**3. Advantages of waterborne HEVNP for vaccine development**

As mentioned above, 30 dimers of the truncated CP of HEV will spontaneously self-assemble into HEVNP following heterologous expression in insect cells or bacteria. Unlike HEV, HEVNP does not encapsulate genomic RNA and is, thus, incapable of replication. HEVNP is, however, capable of encapsulating foreign RNA or DNA. As a vaccine delivery vehicle, HEVNP possesses a combination of advantageous characteristics including surface plasticity, stability within the harsh conditions of the GI tract, significant payload capacity, and platform sustainability.

#### **3.1 Surface plasticity**

The utility of the HEVNP as a mucosa-penetrating vaccine delivery platform was successful demonstrated by the development of an orally administered HEVNPbased HIV vaccine [21]. In this groundbreaking study, a 15-amino-acid-long peptide from the V3 loop of HIV-1 gp120 (called P18) was genetically inserted on the surface of HEVNP, generating the HEVNP-P18 construct (also known as 18-VLP). This insertion was successfully made after residue Y485 of the truncated CP, a location that is within the antibody-binding site of HEVNP. Cryo-EM studies revealed that the HEVNP capsid maintained its icosahedral shape and was not disrupted by the P18 insertion. The successful insertion after Y485 resulted in fully formed, stable HEVNP. In contrast, attempts of insertion after aa residues A179, R366, A507, and R542 all failed to achieve the quaternary assembly of HEVNP. Clearly, surface modification of HEVNP via modulation at Y485 by peptide insertion does not interfere with capsid stability or the formation of T = 1 icosahedral organization.

Following the successful insertion of P18 after Y485, four additional aa residues (T489, S533, N573, and T586) have been identified as targets for modulating the surface of HEVNP [30]. These sites are found within four surface-accessible loops (L1, aa 483–491; L2, aa 530–535; L3, aa 554–561; and L4, aa 582–593) that are found on the P domain (**Figure 1**). These sites (as well as Y485) were identified based on (i) their three-dimensional localization on the surface of HEVNP and (ii) the likelihood that mutation would result in minimal or no distortion of the HEVNP structure. In order to test the hypotheses generated by the structural analyses regarding these sites, site-directed mutagenesis was performed in order to replace these residues with a cysteine residue. All of the cysteine mutation constructs successfully assembled into stable icosahedral capsids and were subjected to surface modulation through covalent chemical conjugation. The conjugations were performed via a cysteine acylation reaction with maleimide-linked biotin, and the conjugation efficiency at each site was determined using labeled streptavidin. Of the mutations that were generated (HEVNP-485C, HEVNP-489C, HEVNP-533C, HEVNP-573C, and HEVNP-586C), the HEVNP-573C construct showed the greatest streptavidin signal. This indicated that the N573C mutation of HEVNP-573C is the most surface-visible site for modulation. More recent structural analysis has identified additional aa residues that are found on the P domain (aa residues 510–514 and 520–525) as well as the M domain (residues 342–344 and 402–408) that may be utilized as conjugation sites in a future study.

In order to demonstrate the functionality of the HEVNP-573C construct, a breast cancer cell-targeting ligand LXY30 [31] was chemically conjugated to HEVNP-573C in order to generate HEVNP-573C-LXY30 [30]. The HEVNP-573C-LXY30 construct

**125**

functionalization.

**Figure 2.**

**3.2 Gastrointestinal tract stability**

*BALB/c mice were injected with MDA-MB-231 cells (5 x 105*

*prior to degradation (B). Modified from Chen et al., 2016.*

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

selectively binds to cells in the breast cancer cell line MDA-MB-231 (**Figure 2A**). Furthermore, in vivo fluorescence microscopy demonstrates that HEVNP-573C-LYX30, unlike HEVNP-573C, is selectively delivered to breast cancer tumors (formed following the subcutaneous injection of MDA-MB-231 cells in female SPF BLAB/c mice) (**Figure 2B**). These findings demonstrate that HEVNP can be engineered for surface modulation by covalent attachment of a small molecule while maintaining the integrity of the capsid structure. HEVNP thus presents a unique platform for surface

*Selective binding and internalization of LXY30-tagged HEVNP. Breast cancer cells (MDA-MB-231) were inoculated with Cy5.5-labeled HEVNP (row H) or Cy5.5-labeled HEVNP tagged with LXY30 (row LXY). At 1 h post inoculation, the cells were visualized for nuclear dsDNA (DAPI) or Cy5.5. Cy5.5 staining is significantly higher in the cytoplasm of cells inoculated with HEVNP tagged with LXY30 (A). Female SPF* 

*arrows), 0.1 nmoles of Cy5.5-labeled HEVNP (row H) or Cy5.5-labeled HEVNP tagged with LXY30 (row LXY) was injected into the tail vein. Optical imaging of live mice at 1, 6, 24, and 48 h post injection (p.i.) showed that LXY30-tagged HEVNP selectively binds to the tumor at 1 h p.i. Staining was also seen in the abdominal regions at 1 and 6 h p.i., likely due to the accumulation of HEVNPs in the liver or other organs* 

*). Following the formation of tumors (white* 

The human GI tract is divided into the upper tract and lower tract with the upper tract consisting of the mouth, pharynx, esophagus, stomach, and first part of the small intestine (i.e., the duodenum) and the lower tract consisting of the remainder of the small intestine (i.e., jejunum and ileum) and large intestine [32]. Although the duodenum is the shortest portion of the small intestine, it is connected to and/

*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*

#### **Figure 2.**

*Synthetic Biology - New Interdisciplinary Science*

molecules, and immunogenic peptides.

**3.1 Surface plasticity**

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

As mentioned above, 30 dimers of the truncated CP of HEV will spontaneously self-assemble into HEVNP following heterologous expression in insect cells or bacteria. Unlike HEV, HEVNP does not encapsulate genomic RNA and is, thus, incapable of replication. HEVNP is, however, capable of encapsulating foreign RNA or DNA. As a vaccine delivery vehicle, HEVNP possesses a combination of advantageous characteristics including surface plasticity, stability within the harsh conditions of the GI tract, significant payload capacity, and platform sustainability.

The utility of the HEVNP as a mucosa-penetrating vaccine delivery platform was

successful demonstrated by the development of an orally administered HEVNPbased HIV vaccine [21]. In this groundbreaking study, a 15-amino-acid-long peptide from the V3 loop of HIV-1 gp120 (called P18) was genetically inserted on the surface of HEVNP, generating the HEVNP-P18 construct (also known as 18-VLP). This insertion was successfully made after residue Y485 of the truncated CP, a location that is within the antibody-binding site of HEVNP. Cryo-EM studies revealed that the HEVNP capsid maintained its icosahedral shape and was not disrupted by the P18 insertion. The successful insertion after Y485 resulted in fully formed, stable HEVNP. In contrast, attempts of insertion after aa residues A179, R366, A507, and R542 all failed to achieve the quaternary assembly of HEVNP. Clearly, surface modification of HEVNP via modulation at Y485 by peptide insertion does not interfere with capsid stability or the formation of T = 1 icosahedral organization. Following the successful insertion of P18 after Y485, four additional aa residues (T489, S533, N573, and T586) have been identified as targets for modulating the surface of HEVNP [30]. These sites are found within four surface-accessible loops (L1, aa 483–491; L2, aa 530–535; L3, aa 554–561; and L4, aa 582–593) that are found on the P domain (**Figure 1**). These sites (as well as Y485) were identified based on (i) their three-dimensional localization on the surface of HEVNP and (ii) the likelihood that mutation would result in minimal or no distortion of the HEVNP structure. In order to test the hypotheses generated by the structural analyses regarding these sites, site-directed mutagenesis was performed in order to replace these residues with a cysteine residue. All of the cysteine mutation constructs successfully assembled into stable icosahedral capsids and were subjected to surface modulation through covalent chemical conjugation. The conjugations were performed via a cysteine acylation reaction with maleimide-linked biotin, and the conjugation efficiency at each site was determined using labeled streptavidin. Of the mutations that were generated (HEVNP-485C, HEVNP-489C, HEVNP-533C, HEVNP-573C, and HEVNP-586C), the HEVNP-573C construct showed the greatest streptavidin signal. This indicated that the N573C mutation of HEVNP-573C is the most surface-visible site for modulation. More recent structural analysis has identified additional aa residues that are found on the P domain (aa residues 510–514 and 520–525) as well as the M domain (residues 342–344 and 402–408) that may be utilized as conjugation sites in a future study. In order to demonstrate the functionality of the HEVNP-573C construct, a breast cancer cell-targeting ligand LXY30 [31] was chemically conjugated to HEVNP-573C in order to generate HEVNP-573C-LXY30 [30]. The HEVNP-573C-LXY30 construct

**3. Advantages of waterborne HEVNP for vaccine development**

**124**

*Selective binding and internalization of LXY30-tagged HEVNP. Breast cancer cells (MDA-MB-231) were inoculated with Cy5.5-labeled HEVNP (row H) or Cy5.5-labeled HEVNP tagged with LXY30 (row LXY). At 1 h post inoculation, the cells were visualized for nuclear dsDNA (DAPI) or Cy5.5. Cy5.5 staining is significantly higher in the cytoplasm of cells inoculated with HEVNP tagged with LXY30 (A). Female SPF BALB/c mice were injected with MDA-MB-231 cells (5 x 105 ). Following the formation of tumors (white arrows), 0.1 nmoles of Cy5.5-labeled HEVNP (row H) or Cy5.5-labeled HEVNP tagged with LXY30 (row LXY) was injected into the tail vein. Optical imaging of live mice at 1, 6, 24, and 48 h post injection (p.i.) showed that LXY30-tagged HEVNP selectively binds to the tumor at 1 h p.i. Staining was also seen in the abdominal regions at 1 and 6 h p.i., likely due to the accumulation of HEVNPs in the liver or other organs prior to degradation (B). Modified from Chen et al., 2016.*

selectively binds to cells in the breast cancer cell line MDA-MB-231 (**Figure 2A**). Furthermore, in vivo fluorescence microscopy demonstrates that HEVNP-573C-LYX30, unlike HEVNP-573C, is selectively delivered to breast cancer tumors (formed following the subcutaneous injection of MDA-MB-231 cells in female SPF BLAB/c mice) (**Figure 2B**). These findings demonstrate that HEVNP can be engineered for surface modulation by covalent attachment of a small molecule while maintaining the integrity of the capsid structure. HEVNP thus presents a unique platform for surface functionalization.

#### **3.2 Gastrointestinal tract stability**

The human GI tract is divided into the upper tract and lower tract with the upper tract consisting of the mouth, pharynx, esophagus, stomach, and first part of the small intestine (i.e., the duodenum) and the lower tract consisting of the remainder of the small intestine (i.e., jejunum and ileum) and large intestine [32]. Although the duodenum is the shortest portion of the small intestine, it is connected to and/

or associated with the liver, gallbladder, and pancreas through various ducts, veins, and arteries. Following ingestion of HEV, the virus capsid will be exposed to extreme conditions including highly acidic and then alkaline pH, a wide range of digestive enzymes, bile, bacteria and other microorganisms, thick mucosal layers, and mucosal flow throughout the 5-plus-meter length of the human digestive tract. At the cellular level, the inner surface of the intestines is lined with a layer of several types of simple columnar cells including villi and goblet cells that face the lumen. The villi and goblet cells are primarily involved in the absorption of digested nutrients and secretion of a thick (ca. 200 μm) layer of mucosa composed primarily of mucin, respectively. Microfold cells (M cells) are also found in the intestines which play important roles in the initiation of mucosal immunity and the transport of antigens across the epithelial cell layer. HEV has evolved to efficiently overcome these barriers (chemical, enzymatic, mechanical, physical, immunological, etc.) and to eventually initiate a productive infection of cells of the liver and other tissues. Although HEVNP is unable to replicate, it retains the inherent ability of HEV to efficiently target and deliver therapeutic agents through the GI tract with little or no toxicity [1, 2]. This ability of HEVNP to deliver, through oral dosing, a therapeutic payload in a targeted manner using a modular format is currently unavailable through other nanocarrier platforms. Additionally, HEVNP is highly stable to long-term storage at room temperature. Thus, the need for a temperature-controlled supply chain for storage and distribution of HEVNP is minimized or eliminated. This makes the storage and distribution of HEVNP significantly less difficult especially in less developed regions. In addition to being stable at room temperature, HEVNP is water soluble and can be formulated as a liquid (as well as a cream, powder, or solid) which allows HEVNP to be administered noninvasively as a drink or droplet.

## **3.3 Significant payload capacity**

HEVNP functions as an epitope nanocarrier through display of the epitope on its surface. HEVNP also has a large hollow core with a width that ranges from approximately 10 to 12 nm (**Figure 3A**) that can be loaded with a payload such as a nucleic acid chain, peptide, or small molecule. This large hollow core results from the space within HEVNP that in HEV encapsulates genomic RNA. A payload can be encapsulated within the hollow core of HEVNP using a simple process that reversibly disassembles HEVNP and then reassembles it in the presence of the payload molecules. This reversible process occurs through chemical reduction, chelation of Ca2+, and the subsequent return of Ca2+ (**Figure 3B**). Specifically, HEVNP disassembles in the presence of DTT and EGTA and reassembles by the slow addition of Ca2+. If peptide molecules such as insulin or inorganic molecules such as ferrite are present during the reassembly process, these molecules are encapsulated within the reassembled HEVNP. Similarly, in the presence of DNA or RNA, the reassembled HEVNP will incorporate the nucleic acid molecule, and HEVNP can function as an orally deliverable DNA vaccine nanocarrier (reviewed in [2]). For example, plasmid DNA-encoding HIV envelope gp120 has been encapsulated by HEV VLP, and this construct has been used to orally deliver the plasmid to the spleen, Peyer's patches, and mesenteric lymph nodes of mice [33]. Cell-mediated immune (specific cytotoxic T-lymphocyte (CTL) response) and specific humoral responses are generated locally and systemically. A payload is not essential for HEVNP capsid formation or capsid stability, but having this capacity offers a way to further increase the epitope signal beyond the 60 epitope copies that can be placed through chemical conjugation or genetic insertion on the HEVNP surface via the P domains.

**127**

**Figure 3.**

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

**3.4 Platform sustainability by prevention of self-immunity**

*reassembly following the addition of calcium (B).*

While HEVNP exhibits natural tolerance against the harsh enzymatic environment associated with the digestive tract, its repeated use as a drug delivery vehicle will quickly result in self-immunity if a mechanism is not in place to avoid this common problem. As discussed above, insertion of the HIV-1 P18 peptide onto the surface P domain maintains the icosahedral arrangement of P18-HEVNP and indicates that intermolecular forces between the truncated CP of the recombinant nucleocapsid are not disrupted by the insertion of P18. Additionally, since the antigenicity of HEVNP lies specifically within the P domain, the insertion of the P18 peptide significantly lowers immune detection of the HEVNP vehicle [21] (**Figure 4A**). The immune reactivity of P18-HEVNP has been tested by two antibodies, 447-52D and HEP224. Antibody 447-52D specifically targets the V3 loop of HIV-1 gp120, and monoclonal antibody HEP224 targets the conformational epitope (i.e., the three loops around Y485) of the P domain of HEV CP. Based on ELISA experiments, antibody 447-52D shows preferential binding of P18-HEVNP. On the other hand, the binding of antibody HEP224 to the conformational epitope of the P domain of HEV CP is disrupted by the insertion of HIV P18 without altering the structural characteristic of HEVNP. Thus, insertion of specific peptides into the exposed P domain serves as a practical strategy to escape antibody recognition by the immune system (i.e., issues with self-immunity) while triggering the desired humoral and cellular responses against the attached/inserted antigen. Similarly, an HEV-specific monoclonal antibody, Fab230, fails to recognize HEVNP after maleimide-biotin conjugation at position N573C of HEVNP [30] (**Figure 4B**). Additionally, the geometrical constraints provided by the M domain provide a

*Structure and disassembly/reassembly of HEVNP. The HEVNP monomer is composed of three domains: Shell (S), middle (M), and protrusion (P). The surface and interior localization of these domains is indicated by the color map (A). A large hollow core is found in the interior of HEVNP (right image in A). The hollow core can encapsulate various payloads such as nucleic acids, peptides, small proteins, or small molecules. Electron microscopic images of the process of HEVNP disassembly (following the addition of EGTA and DTT) and* 

*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*

#### **Figure 3.**

*Synthetic Biology - New Interdisciplinary Science*

or associated with the liver, gallbladder, and pancreas through various ducts, veins, and arteries. Following ingestion of HEV, the virus capsid will be exposed to extreme conditions including highly acidic and then alkaline pH, a wide range of digestive enzymes, bile, bacteria and other microorganisms, thick mucosal layers, and mucosal flow throughout the 5-plus-meter length of the human digestive tract. At the cellular level, the inner surface of the intestines is lined with a layer of several types of simple columnar cells including villi and goblet cells that face the lumen. The villi and goblet cells are primarily involved in the absorption of digested nutrients and secretion of a thick (ca. 200 μm) layer of mucosa composed primarily of mucin, respectively. Microfold cells (M cells) are also found in the intestines which play important roles in the initiation of mucosal immunity and the transport of antigens across the epithelial cell layer. HEV has evolved to efficiently overcome these barriers (chemical, enzymatic, mechanical, physical, immunological, etc.) and to eventually initiate a productive infection of cells of the liver and other tissues. Although HEVNP is unable to replicate, it retains the inherent ability of HEV to efficiently target and deliver therapeutic agents through the GI tract with little or no toxicity [1, 2]. This ability of HEVNP to deliver, through oral dosing, a therapeutic payload in a targeted manner using a modular format is currently unavailable through other nanocarrier platforms. Additionally, HEVNP is highly stable to long-term storage at room temperature. Thus, the need for a temperature-controlled supply chain for storage and distribution of HEVNP is minimized or eliminated. This makes the storage and distribution of HEVNP significantly less difficult especially in less developed regions. In addition to being stable at room temperature, HEVNP is water soluble and can be formulated as a liquid (as well as a cream, powder, or solid) which allows HEVNP to be administered noninvasively as a

HEVNP functions as an epitope nanocarrier through display of the epitope on its surface. HEVNP also has a large hollow core with a width that ranges from approximately 10 to 12 nm (**Figure 3A**) that can be loaded with a payload such as a nucleic acid chain, peptide, or small molecule. This large hollow core results from the space within HEVNP that in HEV encapsulates genomic RNA. A payload can be encapsulated within the hollow core of HEVNP using a simple process that reversibly disassembles HEVNP and then reassembles it in the presence of the payload molecules. This reversible process occurs through chemical reduction, chelation of Ca2+, and the subsequent return of Ca2+ (**Figure 3B**). Specifically, HEVNP disassembles in the presence of DTT and EGTA and reassembles by the slow addition of Ca2+. If peptide molecules such as insulin or inorganic molecules such as ferrite are present during the reassembly process, these molecules are encapsulated within the reassembled HEVNP. Similarly, in the presence of DNA or RNA, the reassembled HEVNP will incorporate the nucleic acid molecule, and HEVNP can function as an orally deliverable DNA vaccine nanocarrier (reviewed in [2]). For example, plasmid DNA-encoding HIV envelope gp120 has been encapsulated by HEV VLP, and this construct has been used to orally deliver the plasmid to the spleen, Peyer's patches, and mesenteric lymph nodes of mice [33]. Cell-mediated immune (specific cytotoxic T-lymphocyte (CTL) response) and specific humoral responses are generated locally and systemically. A payload is not essential for HEVNP capsid formation or capsid stability, but having this capacity offers a way to further increase the epitope signal beyond the 60 epitope copies that can be placed through chemical conjuga-

tion or genetic insertion on the HEVNP surface via the P domains.

**126**

drink or droplet.

**3.3 Significant payload capacity**

*Structure and disassembly/reassembly of HEVNP. The HEVNP monomer is composed of three domains: Shell (S), middle (M), and protrusion (P). The surface and interior localization of these domains is indicated by the color map (A). A large hollow core is found in the interior of HEVNP (right image in A). The hollow core can encapsulate various payloads such as nucleic acids, peptides, small proteins, or small molecules. Electron microscopic images of the process of HEVNP disassembly (following the addition of EGTA and DTT) and reassembly following the addition of calcium (B).*

#### **3.4 Platform sustainability by prevention of self-immunity**

While HEVNP exhibits natural tolerance against the harsh enzymatic environment associated with the digestive tract, its repeated use as a drug delivery vehicle will quickly result in self-immunity if a mechanism is not in place to avoid this common problem. As discussed above, insertion of the HIV-1 P18 peptide onto the surface P domain maintains the icosahedral arrangement of P18-HEVNP and indicates that intermolecular forces between the truncated CP of the recombinant nucleocapsid are not disrupted by the insertion of P18. Additionally, since the antigenicity of HEVNP lies specifically within the P domain, the insertion of the P18 peptide significantly lowers immune detection of the HEVNP vehicle [21] (**Figure 4A**). The immune reactivity of P18-HEVNP has been tested by two antibodies, 447-52D and HEP224. Antibody 447-52D specifically targets the V3 loop of HIV-1 gp120, and monoclonal antibody HEP224 targets the conformational epitope (i.e., the three loops around Y485) of the P domain of HEV CP. Based on ELISA experiments, antibody 447-52D shows preferential binding of P18-HEVNP. On the other hand, the binding of antibody HEP224 to the conformational epitope of the P domain of HEV CP is disrupted by the insertion of HIV P18 without altering the structural characteristic of HEVNP. Thus, insertion of specific peptides into the exposed P domain serves as a practical strategy to escape antibody recognition by the immune system (i.e., issues with self-immunity) while triggering the desired humoral and cellular responses against the attached/inserted antigen. Similarly, an HEV-specific monoclonal antibody, Fab230, fails to recognize HEVNP after maleimide-biotin conjugation at position N573C of HEVNP [30] (**Figure 4B**). Additionally, the geometrical constraints provided by the M domain provide a

**Figure 4.**

*3-D modeling of HEVNP surface modulation. Surface modulation through chemical conjugation or genetic modification promotes the escape of HEVNP from immune surveillance (A). Surface modulation of HEVNP with maleimide-biotin or mutation of the P domain at residue N573 dramatically reduces cross reactivity with the HEV-specific monoclonal antibody fab230 by ELISA (B).*

physical barrier for antibody binding which helps HEVNP avoid immune system surveillance by HEV-specific antibodies. These findings show the sustainability of the HEVNP nanocarrier platform.
