Class-B CpG-ODN formulated with a nanostructure induces type I interferons-dependent and CD4+T cell-independent CD8+T-Cell response against unconjugated protein antigen

There is a need for new vaccine adjuvant strategies that offer both vigorous antibody and T-cell mediated protection to combat difficult intracellular pathogens and cancer. To this aim, we formulated class-B synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) with a nanostructure (Coa-ASC16 or coagel) formed by self-assembly of 6-0-ascorbyl palmitate ester. Our previous results demonstrated that mice immunized with ovalbumin (OVA) and CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16) elicited strong antibodies (IgG1 and IgG2a) and Th1/Th17 cellular responses without toxic systemic effects. These responses were superior to those induced by a solution of OVA with CpG-ODN or OVA/CpG-ODN formulated with aluminum salts. In this study, we investigated the capacity of this adjuvant strategy (CpG-ODN/Coa-ASC16) to elicit CD8+ T-cell response and some of the underlying cellular and molecular mechanisms involved in adaptive response. We also analyzed whether this adjuvant strategy allows a switch from an immunization scheme of three-doses to one of single-dose. Our results demonstrated that vaccination with OVA/CpG-ODN/Coa-ASC16 elicited an antigen-specific long-lasting humoral response and importantly-high quality CD8+ T-cell immunity with a single-dose immunization. Moreover, Coa-ASC16 promoted co-uptake of OVA and CpG-ODN by dendritic cells. The CD8+ T-cell response induced by OVA/CpG-ODN/Coa-ASC16 was dependent of type I interferons and independent of CD4+ T-cells, and showed polyfunctionality and efficiency against an intracellular pathogen. Furthermore, the cellular and humoral responses elicited by the nanostructured formulation were IL-6-independent. This system provides a simple and inexpensive adjuvant strategy with great potential for future rationally designed vaccines.Fil: Chiodetti, Ana Laura. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Sánchez Vallecillo, María Fernanda. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Dolina, Joseph S.. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Crespo, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Marin, Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Schoenberger, Stephen P.. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Allemandi, Daniel Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; ArgentinaFil: Palma, Santiago Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; ArgentinaFil: Pistoresi, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Moron, Victor Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Maletto, Belkys Angélica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentin

Liver Is Able to Activate Naïve CD8+ T Cells with Dysfunctional Anti-Viral Activity in the Murine System

The liver possesses distinct tolerogenic properties because of continuous exposure to bacterial constituents and nonpathogenic food antigen. The central immune mediators required for the generation of effective immune responses in the liver environment have not been fully elucidated. In this report, we demonstrate that the liver can indeed support effector CD8+ T cells during adenovirus infection when the T cells are primed in secondary lymphoid tissues. In contrast, when viral antigen is delivered predominantly to the liver via intravenous (IV) adenovirus infection, intrahepatic CD8+ T cells are significantly impaired in their ability to produce inflammatory cytokines and lyse target cells. Additionally, intrahepatic CD8+ T cells generated during IV adenovirus infection express elevated levels of PD-1. Notably, lower doses of adenovirus infection do not rescue the impaired effector function of intrahepatic CD8+ T cell responses. Instead, intrahepatic antigen recognition limits the generation of potent anti-viral responses at both priming and effector stages of the CD8+ T cell response and accounts for the dysfunctional CD8+ T cell response observed during IV adenovirus infection. These results also implicate that manipulation of antigen delivery will facilitate the design of improved vaccination strategies to persistent viral infection

Class-B CpG-ODN Formulated With a Nanostructure Induces Type I Interferons-Dependent and CD4+ T Cell-Independent CD8+ T-Cell Response Against Unconjugated Protein Antigen

There is a need for new vaccine adjuvant strategies that offer both vigorous antibody and T-cell mediated protection to combat difficult intracellular pathogens and cancer. To this aim, we formulated class-B synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) with a nanostructure (Coa-ASC16 or coagel) formed by self-assembly of 6-0-ascorbyl palmitate ester. Our previous results demonstrated that mice immunized with ovalbumin (OVA) and CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16) elicited strong antibodies (IgG1 and IgG2a) and Th1/Th17 cellular responses without toxic systemic effects. These responses were superior to those induced by a solution of OVA with CpG-ODN or OVA/CpG-ODN formulated with aluminum salts. In this study, we investigated the capacity of this adjuvant strategy (CpG-ODN/Coa-ASC16) to elicit CD8+ T-cell response and some of the underlying cellular and molecular mechanisms involved in adaptive response. We also analyzed whether this adjuvant strategy allows a switch from an immunization scheme of three-doses to one of single-dose. Our results demonstrated that vaccination with OVA/CpG-ODN/Coa-ASC16 elicited an antigen-specific long-lasting humoral response and importantly-high quality CD8+ T-cell immunity with a single-dose immunization. Moreover, Coa-ASC16 promoted co-uptake of OVA and CpG-ODN by dendritic cells. The CD8+ T-cell response induced by OVA/CpG-ODN/Coa-ASC16 was dependent of type I interferons and independent of CD4+ T-cells, and showed polyfunctionality and efficiency against an intracellular pathogen. Furthermore, the cellular and humoral responses elicited by the nanostructured formulation were IL-6-independent. This system provides a simple and inexpensive adjuvant strategy with great potential for future rationally designed vaccines

Lipidoid Nanoparticles Containing PD-L1 siRNA Delivered In Vivo Enter Kupffer Cells and Enhance NK and CD8+ T Cell-mediated Hepatic Antiviral Immunity

Effective clinical application of antiviral immunotherapies necessitates enhancing the functional state of natural killer (NK) and CD8+ T cells. An important mechanism for the establishment of viral persistence in the liver is the activation of the PD-1/PD-L1 inhibitory pathway. To examine the role of hepatic myeloid PD-L1 expression during viral infection, we determined the magnitude and quality of antiviral immune responses by administering PD-L1 short-interfering RNA (siRNA) encapsulated in lipidoid nanoparticles (LNP) in mice. Our studies indicate that Kupffer cells (KC) preferentially engulfed PD-L1 LNP within a short period of time and silenced Pdl1 during adenovirus and MCMV infection leading to enhanced NK and CD8+ T cell intrahepatic accumulation, effector function (interferon (IFN)-γ and granzyme B (GrB) production), CD8+ T cell–mediated viral clearance, and memory. Our results demonstrate that PD-L1 knockdown on KCs is central in determining the outcome of liver viral infections, and they represent a new class of gene therapy

Antigen presentation by liver parenchyma cells induces suboptimal differentiation of CD8⁺ T cells.

<p>2×10⁶ CFSE labeled Thy1.1⁺OT-1⁺CD8⁺ T cells were adoptively transferred into naïve Thy1.2⁺ C57BL/6 mice reconstituted with BalbC or C57BL/6 bone marrow and then the recipient mice were infected with 5×10⁸ PFU Ad-OVA via IV injection one day later. (A) At 48 hours p.i., liver cells were isolated and the proliferative response of OVA-specific CD8⁺ T cells was evaluated. The plots are gated on Thy1.1⁺CD8⁺ T cells. (B) The expression of CD25 and OVA tetramer by proliferating OVA-specific CD8⁺ was evaluated directly <i>ex vivo</i>. The ability of dividing Thy1.1⁺CD8⁺ T cells to produce IFN-γ, and granzyme-B following peptide restimulation was assessed using flow cytometry. The plots are gated on Thy1.1⁺CD8⁺ T cells. Data are representative of two independent experiments.</p

IV adenovirus administration results in the diminished CD8⁺ T cell responses in the liver.

<p>(A, B) C57BL/6 mice were infected with 5×10⁸ PFU Ad-OVA via either SubQ or IV inoculation. At days 7 and 14 p.i., liver leukocytes were isolated and the percentage of OVA tet⁺CD8⁺ T cells (A) was determined by direct <i>ex vivo</i> staining. The percentage of CD8⁺ T cells producing IFN-γ (B) was assessed following OVA peptide restimulation for 5 hours in the presence of monensin. Data are representative of at least two independent experiments for each time point (<i>n</i> = 3/group). (C) C57BL/6 mice were infected with 5×10⁸ PFU Ad-LacZ via either SubQ or IV administration. At days 7 and 14 p.i., the percentage of βgal tet⁺CD8⁺ T cells and IFN-γ-producing CD8⁺ T cells was determined. Data are presented as averages ± SEM (<i>n</i> = 3/group). (D) C57BL/6 mice were infected with Ad-LacZ via IV inoculation at 10⁸, 10⁷, 10⁶, or 10⁵ PFU per mouse. The percentage of βgal tet⁺CD8⁺ T cells and IFN-γ⁺CD8⁺ T cells in the liver was determined at day 7 p.i. Data are representative of three independent experiments. (E) C57BL/6 mice were infected with 5×10⁸ PFU Ad-OVA via SubQ, IV, or IN immunization. At 7 days p.i., the percentage of OVA tet⁺CD8⁺ T cells and IFN-γ⁺CD8⁺ T cells resident in the liver was calculated. Data are representative of two independent experiments.</p

IV adenovirus infection causes a robust defect in CD8⁺ T cell effector function with elevated PD-1 expression in the liver.

<p>(A, B) 0.5×10⁶ Thy1.1⁺OT-1⁺CD8⁺ T cells were adoptively transferred into naïve Thy1.2⁺ C57BL/6 mice. These mice were allowed to rest for one day and were then infected with 5×10⁸ PFU Ad-OVA via either SubQ or IV injection (A) or 5×10⁸, 5×10⁷, 5×10⁶ PFU Ad-OVA via IV injection (B). At day 7 p.i., liver leukocytes were isolated and then restimulated directly ex vivo with PMA/ionomycin (A) or OVA peptide in the presence of monensin (B) for 5 hours. The plots are gated on live cells and the numbers represent the percentage of cells within the indicated gates. (C) 2×10⁶ Thy1.1⁺OT-1⁺CD8⁺ T cells were adoptively transferred into naïve Thy1.2⁺ C57BL/6 mice. These mice were allowed to rest for one day and then they were infected with 5×10⁸ PFU Ad-OVA via either SubQ or IV injection. At day 7 p.i., liver leukocytes were isolated and the percentage of Thy1.1⁺CD8⁺ T cells expressing PD-1 was determined. The expression of PD-1 by CD8⁺ T cells in the spleen, lung, and liver following 5×10⁸ PFU Ad-OVA IV infection was also evaluated (right histogram). Both of the plots are gated on CD8⁺ T cells. Data are representative of two independent experiments (<i>n</i> = 2/group). (D) Effect of PD-1 blockade on the liver primed CD8⁺ T cell effector activity. 2×10⁶ CFSE labeled Thy1.1⁺OT-1⁺CD8⁺ T cells were adoptively transferred into Thy1.2⁺C57BL/6 mice treated with anti-B7-H1 or control antibody (200 ug per mouse) one day prior to adoptive transfer. The recipient mice were infected with 5×10⁸ PFU Ad-OVA via IV administration one day later. At 48 hours p.i., liver and spleen cells were isolated and then restimulated directly <i>ex vivo</i> with OVA peptide for 5 hours in the presence of monensin. The production of IFN-γ by proliferating Thy1.1⁺CD8⁺ T cells was analyzed by flow cytometry and the plots are gated on Thy1.1⁺CD8⁺ T cells.</p

Liver primed CD8⁺ T cells do not differentiate into competent effectors.

<p>(A) 2×10⁶ CFSE labeled Thy1.1⁺OT-1⁺CD8⁺ T cells were adoptively transferred into naïve Thy1.2⁺ C57BL/6 mice and then mice were infected with 5×10⁸ PFU Ad-OVA via either SubQ or IV immunization one day later. At 48 hours p.i., liver, spleen, and Ig LN were isolated and leukocytes were stimulated in the presence of OVA peptide and monensin for 5 hours. The ability of dividing Thy1.1⁺CD8⁺ T cells to produce IFN-γ and granzyme-B (GrB) following peptide restimulation was assessed using flow cytometry. The plots are gated on Thy1.1⁺CD8⁺ T cells. (B) 2×10⁶ CFSE labeled Thy1.1⁺OT-1⁺CD8⁺ T cells were adoptively transferred into either splenectomized or sham control Thy1.2⁺ C57BL/6 mice that were treated a day before with Mel-14 Ab. Mice were then infected with 5×10⁸ PFU Ad-OVA via IV administration one day later. At 48 hours p.i., liver and spleen cells were isolated and then restimulated directly ex vivo with OVA peptide for 5 hours in the presence of monensin. The production of IFN-γ and granzyme-B by proliferating Thy1.1⁺CD8⁺ T cells was analyzed by flow cytometry. The plots are gated on Thy1.1⁺CD8⁺ T cells. Data are representative of at least two independent experiments.</p

IV adenovirus administration leads to systemic CD8⁺ T cell proliferation.

<p>(A) CFSE labeled Thy1.1⁺OT-1⁺CD8⁺ T cells (2×10⁶ cells per mouse) were adoptively transferred into naïve Thy1.2⁺ C57BL/6 mice. After 24 hours of adoptive transfer, the recipient mice were then infected with 5×10⁸ PFU Ad-OVA via either SubQ or IV injection. At 36 and 48 hours p.i., the liver, spleen, Ig LN, and liver LN were isolated and the expression of CD25 by proliferating Thy1.1⁺CD8⁺ T cells was analyzed by flow cytometry. The plots are gated on Thy1.1⁺CD8⁺ T cells. Data are representative of three independent experiments. (B) Thy1.2⁺ C57BL/6 mice were infected with 5×10⁸ PFU Ad-OVA via either SubQ or IV inoculation. At 48 hours p.i., liver, spleen, Ig LN, and liver LN cells were isolated and then incubated with 1×10⁵ naïve CFSE labeled Thy1.1⁺OT-1⁺CD8⁺ T cells <i>in vitro</i> for 72 hours. The plots are gated on Thy1.1⁺CD8⁺ T cells and the presence of dividing Thy1.1⁺CD8⁺ T cells was determined. Data are representative of two independent experiments.</p