The primary goal of this work was to investigate the immunomodulatory properties of liposome formulations for pulmonary delivery of protein-based vaccines and test their efficacy on a murine model of S. mansoni infection.
We investigated the immunomodulatory effects of cationic and negatively-charged liposomes, composed of dioleoyl-trimethylammonium-propane (DOTAP) (Chapter III) or phosphatidylglycerol (PG) (Chapter II), respectively. Results indicated that the kinetics and extent of migration of pulmonary cells to the mediastinal lymph nodes is favored by liposomes with a fluid bilayer (Chapter II). We observed that their uptake after pulmonary delivery is decreased in the presence of monosphosphoryl lipid A (MPL), with no significant effect on the extent of leukocyte migration to the lymph nodes. These findings suggest that liposome composition might tailor the adjuvant properties of liposome vaccines delivered to the lung.
Results from in vitro studies (Chapter II) indicated that dendritic cells internalize fluid-phase PG liposomes extensively. Although liposome uptake was significantly increased in the presence of MPL, it resulted in a decreased cell-surface expression of CD86 on dendritic cells. These findings suggest that liposome composition can modulate specific immunologic events and thus, may partly dictate the final immune response.
In this context, we assessed the influence of different liposome-associated antigen conformations on the pattern of antigen processing, and its impact on the development of T-cell repertoires. In this context, the Schistosoma mansoni hybrid immunogen GST1194 was selected as a model antigen (Chapter 2). Results indicated that the delivery of different liposome-associated antigen conformations to dendritic cells results in the development of distinct patterns of GST1194 processing, which translate into the development of distinct T-cell repertoires in vivo. This suggests that DOPG liposomes may affect the processing and presentation of liposome-associated proteins to T cells and thus modulate the generation and type of immune responses at multiple levels.
Results (Chapter III) of in vitro studies suggest that the uptake of DOTAP liposomes by dendritic cells is dependent on charge density and lipid composition. Liposomes with lower cationic charge density induced a higher expression of the costimulatory molecules CD80 and CD86 on the surface of DCs. Results from in vivo studies showed that the uptake of cationic liposomes by pulmonary leukocytes is significantly higher than that of negatively-charged liposomes and mainly mediated by CD11c+ leukocytes, potentially myeloid DCs. Moreover, we observed that the pulmonary uptake of cationic liposomes induced a significant increase in the migration of leukocytes to both draining and non-draining lymph nodes relative to negatively-charged vesicles, suggesting a composition-dependent modulation of dendritic cell migration. Taken together, work presented in Chapters II and III suggest that liposome composition and charge may have immunomodulatory effects on dendritic cells, both in their activation and in their migration to lymph nodes.
Based on this knowledge, we evaluated the impact of various antigen conformational variants mediated by the association of GST1194 with negatively-charged and cationic liposomes (Chapter IV). Our results indicate that the association of GST1194 with liposomes leads to the development of higher cytokine and immunoglobulin isotype ratios toward a Th1 phenotype, opposing the Th2-biased lung environment. However, all conformational variants failed to induce protective immunity in a murine model of Schistosoma mansoni infection.
Alternatively, we evaluated the effect of intramuscular immunization with GST1194 alone or associated with DOPG liposomes, on the maturation and fecundity of females in a Balb/c model of S. mansoni infection (Chapter V). We observed a significant decrease in female maturation in animals immunized with peptide alone, whereas immunization with liposome-associated peptide had no effect. Our results suggest that the impact on female development might be related to the generation of a strong cellular and humoral immune response against both tropomyosin and GSTs. We speculate that the orchestrated development of strong and parallel levels of IgG2a and IgG2b, with the maintenance of moderate IgG1 levels, aided by a strong cellular immune response in the lung mediated by both IFNγ and TNFα, may have been the determining factors.
In conclusion, our findings suggest that lipid composition and charge have a direct impact on the uptake of liposomes by pulmonary leukocytes, including dendritic cells. Furthermore, the above work has shown that the uptake of liposomes by dendritic cells can modulate their activation and subsequent migration to regional lymph nodes.
These results clearly suggest that the immunologic outcome of vaccination is dependent upon the antigen used, its conformation, the specific adjuvant used and the route of administration. Overall we believe that the above findings can contribute to the development of efficacious liposome-based vaccines for pulmonary immunization. Furthermore, we believe that our findings contribute towards a basis for developing a subunit vaccine against schistosomiasis. (Abstract shortened by UMI.)