Limits...
Protection and polyfunctional T cells induced by Ag85B-TB10.4/IC31 against Mycobacterium tuberculosis is highly dependent on the antigen dose.

Aagaard C, Hoang TT, Izzo A, Billeskov R, Troudt J, Arnett K, Keyser A, Elvang T, Andersen P, Dietrich J - PLoS ONE (2009)

Bottom Line: We found that vaccination with the combination of Ag85B-TB10.4 and IC31 resulted in high numbers of polyfunctional CD4 T cells co-expressing IL-2, IFN-gamma and TNF-alpha.Thus, whereas antigen doses of 5 and 15 microg did not induce significant protection against M.tb, reducing the dose to 0.5 microg selectively increased the number of polyfunctional T cells and induced a strong protection against infection with M.tb.In this model a 2.5 fold increase in the antigen dose reduced the protection against infection with M.tb to the level observed in non-vaccinated animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Disease Immunology, Statens Serum Institute, Copenhagen, Denmark.

ABSTRACT

Background: Previously we have shown that Ag85B-TB10.4 is a highly efficient vaccine against tuberculosis when delivered in a Th1 inducing adjuvant based on cationic liposomes. Another Th1 inducing adjuvant, which has shown a very promising profile in both preclinical and clinical trials, is IC31. In this study, we examined the potential of Ag85B-TB10.4 delivered in the adjuvant IC31 for the ability to induce protection against infection with Mycobacterium tuberculosis. In addition, we examined if the antigen dose could influence the phenotype of the induced T cells.

Methods and findings: We found that vaccination with the combination of Ag85B-TB10.4 and IC31 resulted in high numbers of polyfunctional CD4 T cells co-expressing IL-2, IFN-gamma and TNF-alpha. This correlated with protection against subsequent challenge with M.tb in the mouse TB model. Importantly, our results also showed that both the vaccine induced T cell response, and the protective efficacy, was highly dependent on the antigen dose. Thus, whereas antigen doses of 5 and 15 microg did not induce significant protection against M.tb, reducing the dose to 0.5 microg selectively increased the number of polyfunctional T cells and induced a strong protection against infection with M.tb. The influence of antigen dose was also observed in the guinea pig model of aerosol infection with M.tb. In this model a 2.5 fold increase in the antigen dose reduced the protection against infection with M.tb to the level observed in non-vaccinated animals.

Conclusions/significance: Small changes in the antigen dose can greatly influence the induction of specific T cell subpopulations and the dose is therefore a crucial factor when testing new vaccines. However, the adjuvant IC31 can, with the optimal dose of Ag85B-TB10.4, induce strong protection against Mycobacterium tuberculosis. This vaccine has now entered clinical trials.

Show MeSH

Related in: MedlinePlus

Ag85B and TB10.4 specific T cells are polyfunctional.(A). Cytokine profiles of H4 specific CD4 T cells were determined by first dividing the CD4 T cells into IFN-γ positive (+) or IFN-γ negative (-) cells. Both the IFN-γ+ and IFN-γ− cells were analyzed with respect to the production of TNF-α and IL-2. The numbers in the quadrant gates of the plots denominates each distinct population based on their cytokine production and is color coded as shown. (B–D) The pie charts are grouped after vaccination dose and colour coded according to the cytokine production profile and summarizes the fractions of the CD4+ T cell response (out of the antigen specific CD4 T cells) that are positive for a given cytokine production profile. Every possible combination of cytokines is shown on the x-axis of the bar chart and the percentage of Ag85B, TB10.4 or Ag85B-TB10.4 (H4) specific CD4+ T cells expressing any combination of cytokines is given for each immunization group. The antigen used for in vitro stimulation of the PBMC's is indicated. No responses were seen in the CD8+ T cell subset. (E) The mean fluorescence intensity (MFI) of IFN-γ in the subpopulations expressing this cytokine from animals vaccinated with either 0.5 µg H4 or 5 µg H4. Results are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2691953&req=5

pone-0005930-g003: Ag85B and TB10.4 specific T cells are polyfunctional.(A). Cytokine profiles of H4 specific CD4 T cells were determined by first dividing the CD4 T cells into IFN-γ positive (+) or IFN-γ negative (-) cells. Both the IFN-γ+ and IFN-γ− cells were analyzed with respect to the production of TNF-α and IL-2. The numbers in the quadrant gates of the plots denominates each distinct population based on their cytokine production and is color coded as shown. (B–D) The pie charts are grouped after vaccination dose and colour coded according to the cytokine production profile and summarizes the fractions of the CD4+ T cell response (out of the antigen specific CD4 T cells) that are positive for a given cytokine production profile. Every possible combination of cytokines is shown on the x-axis of the bar chart and the percentage of Ag85B, TB10.4 or Ag85B-TB10.4 (H4) specific CD4+ T cells expressing any combination of cytokines is given for each immunization group. The antigen used for in vitro stimulation of the PBMC's is indicated. No responses were seen in the CD8+ T cell subset. (E) The mean fluorescence intensity (MFI) of IFN-γ in the subpopulations expressing this cytokine from animals vaccinated with either 0.5 µg H4 or 5 µg H4. Results are representative of two independent experiments.

Mentions: We next analyzed the cytokine expression of the T cells induced by immunizing with 0.5 µg compared to 5 µg Ag85B-TB10.4/IC31®. In particular, we were interested in analyzing the induction of polyfunctional CD4 T cells as these have been shown to correlate with protective immunity against infections such as Leishmania major and to form the basis for a long lived memory response [14], [15]. PBMCs from vaccinated mice were stimulated in vitro with Ag85B, TB10.4 or Ag85B-TB10.4 and analyzed by flow cytometry for expression of CD4, CD8, IFN-γ, TNF-α, and IL-2. The results showed that immunizing with Ag85B-TB10.4/IC31® induced two major polyfunctional T cell populations; CD4+IFN-γ+IL-2+TNF-α+ and CD4+IL-2+TNF-α+ T cells. Similarly to in the result depicted in figure 1, we observed an increased response in the group vaccinated with 0.5 µg Ag85B-TB10.4 compared to the group vaccinated with 5 µg Ag85B-TB10.4, against both antigen components (Fig. 3). Interestingly, the T cell subsets that were expanded by decreasing the antigen dose from 5 µg to 0.5 µg, were the polyfunctional memory T cell subsets expressing IFN-γ/IL-2/TNF-α (Fig. 3B–D) and this was observed for both Ag85B (2 fold increase in IFN-γ/IL-2/TNF-α cells) and TB10.4 (up to 10 fold increase in IFN-γ/IL-2/TNF-α cells) specific T cells (Fig. 3B and C). We also compared the level of IFN-γ production in all the IFN-γ expressing subpopulations from both vaccine groups by looking at the mean fluorescence intensity (MFI). These results showed that animals vaccinated with 0.5 µg Ag85B-TB10.4 produced significantly more IFN-γ per cell, than the corresponding subpopulation in animals vaccinated with the high dose (5 µg Ag85B-TB10.4). This was observed for both Ag85B and TB10.4 specific T cells (Fig. 3E). In summary, vaccination with Ag85B-TB10.4/IC31® induced polyfunctional CD4 T cells and reducing the dose increased the immunogenicity of the vaccine, specifically in terms of the proportion of polyfunctional T cells within the pool of antigen specific T cells. Moreover, the T cells from animals vaccinated with the low dose also produced more IFN-γ per cell. Thus, the observed increase in IFN-γ production (Fig. 1 and 2) was due to both an increase in T cells numbers and an increase in IFN-γ production by these T cells (Fig. 3).


Protection and polyfunctional T cells induced by Ag85B-TB10.4/IC31 against Mycobacterium tuberculosis is highly dependent on the antigen dose.

Aagaard C, Hoang TT, Izzo A, Billeskov R, Troudt J, Arnett K, Keyser A, Elvang T, Andersen P, Dietrich J - PLoS ONE (2009)

Ag85B and TB10.4 specific T cells are polyfunctional.(A). Cytokine profiles of H4 specific CD4 T cells were determined by first dividing the CD4 T cells into IFN-γ positive (+) or IFN-γ negative (-) cells. Both the IFN-γ+ and IFN-γ− cells were analyzed with respect to the production of TNF-α and IL-2. The numbers in the quadrant gates of the plots denominates each distinct population based on their cytokine production and is color coded as shown. (B–D) The pie charts are grouped after vaccination dose and colour coded according to the cytokine production profile and summarizes the fractions of the CD4+ T cell response (out of the antigen specific CD4 T cells) that are positive for a given cytokine production profile. Every possible combination of cytokines is shown on the x-axis of the bar chart and the percentage of Ag85B, TB10.4 or Ag85B-TB10.4 (H4) specific CD4+ T cells expressing any combination of cytokines is given for each immunization group. The antigen used for in vitro stimulation of the PBMC's is indicated. No responses were seen in the CD8+ T cell subset. (E) The mean fluorescence intensity (MFI) of IFN-γ in the subpopulations expressing this cytokine from animals vaccinated with either 0.5 µg H4 or 5 µg H4. Results are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691953&req=5

pone-0005930-g003: Ag85B and TB10.4 specific T cells are polyfunctional.(A). Cytokine profiles of H4 specific CD4 T cells were determined by first dividing the CD4 T cells into IFN-γ positive (+) or IFN-γ negative (-) cells. Both the IFN-γ+ and IFN-γ− cells were analyzed with respect to the production of TNF-α and IL-2. The numbers in the quadrant gates of the plots denominates each distinct population based on their cytokine production and is color coded as shown. (B–D) The pie charts are grouped after vaccination dose and colour coded according to the cytokine production profile and summarizes the fractions of the CD4+ T cell response (out of the antigen specific CD4 T cells) that are positive for a given cytokine production profile. Every possible combination of cytokines is shown on the x-axis of the bar chart and the percentage of Ag85B, TB10.4 or Ag85B-TB10.4 (H4) specific CD4+ T cells expressing any combination of cytokines is given for each immunization group. The antigen used for in vitro stimulation of the PBMC's is indicated. No responses were seen in the CD8+ T cell subset. (E) The mean fluorescence intensity (MFI) of IFN-γ in the subpopulations expressing this cytokine from animals vaccinated with either 0.5 µg H4 or 5 µg H4. Results are representative of two independent experiments.
Mentions: We next analyzed the cytokine expression of the T cells induced by immunizing with 0.5 µg compared to 5 µg Ag85B-TB10.4/IC31®. In particular, we were interested in analyzing the induction of polyfunctional CD4 T cells as these have been shown to correlate with protective immunity against infections such as Leishmania major and to form the basis for a long lived memory response [14], [15]. PBMCs from vaccinated mice were stimulated in vitro with Ag85B, TB10.4 or Ag85B-TB10.4 and analyzed by flow cytometry for expression of CD4, CD8, IFN-γ, TNF-α, and IL-2. The results showed that immunizing with Ag85B-TB10.4/IC31® induced two major polyfunctional T cell populations; CD4+IFN-γ+IL-2+TNF-α+ and CD4+IL-2+TNF-α+ T cells. Similarly to in the result depicted in figure 1, we observed an increased response in the group vaccinated with 0.5 µg Ag85B-TB10.4 compared to the group vaccinated with 5 µg Ag85B-TB10.4, against both antigen components (Fig. 3). Interestingly, the T cell subsets that were expanded by decreasing the antigen dose from 5 µg to 0.5 µg, were the polyfunctional memory T cell subsets expressing IFN-γ/IL-2/TNF-α (Fig. 3B–D) and this was observed for both Ag85B (2 fold increase in IFN-γ/IL-2/TNF-α cells) and TB10.4 (up to 10 fold increase in IFN-γ/IL-2/TNF-α cells) specific T cells (Fig. 3B and C). We also compared the level of IFN-γ production in all the IFN-γ expressing subpopulations from both vaccine groups by looking at the mean fluorescence intensity (MFI). These results showed that animals vaccinated with 0.5 µg Ag85B-TB10.4 produced significantly more IFN-γ per cell, than the corresponding subpopulation in animals vaccinated with the high dose (5 µg Ag85B-TB10.4). This was observed for both Ag85B and TB10.4 specific T cells (Fig. 3E). In summary, vaccination with Ag85B-TB10.4/IC31® induced polyfunctional CD4 T cells and reducing the dose increased the immunogenicity of the vaccine, specifically in terms of the proportion of polyfunctional T cells within the pool of antigen specific T cells. Moreover, the T cells from animals vaccinated with the low dose also produced more IFN-γ per cell. Thus, the observed increase in IFN-γ production (Fig. 1 and 2) was due to both an increase in T cells numbers and an increase in IFN-γ production by these T cells (Fig. 3).

Bottom Line: We found that vaccination with the combination of Ag85B-TB10.4 and IC31 resulted in high numbers of polyfunctional CD4 T cells co-expressing IL-2, IFN-gamma and TNF-alpha.Thus, whereas antigen doses of 5 and 15 microg did not induce significant protection against M.tb, reducing the dose to 0.5 microg selectively increased the number of polyfunctional T cells and induced a strong protection against infection with M.tb.In this model a 2.5 fold increase in the antigen dose reduced the protection against infection with M.tb to the level observed in non-vaccinated animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Disease Immunology, Statens Serum Institute, Copenhagen, Denmark.

ABSTRACT

Background: Previously we have shown that Ag85B-TB10.4 is a highly efficient vaccine against tuberculosis when delivered in a Th1 inducing adjuvant based on cationic liposomes. Another Th1 inducing adjuvant, which has shown a very promising profile in both preclinical and clinical trials, is IC31. In this study, we examined the potential of Ag85B-TB10.4 delivered in the adjuvant IC31 for the ability to induce protection against infection with Mycobacterium tuberculosis. In addition, we examined if the antigen dose could influence the phenotype of the induced T cells.

Methods and findings: We found that vaccination with the combination of Ag85B-TB10.4 and IC31 resulted in high numbers of polyfunctional CD4 T cells co-expressing IL-2, IFN-gamma and TNF-alpha. This correlated with protection against subsequent challenge with M.tb in the mouse TB model. Importantly, our results also showed that both the vaccine induced T cell response, and the protective efficacy, was highly dependent on the antigen dose. Thus, whereas antigen doses of 5 and 15 microg did not induce significant protection against M.tb, reducing the dose to 0.5 microg selectively increased the number of polyfunctional T cells and induced a strong protection against infection with M.tb. The influence of antigen dose was also observed in the guinea pig model of aerosol infection with M.tb. In this model a 2.5 fold increase in the antigen dose reduced the protection against infection with M.tb to the level observed in non-vaccinated animals.

Conclusions/significance: Small changes in the antigen dose can greatly influence the induction of specific T cell subpopulations and the dose is therefore a crucial factor when testing new vaccines. However, the adjuvant IC31 can, with the optimal dose of Ag85B-TB10.4, induce strong protection against Mycobacterium tuberculosis. This vaccine has now entered clinical trials.

Show MeSH
Related in: MedlinePlus