Comparison of two different PEGylation strategies for the liposomal adjuvant CAF09: Towards induction of CTL responses upon subcutaneous vaccine administration.

Using subunit vaccines, e.g., based on peptide or protein antigens, to teach the immune system to kill abnormal host cells via induction of cytotoxic T lymphocytes (CTL) is a promising strategy against intracellular infections and cancer. However, customized adjuvants are required to potentiate antigen-specific cellular immunity. One strong CTL-inducing adjuvant is the liposomal cationic adjuvant formulation (CAF)09, which is composed of dimethyldioctadecylammonium (DDA) bromide, monomycoloyl glycerol (MMG) analogue 1 and polyinosinic:polycytidylic acid [poly(I:C)]. However, this strong CTL induction requires intraperitoneal administration because the vaccine forms a depot at the site of injection (SOI) after subcutaneous (s.c.) or intramuscular (i.m.) injection, and depot formation impedes the crucial vaccine targeting to the cross-presenting dendritic cells (DCs) residing in the lymph nodes (LNs). The purpose of the present study was to investigate the effect of polyethylene glycol (PEG) grafting of CAF09 on the ability of the vaccine to induce antigen-specific CTL responses after s.c. administration. We hypothesized that steric stabilization and charge shielding of CAF09 by PEGylation may reduce depot formation at the SOI and enhance passive drainage to the LNs, eventually improving CTL induction. Hence, the vaccine (antigen/CAF09) was post-grafted with a novel type of anionic PEGylated peptides based on GDGDY repeats, which were end-conjugated with one or two PEG moieties, resulting in mono- and bis-PEG-peptides of different lengths (10, 15 and 20 amino acid residues). For comparison, CAF09 was also grafted by inclusion of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(PEG)-2000 (DSPE-PEG) in the bilayer structure during preparation. Grafting of CAF09 with either type of PEG resulted in charge shielding, evident from a reduced surface charge. Upon s.c. immunization of mice with the model antigen ovalbumin (OVA) adjuvanted with PEGylated CAF09, stronger CTL responses were induced as compared to immunization of mice with unadjuvanted OVA. Biodistribution studies confirmed that grafting of CAF09 with DSPE-PEG improved the passive drainage of the vaccine to LNs, because a higher dose fraction was recovered in DCs present in the draining LNs, as compared to the dose fraction detected for non-PEGylated CAF09. In conclusion, PEGylation of CAF09 may be a useful strategy for the design of an adjuvant, which induces CTL responses after s.c. and i.m. administration. In the present studies, CAF09 grafted with 10 mol% DSPE-PEG is the most promising of the tested adjuvants, but additional studies are required to further elucidate the potential of the strategy.