*4.4. Q*β *Nanoparticle*

The bacteriophage Qβ is a member of the *Leviviridae* family, forming 30 nm icosahedral nanoparticles of 180 copies from the 14 kDa coat protein [105]. The coat protein monomer (Figure 4A) forms Qβ dimers (Figure 4B) that further assemble into pentamers and hexamers by disulfide and hydrogen bond interactions to form a Qβ nanoparticle under physiological conditions (Figure 4C). This nanoparticle is highly stable and is used to display ligands and proteins on its exterior surface [106,107]. Co-expression of Qβ proteins displaying different antigens of interest or engineered Qβ proteins displaying functionalized groups for subsequent direct antigen conjugation can be easily produced in bacterial cells. Of note, the genetic fusion occurs at the C-terminus of the protein, a property that might impair the correct folding of some antigens of interest. Genes encoding the viral Qβ protein have been also successfully expressed in yeast cells but the possibility to generate a Qβ nanoparticle in prokaryotic hosts, maintaining therefore a low production cost, will be of great interest for the generation of a personalized T cell vaccine targeting the proteins associated to latency. Regarding mammalian cell expression, there is currently no evidence that the system is also compatible with it, which is a prerequisite for the production of most of the HCMV glycoprotein antigens.

**Figure 4.** Structure and assembly of Qβ nanoparticle. Qβ protein is depicted as a monomer (**A**) and noncovalent dimer (**B**). (**C**) The fully self-assembled Qβ nanoparticle consists of 20 hexamers and 12 pentamers. The figure was prepared using 1qbe PDB structure and PyMOL software (The PyMOL Molecular Graphics System, Version 4.5 Schrödinger, LLC).

#### *4.5. De Novo Design-Based Nanoparticle*

Structure-based design of nanoparticle immunogens has been limited by the restricted number of scaffolds available and the fact that their physico-chemical properties are fixed (i.e., ferritin, encapsulins). Moreover, most of the self-assembling scaffolds spontaneously do so upon expression in the transfected host cell, a property that might lead to poor production yield. These constraints have pushed the exploration of new structural and functional geometries (i.e., icosahedron, dodecahedron) in the nanoparticle immunogen design field. Recent computational methods for

designing novel self-assembling proteins with atomic-level accuracy offer the possibility to design self-assembling proteins with customized structures, offering new opportunities for structure-based vaccine design [108–110]. For instance, the HIV gp41 ectodomain trimer was displayed onto the I3–01 particle [108,111]. The gp41 antigen exposed on NPs, stimulated mAb-expressing B cells more effectively than the soluble trimers [111]. Two additional publications highlighted the potential of this de novo self-assembling nanoparticle for antigen display. The I5350 nanoparticle (Figure 5) was used to generate an enhanced vaccine against the respiratory syncytial virus (RSV). Self-assembling protein nanoparticles displaying 20 copies of the stabilized version of the RSV fusion glycoprotein trimer (DS-Cav1) induced a neutralizing antibody response ∼10-fold higher than soluble trimeric DS-Cav1 [112]. In the second case, native-like HIV-1 envelope trimer antigens were displayed in a multivalent fashion on the I5350 nanoparticle scaffold and immunization studies revealed a more effective priming compared to the soluble SOSIP trimers [113]. SOSIP corresponds to a stabilized trimer with mutations that cross-link the cleaved gp120 and gp41. In contrast, the natural HIV trimer is comprised of three copies of a non-covalently linked gp120/gp41 heterodimer arising from cleavage of the viral gp160 precursor protein. It can be speculated that de novo design-based nanoparticles will have an increasing impact on next generation vaccines since they are customizable at will, supported by a multitude of expression platforms and able to generate multivalent display as well as encapsulation of small proteins or compounds [114,115].

**Figure 5.** Self-assembly of the I5350 nanoparticle. (**A**) Polypeptide I53–50A.1NT1; shown in red is the N-terminus projecting outward, where antigens can be displayed. I53–50A.1NT1 naturally trimerizes forming a 3-fold symmetry axis. (**B**) The I53–50B.4PT1 protein (orange) assembles into a pentamer. (**C**) I53–50A.1NT1 trimer and I53–50B.4PT1 pentamer self-assemble as a nanoparticle. (**D**) Assembled nanoparticle formed by 20 trimers and 12 pentamers, the N-termini of I53–50A.1NT1 is shown in red, and the black triangle represents the 3-fold symmetry axis of each pair of trimers. The figure was prepared using PyMOL software (The PyMOL Molecular Graphics System, Version 4.5 Schrödinger, LLC).

Ideally, I5350 particles should present on their surface the pentamer and gB to generate a humoral response against the gH, gL, pULs, and gB molecules. Moreover, as I5350 nanoparticles are hollow, the in vitro assembly step with I53–50B.4PT1 could be performed in the presence of T-cell stimulating peptides (i.e., pp65, IE1 and US28) that would be encapsulated inside the particles. The resulting vaccine candidates should in this way stimulate both arms of the immune system, offering a maximal protection.
