Nanoparticles as a delivery strategy for HIV antigens to pulse monocyte derived dendritic cells: potential application in anti HIV immunotherapy

Author(s)

Bruna Tiaki Tiyo

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2023-12-11

Examining board members

Alberto Jose da Silva Duarte; Cyro Alves de Brito; Jonnatan Julival dos Santos; Maria Notomi Sato

• Advisor: Alberto Jose da Silva Duarte; Telma Miyuki Oshiro Sumida

Abstract

Immunotherapy based on dendritic cells (DC) is a potential tool for improving the treatment of HIV infection, whose aim is to stimulate a direct and specific immune response against viral infection. The most commonly used protocol for obtaining DCs involve s culturing monocytes in the presence of IL 4 and GM CSF and subsequent activation of immature DCs with pro inflammatory cytokines. aDC1, unlike conventional monocyte derived DCs, exhibits a superior ability to induce a CD8+ T lymphocyte response capable o f eliminating HIV 1 infected cells. In addition to the importance of DC quality, carrier agents have gained notoriety for improving antigen delivery to the cells or tissues of interest. Chitosan nanoparticles (QS) have been used because they are from a nat ural source, non toxic, biocompatible, and biodegradable polymer, while silica nanoparticles have been widely applied in the context of drug and antigen delivery owing to their stability and the possibility of different formulations. Therefore, the objecti ve of this study was to evaluate the effect of nanoparticles on delivery of HIV antigens to aDC1 type dendritic cells. In the initial tests, the viability of RAW 264.7 cells, stimulated with QS and silica nanoparticles was evaluated by MTT. However, challe nges, such as instability and reproducibility in the production of QS nanoparticles, were decisive in ending QS trials. Therefore, we chose to continue using only silica nanoparticles. The ratio calculated for the conjugation of silica nanoparticles to HIV peptides was 10:1. Evaluation of the viability and phenotypic analysis of aDC1 pulsed with silica nanoparticles was carried out by flow cytometry, as well as evaluation of the different internalization times of the particles by the cells. In contrast, sil ica nanoparticles, at a concentration of 10 µg/ml, conjugated to 1 µg/ml of the HIV Gag.27 peptide, did not seem to interfere with the viability of aDC1, when compared to nanoparticles without conjugation to antigens. Regarding the evaluation of the phenot ypic profile of aDC1, in which the activation and maturation markers CD40, CCR7, CD80, CD83, and HLA DR were used, no significant difference was observed between the cells that received nanoparticles and the controls. An unexpected finding was the increase in the expression of the CD14 molecule in aDC1 pulsed with silica nanoparticles, especially when not associated with peptides, which may be related to cell apoptosis. Finally, 8 h seemed to be sufficient for the internalization of silica nanoparticles con jugated to HIV peptides by aDC1, as demonstrated by cytometry and fluorescence microscopy. In summary, we reiterated the potential use of silica nanoparticles conjugated to HIV peptides in aDC1 cell cultures. (AU)

• FAPESP's process: 19/24849-1 - Nanoparticles as HIV antigen delivery: potential application in anti-HIV immunotherapy using monocyte-derived dendritic cells
• Grantee: Bruna Tiaki Tiyo
• Support Opportunities: Scholarships in Brazil - Doctorate