Scientists report on the development of cytokine-loaded nanoparticles that act as lymph-node targeting vaccine adjuvants capable of boosting antibody- and cell-mediated immune responses against lethal infections in mice. The design of the nanoparticles was inspired by the body’s own mast cells, which release inflammatory mediator-loaded granules that rapidly traverse to draining lymph nodes following infection, and accelerate the reorganization of lymph nodes and the development of a robust antibody response.
The Duke University team that developed the technology found that nanoparticles loaded with tumor necrosis factor (TNF) boosted the production of antigen-specific antibodies when administered in conjunction with a flu antigen, and also prolonged survival. When nanoparticles loaded with IL-12 were injected alongside the soluble antigen ovalbumin, recipient mice demonstrated significantly increased numbers of lymph node T cells that produced IFN-γ.
Reporting their results in Nature Materials, Soman N. Abraham and colleagues say their nanoparticles appear to replicate the structure, biochemical attributes, and functional capabilities of mast cell granules. Moreover, the ability to load the structures with different combinations of cytokines could provide a way of steering the direction of an immune response, dependent on the vaccine candidate. Their paper is titled “Synthetic mast-cell granules as adjuvants to promote and polarize immunity in lymph nodes.”
Natural immune responses to infection rely on fast communication between the periphery and the draining lymph nodes, and this rapid response is in part facilitated through the actions of mast cells. Nast cells influence dendritic cell migration, and previous work by the Duke University team has shown that particles released by mast cells in response to infection retain inflammatory mediators and travel to the draining lymph nodes, where they are believed to heighten lymph node response to infection, and the development of antibodies.
These mast cell granules can thus be thought of as physiological drug-delivery particles that efficiently transport pro-inflammatory mediators directly to draining lymph nodes in a form that is protected from degradation and dilution, in order to promote the adaptive immune process. In effect, mast cell granules represent the ideal vaccine adjuvant.
The team explains that existing adjuvant approaches designed by man don’t come close to the effectiveness of mast cell granules, and all currently approved adjuvants are thought to enhance immunity by exerting their effects at the site of vaccine injection, the team continues. Moreover, while mast cell granules can influence the character of the natural immune response, most adjuvant development is still focused on enhancing the magnitude of an immune response.
The Duke team’s latest work was focused on mimicking the function of mast cell granules by developing nanoparticles that contain inflammatory mediators, which can directly target thedraining lymph nodes. Natural MC-derived particles consist primarily of carbohydrates—specifically heparin—and proteases, so the researchers engineered their particles to comprise heparin complexed with the crustacean shell- derived carbohydrate chitosan, which is nonimmunogenic. “We proposed that targeted delivery of cytokines in heparin-chitosan complexes would recapitulate the adjuvant activity of natural mast cell activation during infection in vivo,” they explain.
The resulting nanoparticles, at 200–1,000 nm in diameter, approximated to the size of MC-derived particles, and were stable without crosslinking at a physiological pH range. The particle formation process was expected to facilitate the entrapment of a cytokine, via cytokine binding to heparin in solution, and entrapment within the forming heparin-chitosan matrix. In fact, tests indicated that that particles formed could entrap about 50% of the functional TNF available in the reaction mixture. Measuring the activity of the particles demonstrated that particulate TNF was much more effective in promoting cytotoxicity than soluble TNF, while additional studies confirmed that the particles slowly released TNF in a soluble form over an extended time course.
The researchers then injected fluorescent TNF-nanoparticles into the hind foot pads of mice, and subsequently isolated and examined draining popliteal lymph nodes. Microscopic evaluation confirmed that after footpad injection, the particles traveled quickly to the lymph nodes, and could be observed in subcapsular and medullary sinuses within minutes, and in "striking quantities" within just 45 minutes. Dendritic cells in particular, but also macrophages, were shown to take up the particles within the lymph nodes.
Importantly, injecting particulate TNF in combination with a low dose of antigen induced the formation of lymph node germinal centers that are characteristic of lymph node remodeling and swelling in response to infection. In contrast, administration of the same dose of antigen alone wasn’t sufficient to induce germinal center formation. “Germinal centers contain activated B cells, as well as some dendritic cells and T cells, and are highly consequential to the development of adaptive immune responses and to the production of high-affinity antibodies of multiple subclasses,” the authors note.
An important test was to see whether particulate TNF could act as an adjuvant and boost immune responses to vaccination. Initial tests compared antibody production in response to the soluble HA antigen derived from flu, injected either in combination with particulate TNF or with soluble TNF. Mice were vaccinated twice, 14 days apart, and serum was collected at 21 days to assess the resulting antibodies. Measurement of total HA-specific IgG confirmed that particulate TNF, but not soluble TNF or "empty" heparin-chitosan nanoparticles, showed adjuvant activity.
And when compared with antibody responses following administration of an alum adjuvant, those resulting from particulate TNF administration appeared to promote a broader specific antibody response. “Individual antibody subclasses have been shown to have unique activities in vivo and differing effectivenesses against individual challenges, therefore, the ability to promote antibody diversity is an important attribute of this adjuvant system,” the authors state.
Functional improvements were also demonstrated using the particulate TNF adjuvant. Analyses using a modified a modified ELISA procedure demonstrated that avidity of the antigen-specific antibodies was much higher in mice given HA and particulate TNF than mice given either HA alone or with alum.
The ultimate evaluation was to see whether the adjuvant system improved protection against a lethal infection, of flu in this case. Animals were vaccinated twice, 14 days apart, and then given a lethal dose of flu. As hoped, vaccination with HA plus particulate TNF led to a significant increase in survival of flu-challenged animals, to levels that were no different from those among animals receiving the positive control adjuvant, alum.
Having generated positive data using the TNF-encapsulating nanoparticles, the Duke team then tested particles encapsulating IL-12, “because this cytokine, which is produced by dendritic cells, is a key driver of cell-mediated immune responses needed to clear many viral infections. In vivo tests using this system showed that in contrast with soluble IL-12, injection of particulate IL-12 in conjunction with the soluble antigen ovalbumin (OVA) greatly increased the number of lymph node T cells that produced IFN-γ. This effect was further augmented in combination with particulate TNF.
“These results demonstrate that differential loading of our synthetic particles may be an effective way to target minute quantities of cytokines to the draining LN during vaccination, and that the loaded cytokines need not conform to the template provided by MC granules, Dr. Abraham and colleagues point out. “As a result, the cytokines delivered in particulate form and the resulting polarization of immune responses in the draining lymph nodes could be tailored to meet the requirements for protection against an individual challenge.”
The team doesn’t foresee major problems in progressing their technology toward the clinic. “It should not be long because all the individual cytokines and additional materials loaded into these particles are already FDA-approved for use in humans,” Dr. Abraham states. “There is a lot of interest in nanoparticle-based therapy, but we are basing our materials on our observation of mast cells in nature. This is an informed application to deliver the right material to the right place in the body to get the most effective immune response.”