Immunoinformatics Analysis of Potent Therapeutic Formulations for the Development of HIV-1 Nefmut-Carrying Engineered Exosomes.

Background: The concept of designer exosomes involves developing engineered ex-osomes to overcome the limitations of natural exosomes in targeted drug delivery and vaccine development.

Methods: In this study, the multiepitope constructs were designed based on immunogenic regions of mutant Nef protein of Human Immunodeficiency Virus-1 (HIV-1 Nefmut) that were prone to high Post-Translational Modifications (PTMs), such as palmitoylation and myristoylation. These constructs with high scores in PTMs were selected for interactions with molecules involved in exosome biogenesis, anchoring of a protein in membranes, and enzymes involved in PTMs (e.g., the mutant enzyme ZDHHC21 p.T209S). Moreover, the selected multiepitope construct with the highest PTM score and stable linkage with these molecules was fused to the first exon of the HIV-1 Tat protein as an antigen candidate, and to GFP as a tracking tool for evaluating their effects on the PTM scores and affinity binding with various molecules.

Results: Our data demonstrated that the multiepitope construct No.13 had better scores for incor-poration into exosomes compared to the whole sequences of Nefmut and wild-type Nef protein (Nefwt). Furthermore, the linkage of Tat protein to construct No. 13 did not hinder its loading in exosomes compared to GFP, suggesting the use of this construct in vaccine development.

Conclusion: The multiepitope construct No.13 harboring potent Nef mut epitopes can be applied for linkage with other viral antigens, enhancing their delivery into exosomes for therapeutic ap-plications.