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Long-term programming of antigen-specific immunity from gene expression signatures in the PBMC of rhesus macaques immunized with an SIV DNA vaccine.

Belisle SE, Yin J, Shedlock DJ, Dai A, Yan J, Hirao L, Kutzler MA, Lewis MG, Andersen H, Lank SM, Karl JA, O'Connor DH, Khan A, Sardesai N, Chang J, Aicher L, Palermo RE, Weiner DB, Katze MG, Boyer J - PLoS ONE (2011)

Bottom Line: We observed that the RANTES-adjuvanted animals were significantly better at suppressing viral replication during chronic infection and exhibited a distinct pattern of gene expression which included immune cell-trafficking and cell cycle genes.Furthermore, a greater percentage of vaccine-induced central memory CD8+ T-cells capable of an activated phenotype were detected in these animals as measured by activation analysis.Thus, co-immunization with the RANTES molecular adjuvant followed by EP led to the generation of cellular immunity that was transcriptionally distinct and had a greater protective efficacy than its DNA alone counterpart.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
While HIV-1-specific cellular immunity is thought to be critical for the suppression of viral replication, the correlates of protection have not yet been determined. Rhesus macaques (RM) are an important animal model for the study and development of vaccines against HIV/AIDS. Our laboratory has helped to develop and study DNA-based vaccines in which recent technological advances, including genetic optimization and in vivo electroporation (EP), have helped to dramatically boost their immunogenicity. In this study, RMs were immunized with a DNA vaccine including individual plasmids encoding SIV gag, env, and pol alone, or in combination with a molecular adjuvant, plasmid DNA expressing the chemokine ligand 5 (RANTES), followed by EP. Along with standard immunological assays, flow-based activation analysis without ex vivo restimulation and high-throughput gene expression analysis was performed. Strong cellular immunity was induced by vaccination which was supported by all assays including PBMC microarray analysis that identified the up-regulation of 563 gene sequences including those involved in interferon signaling. Furthermore, 699 gene sequences were differentially regulated in these groups at peak viremia following SIVmac251 challenge. We observed that the RANTES-adjuvanted animals were significantly better at suppressing viral replication during chronic infection and exhibited a distinct pattern of gene expression which included immune cell-trafficking and cell cycle genes. Furthermore, a greater percentage of vaccine-induced central memory CD8+ T-cells capable of an activated phenotype were detected in these animals as measured by activation analysis. Thus, co-immunization with the RANTES molecular adjuvant followed by EP led to the generation of cellular immunity that was transcriptionally distinct and had a greater protective efficacy than its DNA alone counterpart. Furthermore, activation analysis and high-throughput gene expression data may provide better insight into mechanisms of viral control than may be observed using standard immunological assays.

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Related in: MedlinePlus

RANTES increases central memory CD8+ T formation during DNA vaccination.PBMCs isolated before the first immunization (Pre-bleed) and 2 weeks following the fourth (Post-imm.) were stained without ex vivo stimulation and analyzed by flow cytometry. (A) Strategy showing gating of total lymphocytes, live CD3+ cells, CD4+ and CD8+ T-cells, and activated cells (HLA-DR+CD95+). (B) Vaccine-induced activation from before (Pre-bleed) and after immunization (Post-imm.) are shown as dot plots with total CD8+ cells (red) overlayed by activated ones (blue) as a function of CD28 and CD45RA. The average percentage of activation per group of animals (Post-imm. – Pre-bleed) is displayed to the right for total CD8+ (red bars) and CD4+ (blue bars) T-cells. (C) Activated memory T-cell subsets were determined by gating as a function of CD28 and CD45RA, which differentiate naïve (N; CD28+CD45RA+), effectors/terminally differentiated effectors (TE; CD28−CD45RA+), central memory (TEM; CD28+CD45RA−) and effector memory (TEM; CD28−CD45RA−). The average percentage of activated cells for each phenotype is enumerated on right for DNA (white bars) versus DNA+RANTES (black bars) vaccinated animals. Graphs are pseudo-color and contour plots with outliers shown and data are from a representative animal. Error bars represent SD; *P<0.05, and **P<0.005 per the unpaired t test.
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pone-0019681-g003: RANTES increases central memory CD8+ T formation during DNA vaccination.PBMCs isolated before the first immunization (Pre-bleed) and 2 weeks following the fourth (Post-imm.) were stained without ex vivo stimulation and analyzed by flow cytometry. (A) Strategy showing gating of total lymphocytes, live CD3+ cells, CD4+ and CD8+ T-cells, and activated cells (HLA-DR+CD95+). (B) Vaccine-induced activation from before (Pre-bleed) and after immunization (Post-imm.) are shown as dot plots with total CD8+ cells (red) overlayed by activated ones (blue) as a function of CD28 and CD45RA. The average percentage of activation per group of animals (Post-imm. – Pre-bleed) is displayed to the right for total CD8+ (red bars) and CD4+ (blue bars) T-cells. (C) Activated memory T-cell subsets were determined by gating as a function of CD28 and CD45RA, which differentiate naïve (N; CD28+CD45RA+), effectors/terminally differentiated effectors (TE; CD28−CD45RA+), central memory (TEM; CD28+CD45RA−) and effector memory (TEM; CD28−CD45RA−). The average percentage of activated cells for each phenotype is enumerated on right for DNA (white bars) versus DNA+RANTES (black bars) vaccinated animals. Graphs are pseudo-color and contour plots with outliers shown and data are from a representative animal. Error bars represent SD; *P<0.05, and **P<0.005 per the unpaired t test.

Mentions: Since standard immunological assays evaluating Ag-specific immune responses using ex vivo stimulation may be limiting, we expanded our analysis of vaccine-induced immunity by examining the total level of cellular activation following immunization in the absence of ex vivo stimulation and primary cell culture. PBMC samples from before (Pre-bleed) and after the fourth immunization (Post-imm.) were stained and analyzed by flow cytometry to determine the overall change in total T-cell activation after vaccination (Fig. 3). Since HLA-DR, major histocompatibility complex class II cell surface receptor, molecules are up-regulated on the surface of T-cells in response to signaling, we gated HLA-DR+CD95+ T-cells to assess changes in T-cell activation after vaccination (Fig. 3A). Increases in vaccine-induced activation for CD4+ and CD8+ T-cells was determined for each animal by subtracting basal activation levels during the Pre-bleed from levels after the fourth immunization (Fig. 3B). Little increase in T-cell activation was observed on average for the control group; average activation levels for the control group were likely increased due to animal M4399, which exhibited an increase in CD8+ T-cell activation of almost 10% (data not shown) and may likely be the result of a common infection. However, vaccination induced measurable increases in the overall levels of CD4+ and CD8+ T-cell activation in both the DNA and the DNA+RANTES groups; vaccination with DNA alone induced a significant (P<0.005) 3.7-fold increase in the average percentage of CD8+ T-cell activation to a value of 10.4% while the DNA+RANTES vaccine led to a 2.4-fold increase in activation to a value of 6.8%. However, no significant increase in the level of CD4+ T-cell activation was observed in either vaccinated group. When the phenotype of activated T-cells was investigated by staining with CD28 and CD45RA, which differentiates naïve (CD28+CD45RA+), effectors/terminally differentiated effectors (CD28−CD45RA+), central memory (CD28+CD45RA−) and effector memory (CD28−CD45RA−), the DNA+RANTES vaccinated animals were observed to have greater percentages of activated central memory CD8+ T-cells on average (1.9-fold higher), but lower percentages of effector memory cells (1.3-fold lower) when compared with DNA vaccinated RMs (Fig. 3C). While no significant difference in CD4+ central memory or effectors between the vaccinated groups was found, there was a 2.2-fold higher percentage of effector memory cells in the DNA-vaccinated animals on average (data not shown). Altogether, these data show that vaccination induced measurable amounts of activation in the T-cell compartment when estimated longitudinally in the absence of ex vivo stimulation, but that inclusion of the RANTES adjuvant during vaccination shifted the activation phenotype of memory cells from effector to the central memory subset. Therefore, while DNA vaccination alone induced a greater amount of SIV-specific effectors as measured by standard immunological assays, DNA vaccination adjuvanted by RANTES generated a greater percentage of central memory CD8+ T-cells capable of an activated phenotype following the final immunization.


Long-term programming of antigen-specific immunity from gene expression signatures in the PBMC of rhesus macaques immunized with an SIV DNA vaccine.

Belisle SE, Yin J, Shedlock DJ, Dai A, Yan J, Hirao L, Kutzler MA, Lewis MG, Andersen H, Lank SM, Karl JA, O'Connor DH, Khan A, Sardesai N, Chang J, Aicher L, Palermo RE, Weiner DB, Katze MG, Boyer J - PLoS ONE (2011)

RANTES increases central memory CD8+ T formation during DNA vaccination.PBMCs isolated before the first immunization (Pre-bleed) and 2 weeks following the fourth (Post-imm.) were stained without ex vivo stimulation and analyzed by flow cytometry. (A) Strategy showing gating of total lymphocytes, live CD3+ cells, CD4+ and CD8+ T-cells, and activated cells (HLA-DR+CD95+). (B) Vaccine-induced activation from before (Pre-bleed) and after immunization (Post-imm.) are shown as dot plots with total CD8+ cells (red) overlayed by activated ones (blue) as a function of CD28 and CD45RA. The average percentage of activation per group of animals (Post-imm. – Pre-bleed) is displayed to the right for total CD8+ (red bars) and CD4+ (blue bars) T-cells. (C) Activated memory T-cell subsets were determined by gating as a function of CD28 and CD45RA, which differentiate naïve (N; CD28+CD45RA+), effectors/terminally differentiated effectors (TE; CD28−CD45RA+), central memory (TEM; CD28+CD45RA−) and effector memory (TEM; CD28−CD45RA−). The average percentage of activated cells for each phenotype is enumerated on right for DNA (white bars) versus DNA+RANTES (black bars) vaccinated animals. Graphs are pseudo-color and contour plots with outliers shown and data are from a representative animal. Error bars represent SD; *P<0.05, and **P<0.005 per the unpaired t test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3119060&req=5

pone-0019681-g003: RANTES increases central memory CD8+ T formation during DNA vaccination.PBMCs isolated before the first immunization (Pre-bleed) and 2 weeks following the fourth (Post-imm.) were stained without ex vivo stimulation and analyzed by flow cytometry. (A) Strategy showing gating of total lymphocytes, live CD3+ cells, CD4+ and CD8+ T-cells, and activated cells (HLA-DR+CD95+). (B) Vaccine-induced activation from before (Pre-bleed) and after immunization (Post-imm.) are shown as dot plots with total CD8+ cells (red) overlayed by activated ones (blue) as a function of CD28 and CD45RA. The average percentage of activation per group of animals (Post-imm. – Pre-bleed) is displayed to the right for total CD8+ (red bars) and CD4+ (blue bars) T-cells. (C) Activated memory T-cell subsets were determined by gating as a function of CD28 and CD45RA, which differentiate naïve (N; CD28+CD45RA+), effectors/terminally differentiated effectors (TE; CD28−CD45RA+), central memory (TEM; CD28+CD45RA−) and effector memory (TEM; CD28−CD45RA−). The average percentage of activated cells for each phenotype is enumerated on right for DNA (white bars) versus DNA+RANTES (black bars) vaccinated animals. Graphs are pseudo-color and contour plots with outliers shown and data are from a representative animal. Error bars represent SD; *P<0.05, and **P<0.005 per the unpaired t test.
Mentions: Since standard immunological assays evaluating Ag-specific immune responses using ex vivo stimulation may be limiting, we expanded our analysis of vaccine-induced immunity by examining the total level of cellular activation following immunization in the absence of ex vivo stimulation and primary cell culture. PBMC samples from before (Pre-bleed) and after the fourth immunization (Post-imm.) were stained and analyzed by flow cytometry to determine the overall change in total T-cell activation after vaccination (Fig. 3). Since HLA-DR, major histocompatibility complex class II cell surface receptor, molecules are up-regulated on the surface of T-cells in response to signaling, we gated HLA-DR+CD95+ T-cells to assess changes in T-cell activation after vaccination (Fig. 3A). Increases in vaccine-induced activation for CD4+ and CD8+ T-cells was determined for each animal by subtracting basal activation levels during the Pre-bleed from levels after the fourth immunization (Fig. 3B). Little increase in T-cell activation was observed on average for the control group; average activation levels for the control group were likely increased due to animal M4399, which exhibited an increase in CD8+ T-cell activation of almost 10% (data not shown) and may likely be the result of a common infection. However, vaccination induced measurable increases in the overall levels of CD4+ and CD8+ T-cell activation in both the DNA and the DNA+RANTES groups; vaccination with DNA alone induced a significant (P<0.005) 3.7-fold increase in the average percentage of CD8+ T-cell activation to a value of 10.4% while the DNA+RANTES vaccine led to a 2.4-fold increase in activation to a value of 6.8%. However, no significant increase in the level of CD4+ T-cell activation was observed in either vaccinated group. When the phenotype of activated T-cells was investigated by staining with CD28 and CD45RA, which differentiates naïve (CD28+CD45RA+), effectors/terminally differentiated effectors (CD28−CD45RA+), central memory (CD28+CD45RA−) and effector memory (CD28−CD45RA−), the DNA+RANTES vaccinated animals were observed to have greater percentages of activated central memory CD8+ T-cells on average (1.9-fold higher), but lower percentages of effector memory cells (1.3-fold lower) when compared with DNA vaccinated RMs (Fig. 3C). While no significant difference in CD4+ central memory or effectors between the vaccinated groups was found, there was a 2.2-fold higher percentage of effector memory cells in the DNA-vaccinated animals on average (data not shown). Altogether, these data show that vaccination induced measurable amounts of activation in the T-cell compartment when estimated longitudinally in the absence of ex vivo stimulation, but that inclusion of the RANTES adjuvant during vaccination shifted the activation phenotype of memory cells from effector to the central memory subset. Therefore, while DNA vaccination alone induced a greater amount of SIV-specific effectors as measured by standard immunological assays, DNA vaccination adjuvanted by RANTES generated a greater percentage of central memory CD8+ T-cells capable of an activated phenotype following the final immunization.

Bottom Line: We observed that the RANTES-adjuvanted animals were significantly better at suppressing viral replication during chronic infection and exhibited a distinct pattern of gene expression which included immune cell-trafficking and cell cycle genes.Furthermore, a greater percentage of vaccine-induced central memory CD8+ T-cells capable of an activated phenotype were detected in these animals as measured by activation analysis.Thus, co-immunization with the RANTES molecular adjuvant followed by EP led to the generation of cellular immunity that was transcriptionally distinct and had a greater protective efficacy than its DNA alone counterpart.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
While HIV-1-specific cellular immunity is thought to be critical for the suppression of viral replication, the correlates of protection have not yet been determined. Rhesus macaques (RM) are an important animal model for the study and development of vaccines against HIV/AIDS. Our laboratory has helped to develop and study DNA-based vaccines in which recent technological advances, including genetic optimization and in vivo electroporation (EP), have helped to dramatically boost their immunogenicity. In this study, RMs were immunized with a DNA vaccine including individual plasmids encoding SIV gag, env, and pol alone, or in combination with a molecular adjuvant, plasmid DNA expressing the chemokine ligand 5 (RANTES), followed by EP. Along with standard immunological assays, flow-based activation analysis without ex vivo restimulation and high-throughput gene expression analysis was performed. Strong cellular immunity was induced by vaccination which was supported by all assays including PBMC microarray analysis that identified the up-regulation of 563 gene sequences including those involved in interferon signaling. Furthermore, 699 gene sequences were differentially regulated in these groups at peak viremia following SIVmac251 challenge. We observed that the RANTES-adjuvanted animals were significantly better at suppressing viral replication during chronic infection and exhibited a distinct pattern of gene expression which included immune cell-trafficking and cell cycle genes. Furthermore, a greater percentage of vaccine-induced central memory CD8+ T-cells capable of an activated phenotype were detected in these animals as measured by activation analysis. Thus, co-immunization with the RANTES molecular adjuvant followed by EP led to the generation of cellular immunity that was transcriptionally distinct and had a greater protective efficacy than its DNA alone counterpart. Furthermore, activation analysis and high-throughput gene expression data may provide better insight into mechanisms of viral control than may be observed using standard immunological assays.

Show MeSH
Related in: MedlinePlus