(353c) Award Submission: Rational Design of Pathogen Mimicking Amphiphilic Nanoparticle Adjuvants

The natural infection of a pathogen stimulates a protective and enduring immune response. Thus, an ideal approach to vaccine design is to mimic the immune response associated with a natural infection while avoiding the undesirable side effects of the disease. Vaccines have proven to be the most effective method of preventing infectious diseases, yet many problems still exist including multi-dose administration, poor immunogenicity of vaccine antigens, and undesirable side effects of immunization with adjuvants which can all lead to poor patience compliance and poor vaccine efficacy. Thus there is a need for the development of new vaccine adjuvants and delivery vehicles capable of mimicking the properties of the natural infection while limiting the undesirable side effects and need for repeated administrations.

Polyanhydrides are a class of biomaterials that have demonstrated biocompatibility, controlled antigen release kinetics, antigen stabilization, and immune modulation, making them excellent vaccine delivery vehicles. Furthermore, they can be fabricated into nanoparticles capable of being inhaled or injected for ease of administration. This work is focused on polyanhydride copolymers based on sebacic acid (SA), 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH). While many chemistry-dependent trends associated with protein stabilization, protein release, phagocytosis, and immune modulation have been identified, little work to date has investigated the specific polymer characteristics responsible for these trends. By identifying these properties, novel adjuvants can be designed that more closely mimic infections with live pathogens. Therefore, this research is focused on the rational design of pathogen-mimicking adjuvants for vaccine delivery by utilizing flow cytometry and confocal microscopy in concert with materials informatics to identify key properties responsible for nanoparticle uptake and downstream activation of murine bone marrow derived dendritic cells (BMDCs). These internalization patterns were compared to that of live pathogens (Yersinia pestis and Escherichia coli) and the activation patterns compared to that of lipopolysaccharide (LPS, an endotoxin found on the surface of gram negative bacteria).

This investigation identified that the least hydrophilic (CPTEG-rich and the SA-rich) nanoparticles were readily internalized by BMDCs. Upon further assessment of the nanoparticle positive and negative BMDC populations, it was discovered that nanoparticle internalization was required for enhanced expression of CD40 and production of IL-12p40 and IL-6 but not for the expression of CD86 and MHC II. Interestingly, bacteria have been reported to have very similar internalization patters in which association is required for production of IL-12p40 and IL-6 and expression of CD40 but less dependent for expression of CD86 and MHC II. Additional flow cytometric analysis of the most adjuvanting nanoparticle chemistries (poly(SA) and 50:50 CPTEG:CPH) and the positive control (LPS) revealed stark similarities of double positive DC populations for cytokines and cell markers between the amphiphilic 50:50 CPTEG:CPH nanoparticles and LPS. Further pathogen mimicking patterns were observed with confocal microscopy in which internalization and persistence were compared between the bacteria, E. coli and Y. pestis, and the 50:50 CPTEG:CPH nanoparticles. Microscopic observations at both 2 and 48 h indicated that the nanoparticles were found inside late endosomes, consistent with phagosome–lysosome fusion events of antigen processing and presentation. When compared to the internalization of Y. Pestis and E. coli, the amphiphilic 50:50 CPTEG:CPH nanoparticles appeared to be similarly internalized and persist at 2 and 48 h, respectively, suggesting that the stimuli provided by these particles to activate DCs do not rapidly degrade, much like that provided by persisting bacteria.

To validate and assess these multiple pathogen mimicking observations, materials informatics analysis, including principal component analysis (PCA) and partial least squares (PLS), were employed. The PCA confirmed that 50:50 CPTEG:CPH nanoparticles were very similar in the overall activation, internalization, and persistence patterns to the pathogenic correlates. Furthermore, a PLS analysis was employed, which identified the polymer properties responsible for these pathogen mimicking patterns to be i) percentage of hydroxyl end groups, ii) hydrophobicity, iii) alkyl ethers, iv) aliphatic carbons, and v) backbone oxygen moieties. The approach described here will facilitate the rational design of single-dose, pathogen-mimicking vaccine adjuvants that could be tailored to provide immune modulation and protection against a wide range of emerging and re-emerging diseases.