Control of vaccinia virus skin lesions by long-term-maintained IFN-gamma+TNF-alpha+ effector/memory CD4+ lymphocytes in humans

Abstract

Vaccinia virus (VV) vaccination is used to immunize against smallpox and historically was considered to have been successful if a skin lesion formed at the vaccination site. While antibody responses have been widely proposed as a correlate of efficacy and protection in humans, the role of cellular and humoral immunity in VV-associated skin lesion formation was unknown. We therefore investigated whether long-term residual humoral and cellular immune memory to VV, persisting 30 years after vaccination, could control VV-induced skin lesion in revaccinated individuals. Here, we have shown that residual VV-specific IFN-gamma+TNF-alpha+ or IFN-gamma+IL-2+ CD4+ lymphocytes but not CD8+ effector/memory lymphocytes expressing a skin-homing marker are inversely associated with the size of the skin lesion formed in response to revaccination. Indeed, high numbers of residual effector T cells were associated with lower VV skin lesion size after revaccination. In contrast, long-term residual VV-specific neutralizing antibody (NAbs) titers did not affect skin lesion formation. However, the size of the skin lesion strongly correlated with high levels of NAbs boosted after revaccination. These findings demonstrate a potential role for VV-specific CD4+ responses at the site of VV-associated skin lesion, thereby providing new insight into immune responses at these sites and potentially contributing to the development of new approaches to measure the efficacy of VV vaccination.

Figure 1. Induction of high titers of neutralizing Ab after vaccination correlates with large cutaneous lesion formation.

Neutralizing VV-specific antibody (NAb) titers (IU/ml) (A) and NT50 scores (B) in previously vaccinated volunteers at different time points after revaccination (kinetics from W0 to W8 were available for n = 36): W0, W4, W6, W8. The box-and-whisker plots show the median values and the 10th, 25th, 75th, and 90th percentiles. Statistical analyses were performed using the Wilcoxon matched pairs test; **P < 0.001, ***P < 0.0001. Dot plots of the size of skin lesions at day 8 (in mm) and the amplitude of NAb responses (IU/ml) in previously vaccinated individuals at W0 (n = 85) (C) and recently revaccinated individuals at W4 (n = 53) (D). (E) Dot plots of the size of skin lesions at day 8 (in mm) and the neutralizing titers of VV-specific Abs (NT50 scores) in previously vaccinated individuals at W0 (n = 55) (white circles) and recently revaccinated individuals at W4 (n = 53, black circles). Each circle represents data from 1 individual. All data are shown in the dot plot. Statistical analyses were performed using the Spearman test.

Figure 2. Control of skin lesion formation by residual effector/memory T cell responses.

(A) IFN-γ ELISpot assays (SFU/million PBMCs) were performed prior to and after revaccination at W0 and W4 (n = 65). Assays were also performed in control unvaccinated individuals (Unvac, n = 10). Levels of unstimulated cells were subtracted as background. The threshold of positivity of VV-specific IFN-γ⁺ T cell frequency was set greater than 50 SFU/million PBMCs after subtraction of background values. Data represent the median values and the 10th, 25th, 75th, and 90th percentiles. The P value between groups was calculated using Wilcoxon matched pairs test; **P < 0.001, ***P < 0.0001. (B and C) Dot plots of the size of the skin lesions at day 8 (in mm) and IFN-γ ELISpot responses at W0 (B) and W4 (C). Each circle represents data from 1 individual. All data are shown in the figures. Statistical analyses were performed using the Spearman test.

Figure 3. Control of skin lesion formation by residual proliferative memory T cell responses.

(A) Index of proliferation to VV was calculated prior to and after revaccination (n = 83) at W0 and W4. Assays were also performed in control unvaccinated individuals (n = 10). The threshold of a positive response was set at an index of proliferation of at least 3. Data represent the median values and the 10th, 25th, 75th, and 90th percentiles. The P value between groups was calculated using the Wilcoxon matched pairs test; ***P < 0.0001. (B and C) Dot plots of VV-specific T cell proliferation index at W0 (B) and W4 (C). Each circle represents data from 1 individual. Statistical analyses were performed using the Spearman test.

Figure 4. Influence of long-term residual VV virus effector/memory T cell response on the boosting of humoral responses after VV vaccination.

IFN-γ ELISpot assays (SFU/million PBMCs) were performed prior to revaccination (W0) and neutralizing VV-specific antibody (NAb) titers (IU/ml) were determined at W4 after revaccination (n = 52). Each symbol represents data from 1 individual. Statistical analyses were performed using the Spearman test.

Figure 5. Residual IFN-γ⁺TNF-α⁺ long-term effector/memory CD4 lymphocytes control VV skin lesion formation after viral challenge in humans.

(A and B) PBMCs from previously vaccinated volunteers were stimulated for 16 hours with VV and harvested for flow cytometric intracellular cytokine staining. Boolean gating using FlowJo software was performed to calculate single producers, double producers, and triple producers cells in regard to IFN-γ⁺, IL-2⁺, and/or TNF-α⁺ as indicated. Pie chart analyses are shown for CD3⁺CD4⁺ (A) and CD3⁺CD8⁺ cells (B). Mean of percentages for each sub-population of VV-specific T cells that produce cytokines are indicated in the pie chart for 23 volunteers tested at W0 (prior to vaccination). (C and D) Dot plot representation of correlation between the size of skin lesions at day 8 after vaccination and residual VV-specific T cell responses prior to revaccination: percentage of residual VV-specific effector T cells as shown by IFN-γ⁺TNF-α⁺ or IFN-γ⁺IL-2⁺ CD3⁺CD4⁺ (C) or CD8⁺CD3⁺ (D) lymphocytes are shown. All data are shown (n = 23). Each symbol represents data from 1 individual. Statistical analyses were performed using the Spearman test.

Figure 6. CLA and CD107a cytotoxic effector molecule on VV-specific CD4 cells in previously vaccinated volunteers.

(A) PBMCs prior to revaccination were thawed and stimulated for 16 hours with VV and harvested for flow cytometric assays. Multiparametric flow cytometric analyses of IL-2, IFN-γ, and/or TNF-α and gated on CD3⁺CD4⁺ as indicated using FlowJo software. Flow cytometric analyses of the basal level of cytokine production by unstimulated CD3⁺CD4⁺ cells are shown in the upper panels. We showed percentages of VV-specific CD3⁺CD4⁺ cells producing IFN-γ and/or IL-2 (middle, left) and of CD3⁺CD4⁺ cells producing IFN-γ and/or TNF-α (middle, right) for a representative individual. Flow cytometric analyses of CLA-expressing cells among VV-specific CD4⁺ cells producing IFN-γ (bottom). Data are representative of 10 individuals tested. (B) Box-and-whiskers representation of the frequency of VV-specific CD3⁺CD4⁺ lymphocytes expressing CLA markers is presented for 10 vaccinated volunteers. The data show the median values and the 10th, 25th, 75th, and 90th percentiles. (C) The expression of CD107a cytotoxic effector molecule was analyzed for 3 vaccinated volunteers. This latter population represents 79%–85% of VV-specific CD4⁺ cells producing IFN-γ and/or IL-2 in all volunteers tested. Representative plot for CD107a marker gated on CD3⁺CD4⁺ cells producing IFN-γ (C, bottom).