**2. Structure of hepatitis E virus capsid and HEVNP**

Significant effort has been invested in characterizing the structure of the capsid of HEV by biochemical methods, imaging (X-ray crystallography and

**123**

**Figure 1.**

*A Noninvasive, Orally Stable, Mucosa-Penetrating Polyvalent Vaccine Platform Based…*

cryogenic electron microscopy (cryo-EM)), and molecular biological techniques [15–22]. These studies have revealed the underlying architecture and biochemical composition of HEV (reviewed in [1, 2, 23, 24]). The authentic HEV is composed of 180 monomers of capsid protein (CP) that are assembled into an icosahedral cage in an RNA-dependent manner with a triangulation number of 3 (T = 3). Native HEV has a virion diameter of approximately 45 nm. When the native CP is truncated (leaving amino acid (aa) residues 112–608), this truncated CP forms a smaller particle with a diameter of approximately 27 nm. This structure, known as the HEV nanoparticle (HEVNP), is composed of 60 monomers (i.e., 30 CP dimers) of the truncated CP and forms a T = 1 icosahedral conformation. The CP is comprised of three domains: S (shell domain, aa 118–317), M (middle domain, aa 318–451), and P (protrusion domain, aa 452–606) (**Figure 1**). The S domain is the most conserved region among HEV genotypes and, along with the M domain, is responsible for the formation of the HEV capsid base [17, 19, 25]. The P domain, as the name suggests, protrudes from the capsid surface and plays a role in CP dimerization [18, 26], HEV capsid antigenicity [19, 27, 28], and recognition by the host cell receptor [29]. The M domain interacts strongly with the P domain through a long proline-rich hinge; however, the biological roles of the S, M, and P domains are independent [19, 22, 25]. This modular functionality allows the P domain to be genetically modified while (i) causing no or minimal effects on capsid formation and (ii) retaining capsid stability and resistance to acidic and proteolytic conditions found in the mammalian GI tract. Additionally, genetic modification of the P domain results in invisibility of the capsid to host immune surveillance as will be discussed below. Since the P domain of HEVNP is repeated

*Modular composition of HEVNP. HEVNP is formed by 30 homodimers of the HEVNP monomer. The dimer is the building block of HEVNP. The HEVNP monomer is composed of three domains: Shell (S), middle (M), and protrusion (P). The P domain has four surface-exposed loops (L1–L4) and a C-terminus that can be genetically or chemically (e.G., at N573C) modified in order to functionalize the HEVNP surface.*

*DOI: http://dx.doi.org/10.5772/intechopen.86830*

*A Noninvasive, Orally Stable, Mucosa-Penetrating Polyvalent Vaccine Platform Based… DOI: http://dx.doi.org/10.5772/intechopen.86830*

cryogenic electron microscopy (cryo-EM)), and molecular biological techniques [15–22]. These studies have revealed the underlying architecture and biochemical composition of HEV (reviewed in [1, 2, 23, 24]). The authentic HEV is composed of 180 monomers of capsid protein (CP) that are assembled into an icosahedral cage in an RNA-dependent manner with a triangulation number of 3 (T = 3). Native HEV has a virion diameter of approximately 45 nm. When the native CP is truncated (leaving amino acid (aa) residues 112–608), this truncated CP forms a smaller particle with a diameter of approximately 27 nm. This structure, known as the HEV nanoparticle (HEVNP), is composed of 60 monomers (i.e., 30 CP dimers) of the truncated CP and forms a T = 1 icosahedral conformation. The CP is comprised of three domains: S (shell domain, aa 118–317), M (middle domain, aa 318–451), and P (protrusion domain, aa 452–606) (**Figure 1**). The S domain is the most conserved region among HEV genotypes and, along with the M domain, is responsible for the formation of the HEV capsid base [17, 19, 25]. The P domain, as the name suggests, protrudes from the capsid surface and plays a role in CP dimerization [18, 26], HEV capsid antigenicity [19, 27, 28], and recognition by the host cell receptor [29]. The M domain interacts strongly with the P domain through a long proline-rich hinge; however, the biological roles of the S, M, and P domains are independent [19, 22, 25]. This modular functionality allows the P domain to be genetically modified while (i) causing no or minimal effects on capsid formation and (ii) retaining capsid stability and resistance to acidic and proteolytic conditions found in the mammalian GI tract. Additionally, genetic modification of the P domain results in invisibility of the capsid to host immune surveillance as will be discussed below. Since the P domain of HEVNP is repeated

#### **Figure 1.**

*Synthetic Biology - New Interdisciplinary Science*

developments and additionally show diminished toxicity in nontargeted cells and tissues. Despite these early successes, instability under physiological conditions, inefficient targeting, toxicity, and lack of bioavailability impose serious limitations

[3, 8, 9]. The size and exact composition of these nanocarriers are commonly altered and optimized based on their intended application. The key factors required for entry and distribution of theranostics include high degrees of bioavailability, the ability to withstand physiological conditions without degradation or premature exposure of the drug, and efficient distribution by overcoming the physical and

Evolutionarily, viruses adapt and coevolve with their host. Genetic engineering techniques and elucidation of viral structures have enabled virologist to generate empty capsids, called VLPs, which retain the physical characteristics of the capsid structure but lack the viral genome. VLPs thus exhibit the structural characteristics of the authentic virus but are incapable of replicating. In addition to being noninfectious, VLPs are generally nontoxic, biodegradable, and highly biocompatible. Structurally, the symmetrical configuration of VLPs allows them to be developed as nanocarrier systems that can entrap not only foreign nucleic acids but also peptides and imaging agents within their internal cavity. The exterior surface of VLPs, in some cases, can be tagged with targeting ligands without disruption of the VLP structure. The VLP assembles spontaneously and forms highly ordered structures following recombinant expression of the capsid protein (CP) in prokaryotic, eukaryotic, and cell-free protein expression systems. Currently, there are numerous ongoing VLP-based clinical trials worldwide [10–13]. From these clinical trials, a handful of VLP-based vaccines have been approved by the US FDA and other governmental regulatory agencies. For example, VLPs of hepatitis B virus (HBV), human papillomavirus (HPV), influenza virus, human parvovirus, and Norwalk virus have shown success in clinical trials or have been commercially developed as vaccines. The effectiveness of the delivery of therapeutic and/or diagnostic payloads using VLPs, as well as VLP surface modulation by attachment of ligands and tracking molecules, has been recently reviewed [10, 11, 14]. Here, a description of the key advantages and application of hepatitis E virus

While there are several routes of mucosal drug delivery, the oral and nasal routes are among the most safe and preferred by patients. The inherent characteristics of a nanocarrier such as structural composition, size, and natural stability play major roles in the potential success of a drug delivery system (reviewed in [1–4, 7–9]). For many nanocarrier platforms, problems with enzymatic degradation, limited penetration of the thick mucosal layer, and subsequently transportation of drugs through transcellular or paracellular routes are major shortcomings. The majority of currently available nanocarriers consist of simple structures that are on the nanometer to micrometer scale. Theranostic delivery vehicles that are currently used or considered for use fall into a handful of categories including polymers, lipids, solid-lipid carriers, gold carriers, nanotubes, immunostimulant complexes,

for the development of an effective mucosal delivery platform.

magnetic carriers, and virus-like particles (VLPs)

enzymatic barriers through noninvasive routes.

**1.2 Nanocarrier platform based on viral capsids**

nanoparticles for use in vaccine development will be discussed.

**2. Structure of hepatitis E virus capsid and HEVNP**

Significant effort has been invested in characterizing the structure of the capsid of HEV by biochemical methods, imaging (X-ray crystallography and

**122**

*Modular composition of HEVNP. HEVNP is formed by 30 homodimers of the HEVNP monomer. The dimer is the building block of HEVNP. The HEVNP monomer is composed of three domains: Shell (S), middle (M), and protrusion (P). The P domain has four surface-exposed loops (L1–L4) and a C-terminus that can be genetically or chemically (e.G., at N573C) modified in order to functionalize the HEVNP surface.*

60 times on the surface of the capsid, it provides high accessibility for surface modulations that may include targeting ligands, imaging molecules, tracking molecules, and immunogenic peptides.
