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. 2018 Oct 10:9:2319.
doi: 10.3389/fimmu.2018.02319. eCollection 2018.

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

Ana L Chiodetti  1   2 , María F Sánchez Vallecillo  1   2 , Joseph S Dolina  3 , María I Crespo  1   2 , Constanza Marin  1   2 , Stephen P Schoenberger  3 , Daniel A Allemandi  4   5 , Santiago D Palma  4   5 , María C Pistoresi-Palencia  1   2 , Gabriel Morón  1   2 , Belkys A Maletto  1   2
Affiliations

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

Ana L Chiodetti et al. Front Immunol. .

Abstract

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.

Keywords: CD8+ T-cell response; CpG-ODN; adjuvant; ascorbyl palmitate ester; nanostructure; type I interferons; vaccine.

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Figures

Figure 1
Figure 1
The formulation of OVA and CpG-ODN with the nanostructure. (A) Schematic representation of Coa-ASC16 and photographic image. (B) Schematic representation of OVA/CpG-ODN/Coa-ASC16. (C) Native-PAGE of formulations using IRDye® 680RD OVA and 5′ IRDye® 800CW CpG-ODN comparing non-treated and heat-treated samples (marked with asterisk).
Figure 2
Figure 2
Formulation of OVA/CpG-ODN with Coa-ASC16 optimizes humoral and CD8+ T-cell responses independently of IL-6. WT or Il6−/− mice were immunized with OVA/Coa-ASC16, OVA/CpG-ODN, OVA/CpG-ODN/Coa-ASC16 (on days 0, 7, and 14) or OVA/CFA (days 0, 15, and 30). (A,C) In vivo killing assay and (B,D) ex vivo IFN-γ secretion by splenocytes after stimulation with SIINFEKL peptide determined by ELISA on day 21. (E) Titers of OVA-specific IgG1 and IgG2c in plasma determined by ELISA on day 21. (F) WT mice. Avidity OVA-specific IgG in plasma determined by ELISA using KSCN elution seven days after the last immunization. The data show the mean ± SEM of individual values (3-4 mice/treatment group in each experiment) and are representative of two independent experiments performed. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
The nanostructured formulation allows antigen dose-sparing without compromise the resulting antigen-specific immune response. C57BL/6 mice were immunized on day 0, 7, and 14 with OVA (2 μg)/CpG-ODN/Coa-ASC16 or OVA (6 μg)/CpG-ODN/Coa-ASC16. Seven days after the last immunization, plasma and spleen were obtained. (A) Titers of OVA-specific IgG1 and IgG2c in plasma determined by ELISA. (B) Ex vivo IFN-γ secretion by splenocytes after stimulation with OVA determined by ELISA. The data show the mean ± SEM of individual values (4 mice/treatment group in each experiment) and are representative of two independent experiments performed. n.s., not significant.
Figure 4
Figure 4
The nanostructured formulation elicits long-lasting humoral response with a single-dose immunization. C57BL/6 mice were immunized on day 0 with OVA/CpG-ODN or OVA/CpG-ODN/Coa-ASC16. On day 147 after immunization, mice were intraperitoneally challenged with OVA. Plasma samples were collected at different time points for determination of OVA-specific antibodies titers by ELISA. (A) Kinetics in plasma of OVA-specific IgG. (B) OVA-specific IgG2c on days 140 (pre-challenge) and 154 (post-challenge) after immunization. The data show the mean ± SEM of individual values (4 mice/treatment group in each experiment) and are representative of two independent experiments performed. ***p < 0.001, n.s., not significant.
Figure 5
Figure 5
CD8+ T-cell response elicited by the nanostructured formulation is CD4+ T-cell independent and IFN-I dependent. WT (A–D) or Ifnar1−/− (E–F) mice were immunized with a single-dose (day 0) of the indicated formulations. (A,C,E) In vivo killing assay and (B, D and F) ex vivo IFN-γ secretion by splenocytes after stimulation with SIINFEKL peptide determined by ELISA on day 7. In C and D, mice were treated on days −2, −1, 0, and 2 with anti-CD4 or isotype control (IgG) antibodies. The data show the mean ± SEM of individual values (3-4 mice/treatment group in each experiment) and are representative of two independent experiments performed. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6
Figure 6
The formulation of OVA/CpG-ODN with Coa-ASC16 enhances in vivo co-uptake of OVA and CpG-ODN by DCs. C57BL/6 mice were immunized in both hind limbs with Alexa Fluor 647-OVA/Alexa Fluor 488-CpG-ODN or Alexa Fluor 647-OVA/Alexa Fluor 488-CpG-ODN/Coa-ASC16. Seventy-two h later, cell suspension from inguinal LN were analyzed by flow cytometry. (A) Percentage and (B) absolute number of total CD11c+ DCs. (C) Percentage and (D) absolute number of total OVA+ CD11c+ DCs. (E) Percentage and (F) absolute number of total CpG-ODN+ CD11c+ DCs. (G) Percentage and (H) absolute number of OVA+ or CpG-ODN+ simple positive and OVA+ CpG-ODN+ double positive within CD11c+ DCs. The data show the mean ± SEM of individual values (3-4 mice/treatment group in each experiment) and are representative of two independent experiments performed. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 7
Figure 7
The formulation of OVA/CpG-ODN with Coa-ASC16 enhances expansion and polyfunctionality of effector CD8+ T-cells. C57BL/6 mice were immunized on day 0 with OVA/CpG-ODN, OVA/CpG-ODN/Coa-ASC16 or ΔactA Lm-OVA and euthanized on day 7 for spleen extraction. (A) Percentage of SIINFEKL-Kb tetramer+ CD8+ T-cells. (B) Total number of SIINFEKL-Kb tetramer+ CD8+ T-cells/spleen. (C–E) CD8+ T-cell cytokine production from splenocytes after in vitro stimulation with SIINFEKL peptide. Multiparameter flow cytometry was used to determine (C) percentage and (D) total number of cytokine producing CD8+ T-cells expressing each of the seven possible combinations of IFN-γ, TNF-α, and IL-2, (E) fraction of total CD8+ T-cell response comprising cells expressing each of the seven combinations of IFN- γ, TNF-α, and IL-2. For gating strategy and representative flow cytometry data, see Supplementary Figure 3. The data show the mean ± SEM of individual values (4 mice/treatment group in each experiment) and are representative of two independent experiments performed. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 8
Figure 8
CD8+ T-cell response induced by the nanostructured formulation protects against Lm-OVA infection. C57BL/6 mice were immunized on day 0 with OVA/CpG-ODN, OVA/CpG-ODN/Coa-ASC16 or CpG-ODN/Coa-ASC16. Seven days later, immunized and non-immunized mice were intravenously infected with 1 × 105 CFU of Lm-OVA. Two days after infection, mice were euthanized for liver and spleen extraction. (A) Percentage of SIINFEKL-Kb tetramer+ CD8+ T-cells in spleen. (B) Total number of SIINFEKL-Kb tetramer+ CD8+ T-cells/spleen. (C–E) CD8+ T-cell cytokine production from splenocytes after in vitro stimulation with SIINFEKL peptide. Multiparameter flow cytometry was used to determine (C) percentage and (D) total number of CD8+ T-cells expressing each of the seven possible combinations of IFN-γ, TNF-α, and CD107a, (E) the fraction of the total CD8+ T-cell response comprising cells expressing each of the seven combinations of IFN-γ, TNF-α and CD107a. For gating strategy and representative flow cytometry data, see Supplementary Figure 4. (F) Bacterial load in liver. The data show the mean ± SEM of individual values (3–4 mice/treatment group in each experiment) and are representative of two independent experiments performed. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
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