Montanide, Poly I:C and nanoparticle based vaccines promote differential suppressor and effector cell expansion: a study of induction of CD8 T cells to a minimal Plasmodium berghei epitope

Kirsty L Wilson, Sue D Xiang, Magdalena Plebanski, Kirsty L Wilson, Sue D Xiang, Magdalena Plebanski

Abstract

The development of practical and flexible vaccines to target liver stage malaria parasites would benefit from an ability to induce high levels of CD8 T cells to minimal peptide epitopes. Herein we compare different adjuvant and carrier systems in a murine model for induction of interferon gamma (IFN-γ) producing CD8 T cells to the minimal immuno-dominant peptide epitope from the circumsporozoite protein (CSP) of Plasmodium berghei, pb9 (SYIPSAEKI, referred to as KI). Two pro-inflammatory adjuvants, Montanide and Poly I:C, and a non-classical, non-inflammatory nanoparticle based carrier (polystyrene nanoparticles, PSNPs), were compared side-by-side for their ability to induce potentially protective CD8 T cell responses after two immunizations. KI in Montanide (Montanide + KI) or covalently conjugated to PSNPs (PSNPs-KI) induced such high responses, whereas adjuvanting with Poly I:C or PSNPs without conjugation was ineffective. This result was consistent with an observed induction of an immunosuppressed environment by Poly I:C in the draining lymph node (dLN) 48 h post injection, which was reflected by increased frequencies of myeloid derived suppressor cells (MDSCs) and a proportion of inflammation reactive regulatory T cells (Treg) expressing the tumor necrosis factor receptor 2 (TNFR2), as well as decreased dendritic cell (DC) maturation. The other inflammatory adjuvant, Montanide, also promoted proportional increases in the TNFR2(+) Treg subpopulation, but not MDSCs, in the dLN. By contrast, injection with non-inflammatory PSNPs did not cause these changes. Induction of high CD8 T cell responses, using minimal peptide epitopes, can be achieved by non-inflammatory carrier nanoparticles, which in contrast to some conventional inflammatory adjuvants, do not expand either MDSCs or inflammation reactive Tregs at the site of priming.

Keywords: CD8 peptide; MDSC; Treg; adjuvant; malaria; nanoparticle.

Figures

FIGURE 1
FIGURE 1
Size distribution for PSNPs-KI formulations. SYIPSAEKI peptides were covalently conjugated to PSNPs, and the final sizes were measured by dynamic light scattering instruments (Zetasizer).
FIGURE 2
FIGURE 2
Antigen specific CD8 T cell responses induced by SYIPSAEKI peptide vaccines in combination with PSNPs. Mice (BALB/c) were immunized with SYIPSAEKI peptide mixed with or conjugated to PSNPs intradermally at the base of tail. 14 days after the last immunization, spleen cells were collected and assessed for IFN-γ production by ELISpot assay. (A) KI peptides conjugated to, but not mixed with, PSNPs induced high levels of KI specific CD8 T cell responses after two immunizations (2 weeks apart). Data presented as mean ± SD of SFU/million cells from each group (n = 3 mice/group). (B) Immunogenicity of PSNPs-KI formulation after one immunization (n = 4 mice per group). Data presented as mean ± SD of SFU/million cells (pooled for each group) from the triplicated wells in ELISpot assay. Statistical analysis was performed via ANOVA, ***p ≤ 0.001.
FIGURE 3
FIGURE 3
Induction of SYIPSAEKI specific CD8 T cells by different adjuvants. BALB/c mice were immunized twice, intradermally, 2 weeks apart, with SYIPSAEKI peptides incorporated with respective adjuvants. 14 days after the last immunization, spleen cells were collected and assessed for IFN-γ production by ELISpot assay. Data presented as mean ± SD of SFU/million cells from each group (n = 3 mice/group). Statistical analysis was performed via ANOVA, ***p ≤ 0.001.
FIGURE 4
FIGURE 4
Dendritic cell activation in dLNs after injection with PSNPs, Montanide, and Poly I:C. Mice (BALB/c) were injected once intradermally at the base of tail with the different adjuvants alone. 48 h after injection, mice were sacrificed, local (inguinal) dLNs were harvested and the levels of CD11c+ DCs and various activation markers were assessed by flow cytometry. (A) gating strategy; (B) frequency of GR-1-CD11c+ cells; (C) Mean fluorescent intensity (MFI) of CD80 and CD86 on GR-1-CD11c+ cells; (D) MFI of CD40 and MHCII on GR-1-CD11c+ cells. Data presented as mean ± SD of MFI for each group of treatment (n = 3 mice/group). Statistical analysis was performed via t-tests, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
FIGURE 5
FIGURE 5
Differential expression of GR-1+ MDSCs and DCs in the local dLN. Mice (BALB/c) were injected once intradermally at the base of the tail with the different adjuvants alone. 48 h after injection, mice were sacrificed, local (inguinal) dLNs were harvested and levels of GR-1+ MDSCs and DCs, and their ratios, were assessed by flow cytometry. (A) gating strategy; (B) ratio of GR-1+ MDSCs: CD11c+ cells; (C) ratio of moMDSCs: CD11c+ cells; (D) ratio of gMDSCs: CD11c+ cells. Data presented as mean ± SD of ratio for each group of treatment (n = 3 mice/group). Statistical analysis was performed via t-tests, *p ≤ 0.05, **p ≤ 0.01.
FIGURE 6
FIGURE 6
Differential expression of CD4+ Treg and effector T cells in the local dLN. Mice (BALB/c) were injected once intradermally at the base of tail with the different adjuvants alone. 48 h after injection, mice were sacrificed, local (inguinal) dLNs were harvested and the levels of CD4+ Treg and effector T cells, and their ratios, were assessed by flow cytometry. (A) gating strategy; (B) ratio of FoxP3+CD25+: FoxP3-CD25- cells; (C) ratio of FoxP3+CD25+TNFR2+: FoxP3+CD25+TNFR2- cells. Data presented as mean ± SD of ratio for each group of treatment (n = 3 mice/group). Statistical analysis was performed via t-tests, *p ≤ 0.05.

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