**3.5 Safety of HEVNP**

HEV annually causes acute and self-limiting infection in about 20 million people worldwide [34–36]. The majority of people infected with HEV show clinical symptoms that are relatively mild, and death rates from hepatitis E are low. The disease, however, is more severe in pregnant women, and chronic infection may occur in immunocompromised individuals. Although the exact mechanism of the increased severity of the disease during pregnancy is unknown, there is some evidence that increased viral replication in placental tissues plays a role [34, 35]. Thus, in a large proportion of the population, HEV is naturally a low-virulence pathogen. The low virulence of HEV and the inability of HEVNP to replicate (because it does not carry HEV genomic RNA) suggest that an HEVNP-based nanocarrier will not induce undue virulence in patients.

## **3.6 Established production and engineering technology**

A common eukaryotic cell-based technology for vaccine production utilizes recombinant baculoviruses and insect cells. Baculoviruses are arthropod-specific viruses that are commonly used to produce recombinant proteins for basic research and commercial applications. Baculoviruses have been successfully used to produce human therapeutics and diagnostics since the late 1990s [12]. A recent example of a baculovirus-based vaccine is Flublok (released in 2013 by Protein Sciences Corporation), a vaccine against human influenza virus. The baculovirus expression vector system is also used to express the major capsid protein L1 of human papilloma virus. The recombinant L1 capsid protein forms a VLP-based vaccine (Cervarix™) that protects against cervical cancer [12, 37]. The commercial GMP technology that is currently used to express and purify these vaccines and others can be easily adapted for the production of recombinant truncated CP and the engineering of HEVNP-based vaccine delivery nanocarriers.

**129**

*Chlamydia*.

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

**4. Single epitope modification of HEVNP: HIV-1 GP120 P18 example**

system response against HEVNP. When the P18-HEVNP construct is orally inoculated into mice, it induces strong and specific cell-mediated and humoral responses in comparison to immunization with HEVNP [21]. After three rounds of oral immunization, the cell-mediated response includes the lysis of cytotoxic T lymphocytes (CTLs) in three immune system-associated organs. Similarly, humoral responses (IgG, IgA, and IgM induction) in the sera and intestinal fluids are detected by ELISA. These responses were generated by P18-HEVNP without the

**5. Multiple epitope modifications of HEVNP: MOMP example**

Over 120 million people are annually infected with *Chlamydia trachomatis*. Because the initial stages of chlamydia are generally asymptomatic, many individuals are unaware that they are infected and do not seek antibiotic treatment. As the infection spreads, chronic abdominal pain, pelvic inflammatory disease, ectopic pregnancy, and infertility can result. The major outer membrane protein (MOMP) of *Chlamydia*, in its native trimeric form (nMOMP), has been demonstrated to impart significant protection against chlamydial infection and disease in a mouse model. MOMP is a structurally rigid, 40 kDa trimer-forming protein that makes up about 60% of the total mass of the outer membrane of *Chlamydia* [38, 39]. MOMP itself is characterized by five constant domains (CDs) and four variable domains (VDs) which help to define the immunogenicity of various serovars of *Chlamydia* [40, 41]. MOMP is the immunodominant antigen of *Chlamydia* and has multiple epitopes for T-cell and B-cell activation; thus, it induces both cell-mediated and humoral immunity [42–46]. Mice that are vaccinated with the nMOMP with Freund's adjuvant are significantly protected from the effects of *Chlamydia* in terms of a shedding assay and infertility [47]. In contrast, denatured MOMP does not offer this protection. The denatured MOMP, however, induces a greater humoral response than nMOMP. Robust protective activity following vaccination with nMOMP and other adjuvants has also been reported [45, 48]; this activity was similar to that of mice that were immunized intranasally with live *Chlamydia* elementary bodies (EBs). These and other studies [47, 49–52] using various readouts (i.e., body weight, lung weight, number of inclusions forming units recovered, length of shedding, etc.) demonstrate the important role of MOMP in inducing protection against

Formulating a vaccine with a properly folded membrane protein such as MOMP remains a genetic engineering challenge. The use of membrane proteins for vaccine applications requires a platform that can be engineered to enable proper folding of the membrane protein, potentially allow for adjuvant incorporation, and be amenable to the display of multiple epitopes employing multiple display strategies. In the past 10 years, incorporating membrane proteins into nanolipoprotein particles for both solubility and stabilization has become increasingly common with varied success. In our laboratory, HEVNP has been used as a platform to display two MOMP

VD epitope sequences (VD1 and VD4). With this construct, HEVNP-VD1/

HEVNP-VD4, the VD1 and VD4 peptide sequences were genetically incorporated at S533 and T485, respectively, of the truncated capsid protein, a region corresponding

As discussed earlier, insertion of the P18 peptide from the GP120 protein of HIV-1 results in a stable HEVNP that displays P18 on its surface (in the P domain after residue Y485). Additionally, the P18 insertion significantly lowers the immune

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

need for an external adjuvant coadministration.

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