Exosome-Inspired Functionalized Calcium Phosphate Nano-Probes for Vaccine Application

Subunit vaccines have emerged as a promising strategy in preventing infectious diseases, offering a safer alternative to conventional live attenuated or inactivated virus/bacterial vaccines. Additionally, their cost-effectiveness and ease of mass production give them a significant advantage over bacterial exosome-based vaccines. Nonetheless, challenges persist, such as limited immunogenicity and inadequate antigen stability within these formulations. To overcome these hurdles, nanoparticles (NPs) can serve as an effective delivery platform for vaccines. NPs exhibit the capacity to shield the antigen cargo, augment immunogenicity, and precisely deliver the cargo to the intended site [1].<br/>Among the various NP delivery systems, calcium phosphate (CaP) NPs emerge as exceptionally promising candidates. Their biocompatibility and biodegradability render them well-tolerated by the body. Notably, CaP NPs demonstrate potential as vaccine adjuvants, capable of provoking both cell-mediated and humoral immune responses [2]. Furthermore, multiple antigens can be conjugated to CaP NPs due to their high loading capacity for diverse proteins and peptides [3], positioning them as a favorable nanocarrier for protein-based vaccines.<br/>CaP exists as a polymorph and a precise control of its composition, size and crystallinity is crucial for its translation for vaccine application. Therefore, we use flame spray pyrolysis, a single step and scalable aerosol synthesis technique which allows easy tuning of the mentioned NPs properties. We screened three different types of CaP NPs (named as S-CaP, M-CaP and L-CaP) varying in particle size and crystallinity to evaluate their immunomodulatory properties. Medium (M-CaP) and large (L-CaP) sized NPs were crystalline whereas the smallest NPs (S-CaP) exhibit amorphous nature. We achieved a high specific surface area of 232 m²/g for the S-CaP. Upon loading ovalbumin (OVA), a model protein antigen, we achieved loading capacity values with S-CaP NPs reaching more than 400 µg/mg. Moreover, upon incubation with Proteinase K, OVA conjugated with the NPs resisted degradation until 3 hours compared to free OVA degrading within the 1^st hour. Furthermore, all three sizes of CaP NPs enhanced the OVA uptake and processing by the DCs. We observed a significant upregulation of CD80 and CD86 expression on BMDCs in the case of the smallest CaP NPs (S CaP-OVA) conjugates, as compared to water and OVA alone thus indicating immunomodulation via DC activation.<br/>S-CaP-OVA had the best immunogenic performance, so these amorphous particles were further studied with pneumococcal extracellular vesicle (referred as exosomes). The identified protective antigens, MalX and PrsA [4], from pneumococcal exosomes are decorated on these NPs and further tested <i>in vitro</i> and <i>in vivo</i>.<br/><br/>This research is funded by the Swedish Foundation for Strategic Research (FFL18-0043, RMX18-0041) and European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC Grant agreement n° 758705). Funding from Karolinska Institutet Faculty Board, Swedish Research Council (2018-05798 and 2021-02059) is kindly acknowledged.<br/><br/>[1] Pati, R., et al. (2018). Frontiers in immunology, 9, 2224.<br/>[2] Lin, Y., et al (2017). Expert review of vaccines, 16(9), 895-906.<br/>[3] Tsikourkitoudi, V., et al. (2020) Molecules, 25(7), 1747.<br/>[4] Narciso, AR., et al. (2022) PNAS, 119(23), e2122386119.