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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

In vitro stability of the BCG.HIVA2auxo strain. (a) In vitro persistence of the p2auxo.HIVA in BCG lysA- grown for successive passages on selective (no lysine) or nonselective (supplemented with lysine) media. The percentage represents the cfu (titer) that maintained the vector containing the lysine complementing gene (grown on selective medium) versus to the total cfu. For BCG, the generation time is ~24 h. Thus, four subcultures represent ~30 BCG generations. The most representative of two experiments is shown. (b) Immunodot of BCG colonies lysates that were grown on selective (41, 42, 43, 44, and 45) and nonselective medium (1, 7, 13, 17, and 24); lysates of BCG wild type were used as negative control and lysates of BCG.HIVA2auxo WVS (before subculturing) as positive control. (c) HIVA PCR from purified plasmids of five individual colonies that were grown on selective (41, 42, 43, 44, and 45) and nonselective (1, 7, 13, 17, and 24) medium. Plasmid DNA p2auxo.HIVA pretransformation in BCG was used as positive control. Distilled water (negative control).
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fig3: In vitro stability of the BCG.HIVA2auxo strain. (a) In vitro persistence of the p2auxo.HIVA in BCG lysA- grown for successive passages on selective (no lysine) or nonselective (supplemented with lysine) media. The percentage represents the cfu (titer) that maintained the vector containing the lysine complementing gene (grown on selective medium) versus to the total cfu. For BCG, the generation time is ~24 h. Thus, four subcultures represent ~30 BCG generations. The most representative of two experiments is shown. (b) Immunodot of BCG colonies lysates that were grown on selective (41, 42, 43, 44, and 45) and nonselective medium (1, 7, 13, 17, and 24); lysates of BCG wild type were used as negative control and lysates of BCG.HIVA2auxo WVS (before subculturing) as positive control. (c) HIVA PCR from purified plasmids of five individual colonies that were grown on selective (41, 42, 43, 44, and 45) and nonselective (1, 7, 13, 17, and 24) medium. Plasmid DNA p2auxo.HIVA pretransformation in BCG was used as positive control. Distilled water (negative control).

Mentions: To evaluate the in vitro stability of the p2auxo.HIVA plasmid DNA harboring the auxotrophic complementation lysA gene, subcultures on media with and without selection were carried out. All BCG.HIVA2auxo colonies that were grown on selective medium (without lysine supplementation) maintained the vector for over four subcultures (~30 bacterial generations). In contrast, when bacteria were grown without selective pressure (with lysine supplementation), only 9% of the BCG colonies were harboring the plasmid DNA after the first subculture with an average of 17% maintenance over the subsequent subculturing passages. The differences between both groups were statistically significant (P < 0.05; Figure 3a).


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)

In vitro stability of the BCG.HIVA2auxo strain. (a) In vitro persistence of the p2auxo.HIVA in BCG lysA- grown for successive passages on selective (no lysine) or nonselective (supplemented with lysine) media. The percentage represents the cfu (titer) that maintained the vector containing the lysine complementing gene (grown on selective medium) versus to the total cfu. For BCG, the generation time is ~24 h. Thus, four subcultures represent ~30 BCG generations. The most representative of two experiments is shown. (b) Immunodot of BCG colonies lysates that were grown on selective (41, 42, 43, 44, and 45) and nonselective medium (1, 7, 13, 17, and 24); lysates of BCG wild type were used as negative control and lysates of BCG.HIVA2auxo WVS (before subculturing) as positive control. (c) HIVA PCR from purified plasmids of five individual colonies that were grown on selective (41, 42, 43, 44, and 45) and nonselective (1, 7, 13, 17, and 24) medium. Plasmid DNA p2auxo.HIVA pretransformation in BCG was used as positive control. Distilled water (negative control).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362382&req=5

fig3: In vitro stability of the BCG.HIVA2auxo strain. (a) In vitro persistence of the p2auxo.HIVA in BCG lysA- grown for successive passages on selective (no lysine) or nonselective (supplemented with lysine) media. The percentage represents the cfu (titer) that maintained the vector containing the lysine complementing gene (grown on selective medium) versus to the total cfu. For BCG, the generation time is ~24 h. Thus, four subcultures represent ~30 BCG generations. The most representative of two experiments is shown. (b) Immunodot of BCG colonies lysates that were grown on selective (41, 42, 43, 44, and 45) and nonselective medium (1, 7, 13, 17, and 24); lysates of BCG wild type were used as negative control and lysates of BCG.HIVA2auxo WVS (before subculturing) as positive control. (c) HIVA PCR from purified plasmids of five individual colonies that were grown on selective (41, 42, 43, 44, and 45) and nonselective (1, 7, 13, 17, and 24) medium. Plasmid DNA p2auxo.HIVA pretransformation in BCG was used as positive control. Distilled water (negative control).
Mentions: To evaluate the in vitro stability of the p2auxo.HIVA plasmid DNA harboring the auxotrophic complementation lysA gene, subcultures on media with and without selection were carried out. All BCG.HIVA2auxo colonies that were grown on selective medium (without lysine supplementation) maintained the vector for over four subcultures (~30 bacterial generations). In contrast, when bacteria were grown without selective pressure (with lysine supplementation), only 9% of the BCG colonies were harboring the plasmid DNA after the first subculture with an average of 17% maintenance over the subsequent subculturing passages. The differences between both groups were statistically significant (P < 0.05; Figure 3a).

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