Title:Heterologous DNA Prime/Protein Boost Immunization Targeting Nef-Tat
Fusion Antigen Induces Potent T-cell Activity and in vitro Anti-SCR
HIV-1 Effects
Volume: 22
Issue: 2
Author(s): Leila Sadeghi, Azam Bolhassani*, Elham Mohit*, Kazem Baesi and Mohammad Reza Aghasadeghi
Affiliation:
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
- Department of Pharmaceutical Biotechnology,
School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
- Protein Technology
Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Keywords:
HIV-1, therapeutic vaccine, heterologous regimen, DNA vaccine, protein vaccine, SCR HIV-1.
Abstract:
Background: Heterologous combinations in vaccine design are an effective approach
to promote T cell activity and antiviral effects. The goal of this study was to compare the homologous
and heterologous regimens targeting the Nef-Tat fusion antigen to develop a human immunodeficiency
virus-1 (HIV-1) therapeutic vaccine candidate.
Methods: At first, the DNA and protein constructs harboring HIV-1 Nef and the first exon of Tat
as linked form (pcDNA-nef-tat and Nef-Tat protein) were prepared in large scale and high purity.
The generation of the Nef-Tat protein was performed in the E. coli expression system using an
IPTG inducer. Then, we evaluated and compared immune responses of homologous DNA prime/
DNA boost, homologous protein prime/ protein boost, and heterologous DNA prime/protein boost
regimens in BALB/c mice. Finally, the ability of mice splenocytes to secret cytokines after exposure
to single-cycle replicable (SCR) HIV-1 was compared between immunized and control
groups in vitro.
Results: The nef-tat gene was successfully subcloned in eukaryotic pcDNA3.1 (-) and prokaryotic
pET-24a (+) expression vectors. The recombinant Nef-Tat protein was generated in the E. coli
Rosetta strain under optimized conditions as a clear band of ~ 35 kDa detected on SDS-PAGE.
Moreover, transfection of pcDNA-nef-tat into HEK-293T cells was successfully performed using
Lipofectamine 2000, as confirmed by western blotting. The immunization studies showed that
heterologous DNA prime/protein boost regimen could significantly elicit the highest levels of Ig-
G2a, IFN-γ, and Granzyme B in mice as compared to homologous DNA/DNA and protein/protein
regimens. Moreover, the secretion of IFN-γ was higher in DNA/protein regimens than in DNA/DNA
and protein/protein regimens after exposure of mice splenocytes to SCR HIV-1 in vitro.
Conclusion: The chimeric HIV-1 Nef-Tat antigen was highly immunogenic, especially when applied
in a heterologous prime/ boost regimen. This regimen could direct immune response toward
cellular immunity (Th1 and CTL activity) and increase IFN-γ secretion after virus exposure.
