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A DNA vaccine encoding multiple HIV CD4 epitopes elicits vigorous polyfunctional, long-lived CD4+ and CD8+ T cell responses.

Rosa DS, Ribeiro SP, Almeida RR, Mairena EC, Postól E, Kalil J, Cunha-Neto E - PLoS ONE (2011)

Bottom Line: Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection.The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines.By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses- elicited by other HIV immunogens.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Clinical Immunology and Allergy-LIM60, Division of Clinical Immunology and Allergy, Department of Medicine, University of São Paulo School of Medicine, São Paulo, Brazil.

ABSTRACT
T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+) T cells are important for the generation and maintenance of functional CD8(+) cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4(+) T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+)/CD8(+) T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+) and CD8(+) T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2) simultaneously in response to HIV-1 peptides. For CD4(+) T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2). The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses- elicited by other HIV immunogens.

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Immunization with HIVBr18 induces proliferating T cells with a polyfunctional type 1 cytokine profile.Two weeks after the last immunization with HIVBr18 or the empty vector pVAX1, spleen cells from 6 BALB/c mice were collected, labeled with CFSE (1.25 µM) and cultured for 4 days in the presence of pooled HIV-1 peptides or medium only. On day 4, cells were pulsed for 12 hours with pooled peptides in the presence of costimulatory antibody and Brefeldin A. Cells were then surface stained with antibodies to CD4 and CD8, permeabilized and stained for intracellular cytokines (IFNγ, TNFα and IL-2) and CD3. (A) Multiparameter flow cytometry was used to determine the frequency of IFNγ, IL-2 or TNFα CD4+ and CD8+ T cells. (B) Total frequencies of proliferating (CFSElow) and cytokine-producing CD4+ and CD8+ T cells; (C) After gating on proliferating (CFSElow) and cytokine-producing cells, Boolean combinations were then created using FlowJo software to determine the frequency of each response based on all possible combinations of cytokine expression. Background responses detected in negative control tubes were subtracted from those detected in stimulated samples for every specific functional combination. Negative control tubes include cells stimulated with medium and cells from pVAX1 immunized mice stimulated with pooled peptides. For each sample 500,000 events were collected in the live lymphocyte gate. Results are representative of two to three independent experiments.
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pone-0016921-g003: Immunization with HIVBr18 induces proliferating T cells with a polyfunctional type 1 cytokine profile.Two weeks after the last immunization with HIVBr18 or the empty vector pVAX1, spleen cells from 6 BALB/c mice were collected, labeled with CFSE (1.25 µM) and cultured for 4 days in the presence of pooled HIV-1 peptides or medium only. On day 4, cells were pulsed for 12 hours with pooled peptides in the presence of costimulatory antibody and Brefeldin A. Cells were then surface stained with antibodies to CD4 and CD8, permeabilized and stained for intracellular cytokines (IFNγ, TNFα and IL-2) and CD3. (A) Multiparameter flow cytometry was used to determine the frequency of IFNγ, IL-2 or TNFα CD4+ and CD8+ T cells. (B) Total frequencies of proliferating (CFSElow) and cytokine-producing CD4+ and CD8+ T cells; (C) After gating on proliferating (CFSElow) and cytokine-producing cells, Boolean combinations were then created using FlowJo software to determine the frequency of each response based on all possible combinations of cytokine expression. Background responses detected in negative control tubes were subtracted from those detected in stimulated samples for every specific functional combination. Negative control tubes include cells stimulated with medium and cells from pVAX1 immunized mice stimulated with pooled peptides. For each sample 500,000 events were collected in the live lymphocyte gate. Results are representative of two to three independent experiments.

Mentions: Since the quality of the immune responses has been associated with vaccine-mediated protection against certain pathogens, we subsequently characterized the phenotype and functional profile of the induced T cells. Using multiparameter flow cytometry, we sought to characterize antigen-specific T cells (CD4+ and CD8+) based on their ability to proliferate (CFSE dilution assay) and produce the effector cytokines IFNγ, TNFα and IL-2 at a single cell level. As shown in Figure 3A, immunization with HIVBr18 induced HIV-1 peptide-specific production of IFNγ, IL-2 and TNFα by CD4+ and to a lesser extent by CD8+ T cells. We also observed that both T cell subsets showed a higher proportion of of IFNγ+ and TNFα+ cells than IL-2+ cells. A simultaneous analysis of proliferation and intracellular cytokine production demonstrated that 2.3% of CD4+ and 1.0% of CD8+ T cells proliferated (CFSElow) and produced any cytokine tested in response to pooled HIV-1 peptides (Figure 3B). Boolean combinations of proliferating (CFSElow) and cytokine-positive populations indicated that HIVBr18 immunization induced polyfunctional CD4+ and CD8+ T cells, i.e., that proliferate (CFSElow) and produce IFNγ/TNFα simultaneously (Figure 3C). We also observed high proportions of CFSElow/IFNγ or CFSElow/TNFα in CD4+ and CD8+ T cells from mice that received HIVBr18. For CD4+ T cells exclusively, we also detected CFSElow cells that simultaneously produced all three tested cytokines (IFNγ/TNFα/IL-2). Splenocytes from the pVAX1 immunized group produced negligible levels of cytokines. Furthermore, triple-cytokine producing cells produced more IFNγ and TNFα than single-cytokine producing cells, as determined by median fluorescent intensity (MFI) (Figure S3). In contrast, there was no difference in the MFI for IL-2 in the triple-cytokine producing cells when compared to cells producing IL-2 alone.


A DNA vaccine encoding multiple HIV CD4 epitopes elicits vigorous polyfunctional, long-lived CD4+ and CD8+ T cell responses.

Rosa DS, Ribeiro SP, Almeida RR, Mairena EC, Postól E, Kalil J, Cunha-Neto E - PLoS ONE (2011)

Immunization with HIVBr18 induces proliferating T cells with a polyfunctional type 1 cytokine profile.Two weeks after the last immunization with HIVBr18 or the empty vector pVAX1, spleen cells from 6 BALB/c mice were collected, labeled with CFSE (1.25 µM) and cultured for 4 days in the presence of pooled HIV-1 peptides or medium only. On day 4, cells were pulsed for 12 hours with pooled peptides in the presence of costimulatory antibody and Brefeldin A. Cells were then surface stained with antibodies to CD4 and CD8, permeabilized and stained for intracellular cytokines (IFNγ, TNFα and IL-2) and CD3. (A) Multiparameter flow cytometry was used to determine the frequency of IFNγ, IL-2 or TNFα CD4+ and CD8+ T cells. (B) Total frequencies of proliferating (CFSElow) and cytokine-producing CD4+ and CD8+ T cells; (C) After gating on proliferating (CFSElow) and cytokine-producing cells, Boolean combinations were then created using FlowJo software to determine the frequency of each response based on all possible combinations of cytokine expression. Background responses detected in negative control tubes were subtracted from those detected in stimulated samples for every specific functional combination. Negative control tubes include cells stimulated with medium and cells from pVAX1 immunized mice stimulated with pooled peptides. For each sample 500,000 events were collected in the live lymphocyte gate. Results are representative of two to three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037933&req=5

pone-0016921-g003: Immunization with HIVBr18 induces proliferating T cells with a polyfunctional type 1 cytokine profile.Two weeks after the last immunization with HIVBr18 or the empty vector pVAX1, spleen cells from 6 BALB/c mice were collected, labeled with CFSE (1.25 µM) and cultured for 4 days in the presence of pooled HIV-1 peptides or medium only. On day 4, cells were pulsed for 12 hours with pooled peptides in the presence of costimulatory antibody and Brefeldin A. Cells were then surface stained with antibodies to CD4 and CD8, permeabilized and stained for intracellular cytokines (IFNγ, TNFα and IL-2) and CD3. (A) Multiparameter flow cytometry was used to determine the frequency of IFNγ, IL-2 or TNFα CD4+ and CD8+ T cells. (B) Total frequencies of proliferating (CFSElow) and cytokine-producing CD4+ and CD8+ T cells; (C) After gating on proliferating (CFSElow) and cytokine-producing cells, Boolean combinations were then created using FlowJo software to determine the frequency of each response based on all possible combinations of cytokine expression. Background responses detected in negative control tubes were subtracted from those detected in stimulated samples for every specific functional combination. Negative control tubes include cells stimulated with medium and cells from pVAX1 immunized mice stimulated with pooled peptides. For each sample 500,000 events were collected in the live lymphocyte gate. Results are representative of two to three independent experiments.
Mentions: Since the quality of the immune responses has been associated with vaccine-mediated protection against certain pathogens, we subsequently characterized the phenotype and functional profile of the induced T cells. Using multiparameter flow cytometry, we sought to characterize antigen-specific T cells (CD4+ and CD8+) based on their ability to proliferate (CFSE dilution assay) and produce the effector cytokines IFNγ, TNFα and IL-2 at a single cell level. As shown in Figure 3A, immunization with HIVBr18 induced HIV-1 peptide-specific production of IFNγ, IL-2 and TNFα by CD4+ and to a lesser extent by CD8+ T cells. We also observed that both T cell subsets showed a higher proportion of of IFNγ+ and TNFα+ cells than IL-2+ cells. A simultaneous analysis of proliferation and intracellular cytokine production demonstrated that 2.3% of CD4+ and 1.0% of CD8+ T cells proliferated (CFSElow) and produced any cytokine tested in response to pooled HIV-1 peptides (Figure 3B). Boolean combinations of proliferating (CFSElow) and cytokine-positive populations indicated that HIVBr18 immunization induced polyfunctional CD4+ and CD8+ T cells, i.e., that proliferate (CFSElow) and produce IFNγ/TNFα simultaneously (Figure 3C). We also observed high proportions of CFSElow/IFNγ or CFSElow/TNFα in CD4+ and CD8+ T cells from mice that received HIVBr18. For CD4+ T cells exclusively, we also detected CFSElow cells that simultaneously produced all three tested cytokines (IFNγ/TNFα/IL-2). Splenocytes from the pVAX1 immunized group produced negligible levels of cytokines. Furthermore, triple-cytokine producing cells produced more IFNγ and TNFα than single-cytokine producing cells, as determined by median fluorescent intensity (MFI) (Figure S3). In contrast, there was no difference in the MFI for IL-2 in the triple-cytokine producing cells when compared to cells producing IL-2 alone.

Bottom Line: Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection.The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines.By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses- elicited by other HIV immunogens.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Clinical Immunology and Allergy-LIM60, Division of Clinical Immunology and Allergy, Department of Medicine, University of São Paulo School of Medicine, São Paulo, Brazil.

ABSTRACT
T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+) T cells are important for the generation and maintenance of functional CD8(+) cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4(+) T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+)/CD8(+) T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+) and CD8(+) T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2) simultaneously in response to HIV-1 peptides. For CD4(+) T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2). The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses- elicited by other HIV immunogens.

Show MeSH
Related in: MedlinePlus