**Acknowledgements**

*Synthetic Biology - New Interdisciplinary Science*

*respectively, right panel) to the P domain of HEVNP.*

induced by a whole cell vaccine.

**6. Conclusions**

**Figure 5.**

to surface-exposed loops L2 and L1, respectively. Analysis of the visibility of these epitopes by structural modeling (**Figure 5**) and by ELISA using VD1- or VD4 specific antibodies (data not shown) indicates that all three of these constructs are highly immunogenic, suggesting that the epitopes are authentically displayed on the surface of HEVNP. Furthermore, in preliminary animal experiments, anti-MOMP IgG levels in the serum of mice that are immunized by HEVNP-VD1/HEVNP-VD4 (prime and two boosts) are like those found following immunization with a whole *Chlamydia* cell vaccine. These findings are highly exciting; and we are currently investigating whether there is also a cell-mediated immune response induced by HEVNP-VD1/HEVNP-VD4 and, if there is, how this response compares with that

*Chlamydial vaccine design. Peptide sequences from variable domain 1 (VD1, yellow epitopes) and/or VD4 (orange epitopes) from the major outer membrane protein of* Chlamydia *were chemically conjugated to amino acid residue N573C (left and center panels) or genetically inserted (after amino acid residues S533 and T485,* 

As a nanocarrier, HEVNP is a structure that can display multiple epitopes on its surface; and simultaneously it can deliver a payload, for example, an epitope encoding nucleotide sequence, peptide, or small molecule. Unlike nanoparticles generated from polymers, lipids, nanotubes, or other carriers, HEVNP delivers epitopes and payload through the mucosa of the GI tract without the need for any, potentially deleterious, exogenous enhancers such as a mucosal breakdown enzyme, pH regulator, or uptake cofactor. The key characteristics that make HEVNP an ideal and unique vehicle for vaccine delivery include: (i) *Surface plasticity*. Sites on the P domain can be engineered for site-specific attachment or insertion of the epitope(s). Even when the surface of HEVNP is genetically or chemically modified, the core structure of HEVNP remains intact. (ii) *GI tract stability*. Even when surface modified, HEVNP is stable to the harsh conditions of low pH and proteolytic enzymes that are found in the GI tract. This allows HEVNP to deliver epitopes orally. HEVNP has the capability to penetrate the mucosal lining of the entire GI tract and other mucosa-lined cavities or organs and directly target cells of the basement membrane. (iii) *Significant payload capacity*. The large hollow core of HEVNP can package and protect large biological molecules including DNA and RNA. (iv) *Platform sustainability*. Immune recognition of the carrier platform is negated with HEVNP. The surface P domain carries the primary antigenic sites of HEV (and HEVNP). Thus, modification of the P domain by chemical conjugation or genetic insertion of a vaccine epitope completely neutralizes endogenous immunogenicity

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This work was partially supported by grant from the National Institutes of Health (AI095382, EB21230, and CA198880) and National Institute of Food and Agriculture. RHC is a Finland distinguished professor.
