Immunisation with BCG and recombinant MVA85A induces long-lasting, polyfunctional Mycobacterium tuberculosis-specific CD4+ memory T lymphocyte populations

Abstract

In the search for effective vaccines against intracellular pathogens such as HIV, tuberculosis and malaria, recombinant viral vectors are increasingly being used to boost previously primed T cell responses. Published data have shown prime-boost vaccination with BCG-MVA85A (modified vaccinia virus Ankara expressing antigen 85A) to be highly immunogenic in humans as measured by ex vivo IFN-gamma ELISPOT. Here, we used polychromatic flow cytometry to investigate the phenotypic and functional profile of these vaccine-induced Mycobacterium tuberculosis (M.tb) antigen 85A-specific responses in greater detail. Promisingly, antigen 85A-specific CD4(+) T cells were found to be highly polyfunctional, producing IFN-gamma, TNF-alpha, IL-2 and MIP-1beta. Surface staining showed the responding CD4(+) T cells to be relatively immature (CD45RO(+) CD27(int)CD57(-)); this observation was supported by the robust proliferative responses observed following antigenic stimulation. Furthermore, these phenotypic and functional properties were independent of clonotypic composition and epitope specificity, which was maintained through the different phases of the vaccine-induced immune response. Overall, these data strongly support the use of MVA85A in humans as a boosting agent to expand polyfunctional M.tb-specific CD4(+) T cells capable of significant secondary responses.

Figure 1

85A-specific CD4⁺ T cells are polyfunctional. Production of CD107a, IFN-γ, IL-2, MIP-1β and TNF-α was assessed following antigenic stimulation of cryopreserved PBMC using polychromatic flow cytometry. (A) Representative bivariate plots from subject 501 showing M.tb antigen 85A-specific functional responses (upper panels) compared to background responses (lower panels). (B) Frequency of responding CD4⁺ T cells positive for IFN-γ, IL-2, MIP-1β and TNF-α pre-MVA85A vaccination and at wk 1, 8 and 24 post-MVA85A vaccination. All subjects are combined into one group for analysis. Boxes represent interquartile ranges; the line in the middle of the box represents the median, and minimum/maximum lines are shown. (C) Functional composition of the CD4⁺ T cell response. Responses are grouped and colour-coded according to the number of functions. The pie charts summarize the fractions of the total response that are positive for a given number of functions. Every possible combination of functions is shown on the x-axis. Individual data points and median percentage of the total CD4⁺ response (open bars) with 3^rd quartile are shown for each of the functional species. As in (B), all subjects are combined into one group. No responses were seen in the CD8⁺ T cell subset (data not shown).

Figure 2

Polyfunctional 85A-specific CD4⁺ T cell populations are relatively immature. Surface markers were included in the polychromatic flow cytometry panel to investigate the phenotype of responding 85A-specific CD4⁺ T cells. (A) Representative individual responses for subject 514 are shown. Every possible functional combination is shown on the x-axis; the absolute frequencies (total CD4⁺ T cell response) of the four most frequently occurring dominant functional species are shown as colour-coded bars. (B) For subject 514, the CD27 versus CD45RO phenotype of the dominant response profiles are shown as colour-coded dots overlaid on density plots showing the phenotype of the total CD4⁺ population. Comparable results were obtained in all six subjects. (C) The median fluorescence intensity of CD27 expression within the four colour-coded dominant responding populations for each of the six subjects (with median line) is shown. (D) Again for subject 514, the CD27 versus CD57 phenotype of the dominant response profiles are shown as colour-coded dots overlaid on density plots showing the phenotype of the total CD4⁺ population. Comparable results were obtained in all six subjects. (E) CD27 versus CD57 phenotype of a CD8⁺ T cell subset to allow comparison of (B) with a CD57⁺ phenotype (not seen in CD4⁺ T cell subset).

Figure 3

M.tb antigen-specific CD4⁺ T cells are capable of robust proliferative responses. CFSE dilution assays were performed on cryopreserved PBMC. (A) Representative histogram plots from subject 503 showing PPD-T- (upper panels) and 85A- (lower panels) stimulated proliferative responses (black line) overlaid on the unstimulated response (solid grey). (B) Median responses to PPD-T (solid line) and 85A (broken line); data are presented as CDI values. (C) Individual CDI values for all subjects at all time points analysed, with median values and interquartile ranges. (D) Median differences between time points with 95% confidence intervals and p values are provided. Missing data values indicate that stored PBMC were not available.

Figure 4

Clonotype composition analyses for subjects 501 and 514. Molecular analysis of TCRB gene expression was conducted on antigen 85A-stimulated CD4⁺CD25⁺CD69⁺ T cells. The percentage frequency of each clonotype and the total number of sequenced clones are shown, together with CDR3 amino acid sequence, TCRBV and TCRBJ usage. For subject 501 in the antigen 85A-stimulated sample, 582 events (0.42% of CD4⁺ T cells) were collected; in the unstimulated control, 18 events (0.036% of CD4⁺ T cells) were collected. For subject 514 in the antigen 85A-stimulated sample, 1197 events (0.44% of CD4⁺ T cells) were collected; in the unstimulated control, 37 events (0.044% of CD4⁺ T cells) were collected.

Figure 5

Differential clonotype usage is reflected in epitope targeting patterns. IFN-γ ELISPOT was performed on cryopreserved PBMC using individual 85A 15mer peptides. Fine specificity epitope mapping of 85A responses are shown for subject 501 at (A) wk 1 and (C) wk 24 and for subject 514 at (B) wk 1 and (D) wk 24 post-boost.