Recent studies have demonstrated a simple, potentially universal strategy to enhance vaccines via intra-lymph node (i.LN.) injection. Lymph nodes (LNs) are a key site of immune response generation, where dendritic cells (DCs) process and present antigens to resident T cells and B cells, which then migrate to the periphery to carry out mechanisms of adaptive immune response. Since direct administration of antigens or adjuvants in LNs concentrates vaccines at this key site, i.LN. immunization with soluble vaccines has demonstrated great promise in recent human clinical trials. However, these strategies require complex, multiple injection regimens owing to rapid vaccine clearance from LNs. We hypothesized that combining i.LN. injection with controlled release biomaterials permitting sustained, concentrated dosing to local LN environments might augment the function of adjuvants or other drugs, providing a route for improved vaccination or immunotherapy. In this talk I will discuss two components of a new materials-based vaccination strategy aimed at 1) increasing vaccine potency and 2) tailoring specific aspects of immune response.
We first encapsulated poly(inosinic:cytidylic acid) (polyIC) – a potent nucleic acid adjuvant undergoing clinical trials – in biodegradable poly(lactide-co-glycolide) (PLGA) microparticles (MPs) or nanoparticles (NPs) designed to release polyIC in LNs over several days. We hypothesized particle size would allow control over intra-LN particle fate, with NPs being internalized by LN-resident cells, and MPs remaining extracellular and releasing encapsulated cargo within the LN. MPs and NPs were synthesized by double emulsion and lipids were used to stabilize the aqueous/organic interface, and to incorporate functionalized lipids (e.g., poly(ethylene glycol) to control aggregation). Properties such as particle size (0.2-15 µm), polyIC encapsulation (0.5-4% wt/wt), and release kinetics (2-25 days) were tunable by controlling emulsion conditions. To identify the target LN injection sites we administered a non-toxic tracer at the tail base of mice. Trafficking of the dye to the draining LN allowed subsequent identification of vaccine injection sites. Following injection of fluorescently-labeled particles, live animal imaging and histological analysis confirmed particles were localized within LNs. We used this approach to inject polyIC i.LN. in soluble form or encapsulated in MPs (MP-polyIC) or NPs (NP-polyIC) and discovered that MP-polyIC conferred a 4.5-fold increase in polyIC persistence four days after injection compared to soluble polyIC, with polyIC-NPs mediating an intermediate enhancement less than MP-polyIC, but greater than soluble polyIC. The enhancement observed with MP-polyIC mediated a dramatic increase in polyIC uptake by LN-resident DCs over 4 days, in contrast to soluble polyIC which was flushed away one day after injection. Mice vaccinated with antigen mixed with MP-polyIC exhibited antigen-specific T cell (CD8+) levels as high as 18% (7.6-fold greater than MP-polyIC given intramuscularly, i.m.; 4.0-fold greater than soluble polyIC given i.LN.). This increase indicates potent “arming” of the immune system, unmatched by i.LN. injection of antigen mixed with the same dose of NP-polyIC, or of a 10-fold greater doses of soluble polyIC. When T-cells were restimulated with antigen, MP-polyIC enhanced secretion of key cytokines (i.e., IFN-γ, TNF-α) involved in generating immunity, suggesting a functional or protective capacity that we are now exploring.
We have extended this initial approach to tailor immune response by sustained delivery of small molecule immunomodulators – molecules able to bias the specific nature or duration of immune response. A number of such agents have been described, but the potential of these compounds is limited by poor water solubility and rapid clearance in vivo. Thus treatment with these drugs typically involves high doses administered at frequent intervals. Since many immunomodulators act in LNs, polymeric particles designed to slowly release encapsulated small molecules in the LN could allow sustained stimulation, enabling this class of drugs to be effectively employed without continuous re-dosing. Daily, low-dose peripheral injections of Rapamycin –a potent small molecule drug – have recently been found to greatly enhance T cell response. Using modified solvent evaporation, we prepared rapamycin PLGA MPs (MP-rapa) and conducted biodistribution studies using i.m. or i.LN. injections of soluble rapamycin or MP-rapa. HPLC analysis of drug extracts prepared from blood and excised LNs demonstrates MP-rapa permits continuous dosing of rapamycin in LNs for 1 week, whereas injections of soluble rapamycin are undetectable at times longer than 24 hours after injection. In vaccination studies with model antigens, MP-rapa significantly increased the duration of CD8 T cell response, and induced a 6-fold increase in central memory T cells – cells critical for long-term vaccine protection. Strikingly, this enhancement was mediated by just three i.LN. injections of MP-rapa, and these benefits were unmatched by 150 daily peripheral injections of rapamycin in soluble form – the “gold” standard injection regimen for rapamycin. In preliminary studies with clinically-relevant HIV gag antigens, MP-Rapa induced a 2.2-fold increase in CD8 memory populations compared with soluble drug formulations, and these studies are currently being extended to small animal challenge models of HIV.