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Background: Heat shock proteins (HSPs) as an adjuvant induce antigen‐specific immunity through facilitating antigen presentation and stimulating T cells. In this study, the immunostimulatory properties of two major fragments of Hsp70 (N‐Hsp70(aa 1–387) with ATPase property and C‐Hsp70 (aa 508–641) with peptide‐binding capacity) and the full length of Hsp27 as vaccine adjuvants were evaluated to boost HIV‐1 Nef antigen‐specific immunity in both in vitro and in vivo experiments. Method s : At first, the nanoparticles harbouring DNA fusion constructs (i.e. N‐Hsp70‐Nef, C‐Hsp70‐Nef and Hsp27‐Nef) complexed with HIV Rev (34–50) cell‐penetrating peptide were generated to deliver DNA into the cells. Then, the recombinant Nef, Hsp27‐Nef, N‐Hsp70‐Nef and C‐Hsp70‐Nef proteins were generated in E.coli expression system. Next, the immunostimulatory properties of these fusion constructs were evaluated in both in vitro and in vivo studies. Finally, the secretion of main cytokines from single‐cycle replicable (SCR) HIV‐1 virion‐exposed splenocytes was investigated. Results: Our data showed that the stable and non‐toxic DNA/Rev nanoparticles could successfully deliver the genes of interest into the cells. Moreover, higher secretion of antibodies and cytokines was detected in mice receiving the Hsp‐Nef constructs than in mice receiving Nef antigen. The C‐Hsp70 was also superior for inducing Nef‐specific Th1 and CTL immunity compared with N‐Hsp70 and Hsp27. The T‐cell activity was maintained in the SCR‐exposed splenocytes, especially the splenocytes of mice receiving the C‐Hsp70‐Nef regimen. Conclusion: Altogether, these findings demonstrate the significance of Hsps as enhancers of antigen‐specific immunity. Notably, the C‐Hsp70 region showed better adjuvant properties for inducing cellular immunity in the improvement of HIV‐1 therapeutic vaccines. [ABSTRACT FROM AUTHOR]

Therapeutic human immunodeficiency virus (HIV) vaccines can boost the anti-HIV host immunity to control viral replication and eliminate viral reservoirs in the absence of anti-retroviral therapy. In this study, two computationally designed multiepitope Gag-Pol-Env-Nef-Rev and Hsp70-Gag-Pol-Env-Nef-Rev constructs harboring immunogenic and highly conserved HIV T cell epitopes were generated in E. coli as polypeptide vaccine candidates. Furthermore, the multiepitope gag-pol-env-nef-rev and hsp70-gag-pol-env-nef-rev DNA vaccine constructs were prepared and complexed with MPG cell-penetrating peptide. The immunogenicity of the multiepitope constructs were evaluated using the homologous and heterologous prime/boost strategies in mice. Moreover, the secretion of IFN-γ was assessed in infected lymphocytes in vitro. Our data showed that the homologous polypeptide regimens could significantly induce a mixture of IgG1 and IgG2a antibody responses, activate T cells to secret IFN-γ, IL-5, IL-10, and generate Granzyme B. Moreover, IFN-γ secretion was significantly enhanced in single-cycle replicable (SCR) HIV-1 virions -infected splenocytes in these groups compared to uninfected splenocytes. The linkage of heat shock protein 70 (Hsp70) epitopes to Gag-Pol-Env-Nef-Rev polypeptide in the homologous regimen increased significantly cytokines and Granzyme B levels, and IFN-γ secretion in virions-infected splenocytes. Briefly, both designed constructs in the homologous regimens can be used as a promising vaccine candidate against HIV infection. [ABSTRACT FROM AUTHOR]

Objectives: Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection. Methods: Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting. Results: The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56–60% as the bands of ~ 63 and ~ 72 kDa confirmed in western blotting, respectively. Conclusion: The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development. [ABSTRACT FROM AUTHOR]