*3.1. DNA-Based Immunization*

Alphavirus-based DNA plasmids have been frequently used for immunization studies in animal models targeting infectious agents and different types of cancers (Table 1). For instance, a Sindbis virus (SIN) DNA vector expressing the herpes simplex virus type 1 glycoprotein B (HSV-1-gB) elicited a broad spectrum of immune responses including virus-specific antibodies and cytotoxic T cells in mice [13]. Furthermore, a single intramuscular immunization with SIN-HSV-1-gB protected mice from lethal challenges with HSV-1. In another study, a Semliki Forest virus (SFV) DNA vector expressing the bovine viral diarrhea virus (BVDV) p80 (NS3) was evaluated in BALB/c mice [14]. The administration of SFV-BVDV p80 DNA into the quadricep muscles of mice generated statistically significant cytotoxic T-lymphocyte (CTL) activity and cell mediated immune (CMI) responses against cytopathic and noncytopathic BVDV. Related to measles virus (MV), SIN DNA vectors expressing the MV hemagglutinin (pMSIN-H) and fusion protein (pMSINH-FdU) were administered either alone or boosted with a live measles virus vaccine in cotton rats [15]. The study demonstrated that neutralizing antibodies, mucosal and systemic antibody-secreting cells, memory B cells, and interferon-γ (IFN-γ)-secreting T cells were obtained after priming, further enhanced after boosting.


*Vaccines* **2019** , *7*, 29

Toxoplasma gondii nucleoside triphosphate growth factor receptor-2; VP40, matrix viral protein.

 hydrolase-II;

 TP,

toxoplasmosis;

 TRP1, tyrosine related protein-1; VEE, Venezuelan equine encephalitis virus; VEGFR2, vascular epithelial

Protection against pulmonary measles was achieved after immunization with pMSIN-H, whereas pMSINH-FdU provided protection only after boosting with a live measles virus vaccine. In another approach, an SFV DNA vector was compared to a recombinant adenovirus expressing the classical swine fever virus (CSFV) E2 glycoprotein in pigs [16]. Significantly higher titers of CSFV-specific neutralizing antibodies were obtained after a pSFV1CS-E2/rAdV-E2 heterologous prime-boost immunization strategy compared to double immunizations with rAdV-E2 alone. Moreover, the heterologous prime-boost immunization regimen prevented viremia and clinical symptoms in pigs. In contrast, these symptoms were seen in one of five pigs vaccinated with rAdV-E2 alone. Related to HIV vaccines, an SFV DNA plasmid and a poxvirus Ankara (MVA) vector expressing an HIV Env and a Gag-Pol-Nef fusion protein were subjected to a prime-boost study [17]. It was revealed that efficient priming of HIV-specific T cell and IgG responses was achieved with a low dose of 0.2 μg SFV DNA and the priming effect seemed to relate to the number of prime administrations rather than dose. In another prime-boost study, four novel alphavirus DNA replicon vectors were engineered to express structural Core-E1-E2 or nonstructural p7-NS2-NS3 hepatitis C virus (HCV) [18]. Prime immunization with alphavirus DNA-HCV vectors followed by a heterologous boost with a vaccinia virus expressing the nearly full-length HCV genome (MVA-HCV) elicited long-lasting HCV-specific CD4+ and CD8<sup>+</sup> T cell responses in mice presenting a promising approach for prophylactic and therapeutic HCV vaccine development. Moreover, alphavirus DNA vectors were subjected to the expression of the Ebola virus (EBOV) glycoprotein (GP) gene alone or together with the EBOV VP40 gene of Sudan or Zaire EBOV strains [19]. Both binding and neutralizing antibodies were detected in immunized mice. The alphavirus-based DNA vaccine showed superior immunogenicity in comparison to recombinant MVA vaccines. In another study, the co-expression of EBOV GP and VP40 elicited significantly higher antibody levels than for immunization with GP or VP40 alone [20]. SFV-DNA EBOV GP and VP40 co-vaccination induced EBOV-specific humoral and cellular immune responses in mice [20].

In the context of *Mycobacterium tuberculosis*, a SIN DNA vector expressing the p85 antigen (Ag85) was highly immunogenic in mice and provided enhanced long-term protection against challenges with *M. tuberculosis* [21]. In another study, the alphavirus-based Venezuelan equine encephalitis virus (VEE) DNA vector expressing a fusion of the *M. tuberculosis* antigens α-crystallin (Acr) and Ag85B named Vrep-Acr/Ag85B was evaluated in a mouse model of pulmonary tuberculosis [22]. Immunization studies elicited antigen-specific CD4<sup>+</sup> and CD8+ T cell responses, which persisted for at least ten weeks and also induced T cell responses in lung tissues. Moreover, bacterial growth was inhibited in lungs and spleen after aerosol challenges with *M. tuberculosis*. Related to toxoplasmosis, the *Toxoplasma gondii* nucleoside triphosphate hydrolase-II (TgNTPase-II) gene expressed from an SFV DNA vector was intramuscularly delivered to mice [23]. Specific humoral responses were obtained as well as cellular immune responses associated with high levels of IFN-γ, IL-2, and IL-10 cytokines and low levels of IL-4. Partial protection against acute infection with the virulent RH strain and chronic infection with the PRU cyst strain of *T. gondii* was obtained in immunized mice.

Related to toxins, alphavirus DNA vectors expressing the Hc gene of botulinum neurotoxin serotype A (BoNT/A) demonstrated specific antibody and lymphoproliferative responses in immunized BALB/c mice [24]. Co-delivery or co-expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the immunogenicity and survival rates in immunized mice were significantly prolonged after challenges with BoNT/A. Furthermore, co-immunization with aluminum phosphate adjuvant improved the survival.

In the context of cancer, an SFV DNA vector expressing the human papilloma virus type 16 (HPV-16) E7 protein as a fusion protein with the *M. tuberculosis* heat shock protein 70 (Hsp70) elicited significantly higher E7-specific T cell-mediated immune responses in comparison to E7 expressed alone in mice [25]. Moreover, the E7/Hsp70 fusion construct showed superior potency against established E7-expressing metastatic tumors. In another study on HPV, the SFV based DNA encoding the HPV E6 and E7 antigens was subjected to intradermal administration followed by electroporation, which provided effective and therapeutic anti-tumor activity resulting in approximately 85% tumor-free

mice [26]. Related to breast cancer, the HER2/neu gene was targeted due to its role in increased metastasis and poor prognosis [27]. Intramuscular administration of SIN-neu DNA elicited strong antibody responses against the A2L2 mouse breast cancer cell line expressing neu. Moreover, challenges with A2L2 cells reduced tumor incidence and tumor mass in immunized mice. Intradermal vaccination required 80% less SIN-neu DNA to reach the same efficacy compared to intramuscular administration. Furthermore, the vaccination protected against development of spontaneous breast tumors and reduction in metastasis from HER2/neu expressing tumors. In another study, mice injected in the mammary fat pad with A2L2 tumor cells were evaluated for the combination treatment of SIN-neu DNA and chemotherapy [28]. Neither immunization with SIN-neu DNA nor chemotherapy with doxorubicin or paclitaxel alone reduced tumor growth. In contrast, chemotherapy followed by vaccination with SIN-neu DNA reduced tumor growth significantly. In another study, the effect of SIN-DNA immunizations was evaluated in a solid mammary tumor model and a lung metastasis model [29]. When mice were immunized with SIN-neu DNA or an Adenovirus (Ad-neu) vector prior to challenges with A2L2 tumor cells, tumor growth was significantly inhibited. In contrast, vaccination two days after tumor cell challenges was ineffective. However, in a regimen with SIN-neu DNA priming and Ad-neu boosting, significantly prolonged survival of mice was observed.

In an immunotherapy approach SIN-DNA expressing the self/tumor antigen tyrosine-related protein-1 (TRP1) was demonstrated to activate innate immune pathways providing improved immunization efficacy of naked DNA [30]. Related to melanoma, the melanoma cell adhesion molecule /MCAM/MUC18) was expressed from a SIN DNA plasmid (SIN-MUC18) and mice were vaccinated against B16F10 mouse melanoma cells [31]. The immunization provided protection of mice from lethal challenges with melanoma expressing mouse MUC18 in both primary and metastatic tumor models. In the context of brain tumors, immunization with SIN DNA expressing human gp100 and interleukin-18 (IL-18) enhanced both protective and therapeutic effects on malignant brain tumors [33]. The anti-tumor and protective effects were mediated by both CD4+/CD8+ T cells and IFN-γ and the survival rate was significantly improved in mice with implanted B16 tumors. The synergistic approach of targeting tumor cells and angiogenesis was simultaneously executed by co-immunization studies with an SFV DNA replicon vector carrying 1-4 domains of murine vascular epidermal growth factor receptor-2 (VEGFR2) and IL-12 and another SFV DNA replicon expressing the survivin and β-hCG antigens [32]. The combined vaccines elicited strong humoral and cellular immune responses against survivin, β-hCG and VEGFR2, inhibited tumor growth and prolonged survival in a B16 melanoma mouse model.
