Table 1.

A summary of studies on DNA-based vaccines for HCV and their efficiency in animal models

Row	Author(s)	Year	Host	Genomic region for design	Route	Prescribed number	Adjuvant	Laboratory method	Efficiency	References
1	Pouriayevali, M. H., et al	2019	BALB/c mice	NS3 gene sequence (1095-1380aa)	ID	three doses in two-week intervals	Listeriolysin O (LLO)	PCR, western blot, lymphocyte proliferation, cytotoxicity, and cytokine levels assays	Enhancement of total IgG in mixed responses with Th1 dominancy	[56]
2	Lee, H et al	2017	BALB/c mice	NS3 to NS5A	IM	three doses in two-week intervals	murine IL-28B	Body weight change, necropsy, hematological and serum biochemical evaluation, LD50 determination, IFN-γ ELISpot assay	Increased NS3,4, and 5 IFN-γ spots in ELIspot assay Murine IL-28B resulted in more than twice NS3/4A specific IFN-γ spots per million splenocytes	[75]
3	Pouriayevali, M. H., et al	2016	BALB/c mice	NS3 gene (1095—1379 aa)	ID	four doses in two-weeks intervals	N/A	RNA Extraction and cDNA Synthesis, total and subtypes of IgG antibody assay, cell proliferation assay, western blot and ELISPOT	induced significant levels of total antibody, IgG2a, IFN-γ and IL-4	[12]
4	Levander, S., et al	2016	C57BL/6 J mice	NS3/4A-stork-HBcAg	IM	one to five times with doses of 50, 5, 0.5, 0.05, and 0.005 µg	HBcAg	In vitro transcription and translation assay, Transient transfection, western blot and ELISpot assay	Increased production of IFN-γ, IL-2 and	[19]
5	Behzadi, M. A., et al	2016	C57BL/6 mice	NS3/NS4A	IM	Three injection, 2 weeks apart	Freund’s adjuvant, MPL	flow cytometry, T helper frequency using cell staining	Increased production of Th1/ Th2 and T-CD8 + cells	[26]
6	Yazdanian, M., et al	2015	BALB/C mice	HBsAg and core (amino acids 2–120)	IV	Three doses	pluronic acid (F127)	CTL assay, SDS–PAGE and western blotting for plasmid expression, Antigen specific proliferation assay, and ELISpot	shifting the immune responses pathway to Th1 by enhancing the IFN‑γ cytokine level	[1]
7	sun, w.,et al	2015	BALB/C mice	HCV E2 with an immunoglobulin Fc fusion tag	SC	Approximately Four doses in two-weeks intervals	CpG ODN/Quil A	ELISA assay, Lymphocyte Proliferation Assay, Immunofluorescence Assay and HCVpp Neutralization Assay	Enhancement of E2-specific humoral and cellular immune response	[46]
8	Pishraft Sabet, L., et al	2015	CB6F1 mice	Core(132–142),, E2(412–426), NS3(1073–1081)(1248–1262) and NS5B(2727–2735)	IM	three times at 2-week intervals,	heat shock protein gp96 (NT(gp96))	flow cytometry and ELISA	Induction of T-cell and antibody responses	[21]
9	Masalova, O. V., et al	2015	DBA/2 J mice	NS3–NS5B	IM	three times with an interval of 2 weeks	pcGM–CSF	ELISA, ELISpot	Enhancement of humoral and cellular immune response	[32]
10	Gummow, J., et al	2015	C57BL/6 mice	nonstructural (NS) proteins (3, 4A, 4B, and 5B)	ID	either two or three doses at 2-week intervals	perforin	Cell culture., ELISpot, T-cell proliferation assay, Flow cytometry and Western blot	Enhancement of TNF-a-producing CD4? and CD8? T-cells	[50]
11	Hartoonian, C. et al	2014	BALB/C mice	Core protein	SC	three times in 2 weeks intervals	Macrophage Inflammatory Protein 3-beta (MIP-3beta)	ELISpot and cytotoxic Granzyme B release assays), ELISA	Enhancement of IFN-γ/ IL-4 and anti-core IgG2a/IgG1 ratio, lymphoproliferation, strong cytolytic GrzB release	[55]
12	Hartoonian, C. et al	2014	BALB/c mice	Core protein	SC	three times in 2 weeks intervals	CC-chemokine ligand 20 (CCL20)	ELISA/ELISpot and cytotoxic Grenzyme B (GrzB) release assays	Enhancement of core specific IFN-c/IL-4 ratio, IL-2 release, IFN-c and the core-specific serum total IgG and IgG2a/IgG1 ratio were significantly higher when the pCCL20 was co-inoculated	[22]
13	Ahlen, G. et al	2014	BALB/c (H-2d) and/or C57BL/6 J (H-2b) mice	NS3/4A	gene gun delivery to the skin or by IM	one, two, three or four times at monthly interval	N/A	ELISpot, RMA-S stabilization assay and cytotoxicity assay	Production of NS3/4A-specific T cells in vitro	[76]
14	Gorzin, Z. et al	2013	C57BL/6 mice	non-Structural Protein 2 (NS2)	IM	three times with an interval of 2 weeks	IL-12	ELISA, Cytokine secretion assay, MTT	Enhancement of CTL response, interferon-γ production, and lymphocyte proliferation compared to negative control	[77]
15	Wada, T. et al	2013	C57BL/6 mice	structural protein (CN2), non-structural protein (N25)	IP	three times at 48-h intervals	N/A	cytotoxicity assay, ELISPOT assay, Generation of CTL effector cells and cytotoxicity assay, Histopathological examination and ELISA	significant decrease in the expression of HCV protein in mice administered the N25 DNA vaccine and improvement of pathological changes in the liver by N25 DNA vaccine	[78]
16	Naderi, M. et al	2013	C57BL/6 mice	NS3	IM	Three times	IL-12	RT-PCR, western blot, ELISA, Lymphocyte proliferation assay	production of significant levels of both IL-4 and interferon (IFN)-γ and enhancement of cytotoxicity and lymphocyte proliferation responses of vaccinated mice	[23]
17	Holmstorm, F. et al	2013	C57BL/6 J mice	Optimized Synthetic Codon- of NS5A	IM	One to three times at monthly intervals	N/A	ELISA, western blot, ELISPOT assay, cytotoxicity assay and RMA-S stabilization assay	Production of high Ab levels, IFN-γ and lytic cytotoxic T cells	[79]
18	Park, S. et al	2010	monkeys as the naive group	E2	IM	6 times at the indicated months	IL-7	ELISA, ELISPOT assay	Enhancement of antibody responses specific for HCV E2 protein and specific T cell responses by codelivery of hIL-7 DNA	[80]
19	Masalova, O. V. et al	2010	Female DBA/2 J mice	Full-size NS5A protein	IM	thrice with a month’s interval	Immunomax	Eukaryotic Cell Transfection, ELISA	Enhancement of cellular immune response, secretion of antiviral cytokines IFN-γ and IL-2	[81]
20	Martin, P. et al	2008	HLA-A2 transgenic mic	NS3, NS4 and NS5B proteins	IV	twice, 2 weeks apart	N/A	ELISpot and CTL assays	Induction of strong and broader IFN-γ ELISpot and CTL responses	[13]
21	Long, K. L. et al	2008	C57BL/6 mice and monkey	NS3/NS4A	IM	Three times, 2 weeks apart in mice and two times, 4 weeks apart in the monkeys	N/A	Immunofluorescenc, ELISpot,	Induction of strong anti-NS3/NS4A T cell responses	[82]
22	Folgori, A. et al	2006	Chimpanzees (Pan troglodytes)	NS3- NS5B region	IM	Adenovirus injection: Two times, 4 weeks apart administered bilateraly, booster injected at week 25. plasmid DNA injection at 35, 37,39 weeks	N/A	IFN-γ intracellular staining, cytotoxicity assay	High CMI in 4 out of 5 subjects, no increase of hepatic enzymes in vaccinated chimpanzees. HCV specific intrahepatic CD8 + T-cell response	[83]
23	LI, Y. P. et al	2006	B6C3F1 mice and piglets	E2	ID and SC	Three times, 3 weeks apart	N/A	CTL assay and ELISA	Strong stimulation of strong Th1-like immune responses in mice, more balanced immune responses in piglets, production of higher E2-specific antibody levels and shifting the immune response towards Th2-like ones in piglets	[84]
24	Ahlen, G. et al	2005	wild- type or CD8-/- C57BL/6 mice	NS3/4A	IV	Two times, 2 weeks apart	N/A	SDS PAGE and Western blot	Enhancement of T specific cells by transdermal DNA-based vaccination entered the liver and Clearance of NS3/4A-expressing hepatocytes in transiently trans- genic CD8 + / +  mice but not in CD8−/− mice	[85]
25	Zhu, M. et al	2004	BALB/c mice	E1/E2	quadriceps muscles	Three times, two weeks apart	CpG	HCV antigen specific proliferation assays and cytokine secretion assays	Enhancement of humoral and cellular immune responses. CpG adjuvant significantly enhances the cellular immune response	[86]
26	Ou-Yang, P. et al	2002	BALB/c mice	Core protein	IM	Two times, seven days apart	GM-CSF	ELISA, Immunofluorescence, proliferative assay	Higher Antibody titer and cytotoxic T cell and protein expressing CD11c+ Dendritic cells	[87]
27	Ma, X. et al	2002	BALB/c (H-2d) mice	E2	IM	Three times with a month’s interval	CpG	ELISA and CTL assay	superior antibody and CTL responses	[88]

ID; intradermally, IM; intramuscularly, IV; intravenously, MPL; monophosphoryl lipid A, SC; sub-cutaneously, IP; intraperitoneally