The stimulation of CD8+ T cells by dendritic cells pulsed with polyketal microparticles containing ion-paired protein antigen and poly(inosinic acid)-poly(cytidylic acid) - PubMed (original) (raw)

The stimulation of CD8+ T cells by dendritic cells pulsed with polyketal microparticles containing ion-paired protein antigen and poly(inosinic acid)-poly(cytidylic acid)

Michael J Heffernan et al. Biomaterials. 2009 Feb.

Abstract

New adjuvants and delivery strategies are needed to optimize the ability of protein-based vaccines to elicit CD8(+) T cell responses. We have developed a model vaccine formulation containing ovalbumin (OVA) and the double-stranded RNA analog poly(inosinic acid)-poly(cytidylic acid) (poly(I:C)), a TLR3 agonist. OVA and poly(I:C) were each ion-paired to cetyltrimethylammonium bromide (CTAB) to produce hydrophobic complexes, which were co-encapsulated in pH-sensitive polyketal (PK3) microparticles (1-3 microm) using a single emulsion method. Loading levels ranged from 13.6 to 18.8 microg/mg OVA and 4.8 to 10.3 microg/mg poly(I:C). Murine splenic dendritic cells (DCs) pulsed with PK3-OVA-poly(I:C) microparticles, at antigen doses of 0.01 and 0.1 microg/mL, induced a higher percentage of IFNgamma-producing CD8(+) T cells than DCs treated with PK3-OVA particles or soluble OVA/poly(I:C). A higher antigen dose (1 microg/mL) was less effective, which can be attributed to CTAB toxicity. At the lowest antigen dose (0.01 microg/mL), PK3-OVA-poly(I:C) microparticles also enhanced TNF-alpha and IL-2 production in CD8(+) T cells. These data demonstrate the potential of polyketal microparticles in formulating effective CD8(+) T cell-inducing vaccines comprising protein antigens and dsRNA adjuvants.

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Figures

Fig. 1

Fig. 1

Structure of co-polyketal PK3, which is synthesized from 1,4-cyclohexanedimethanol, 1,5-pentanediol, and 2,2-dimethoxypropane via the acetal exchange polymerization [32,34].

Fig. 2

Fig. 2

Biodegradable polyketal microparticles containing protein antigen and poly(I:C) adjuvant are phagocytosed by dendritic cells (DCs). pH-Sensitive polyketal degrades and releases protein antigen and dsRNA analog poly(I:C) in phagolysosome. Poly(I:C) engages TLR3, thereby activating DC and cross-priming CD8+ cytotoxic T lymphocytes.

Fig. 3

Fig. 3

Hydrophobic ion-pairing procedure. (A) Poly(I:C) solution or (B) OVA solution at pH 11 is paired with CTAB in equimolar ratio of opposite charges to form a hydrophobic complex.

Fig. 4

Fig. 4

Scanning electron microscope image of polyketal microparticles containing ion-paired OVA and poly(I:C).

Fig. 5

Fig. 5

Cell viability of RAW264.7 macrophages treated for 5 h with (A) PK3 micro-particles containing OVA and/or poly(I:C) or empty microparticles or (B) soluble OVA, poly(I:C), or CTAB.

Fig. 6

Fig. 6

Representative flow cytometry plots showing percentage of IFNγ-, TNFα- and IL-2-producing CD8+ T cells stimulated by DCs pulsed with PK3-encapsulated OVA and poly(I:C) (0.01 μg/mL antigen dose).

Fig. 7

Fig. 7

In vitro DC-OT-1 cross-priming by soluble and PK3 microparticle vaccine formulations containing OVA or (OVA + poly(I:C)); percentage of IFNγ-producing CD8+ T cells (n = 4 wells per group). Data shown above is one representative experiment out of two independent experiments with similar trends.

Fig. 8

Fig. 8

In vitro DC-OT-1 cross-priming by soluble and PK3 microparticle vaccine formulations containing OVA or (OVA + poly(I:C)); percentage of TNFα-producing CD8+ T cells (n = 4 wells per group). Data shown above is one representative experiment out of two independent experiments with similar trends.

Fig. 9

Fig. 9

In vitro DC-OT-1 cross-priming by soluble and PK3 microparticle vaccine formulations containing OVA or (OVA + poly(I:C)); percentage of IL-2-producing CD8+ T cells (n = 4 wells per group). Data shown above is one representative experiment out of two independent experiments with similar trends.

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