Combination of cell penetrating peptides and heterologous DNA prime/protein boost strategy enhances immune responses against HIV-1 Nef antigen in BALB/c mouse model.

To develop a strong HIV specific T-cell response, the HIV-1 Tat and Nef regulatory proteins have been known as attractive antigenic candidates in vaccine design. A peptide transduction domain of Tat (48-60 aa) could act to deliver other therapeutic molecules into different cells. In this line, several cell-penetrating peptides (CPPs) have been designed to transfer DNA, siRNA, polypeptides and proteins into cells through non-covalent approach such as CADY and PEP families. Some studies showed that the endogenous adjuvants including heat shock protein Gp96 could stimulate antigen-specific T cell immune responses. In this study, different Nef DNA and protein constructs were generated, and their abilities were evaluated to induce T cell immune responses and humoral immunity in mouse model. A novel prime-boost immunization approach was also used in which the priming injections consisted of Nef/Tat (PTD)-Nef DNA plus Gp96 DNA followed by Nef/Tat (PTD)-Nef protein formulated in Freund's adjuvant or the Pep-1 and Cady-2 CPPs. Generally, our results indicated that Tat (PTD)-Nef fusion DNA or protein could significantly elicit higher humoral and cellular immune responses than Nef DNA or protein, respectively. Analysis of the immune responses demonstrated that the Tat (PTD)-Nef+Gp96 DNA prime/Tat (PTD)-Nef protein+Cady-2 boost regimen significantly enhanced the Nef/Tat (PTD)-Nef-specific T cell responses. This modality induced high levels of IgG2a and IFN-γ directed toward Th1 responses, and also Cytotoxic T Lymphocytes (CTLs) activity as compared to other immunization strategies. The immunostimulatory properties of Pep-1 and Freund's adjuvant were almost similar in different immunization strategies. These findings showed that the use of Tat (PTD)-Nef antigen in prime-boost strategy along with Gp96 adjuvant and Cady-2 CPP may have practical implications for developing HIV-1 vaccine in large animal model.

The Efficiency of Tat Cell Penetrating Peptide for Intracellular Uptake of HIV-1 Nef Expressed in E. coli and Mammalian Cell.

BACKGROUND: Cell penetrating peptides (CPPs) or protein transduction domains (PTDs) have been known as a new field in cargo delivery. These peptides such as Tat, Pep-1 and Cady-2 are able to deliver genes and biologically active proteins to cytoplasmic compartments via the plasma membrane. METHODS: In current study, the efficiency of pEGFP-N1 eukaryotic vector for expression of HIV-1 Tat- Nef fusion was evaluated in HEK-293T cells using TurboFect transfection reagent. In addition, the recombinant GST-Tat-Nef protein was generated in E. coli and transfected using two amphipathic CPPs (Pep-1 and Cady-2) into mammalian cells. The size and morphology of the CPP/GST-Tat-Nef complexes were evaluated by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The transfection of HIV-1 GST-Tat-Nef protein was also analyzed using SDS-PAGE and western blotting. RESULTS: Our data indicated that the recombinant GST-Tat-Nef protein generated in BL21 strain migrated as a clear band of ~ 52 kDa in SDS-PAGE. The results of SEM and Zetasizer confirmed the formation of protein/ Pep-1 or protein/ Cady-2 nanoparticles less than 200 nm in diameter. Tat CPP fused to Nef protein could deliver the recombinant Nef protein alone and notably by forming the noncovalent complexes with TurboFect, Pep-1 and Cady-2 as detected in western blotting. Moreover, intracellular uptake of Tat-Nef gene and subsequently its expression in mammalian cells was considerably higher than that for Nef gene. CONCLUSION: This data indicated that the Tat gene sequence could also increase the transfection of Nef gene in vitro. Generally, the Tat-Nef interaction led to enhance further gene expression and also protein delivery.