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VACCINE RESEARCH

Poly(Anhydride) Nanoparticles Act as Active Th1 Adjuvants through Toll-Like Receptor Exploitation

I. Tamayo, J. M. Irache, C. Mansilla, J. Ochoa-Repáraz, J. J. Lasarte, C. Gamazo
I. Tamayo
1Department of Microbiology, University of Navarra, 31008 Pamplona, Spain
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J. M. Irache
2Department of Pharmacy and Pharmaceutical Technology, University of Navarra, 31008 Pamplona, Spain
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C. Mansilla
3Division of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA), 31008 Pamplona, Spain
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J. Ochoa-Repáraz
1Department of Microbiology, University of Navarra, 31008 Pamplona, Spain
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J. J. Lasarte
3Division of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA), 31008 Pamplona, Spain
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C. Gamazo
1Department of Microbiology, University of Navarra, 31008 Pamplona, Spain
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  • For correspondence: cgamazo@unav.es
DOI: 10.1128/CVI.00164-10
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  • FIG. 1.
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    FIG. 1.

    Flow cytometric analysis of BMDC maturation markers. Bars show the fold-increase of mean relative fluorescence over negative control of CD86 and CD54 after coincubation of BMDC with 500 μg of NP, 100 μg of NP, 20 μg of NP, and 4 μg of NP.

  • FIG. 2.
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    FIG. 2.

    Proinflammatory cytokine detection in supernatants of BMDC. Levels of TNF-α (A) and IL-12 (B) released to the BMDC culture supernatant after 24 h of coincubation with 500 μg of NP, 100 μg of NP, 20 μg of NP, and 4 μg of NP. Nonstimulated controls are also included. All values are shown as arithmetic means ± standard errors of the means (SEM) (n = 3).

  • FIG. 3.
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    FIG. 3.

    In vivo induction of CTL activity specific for SIINFEKL peptide. C57BL/6 mice were immunized with OVA, poly(anhydride) NPs, or OVA plus NP; 7 days after immunization, naïve mice were sacrificed, and splenocytes coincubated in the presence or absence of SIINFEKL. Pulsed cells were incubated in the presence of high levels of CFSE, while nonpulsed cells were incubated with low levels of CFSE. After administration of CFSE-high and CFSE-low splenocytes to immunized mice, the specific cytotoxic activity against the SIINFEKL peptide was measured by an in vivo killing assay. The data represent the mean percentage values from triplicate samples.

  • FIG. 4.
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    FIG. 4.

    In vivo induction of OVA-specific IFN-γ-secreting CD8+ T cells. Mice were immunized with OVA, poly(anhydride) NPs, and OVA plus NP and 8 days after immunization sacrificed. Splenocytes were cultured in triplicate in the presence of SIINFEKL or culture medium alone (negative control). Each bar represents the mean value of SFC/106 cells.

  • FIG. 5.
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    FIG. 5.

    Effects of nanoparticles on the activation of TLR signaling. Bars represent engagement to TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 after incubation with positive controls and poly(anhydride) NPs. Names inside the bars of positive controls represent the specific agonist for each TLR used in the study: PAM2 (100 ng/ml) for TLR2, poly(I:C) (100 ng/ml) for TLR3, E. coli K12 LPS (1 μg/ml) for TLR4, flagellin (1 μg/ml) for TLR5, R848 (10 μg/ml) for TLR7 and TLR8, and ODN 2006 (10 μg) for TLR9. A TLR nonexpressing recombinant cell line is also included (TLR−). Results are given in OD values.

  • FIG. 6.
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    FIG. 6.

    Comparative protection against the virulent serovar of S. Enteritidis. Groups of 10 BALB/c mice were i.p. immunized with poly(anhydride) NPs (♦) or PBS (○). Ten days later, mice were challenged i.p. with 1.6 × 102 CFU of the virulent strain 3934. Data are expressed as percentage of survivals after challenge.

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Poly(Anhydride) Nanoparticles Act as Active Th1 Adjuvants through Toll-Like Receptor Exploitation
I. Tamayo, J. M. Irache, C. Mansilla, J. Ochoa-Repáraz, J. J. Lasarte, C. Gamazo
Clinical and Vaccine Immunology Sep 2010, 17 (9) 1356-1362; DOI: 10.1128/CVI.00164-10

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Poly(Anhydride) Nanoparticles Act as Active Th1 Adjuvants through Toll-Like Receptor Exploitation
I. Tamayo, J. M. Irache, C. Mansilla, J. Ochoa-Repáraz, J. J. Lasarte, C. Gamazo
Clinical and Vaccine Immunology Sep 2010, 17 (9) 1356-1362; DOI: 10.1128/CVI.00164-10
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KEYWORDS

Adjuvants, Immunologic
Maleates
Nanoparticles
Polyethylenes
Th1 cells
Toll-Like Receptors

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