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Alternative BCG delivery strategies improve protection against Mycobacterium tuberculosis in non-human primates: Protection associated with mycobacterial antigen-specific CD4 effector memory T-cell populations

View Article: PubMed Central - PubMed

ABSTRACT

Intradermal (ID) BCG injection provides incomplete protection against TB in humans and experimental models. Alternative BCG vaccination strategies may improve protection in model species, including rhesus macaques. This study compares the immunogenicity and efficacy of BCG administered by ID and intravenous (IV) injection, or as an intratracheal mucosal boost (ID + IT), against aerosol challenge with Mycobacterium tuberculosis Erdman strain. Disease pathology was significantly reduced, and survival improved, by each BCG vaccination strategy, relative to unvaccinated animals. However, IV induced protection surpassed that achieved by all other routes, providing an opportunity to explore protective immunological mechanisms using antigen-specific IFN-γ ELISpot and polychromatic flow cytometry assays. IFN-γ spot forming units and multifunctional CD4 T-cell frequencies increased significantly following each vaccination regimen and were greatest following IV immunisation. Vaccine-induced multifunctional CD4 T-cells producing IFN-γ and TNF-α were associated with reduced disease pathology following subsequent M.tb challenge; however, high frequencies of this population following M.tb infection correlated with increased pathology. Cytokine producing T-cells primarily occupied the CD4 transitional effector memory phenotype, implicating this population as central to the mycobacterial response, potentially contributing to the stringent control observed in IV vaccinated animals. This study demonstrates the protective efficacy of IV BCG vaccination in rhesus macaques, offering a valuable tool for the interrogation of immunological mechanisms and potential correlates of protection.

No MeSH data available.


PPD-specific polyfunctional CD4 and CD8 T-cell responses. Bar charts represent vaccination group median values of cytokine producing CD4 T-cells (plot A) and CD8 T cells (plot B) isolated prior to vaccination (PB) and at study weeks 6, 10, 12, 20, 23 and 25. Note that all vaccinated animals received BCG vaccinations at week 0 by intravenous (IV), or intradermal (ID and ID + IT) injection, and that the ID + IT vaccination group received a further intratracheal BCG inoculation at study week 11. Polyfunctional T-cell responses measured in unvaccinated controls are shown at PB and weeks 23 and 25. All animals received aerosol M.tb challenge at study week 21. Shaded areas depict immune responses measured following M.tb challenge. Cytokine frequencies in individual animals are represented by dots. Horizontal bars denote significant changes from mean pre-vaccination values as measured by Wilcoxon signed rank test (p ≤ 0.05).
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fig5: PPD-specific polyfunctional CD4 and CD8 T-cell responses. Bar charts represent vaccination group median values of cytokine producing CD4 T-cells (plot A) and CD8 T cells (plot B) isolated prior to vaccination (PB) and at study weeks 6, 10, 12, 20, 23 and 25. Note that all vaccinated animals received BCG vaccinations at week 0 by intravenous (IV), or intradermal (ID and ID + IT) injection, and that the ID + IT vaccination group received a further intratracheal BCG inoculation at study week 11. Polyfunctional T-cell responses measured in unvaccinated controls are shown at PB and weeks 23 and 25. All animals received aerosol M.tb challenge at study week 21. Shaded areas depict immune responses measured following M.tb challenge. Cytokine frequencies in individual animals are represented by dots. Horizontal bars denote significant changes from mean pre-vaccination values as measured by Wilcoxon signed rank test (p ≤ 0.05).

Mentions: Cells were analysed using a five laser LSRII Fortessa instrument (BD Biosciences, Oxford, UK) and data were analysed using FlowJo (version 9.7.6, Treestar, Ashland, US). Cytokine-producing T-cells were identified using a forward scatter-height (FSC-H) versus side scatter-area (SSC-A) dot plot to identify the lymphocyte population, to which appropriate gating strategies were applied to exclude doublet events, non-viable cells, monocytes (CD14+) and B cells (CD20+). For ICS analysis, sequential gating through CD3+, followed by CD4+ or CD8+ gates were used before individual cytokine gates to identify IFN-γ, IL-2 and TNF-α producing populations. Polyfunctional cells were identified using Boolean gating combinations of individual cytokine-producing CD4 or CD8 T-cells. Antigen-specific T-cell memory profiles were identified by applying a summed CD4 or CD8 cytokine Boolean combination, followed by gating for CD95 surface staining. Differentiation of effector to central memory T-cell populations was established by CD28 and CCR7 expression (Fig. 5, Fig. 6). The software package PESTLE (version 1.7) was used for background subtraction to obtain antigen-specific polyfunctional ICS and memory T-cell cytokine responses, and SPICE (version 5.35) was used to generate graphical representations of flow cytometry data (Mario Roederer, Vaccine Research Centre, NIAID, NIH).


Alternative BCG delivery strategies improve protection against Mycobacterium tuberculosis in non-human primates: Protection associated with mycobacterial antigen-specific CD4 effector memory T-cell populations
PPD-specific polyfunctional CD4 and CD8 T-cell responses. Bar charts represent vaccination group median values of cytokine producing CD4 T-cells (plot A) and CD8 T cells (plot B) isolated prior to vaccination (PB) and at study weeks 6, 10, 12, 20, 23 and 25. Note that all vaccinated animals received BCG vaccinations at week 0 by intravenous (IV), or intradermal (ID and ID + IT) injection, and that the ID + IT vaccination group received a further intratracheal BCG inoculation at study week 11. Polyfunctional T-cell responses measured in unvaccinated controls are shown at PB and weeks 23 and 25. All animals received aerosol M.tb challenge at study week 21. Shaded areas depict immune responses measured following M.tb challenge. Cytokine frequencies in individual animals are represented by dots. Horizontal bars denote significant changes from mean pre-vaccination values as measured by Wilcoxon signed rank test (p ≤ 0.05).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5120991&req=5

fig5: PPD-specific polyfunctional CD4 and CD8 T-cell responses. Bar charts represent vaccination group median values of cytokine producing CD4 T-cells (plot A) and CD8 T cells (plot B) isolated prior to vaccination (PB) and at study weeks 6, 10, 12, 20, 23 and 25. Note that all vaccinated animals received BCG vaccinations at week 0 by intravenous (IV), or intradermal (ID and ID + IT) injection, and that the ID + IT vaccination group received a further intratracheal BCG inoculation at study week 11. Polyfunctional T-cell responses measured in unvaccinated controls are shown at PB and weeks 23 and 25. All animals received aerosol M.tb challenge at study week 21. Shaded areas depict immune responses measured following M.tb challenge. Cytokine frequencies in individual animals are represented by dots. Horizontal bars denote significant changes from mean pre-vaccination values as measured by Wilcoxon signed rank test (p ≤ 0.05).
Mentions: Cells were analysed using a five laser LSRII Fortessa instrument (BD Biosciences, Oxford, UK) and data were analysed using FlowJo (version 9.7.6, Treestar, Ashland, US). Cytokine-producing T-cells were identified using a forward scatter-height (FSC-H) versus side scatter-area (SSC-A) dot plot to identify the lymphocyte population, to which appropriate gating strategies were applied to exclude doublet events, non-viable cells, monocytes (CD14+) and B cells (CD20+). For ICS analysis, sequential gating through CD3+, followed by CD4+ or CD8+ gates were used before individual cytokine gates to identify IFN-γ, IL-2 and TNF-α producing populations. Polyfunctional cells were identified using Boolean gating combinations of individual cytokine-producing CD4 or CD8 T-cells. Antigen-specific T-cell memory profiles were identified by applying a summed CD4 or CD8 cytokine Boolean combination, followed by gating for CD95 surface staining. Differentiation of effector to central memory T-cell populations was established by CD28 and CCR7 expression (Fig. 5, Fig. 6). The software package PESTLE (version 1.7) was used for background subtraction to obtain antigen-specific polyfunctional ICS and memory T-cell cytokine responses, and SPICE (version 5.35) was used to generate graphical representations of flow cytometry data (Mario Roederer, Vaccine Research Centre, NIAID, NIH).

View Article: PubMed Central - PubMed

ABSTRACT

Intradermal (ID) BCG injection provides incomplete protection against TB in humans and experimental models. Alternative BCG vaccination strategies may improve protection in model species, including rhesus macaques. This study compares the immunogenicity and efficacy of BCG administered by ID and intravenous (IV) injection, or as an intratracheal mucosal boost (ID + IT), against aerosol challenge with Mycobacterium tuberculosis Erdman strain. Disease pathology was significantly reduced, and survival improved, by each BCG vaccination strategy, relative to unvaccinated animals. However, IV induced protection surpassed that achieved by all other routes, providing an opportunity to explore protective immunological mechanisms using antigen-specific IFN-γ ELISpot and polychromatic flow cytometry assays. IFN-γ spot forming units and multifunctional CD4 T-cell frequencies increased significantly following each vaccination regimen and were greatest following IV immunisation. Vaccine-induced multifunctional CD4 T-cells producing IFN-γ and TNF-α were associated with reduced disease pathology following subsequent M.tb challenge; however, high frequencies of this population following M.tb infection correlated with increased pathology. Cytokine producing T-cells primarily occupied the CD4 transitional effector memory phenotype, implicating this population as central to the mycobacterial response, potentially contributing to the stringent control observed in IV vaccinated animals. This study demonstrates the protective efficacy of IV BCG vaccination in rhesus macaques, offering a valuable tool for the interrogation of immunological mechanisms and potential correlates of protection.

No MeSH data available.