Improving vaccine potency through the spontaneous assembly of antigen-loaded microgels within the lymph node

Introduction: Vaccines have demonstrated their exceptional ability to prevent mortality due to infectious diseases, saving the lives of more than 150 million people over the last 50 years, and there is hope that they can have an equally transformative impact on cancer. Unfortunately, not all vaccines are sufficiently effective to confer sterilizing immunity or long-lasting protection, resulting in 12.7 million deaths due to infectious diseases each year. Traditional vaccines are injected intramuscularly, after which a fraction of the antigen administered spontaneously traffics to the lymph node (LN) where it briefly resides before washing out. Unfortunately, this strategy poorly recapitulates native infections that the immune system has evolved to respond to, which lead to prolonged antigen exposure in the LN. Direct injections into LNs have been shown to enhance the humoral immune response to vaccines by orders of magnitude but are infeasible to implement clinically due to the physician skill and time required as well as the consequences of off-target injections. As a result, many groups have sought to create antigen-loaded nanoparticles that preferentially traffic to LNs. These systems have been effective but are often engulfed at the injection site by resident dendritic cells, offer a limited duration, and cannot access some areas of the LN due to their size. In this study, we aim to overcome these limitations by forming antigen-loaded microgels inside the lymph node using bioorthogonal click chemistry (Figure 1A) and demonstrate the immunological benefits of implementing this intra-LN assembly strategy.

Materials and

Methods: Eight-arm polyethylene glycol (PEG) functionalized with tetrazine (Tz) and ovalbumin (OVA) was synthesized alongside eight-arm PEG functionalized with trans-cyclooctene (TCO) to serve as bioorthogonal bonding partners. We then evaluated the ability of these materials to rapidly form hydrogels via a reaction between Tz and TCO, which we expected to progress rapidly since the reaction between these compounds is the fastest known bioorthogonal. Complementary materials with fluorescently labeled components were then injected into the tail base and ankle of BALB/c mice and imaged longitudinally using an In Vivo Imaging System (IVIS). Tissues collected at terminal time points were also sectioned and stained using fluorescently labeled antibodies to identify the location of injected materials and characterize the immune cell populations present. In parallel, the humoral and cellular immune responses to this delivery system were evaluated using enzyme-linked immunosorbent assays (ELISAs), and flow cytometry.

Results, Conclusions, and Discussions: Mixing Tz- and TCO-functionalized PEG in vitro resulted in immediate crosslinking in vitro, forming a non-degradable hydrogel that was physically robust, as expected for the fastest bioorthogonal reaction currently known. Injecting one material in the ankle and the other in the tail base resulted in passive lymphatic trafficking to the LN and the formation of crosslinked antigen-loaded hydrogels (Figure 1B). These hydrogels were retained within the LN for up to 6 days, resulting in a significant increase in total antigen residence time within the inguinal LN compared to non-crosslinking formulations, as quantified using the area under the curve of fluorescence intensity in IVIS images. Immunofluorescence staining of explanted LNs revealed that Tz-PEG and TCO-PEG molecules co-localized, assembling into microgels approximately 2-8 µm in diameter that were readily taken up by CD169+ medullary sinus macrophages (Figure 1C). This intra-LN crosslinking system increased total OVA-specific geometric mean titers by a factor of 256 compared to a soluble, dose-matched injection of OVA (Figure 1D) and antibody subclassing then revealed that IgG3 titers were significantly increased compared to materials that were unable to crosslink but were otherwise equivalent. Flow cytometry on cells isolated from draining LNs revealed significantly increased dendritic cell activation in LNs with markers suggesting improved antigen presentation and co-stimulatory capacity in response to the OVA-loaded crosslinkable materials compared to mice receiving soluble and alum-adjuvanted OVA (Figure 1E,F). The number of these cells expressing MHC I presenting SIINFEKL, a T cell epitope from OVA, was also significantly increased compared to soluble and alum-adjuvanted OVA (Figure 1G). Taken together, these results suggest that microgel assembly within the lymph node is an effective method for enhancing both the humoral and cellular immune responses to an antigen of interest. This system is highly conducive to the incorporation of molecular adjuvants and may have the potential to improve the potency of vaccines against infectious disease and cancer alike by preferentially distributing antigens within the LN to increase their immunogenicity.

Acknowledgements (Optional): Funding for this work was provided by the National Institutes of Health (K22AI146215) and the Cancer Prevention and Research Institute of Texas (RR190056).