*4.3. TALENs*

Transcription activator-like effectors (TALEs) are crucial virulence factors that function as transcriptional activators in the cell nucleus of plants, where they directly bind to DNA via a central domain of tandem repeats [53]. Currently, TALENs were shown to be an effective tool for precise genome engineering with low toxicity and could be engineered to adapt for an antiviral strategy [54]. Hence, TALENs are likely to become part of a new approach for the treatment of CMV infections.

Chen et al. utilized three pairs of TALEN plasmids (MCMV1–2, 3–4, and 5–6) to target the MCMV M80 and M80.5 overlapping (M80/80.5) sequence to test their efficacy in blocking MCMV lytic replication in NIH3T3 cells [54]. Using lipofectamine or a specific lipoid NKS11 as transfection reagents, TALEN plasmids could specifically target the M80/80.5 sequence and effectively inhibit MCMV growth in cell culture when the plasmid transfection is prior to the MCMV infection. Using NKS11 which was previously proved to be nontoxic to mice as a transfection reagent, the most specific pairs of TALEN plasmids (MCMV3–4) showed that its competency to inhibit the replication and gene expression of latent MCMV in immunocompetent Balb/c mice. The administration of MCMV3–4 plasmids resulted in significant reduction in the copy number level of immediately early gene-1 DNA which is key to viral latency in mice, compared with the controls. Additionally, the innate immune DNA-sensing pathways of host might be involved in the induction of cytokine secretion such as type I interferon (IFN) (mainly IFN α and β) to fight against invading viruses. The result hinted that TALENs were able to provide an effective strategy to clear latent MCMV in animals [54].

#### **5. HCMV Vaccines**

The vaccines against HCMV are still being developed, no licensed vaccine is available so far. It is necessary to have a specific and strong antibody and cell-mediated immunity to confer protection against HCMV primary infection through the analysis of the immune response to HCMV. Many efforts have been made to produce an HCMV vaccine for years, but a successful vaccine candidate has not yet to be developed, probably due to what immune responses needed for protecting against HCMV infections are still poorly understood [36]. To develop an effective HCMV vaccine, immune responses required to fight against HCMV and how to enhance these specific immune responses requires further study.

Choi et al. used the guinea pig which is a small-animal model to develop a CMV vaccine. A glycoprotein pentamer complex encoded by guinea pig cytomegalovirus (GPCMV) is essential for viral entry into non-fibroblast cells to enable congenital CMV [55]. Like HCMV, GPCMV needs a guinea pig specific cell receptor (platelet-derived growth factor receptor α) for fibroblast entry, but other receptors are required for non-fibroblast cells. A disabled infectious GPCMV vaccine strain induced humoral immune responses against viral pentamers to promote neutralization on non-fibroblast cells; thus, the

vaccinated guinea pigs were protected from congenital CMV infections. The design including the pentamer complex as a part of vaccines may significantly enhance efficacy. This new finding lays stress on the importance of the immune response to the pentamer complex in contributing to the protection against congenital CMV and has opened a new era for the development of CMV vaccines [55].

Liu et al. hypothesized that a vaccine candidate able to elicit immune responses analogous to those of HCMV-seropositive subjects may confer protection against congenital HCMV [56]. The V160 vaccine has been shown to be safe and immunogenic in HCMV-seronegative humans, inducing both humoral and cell-mediated immune responses. In this study, they further demonstrated that sera from V160-immunized HCMV-seronegative subjects had similar quality attributes to those from seropositive subjects, including high avidity antibodies to viral antigens. This vaccine is a promising candidate against HCMV, but further evaluation in clinics for the prevention of congenital HCMV is required to warrant its safety, efficiency, and effectiveness [56].
