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Engineering new mycobacterial vaccine design for HIV-TB pediatric vaccine vectored by lysine auxotroph of BCG.

Saubi N, Gea-Mallorquí E, Ferrer P, Hurtado C, Sánchez-Úbeda S, Eto Y, Gatell JM, Hanke T, Joseph J - Mol Ther Methods Clin Dev (2014)

Bottom Line: HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice.Thus, we engineered a novel, safer, good laboratory practice-compatible BCG-vectored vaccine using prototype immunogen HIVA.This antibiotic-free plasmid selection system based on "double" auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth.

View Article: PubMed Central - PubMed

Affiliation: AIDS Research Group, Hospital Clinic/HIVACAT, School of Medicine, University of Barcelona , Barcelona, Catalonia, Spain.

ABSTRACT
In this study, we have engineered a new mycobacterial vaccine design by using an antibiotic-free plasmid selection system. We assembled a novel Escherichia coli (E. coli)-mycobacterial shuttle plasmid p2auxo.HIVA, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism for plasmid selection and maintenance based on glycine complementation in E. coli and lysine complementation in mycobacteria. This plasmid was first transformed into glycine auxotroph of E. coli strain and subsequently transformed into lysine auxotroph of Mycobacterium bovis BCG strain to generate vaccine BCG.HIVA(2auxo). We demonstrated that the episomal plasmid p2auxo.HIVA was stable in vivo over a 7-week period and genetically and phenotypically characterized the BCG.HIVA(2auxo) vaccine strain. The BCG.HIVA(2auxo) vaccine in combination with modified vaccinia virus Ankara (MVA). HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. Polyfunctional HIV-1-specific CD8+ T cells, which produce interferon-γ and tumor necrosis factor-α and express the degranulation marker CD107a, were induced. Thus, we engineered a novel, safer, good laboratory practice-compatible BCG-vectored vaccine using prototype immunogen HIVA. This antibiotic-free plasmid selection system based on "double" auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth.

No MeSH data available.


Related in: MedlinePlus

Induction of HIV-1- and Mtb-specific T-cells responses by the BCG.HIVA2auxo prime–MVA.HIVA boost regimen. (a) Adult mice (7-weeks-old) were either left unimmunized or primed with 106 cfu of BCG.HIVA2auxo or BCG wild type (intradermally) and boosted with 106 pfu of MVA.HIVA (intramuscularly) 5-weeks post-BCG inoculation. Mice were sacrificed 2 weeks later for T-cell analysis. (b) Analysis of IFN-γ vaccine elicited HIV-1-specific CD8+ T-cell responses. The frequencies of cells producing cytokine are shown. Data are presented as means (SEM; n = 8 for group 1, and n = 5 for groups 2, 3, and 4). (c) The functionality of vaccine-induced CD8+ T-cell responses was assessed in a multicolor intracellular cytokine staining assay. The group mean frequencies of single-, double-, or triple cytokine-producing P18–I10-specific cells are shown for the four vaccination groups. (d) Elicitation of specific T-cell responses was assessed in an ex vivo IFN-γ enzyme linked immunosorbent spot (ELISPOT) assay using the immunodominant P18–I10 CD8+ T-cell epitope peptide. The median spot-forming units (SFU) per 106 splenocytes for each group of mice (n = 8 for group 1, and n = 5 for groups 2, 3, and 4) as well as individual animal responses is shown. (e) Purified protein derivative (PPD)-specific T-cell responses elicited by BCG.HIVA2auxo. Immune responses to BCG were assessed in an ex vivo IFN-γ ELISPOT assay using PPD as the antigen. The median SFU per 106 splenocytes for each group of mice (n = 8 for group 1, and n = 5 for groups 2, 3, and 4) as well as individual animal responses is shown. *P < 0.05, **P < 0.01.
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fig4: Induction of HIV-1- and Mtb-specific T-cells responses by the BCG.HIVA2auxo prime–MVA.HIVA boost regimen. (a) Adult mice (7-weeks-old) were either left unimmunized or primed with 106 cfu of BCG.HIVA2auxo or BCG wild type (intradermally) and boosted with 106 pfu of MVA.HIVA (intramuscularly) 5-weeks post-BCG inoculation. Mice were sacrificed 2 weeks later for T-cell analysis. (b) Analysis of IFN-γ vaccine elicited HIV-1-specific CD8+ T-cell responses. The frequencies of cells producing cytokine are shown. Data are presented as means (SEM; n = 8 for group 1, and n = 5 for groups 2, 3, and 4). (c) The functionality of vaccine-induced CD8+ T-cell responses was assessed in a multicolor intracellular cytokine staining assay. The group mean frequencies of single-, double-, or triple cytokine-producing P18–I10-specific cells are shown for the four vaccination groups. (d) Elicitation of specific T-cell responses was assessed in an ex vivo IFN-γ enzyme linked immunosorbent spot (ELISPOT) assay using the immunodominant P18–I10 CD8+ T-cell epitope peptide. The median spot-forming units (SFU) per 106 splenocytes for each group of mice (n = 8 for group 1, and n = 5 for groups 2, 3, and 4) as well as individual animal responses is shown. (e) Purified protein derivative (PPD)-specific T-cell responses elicited by BCG.HIVA2auxo. Immune responses to BCG were assessed in an ex vivo IFN-γ ELISPOT assay using PPD as the antigen. The median SFU per 106 splenocytes for each group of mice (n = 8 for group 1, and n = 5 for groups 2, 3, and 4) as well as individual animal responses is shown. *P < 0.05, **P < 0.01.

Mentions: In this study, we have evaluated the specific HIV-1 T-cell immune responses in BALB/c mice after immunization with BCG.HIVA2auxo prime and MVA.HIVA boost (Figure 4a). The immunogenicity readout was focused on the P18–I10 epitope, an immunodominant CTL epitope derived from HIV-1 Env and H-2Dd murine restricted, which was fused to HIVA immunogen to evaluate the immunogenicity in mice (Figure 1a). Functional specific T cells in response to peptide stimulation were measured by intracellular cytokine staining and enzyme-linked immunosorbent spot (ELISPOT) assays. We have observed that BCG.HIVA2auxo prime and MVA.HIVA boost elicited the highest proportion of P18–I10 epitope-specific CD8+ T-cells producing interferon-γ (IFN-γ), compared with the BCG wild-type priming and MVA.HIVA boost and with MVA.HIVA alone (Figure 4b). The quality of the elicited CD8+ T cells in terms of their ability to produce IFN-γ and tumor necrosis factor-α and to degranulate (surface expression of CD107a) in response to P18–I10 peptide stimulation was also investigated. We found that BCG.HIVA2auxo prime and MVA.HIVA boost induced higher frequencies of trifunctional specific CD8+ T cells compared with the BCG wild-type priming and MVA.HIVA boost and with MVA.HIVA alone (Figure 4c). The capacity of splenocytes from vaccinated mice to secrete IFN-γ was tested also by ELISPOT assay. We observed the highest frequency of specific cells secreting IFN-γ in mice primed with BCG.HIVA2auxo and boosted with MVA.HIVA (Figure 4d). Further experiments assessing different doses, routes, and immunization schedules should be performed.


Engineering new mycobacterial vaccine design for HIV-TB pediatric vaccine vectored by lysine auxotroph of BCG.

Saubi N, Gea-Mallorquí E, Ferrer P, Hurtado C, Sánchez-Úbeda S, Eto Y, Gatell JM, Hanke T, Joseph J - Mol Ther Methods Clin Dev (2014)

Induction of HIV-1- and Mtb-specific T-cells responses by the BCG.HIVA2auxo prime–MVA.HIVA boost regimen. (a) Adult mice (7-weeks-old) were either left unimmunized or primed with 106 cfu of BCG.HIVA2auxo or BCG wild type (intradermally) and boosted with 106 pfu of MVA.HIVA (intramuscularly) 5-weeks post-BCG inoculation. Mice were sacrificed 2 weeks later for T-cell analysis. (b) Analysis of IFN-γ vaccine elicited HIV-1-specific CD8+ T-cell responses. The frequencies of cells producing cytokine are shown. Data are presented as means (SEM; n = 8 for group 1, and n = 5 for groups 2, 3, and 4). (c) The functionality of vaccine-induced CD8+ T-cell responses was assessed in a multicolor intracellular cytokine staining assay. The group mean frequencies of single-, double-, or triple cytokine-producing P18–I10-specific cells are shown for the four vaccination groups. (d) Elicitation of specific T-cell responses was assessed in an ex vivo IFN-γ enzyme linked immunosorbent spot (ELISPOT) assay using the immunodominant P18–I10 CD8+ T-cell epitope peptide. The median spot-forming units (SFU) per 106 splenocytes for each group of mice (n = 8 for group 1, and n = 5 for groups 2, 3, and 4) as well as individual animal responses is shown. (e) Purified protein derivative (PPD)-specific T-cell responses elicited by BCG.HIVA2auxo. Immune responses to BCG were assessed in an ex vivo IFN-γ ELISPOT assay using PPD as the antigen. The median SFU per 106 splenocytes for each group of mice (n = 8 for group 1, and n = 5 for groups 2, 3, and 4) as well as individual animal responses is shown. *P < 0.05, **P < 0.01.
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Related In: Results  -  Collection

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fig4: Induction of HIV-1- and Mtb-specific T-cells responses by the BCG.HIVA2auxo prime–MVA.HIVA boost regimen. (a) Adult mice (7-weeks-old) were either left unimmunized or primed with 106 cfu of BCG.HIVA2auxo or BCG wild type (intradermally) and boosted with 106 pfu of MVA.HIVA (intramuscularly) 5-weeks post-BCG inoculation. Mice were sacrificed 2 weeks later for T-cell analysis. (b) Analysis of IFN-γ vaccine elicited HIV-1-specific CD8+ T-cell responses. The frequencies of cells producing cytokine are shown. Data are presented as means (SEM; n = 8 for group 1, and n = 5 for groups 2, 3, and 4). (c) The functionality of vaccine-induced CD8+ T-cell responses was assessed in a multicolor intracellular cytokine staining assay. The group mean frequencies of single-, double-, or triple cytokine-producing P18–I10-specific cells are shown for the four vaccination groups. (d) Elicitation of specific T-cell responses was assessed in an ex vivo IFN-γ enzyme linked immunosorbent spot (ELISPOT) assay using the immunodominant P18–I10 CD8+ T-cell epitope peptide. The median spot-forming units (SFU) per 106 splenocytes for each group of mice (n = 8 for group 1, and n = 5 for groups 2, 3, and 4) as well as individual animal responses is shown. (e) Purified protein derivative (PPD)-specific T-cell responses elicited by BCG.HIVA2auxo. Immune responses to BCG were assessed in an ex vivo IFN-γ ELISPOT assay using PPD as the antigen. The median SFU per 106 splenocytes for each group of mice (n = 8 for group 1, and n = 5 for groups 2, 3, and 4) as well as individual animal responses is shown. *P < 0.05, **P < 0.01.
Mentions: In this study, we have evaluated the specific HIV-1 T-cell immune responses in BALB/c mice after immunization with BCG.HIVA2auxo prime and MVA.HIVA boost (Figure 4a). The immunogenicity readout was focused on the P18–I10 epitope, an immunodominant CTL epitope derived from HIV-1 Env and H-2Dd murine restricted, which was fused to HIVA immunogen to evaluate the immunogenicity in mice (Figure 1a). Functional specific T cells in response to peptide stimulation were measured by intracellular cytokine staining and enzyme-linked immunosorbent spot (ELISPOT) assays. We have observed that BCG.HIVA2auxo prime and MVA.HIVA boost elicited the highest proportion of P18–I10 epitope-specific CD8+ T-cells producing interferon-γ (IFN-γ), compared with the BCG wild-type priming and MVA.HIVA boost and with MVA.HIVA alone (Figure 4b). The quality of the elicited CD8+ T cells in terms of their ability to produce IFN-γ and tumor necrosis factor-α and to degranulate (surface expression of CD107a) in response to P18–I10 peptide stimulation was also investigated. We found that BCG.HIVA2auxo prime and MVA.HIVA boost induced higher frequencies of trifunctional specific CD8+ T cells compared with the BCG wild-type priming and MVA.HIVA boost and with MVA.HIVA alone (Figure 4c). The capacity of splenocytes from vaccinated mice to secrete IFN-γ was tested also by ELISPOT assay. We observed the highest frequency of specific cells secreting IFN-γ in mice primed with BCG.HIVA2auxo and boosted with MVA.HIVA (Figure 4d). Further experiments assessing different doses, routes, and immunization schedules should be performed.

Bottom Line: HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice.Thus, we engineered a novel, safer, good laboratory practice-compatible BCG-vectored vaccine using prototype immunogen HIVA.This antibiotic-free plasmid selection system based on "double" auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth.

View Article: PubMed Central - PubMed

Affiliation: AIDS Research Group, Hospital Clinic/HIVACAT, School of Medicine, University of Barcelona , Barcelona, Catalonia, Spain.

ABSTRACT
In this study, we have engineered a new mycobacterial vaccine design by using an antibiotic-free plasmid selection system. We assembled a novel Escherichia coli (E. coli)-mycobacterial shuttle plasmid p2auxo.HIVA, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism for plasmid selection and maintenance based on glycine complementation in E. coli and lysine complementation in mycobacteria. This plasmid was first transformed into glycine auxotroph of E. coli strain and subsequently transformed into lysine auxotroph of Mycobacterium bovis BCG strain to generate vaccine BCG.HIVA(2auxo). We demonstrated that the episomal plasmid p2auxo.HIVA was stable in vivo over a 7-week period and genetically and phenotypically characterized the BCG.HIVA(2auxo) vaccine strain. The BCG.HIVA(2auxo) vaccine in combination with modified vaccinia virus Ankara (MVA). HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. Polyfunctional HIV-1-specific CD8+ T cells, which produce interferon-γ and tumor necrosis factor-α and express the degranulation marker CD107a, were induced. Thus, we engineered a novel, safer, good laboratory practice-compatible BCG-vectored vaccine using prototype immunogen HIVA. This antibiotic-free plasmid selection system based on "double" auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth.

No MeSH data available.


Related in: MedlinePlus