**2. DNA Vaccine Adjuvants**

The immunogenicity of DNA vaccines is enhanced by CpG motifs present in the plasmid backbone, which can activate APC via toll like receptors (TLR9) [32–34]. Unmethylated CpG motifs have been reported to induce B cell proliferation and secretion of immunoglobulin in vitro and in vivo [33]. Activation of macrophages and DCs results in upregulation of antigen presentation and costimulatory molecules, and secretion of cytokines (IL-12 and IL-18) involved in helper T cells (Th1) response [34]. Thus, CpG motifs in DNA plasmids serve as a 'natural adjuvant' for DNA vaccines. Plasmid DNA can be designed to encode additional adjuvants with the antigen(s) of interest. Molecular adjuvants such as fusion proteins including heat shock protein 70 (HSP70), and vesicular stomatitis virus (VSVG) have been developed and used to enhance vaccine immunogenicity [35–37]. The gene encoding such proteins as adjuvants is either fused with the gene encoding the vaccine antigen to produce a fusion protein driven by a same promoter or as separate proteins driven by different promoters in the same or different plasmid. Co-encoding of genes creates a suitable cellular micro environment such as sustained antigen release and/or upregulation of cytokines, enhancing the immunogenicity of DNA vaccines [38].

A majority of the studies on experimental DNA vaccines with genetic adjuvants have been studied in animal models such as mice (Table 1), and very few of these have been clinically tested (Table 2). Limited published data on clinical trials pose difficulty to compare efficacy between adjuvants in animals and humans.


#### **Table 1.** Molecular adjuvants and immunogenicity of DNA vaccines in animals.

**Adjuvants:** LT: ligand/trimer, IL: Interleukin, TNF: Tumor necrosis factor, TRIF: Toll-interleukin-1 receptor domain-containing adaptor-inducing beta interferon, MyD88: myeloid differentiation primary response, FliC: phase-1 flagellin, IRF: Interferon regulatory factor, TBK-1: TANK-binding kinase 1, HMGB1: High-mobility group box 1 protein, DAI: DNA-dependent activator of interferon (IFN) regulatory factors, chMDA5: melanoma differentiation-associated gene 5 product, FrC: Fragment C of tetanus toxin, DTa: Diphtheria toxin subunit A, NSP4: Nonstructural protein 4, li: MHC class II invariant chain, HSP: Heat shock protein, VSVG: Vesicular stomatitis virus, PRF: Perforin; **Antigens:** HIV: Human immunodeficiency virus, Env: Envelope, GAG: Group antigens, Pol: Reverse transcriptase, beta-gal: beta galactosidase, FMDV: Foot and Mouth Disease Virus, VP1: Virus protein 1, HBV: Hepatitis B virus, HA: Haemagglutinin, Sc-fv: Single chain fragment variable, Np: Nucleoprotein, P.f: *Plasmodium falciparum*, SE36: serine repeat antigen 36, HCV: Hepatitis C virus, NS3: Nonstructural protein 3, ME: Multiepitope string fused to the native *P. falciparum* T9/96 strain,NS345B: Nonstructural proteins 3, 4, 5B; **Delivery:** DC: Different constructs, BC: Bicistronic construct, FC: Fusion protein/single construct, IM: Intra muscular, SC: Subcutaneous, GG: Gene gun, EP: Electroporation, ID: Intradermal; Responses: +: Increase, −: Decrease, +/−: No significant change, CMI: T cell responses, Ab: Humoral responses; **Ref.:** References.


**Table 2.** Molecular adjuvants tested with DNA vaccines in humans.

**Adjuvants:** IL: Interleukin, GM-CSF: Granulocyte/macrophage colony-stimulating factor; **Antigens:** HIV: Human immunodeficiency virus, Gag: Group antigens, Her2: Human epidermal growth factor receptor 2, CEA: Human carcinoembryonic antigen, MAG: Multi antigen, Env: Envelope, Pol: Reverse transcriptase, Nef: N-terminally myristoylated protein, Tat: Transactivator of transcription, Vif: viral infectivity factor, PAP: Prostatic Acid Phosphatase, HSP: Heat shock protein, HPV: Human Papilloma Virus; **Delivery:** DC: Different constructs, BC: Bicistronic construct, FC: Fusion protein/single construct, RP: Adjuvant as recombinant protein, IM: Intramuscular, ID: Intradermal; Responses: +: Increase, −: Decrease, +/−: No significant change, Ab: Humoral responses, CMI: T cell responses; **Ref.:** References.

#### *2.1. Cytokines*

Different cytokines, such as interleukins (IL-2, IL-6, IL-12), chemokines, granulocyte/macrophage colony-stimulating factor (GM-CSF), costimulatory molecules (CD40, CD80, and CD86), and signaling molecules (Interferon regulatory factor -3) have been used as genetic adjuvants with DNA vaccines [39,40,42–44,48,68]. Genes expressing IFN-γ IL-2, IL-12, IL-15, and IL-18 have been used to stimulate Th1 responses [44,45,70], and IL-4, IL-6, IL-10, IL-13, for Th2 stimulation [42,43,71–73]. The inclusion of genes encoding cytokines, like IL-2 or IL-12, as adjuvants for HIV-1 DNA vaccines is known to increase cell mediated immunity (CMI) [74,75]. However, a bicistronic HIV DNA encoding gp120 and IL-2 elicited weaker specific immune response than monocistronic HIV-1 gp120 DNA [76]. Combinations of genetic adjuvants like IL-2 and IL-15 with HIV-1 DNA vaccine have also been used but no synergistic effect on the level of total antibody to HIV-1 antigen was reported [77]. A phase I/IIa trial showed that coadministration of DNA vaccine encoding prostatic acid phosphatase (PAP) with GM-CSF elicited PAP-specific CD4<sup>+</sup> and/or CD8<sup>+</sup> T cell responses [68]. However, GM-CSF was administered as a recombinant protein.
