**3. HCMV Antigens as Vaccine Candidates**

HCMV is the largest virus infecting humans, with a genome containing a linear double-stranded DNA of about 230 kb, from which at least 170 open reading frames (ORFs) have been identified [63,64]. The virus contains over 30 structural proteins and encodes at least 20 membrane glycoproteins expressed on the virion envelope, which are involved in immune evasion, cell attachment, and viral entry. HCMV exhibits an extremely broad cellular tropism being able to infect different cell types ranging from epithelial, endothelial, fibroblasts, myeloid hematopoietic precursors, monocytes/macrophages to smooth muscle cells, stromal cells, neurons, astrocytes, hepatocytes and glial cells [65]. Identification of the host cellular receptors has been a subject of debate during the last decade. It is now accepted that the two main host cell receptors for HCMV involve the platelet-derived growth factor receptor α (PDGFRα) [66–68] and Neuropilin 2 (Nrp2) [55]. In addition, other cellular host factors were described as facilitators of viral entry such as the olfactory receptor family member OR14I1 [69], the adipocyte plasma membrane-associated protein (APMAP) [70], CD46 [71], and CD147 [72]. However, the biological relevance of this multitude of receptors remains to be understood. In contrast, the viral ligands responsible for the broad cellular tropism were clearly identified and correspond to two viral envelope complexes: the gHgLgO (trimer) and the gHgLpUL128pUL130pUL131A (pentamer) (Figure 1) [73]. The trimer is a heterocomplex, in which the heterodimer consisting of gH (UL75) and gL (UL115) is disulfide-linked to glycoprotein O (gO), a heavily N-glycosylated polypeptide encoded by UL74 [74–76]. The trimer complex binds PDGFRα and is required for entry in all cell types [77], representing an attractive target for vaccine design. However, the gO sequence heterogeneity and the large glycan shield present on this subunit are impairing the generation of potent neutralizing mAbs. Along with others, we speculate that these issues will probably slow down possible efforts to use the trimer as a vaccine candidate [78]. The pentamer complex is composed of the gHgL heterodimer bound to three additional glycoproteins encoded by pUL128, pUL130, and pUL131A. The pentamer complex is required for viral entry in epithelial, endothelial, and myeloid cells and binds Nrp2 [55,65,79]. It represents the main target of HCMV neutralizing antibodies, eliciting a protective response targeting the pULs subunits, which is several orders of magnitude more potent than any other response against HCMV glycoprotein [51,52,80]. However, anti-pULs mAbs are not able to block infection in all cell types, in contrast to mAbs directed against gH and gB subunits [81]. Indeed, membrane fusion and cell entry in all cell types is dictated by the core fusion machinery composed of gHgL and the glycoprotein B (gB) which are thus considered as primary targets for vaccine development [2]. gB is a class III viral fusion protein that forms homotrimers (Figure 1) and was initially reported to bind cell–surface proteins such as PDGFRα and EGFR. However, it is more likely that gB functions as a viral fusogen that is triggered when viral and host membranes are in close proximity [82]. In addition to the HCMV membrane glycoproteins, other viral antigens have been considered as potential vaccine targets. The phosphoprotein 65 (pp65) encoded by pUL83 and the immediate-early protein 1 (IE1) encoded by pUL123, both eliciting a robust T cell response, have undergone evaluation as subunit or vectored vaccines in human [83,84]. In conclusion, the aforementioned vaccine candidates failed to confer an appropriate protection from infection, viral reinfection or reactivation. We believe that the development of a protective vaccine should include antigens from the viral envelope to trigger the humoral response (mainly pentamer and newly designed gB) and antigens from the tegument or proteins that are specifically associated with latency (i.e., US28) to prompt the cellular response able to control viral reactivation.

**Figure 1.** Structural representation of HCMV glycoproteins. Shown is the trimer, composed of the gHgL heterodimer and gO. Representation prepared with PDB: 5voc and EMD-3391. The pentamer is composed of the gHgL heterodimer and the three additional subunits pUL128, pUL130, and pUL131A. Representation prepared with PDB: 5voc. The gB homotrimer in its postfusion conformation is shown based on PDB: 5cxf. The figure was prepared using PyMOL software (The PyMOL Molecular Graphics System, Version 4.5 Schrödinger, LLC).
