Innovative strategies for co-delivering antigens and CpG oligonucleotides

Abstract

Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODN) is a recent class of immunostimulatory adjuvants that includes unmethylated CpG dinucleotide sequences similar to those commonly found in bacterial DNA. CpG ODN specifically triggers toll like receptor 9 (TLR9), which is found within phagoendosomes of antigen presenting cells (APCs) such as dendritic cells (DCs). CpG ODN triggers activation and maturation of DCs and helps to increase expression of antigens. CpG ODN can be used to induce polarized Th1 type immune responses. Several studies have shown that antigens and CpG ODN must be co-localized in the same APC to generate the most potent therapeutic antigen-specific immune responses. Delivery vehicles can be utilized to ensure co-delivery of antigens and CpG ODN to the same APCs and to significantly increase uptake by APCs. These strategies can result in antigen-specific immune responses that are 5 to 500-fold greater than administration of antigen alone. In this review, we discuss several recent and innovative strategies to co-delivering antigens and CpG ODN adjuvants to APCs. These approaches include the utilization of conjugate molecules, multi-component nanorods, liposomes, biodegradable microparticles, pulsatile release chips and cell-microparticle hybrids.

Figure 1

Schematic showing immunostimulatory cascade triggered by co-delivery of antigen and CpG ODN.

Figure 2

Schematic showing some recent innovative strategies to co-delivering antigen and CpG-ODN. These include (A) PLGA microparticles co-entrapping antigen and CpG ODN, (B) pulsatile delivery of antigen and CpG ODN from PDMS microchips with biodegradable PLGA seals, (C) multi-component nanorods functionalized with antigen and CpG ODN in spatially-defined regions, (D) antigen-CpG ODN conjugate molecules and (E) Cell-microparticle hybrids.

Figure 3

Schematic showing synthesis of the OVA-CpG ODN conjugate molecule. Reproduced with permission from [39]. Copyright © Wiley-VCH Verlag CmbH & Co. KHaA.

Figure 4

Graph showing IgG2a antigen (Amb a1) specific IgG2a profile generated by antigen-immunostimulatory ODN (ISS ODN) conjugate, antigen-ISS ODN free solution mixture and antigen-alum mixture. Reproduced with permission from [41]. Copyright © Elsevier B.V.

Figure 5

Ten-fold increase in ovalbumin-specific CD8+ responses are observed in C57BL/6mice immunized with nanorods co-functionalized with OVA and CpG motif (B) in comparison to nanorods functionalized only with OVA (A) or CpG motif (C) alone. Reproduced and adapted with permission from [1]. Copyright © IOP.

Figure 6

Bar charts showing IgG2a and IgG levels (A) and ratio of IgG2a/IgG1 (B) in sera of immunized mice at the end of 14 weeks. The mice were immunized with antigen alone (rLMSTI1 in PBS), empty liposomes, liposomes encapsulating antigen alone (Lip-rLMSTI1),liposomes co-encapsulating both antigen and CpG ODN (Lip-rLMSTI1-CpG ODN), both antigen and CpG ODN in solution (rLMSTI1 + CpG ODN in PBS) or liposomes entrapping antigen alone and co-administering CpG ODN in solution (rLmSTI1 + Non-CpG ODN in PBS). Reproduced with permission from [52]. Copyright © The American Society for Microbiology.

Figure 7

Viable parasite (L. major) in spleen of different groups of mice immunized with antigen alone (rLMSTI1 in PBS), empty liposomes, liposomes encapsulating antigen alone (Lip-rLMSTI1), liposomes co-encapsulating both antigen and CpG ODN (Lip-rLMSTI1-CpG ODN), antigen and CpG ODN in solution (rLMSTI1 + CpG ODN in PBS) or liposomes entrapping antigen alone and co-administering CpG ODN in solution (rLmSTI1 + Non-CpG ODN in PBS). Reproduced with permission from [52]. Copyright © The American Society for Microbiology.

Figure 8

Uptake of rhodamine-loaded PLGA microparticles by DC2.4 dendritic cells (DCs) at the end of 30 minutes and 16 hours as measured by flow cytometry. Reproduced with permission from [26]. Copyright © Lippincott Williams & Wilkins.

Figure 9

Graphs showing data from (A) ELISA assay of anti-OVA IgG response in mouse serum. Mice were inoculated twice on day 1 (week 0) and 14 (week 2) with either empty microparticles (MP), microparticle entrapping OVA alone (MP OVA), or microparticle co-encapsulating OVA and CpG ODN (MP CpG OVA), or with OVA administered as solution) or OVA and CpG co-administered in solution and (B) serial dilution in phosphate buffered saline of week 4 serum from mice immunized at week 0 and week 2. The positive control is a monoclonal anti-Ova antibody (4A9). The negative control is pretreated mouse serum. Reproduced with permission from [26]. Copyright © Lippincort Williams & Wilkins.

Figure 10

Nasal Lavage anti-IgG A titers in rabbits immunized with intranasal formulations of antigen and CpG ODN in solution, alginate microparticles loaded with antigen alone, alginate microparticles co-encapsulating antigen and CpG ODN or antigen co-delivered with the alum as adjuvant. Reproduced with permission from [75]. Copyright © Elsevier B.V.

Figure 11

Serum antitoxin titers in rabbits immunized with intranasal formulations of antigen with or without CpG ODN in solution, alginate microparticles loaded with antigen alone, or alginate microparticles co-encapsulating antigen and CpG ODN [75]. Copyright © Elsevier B.V.

Figure 12

Graph showing IgG2a anti-PA antibody titers (A), and toxin neutralizing activity (TNA) (B), in anthrax-vaccinated mice. Male A/J mice were immunized intraperitoneally with AVA (O) with free CpG ODN (▲) or PLGA-adsorbed CpG ODN (■). Reproduced with permission from [77].Copyright © The American Center for Microbiology.

Figure 13

Graph showing survival of vaccinated mice. A/J mice were immunized intraperitoneally with anthrax vaccine (AVA) plus free CpG ODN (AVA + CpG ODN), AVA and PLGA-adsorbed CpG ODN (AVA + CpG ODN-PLG), AVA alone (AVA) or PLGA. Reproduced with permission from [77]. Copyright © The American Center for Microbiology.

Figure 14

Graph showing pulsatile release of OVA and CpG ODN from PDMS microchips with hydrolytically degrading PLGA seals. Reproduced with permission from [31]. Copyright © Elsevier B.V.

Figure 15

Schematic showing the avidin-biotin mediated self-assembly of hybrid cell-microparticle carrier systems for potential co-delivery of antigen and CpG ODN. Reproduced with permission from [117]. Copyright © Wiley-VCH Verlag CmbH & Co. KGaA.

Figure 16

(A) Light microscopy image of multiple cell-microparticle hybrids cultured on a well plate, (B) SEM image of a cell-microparticle hybrid and (C) rhodamine-loaded microparticles immobilized on the surface of a cell expressing green fluorescent protein. Reproduced with permission from [117]. Copyright © Wiley-VCH Verlag CmbH & Co. KGaA.