Ph.D Thesis


Ph.D StudentAriel Gilert
SubjectDevelopment and Characterization of Polymeric Micro
Particles for Controlled Delivery of Short
Peptides towards Specific Immune
Response
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professors Machluf Marcelle
Full Thesis textFull thesis text - English Version


Abstract

Vaccination is the administration of antigens in order to evoke the immune response against an infection or a disease through APCs. Antigens are generated by two major processes:

1. Proteasomal proteolysis of newly synthesized endogenic proteins that being presented as pMHC-I complexes. This process mediates cellular immunity through the activation of CTLs.

2. Internalization of exogenous peptides and their manifestation on the cell membrane as pMHC-II complexes. This process is responsible for the activation of humoral immunity.

Exogenic cancer vaccination posts many challenges which include: (i) Formation of pMHC-II complexes leading to a humoral response rather than cellular one and (ii) Down-regulation of MHC class I molecules. The latter is influenced by the short duration of pMHC-I complexes' presentation on the cell surface. This obstacle is intensified when soluble synthetic class I antigens, are internalized from the outside - mainly because of antigen instability and degradation.

These problems may be solved or reduced through a process of cross-presentation, in which internalized exogenous proteins leave the phagolysosomes in order to create pMHC-I complexes rather than pMHC-II complexes thus priming a CTL response. However, this process is extremely inefficient as protein tend to stay in the phagolysosomes where they are processed. Therefore, to promote cross-presentation the protein need to leave actively the phagolysosomes. This can be achieved by using vehicles, which carry the antigen to the phagosomal vacuole, and contain proteins that act as membrane poring agents that will perforate the phagosomal membrane, thus, releasing its content to the cytosol.

We have proposed to use PLGA for the fabrication of microspheres which will entrap low molecular weight antigens that will be delivered into APCs cytosol.

Microspheres composed of PLGA were developed for entrapping low molecular antigens. Microspheres' surface morphology, degradation, release kinetics and encapsulation efficiency were studied. The biological activity of antigens loaded in the particles was confirmed in-vitro. Antigens loaded in PLGA microspheres were internalized by APCs and led to extended presentation of pMHC-I complexes, thus, improving the cross-presentation pathway. Cross-priming of specific CTLs, by microspheres loaded APCs, was longer than for free peptides reassuring our results.

Next, we aimed at increasing the efficacy of cytosolic delivery by incorporating membrane poring agents to the PLGA microspheres. We showed that LLO incorporated microspheres, improved the efficacy of antigen delivery to APCs cytosol and enabled stronger presentation of pMHC-I complexes. This enhanced presentation led to stronger and longer cross-priming of specific CTLs.