Improved Tuberculosis DNA Vaccines by Formulation in Cationic Lipids

ABSTRACT Mice were vaccinated with plasmid DNA (pDNA) encoding antigen 85A (Ag85A), Ag85B, or PstS-3 from Mycobacterium tuberculosis either in saline or formulated for intramuscular injections in VC1052:DPyPE (aminopropyl-dimethyl-myristoleyloxy-propanaminium bromide-diphytanoylphosphatidyl-ethanolamine) (Vaxfectin; Vical, Inc., San Diego, Calif.) or for intranasal instillations in GAP-DLRIE:DOPE (aminopropyl-dimethyl-bis-dodecyloxy-propanaminium bromide-dioleoylphosphatidyl-ethanolamine). These two novel cationic and neutral colipid formulations were previously reported to be effective adjuvants for pDNA-induced antibody responses. The levels of Ag85-specific total immunoglobulin G (IgG) and IgG isotypes were all increased 3- to 10-fold by formulation of pDNA in Vaxfectin. The level of production of splenic T-cell-derived Th1-type cytokines (interleukin-2 and gamma interferon) in response to purified Ag85 and to synthetic peptides spanning the entire Ag85A protein was also significantly higher in animals vaccinated with pDNA formulated in Vaxfectin. Cytolytic T-lymphocyte responses generated by pDNA encoding phosphate-binding protein PstS-3 in Vaxfectin were better sustained over time than were those generated by PstS-3 DNA in saline. Intranasal immunization with Ag85A DNA in saline was completely ineffective, whereas administration in GAP-DLRIE:DOPE induced a positive Th1-type cytokine response; however, the extent of the latter response was clearly lower than that obtained following intramuscular immunization with the same DNA dose. Combined intramuscular and intranasal administrations in cationic lipids resulted in stronger immune responses in the spleen and, more importantly, in the lungs as well. Finally, formulation in Vaxfectin increased the protective efficacy of the Ag85B DNA vaccine, as measured by reduced relative light unit counts and CFU counts in the spleen and lungs from mice challenged with bioluminescent M. tuberculosis H37Rv. These results may be of importance for future clinical use of DNA vaccines in humans.

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Coadministration of Interleukin 2 Plasmid DNA with Combined DNA Vaccines Significantly Enhances the Protective Efficacy AgainstMycobacterium tuberculosis

DNA and Cell Biology ◽

10.1089/dna.2005.24.605 ◽

2005 ◽

Vol 24 (10) ◽

pp. 605-613 ◽

Cited By ~ 16

Author(s):

H. Cai ◽

D.H. Yu ◽

X. Tian ◽

Y.X. Zhu

Keyword(s):

Plasmid Dna ◽

Dna Vaccines ◽

Protective Efficacy ◽

Interleukin 2

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Characterization of a factor(s) which synergizes with recombinant interleukin 2 in promoting allogeneic human cytolytic T-lymphocyte responses in vitro

Cellular Immunology ◽

10.1016/0008-8749(88)90049-4 ◽

1988 ◽

Vol 111 (1) ◽

pp. 39-54 ◽

Cited By ~ 37

Author(s):

Henry L. Wong ◽

Darien E. Wilson ◽

James C. Jenson ◽

Philip C. Familletti ◽

Donna L. Stremlo ◽

...

Keyword(s):

T Lymphocyte ◽

Interleukin 2 ◽

Cytolytic T Lymphocyte ◽

Recombinant Interleukin 2 ◽

Lymphocyte Responses

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Tuberculosis DNA Vaccine Encoding Ag85A Is Immunogenic and Protective When Administered by Intramuscular Needle Injection but Not by Epidermal Gene Gun Bombardment

Infection and Immunity ◽

10.1128/iai.68.7.3854-3860.2000 ◽

2000 ◽

Vol 68 (7) ◽

pp. 3854-3860 ◽

Cited By ~ 62

Author(s):

Audrey Tanghe ◽

Olivier Denis ◽

Bénédicte Lambrecht ◽

Vinciane Motte ◽

Thierry van den Berg ◽

...

Keyword(s):

Dna Vaccine ◽

Cytotoxic T Lymphocyte ◽

Protective Efficacy ◽

Interleukin 2 ◽

Gene Gun ◽

Antibody Isotypes ◽

C57bl Mice ◽

Ifn Γ ◽

Lymphocyte Responses ◽

Needle Injection

ABSTRACT Immunogenicity and protective efficacy of a DNA vaccine encoding Ag85A from Mycobacterium tuberculosis were compared in BALB/c and C57BL (B6 and B10) mice immunized by intramuscular (i.m.) needle injection or epidermal gene gun (gg) bombardment. In BALB/c mice, gg immunization could induce elevated antibody and cytotoxic T lymphocyte responses with plasmid doses 50-fold lower than those required for i.m. immunization. Interleukin-2 (IL-2) and gamma interferon (IFN-γ) secretion, however, was much lower in gg-immunized than in i.m.-immunized BALB/c mice. On the other hand, C57BL mice reacted only very weakly to gg immunization, whereas elevated Ag85A-specific antibody, IL-2, and IFN-γ responses (significantly higher than in BALB/c mice) were detected following vaccination by the i.m. route. Antibody isotypes were indicative of Th2 activation following gg injection of BALB/c and of Th1 activation following i.m. injection of C57BL mice. Finally, C57BL but not BALB/c mice were protected by i.m. Ag85A DNA immunization against intravenousM. tuberculosis challenge, as measured by reduced numbers of CFU in spleen and lungs, compared to animals vaccinated with control DNA. Gene gun immunization was not effective in either BALB/c or C57BL mice. These results indicate that i.m. DNA vaccination is the method of choice for the induction of protective Th1 type immune responses with the Ag85A tuberculosis DNA vaccine.

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Improved Immunogenicity and Protective Efficacy of a Tuberculosis DNA Vaccine Encoding Ag85 by Protein Boosting

Infection and Immunity ◽

10.1128/iai.69.5.3041-3047.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3041-3047 ◽

Cited By ~ 144

Author(s):

Audrey Tanghe ◽

Sushila D'Souza ◽

Valérie Rosseels ◽

Olivier Denis ◽

Thomas H. M. Ottenhoff ◽

...

Keyword(s):

T Cells ◽

Dna Vaccine ◽

Protective Efficacy ◽

Interleukin 2 ◽

Relative Light Unit ◽

Dna Immunization ◽

Cytokine Responses ◽

Cytokine Analysis ◽

Protein Boost ◽

Ifn Γ

ABSTRACT C57BL/6 mice were vaccinated with plasmid DNA encoding Ag85 fromMycobacterium tuberculosis, with Ag85 protein in adjuvant, or with a combined DNA prime-protein boost regimen. While DNA immunization, as previously described, induced robust Th1-type cytokine responses, protein-in-adjuvant vaccination elicited very poor cytokine responses, which were 10-fold lower than those observed with DNA immunization alone. Injection of Ag85 DNA-primed mice with 30 to 100 μg of purified Ag85 protein in adjuvant increased the interleukin-2 and gamma interferon (IFN-γ) response in spleen two- to fourfold. Further, intracellular cytokine analysis by flow cytometry also showed an increase in IFN-γ-producing CD4+ T cells in DNA-primed–protein-boosted animals, compared to those that received only the DNA vaccination. Moreover, these responses appeared to be better sustained over time. Antibodies were readily produced by all three methods of immunization but were exclusively of the immunoglobulin G1 (IgG1) isotype following protein immunization in adjuvant and preferentially of the IgG2a isotype following DNA and DNA prime-protein boost vaccination. Finally, protein boosting increased the protective efficacy of the DNA vaccine against an intravenousM. tuberculosis H37Rv challenge infection, as measured by CFU or relative light unit counts in lungs 1 and 2 months after infection. The capacity of exogenously given protein to boost the DNA-primed vaccination effect underlines the dominant role of Th1-type CD4+ helper T cells in mediating protection.

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Crystallization and x-ray diffraction studies of a phosphate-binding protein involved in active transport in Escherichia coli.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)57501-9 ◽

1986 ◽

Vol 261 (17) ◽

pp. 7995-7996

Author(s):

B D Kubena ◽

H Luecke ◽

H Rosenberg ◽

F A Quiocho

Keyword(s):

Escherichia Coli ◽

Active Transport ◽

Binding Protein ◽

X Ray Diffraction ◽

Phosphate Binding ◽

X Ray ◽

Phosphate Binding Protein

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Purification and properties of glucose 1-phosphate binding protein in Agrobacterium tumefaciens

Biochimica et Biophysica Acta (BBA) - Protein Structure ◽

10.1016/0005-2795(73)90337-1 ◽

1973 ◽

Vol 328 (1) ◽

pp. 114-123

Author(s):

Sakuzo Fukui ◽

Kazuko Isobe

Keyword(s):

Agrobacterium Tumefaciens ◽

Binding Protein ◽

Phosphate Binding ◽

Purification And Properties ◽

Phosphate Binding Protein

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Protective Vaccine Efficacy of the Complete Form of PPE39 Protein from Mycobacterium tuberculosis Beijing/K Strain in Mice

Clinical and Vaccine Immunology ◽

10.1128/cvi.00219-17 ◽

2017 ◽

Vol 24 (11) ◽

Cited By ~ 4

Author(s):

Ahreum Kim ◽

Yun-Gyoung Hur ◽

Sunwha Gu ◽

Sang-Nae Cho

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

T Cell ◽

Vaccine Development ◽

Protective Efficacy ◽

Interleukin 2 ◽

T Cell Epitopes ◽

Content Type ◽

Complete Form ◽

Ifn Γ

ABSTRACT The aim of this study was to evaluate the protective efficacy of MTBK_24820, a complete form of PPE39 protein derived from a predominant Beijing/K strain of Mycobacterium tuberculosis in South Korea. Mice were immunized with MTKB_24820, M. bovis Bacilli Calmette-Guérin (BCG), or adjuvant prior to a high-dosed Beijing/K strain aerosol infection. After 4 and 9 weeks, bacterial loads were determined and histopathologic and immunologic features in the lungs and spleens of the M. tuberculosis-infected mice were analyzed. Putative immunogenic T-cell epitopes were examined using synthetic overlapping peptides. Successful immunization of MTBK_24820 in mice was confirmed by increased IgG responses (P < 0.05) and recalled gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-6, and IL-17 responses (P < 0.05 or P < 0.01) to MTBK_24820. After challenge with the Beijing/K strain, an approximately 0.5 to 1.0 log10 reduction in CFU in lungs and fewer lung inflammation lesions were observed in MTBK_24820-immunized mice compared to those for control mice. Moreover, MTBK_24820 immunization elicited significantly higher numbers of CD4+ T cells producing protective cytokines, such as IFN-γ and IL-17, in lungs and spleens (P < 0.01) and CD4+ multifunctional T cells producing IFN-γ, tumor necrosis factor alpha (TNF-α), and/or IL-17 (P < 0.01) than in control mice, suggesting protection comparable to that of BCG against the hypervirulent Beijing/K strain. The dominant immunogenic T-cell epitopes that induced IFN-γ production were at the N terminus (amino acids 85 to 102 and 217 to 234). Its vaccine potential, along with protective immune responses in vivo, may be informative for vaccine development, particularly in regions where the M. tuberculosis Beijing/K-strain is frequently isolated from TB patients.

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The high-affinity phosphate-binding protein PstS is accumulated under high fructose concentrations and mutation of the corresponding gene affects differentiation in Streptomyces lividans

Microbiology ◽

10.1099/mic.0.27983-0 ◽

2005 ◽

Vol 151 (8) ◽

pp. 2583-2592 ◽

Cited By ~ 42

Author(s):

Margarita Díaz ◽

Ana Esteban ◽

José Manuel Fernández-Abalos ◽

Ramón I. Santamaría

Keyword(s):

Culture Media ◽

Binding Protein ◽

Protein Pattern ◽

Phosphate Transport ◽

Streptomyces Lividans ◽

Null Mutant ◽

Phosphate Binding ◽

Genetic Switch ◽

High Affinity ◽

Phosphate Binding Protein

The secreted protein pattern of Streptomyces lividans depends on the carbon source present in the culture media. One protein that shows the most dramatic change is the high-affinity phosphate-binding protein PstS, which is strongly accumulated in the supernatant of liquid cultures containing high concentrations (>3 %) of certain sugars, such as fructose, galactose and mannose. The promoter region of this gene and that of its Streptomyces coelicolor homologue were used to drive the expression of a xylanase in S. lividans that was accumulated in the culture supernatant when grown in the presence of fructose. PstS accumulation was dramatically increased in a S. lividans polyphosphate kinase null mutant (Δppk) and was impaired in a deletion mutant lacking phoP, the transcriptional regulator gene of the two-component phoR-phoP system that controls the Pho regulon. Deletion of the pstS genes in S. lividans and S. coelicolor impaired phosphate transport and accelerated differentiation and sporulation on solid media. Complementation with a single copy in a S. lividans pstS null mutant returned phosphate transport and sporulation to levels similar to those of the wild-type strain. The present work demonstrates that carbon and phosphate metabolism are linked in the regulation of genes and that this can trigger the genetic switch towards morphogenesis.

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