild; for volunteers aged 50+, 87% of adverse events were mild.</p

HTLV-2 integration site and clonal expansion.

<p>(A) The distribution of integration sites according to clonal abundance. Abundance was quantified by the number of copies estimated in a clone per 10,000 PBMCs (based on relative abundance and proviral load). Abundance bins are defined on a logarithmic scale. (B) The proportion of sites within 10 kb of a RefSeq gene for each abundance bin. A significant positive trend (p = 0.04, chi-squared test for trend) was detected for HTLV-1 but not for HTLV-2. (C) Oligoclonality index (OCI) versus log₁₀(proviral load) for each virus. A strong positive correlation (p = 0.0015, Spearman's test) was detected between these parameters for HTLV-2 but not for HTLV-1(p = 0.681, Spearman's correlation). (D) The total number of unique integration sites (UIS) identified in each PBMC sample versus log₁₀(proviral load) for each virus (p<0.001 for HTLV-1, p = 0.0019 for HTLV-2, Spearman's test).</p