A supramolecular system mimicking the infection process of an enveloped virus through membrane fusion.

Membrane fusion is an essential step for the entry of enveloped viruses, such as human immunodeficiency virus and influenza virus, into the host cell, often triggered by the binding of membrane proteins on the viral envelope to host cell membrane. Recently, external stimuli was shown to trigger membrane fusion in an artificial system. Direct observation of artificial membrane fusion using a giant unilamellar vesicle (GUV), which is similar in size to a cell, is useful as a biological model system. However, there are no model systems for studying membrane fusion of enveloped viruses with host cells. Here, we report a supramolecular model system for viral entry into a GUV or cell through membrane fusion. The system was constructed by complexing a cationic lipid bilayer on an anionic artificial viral capsid, self-assembled from viral ß-annulus peptides. We demonstrate that the cationic enveloped artificial viral capsid electrostatically interacts with the anionic GUV or cell, and the capsid enters the GUV or cell through membrane fusion. The model system established in this study will be important for analyzing membrane fusion during infection of a natural virus.

Antigen/Adjuvant-Displaying Enveloped Viral Replica as a Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidate.

We report a promising cancer vaccine candidate comprising antigen/adjuvant-displaying enveloped viral replica as a novel vaccine platform. The artificial viral capsid, which consists of a self-assembled ß-annulus peptide conjugated with an HER2-derived antigenic CH401 peptide, was enveloped within a lipid bilayer containing the lipidic adjuvant α-GalCer. The use of an artificial viral capsid as a scaffold enabled precise control of its size to ∼100 nm, which is generally considered to be optimal for delivery to lymph nodes. The encapsulation of the anionically charged capsid by a cationic lipid bilayer dramatically improved its stability and converted its surface charge to cationic, enhancing its uptake by dendritic cells. The developed CH401/α-GalCer-displaying enveloped viral replica exhibited remarkable antibody-production activity. This study represents a pioneering example of precise vaccine design through bottom-up construction and opens new avenues for the development of effective vaccines.

Anticancer Activity of Reconstituted Ribonuclease S-Decorated Artificial Viral Capsid.

Ribonuclease S (RNase S) is an enzyme that exhibits anticancer activity by degrading RNAs within cancer cells; however, the cellular uptake efficiency is low due to its small molecular size. Here we generated RNase S-decorated artificial viral capsids with a size of 70-170 nm by self-assembly of the ß-annulus-S-peptide followed by reconstitution with S-protein at neutral pH. The RNase S-decorated artificial viral capsids are efficiently taken up by HepG2 cells and exhibit higher RNA degradation activity in cells compared with RNase S alone. Cell viability assays revealed that RNase S-decorated capsids have high anticancer activity comparable to that of standard anticancer drugs.

Embedding a membrane protein into an enveloped artificial viral replica.

Natural enveloped viruses, in which nucleocapsids are covered with lipid bilayers, contain membrane proteins on the outer surface that are involved in diverse functions, such as adhesion and infection of host cells. Previously, we constructed an enveloped artificial viral capsid through the complexation of cationic lipid bilayers onto an anionic artificial viral capsid self-assembled from ß-annulus peptides. Here we demonstrate the embedding of the membrane protein Connexin-43 (Cx43), on the enveloped artificial viral capsid using a cell-free expression system. The expression of Cx43 in the presence of the enveloped artificial viral capsid was confirmed by western blot analysis. The embedding of Cx43 on the envelope was evaluated by detection via the anti-Cx43 antibody, using fluorescence correlation spectroscopy (FCS) and transmission electron microscopy (TEM). Interestingly, many spherical structures connected to each other were observed in TEM images of the Cx43-embedded enveloped viral replica. In addition, it was shown that fluorescent dyes could be selectively transported from Cx43-embedded enveloped viral replicas into Cx43-expressing HepG2 cells. This study provides a proof of concept for the creation of multimolecular crowding complexes, that is, an enveloped artificial viral replica embedded with membrane proteins.

Artificial Nanocage Formed via Self-Assembly of ß-Annulus Peptide for Delivering Biofunctional Proteins into Cell Interiors.

Nanocarriers that deliver functional proteins to cell interiors are an attractive platform for the intracellular delivery of intact proteins without further modification, with in vivo compatibility. Development of efficient methods for cargo protein encapsulation and release in recipient cell cytosol is needed. Herein, we assess the feasibility of the abovementioned requirements using a protein nanocage (artificial nanocage) without compromising the structure and functions of the original protein and allowing for design flexibility of the surfaces and interiors. The protein nanocage formed via the self-assembly of the ß-annulus peptide (24-amino acid peptide) in water was used as a model framework. The nitrilotriacetic acid moiety was displayed on the nanocage lumen for effective encapsulation of hexahistidine-tagged proteins in the presence of Ni2+, and the amphiphilic cationic lytic peptide HAad was displayed on a nanocage surface to attain cell permeability. Successful intracellular delivery of cargo proteins and targeting of cytosolic proteins by a nanobody were achieved, indicating the validity of the approach employed in this study.

Immunological Evaluation of Co-Assembling a Lipidated Peptide Antigen and Lipophilic Adjuvants: Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidates.

Co-assembling vaccines composed of a lipidated HER2-derived antigenic CH401 peptide and either a lipophilic adjuvant, Pam3 CSK4 , α-GalCer, or lipid A 506, were evaluated as breast cancer vaccine candidates. This vaccine design was aimed to inherit both antigen multivalency and antigen-specific immunostimulation properties, observed in reported self-adjuvanting vaccine candidates, by using self-assembly and adjuvant-conjugated antigens. Under vaccination concentrations, respective lipophilic adjuvants underwent co-assembly with lipidated CH401, which boosted the anti-CH401 IgG and IgM production. In particular, α-GalCer was responsible for the most significant immune activation. Therefore, the newly developed vaccine design enabled the optimization of adjuvants against the antigenic CH401 peptide in a simple preparatory manner. Overall, the co-assembling vaccine design opens the door for efficient and practical self-adjuvanting vaccine development.

Enveloped artificial viral capsids self-assembled from anionic ß-annulus peptide and cationic lipid bilayer.

Anionic artificial viral capsids were self-assembled from ß-annulus-EE peptide, then complexed with lipid-bilayer-containing cationic lipids via electrostatic interaction to form enveloped artificial viral capsids. The critical aggregation concentration of the enveloped artificial viral capsid was significantly lower than that of the uncomplexed artificial viral capsid, indicating that the lipid bilayer stabilised the capsid structure.

Super-low-frequency wireless power transfer with lightweight coils for passing through a stainless steel plate.

To achieve wireless power transfer (WPT) through a stainless-steel plate, a super-low frequency (SLF) was used as a resonance frequency. In our previous study of SLF-WPT, heavy coils were prepared. In this study, we designed lightweight coils using a WPT simulator that we developed previously. As a result, the weight was reduced to 1.69 kg from 11.9 kg, the previous coil weight. At a resonance frequency of 400 Hz, the transmission efficiency and output power of advanced SLF-WPT reached 91% and 426 W, respectively, over a transmission distance of 30 mm. Furthermore, 80% efficiency and 317 W output were achieved when transmitting power through a 1 mm-thick stainless-steel plate. This performance is much better than that in previous reports. We show using both calculations and experimental results that a power-to-weight ratio of 252 W/kg is possible even when using a 400 Hz power supply frequency.