Thuong Thi Ho

Porcine Epidemic Diarrhea (PED) has significantly impacted the swine farming industry in several countries worldwide, including Vietnam. Porcine Epidemic Diarrhea Virus (PEDV) has been proven to be the cause of PED. The COE and M proteins are potential candidates for subunit vaccine research against PEDV. The M protein, a largest envelope protein of PEDV, is highly conserved and plays a crucial role in virus assembly as well as in inducing the production of virus-neutralizing antibodies in the presence of complement. In a previous study, we constructed and expressed the M protein fused with the GCN4pII motif and Elastin-Like Polypeptide (M-pII-ELP) separately from the COE/G2a-pII protein in Nicotiana benthamiana. In this study, we assessed the co-expression of M-pII-ELP and COE/G2a-pII proteins in N. benthamiana using SDS-PAGE and Western blot. Next, we verified the assembly of virus-like particles (VLPs) by the M-pII-ELP protein alone through Transmission Electron Microscopy (TEM) analysis after ultracentrifugation with a sucrose gradient. Suitable buffers for the extraction and purification of M-pII-ELP protein using immobilized affinity chromatography (IMAC) were also selected. Results from SDS-PAGE and Western blot confirmed the co-expression of M-pII-ELP and COE/G2a-pII proteins in the plant; however, the expression of COE/G2a-pII protein was suppressed when co-expressed with M-pII-ELP. TEM analysis confirmed the formation of virus-like particles based on the assembly of the M-pII-ELP protein. Among the buffers tested for M-pII-ELP protein extraction, Tris-HCl buffer yielded the highest amount of M-pII-ELP protein. It was determined that the optimal imidazole concentrations for extraction and washing buffers in M-pII-ELP protein purification are 0 mM and 10 mM, respectively. These results lay the groundwork for further studies on developing plant-based subunit vaccines against PEDV.

Since 2003, H5N1 highly pathogenic avian influenza viruses (HPAIV) have not only caused outbreaks in poultry but were also transmitted to humans with high mortality rates. Vaccination is an efficient and economical means of increasing immunity against infections to decrease the shedding of infectious agents in immunized animals and to reduce the probability of further infections. Subunit vaccines from plants are the focus of modern vaccine developments. In this study, plant-made hemagglutinin (H5) trimers were purified from transiently transformed N. benthamiana plants. All chickens immunized with purified H5 trimers were fully protected against the severe HPAIV H5N1 challenge. We further developed a proof-of-principle approach by using disulfide bonds, homoantiparallel peptides or homodimer proteins to combine H5 trimers leading to production of H5 oligomers. Mice vaccinated with crude leaf extracts containing H5 oligomers induced neutralizing antibodies better than those induced by crude leaf extracts containing trimers. As a major result, eleven out of twelve chickens (92%) immunized with adjuvanted H5 oligomer crude extracts were protected from lethal disease while nine out of twelve chickens (75%) vaccinated with adjuvanted H5 trimer crude extracts survived. The solid protective immune response achieved by immunization with crude extracts and the stability of the oligomers form the basis for the development of inexpensive protective veterinary vaccines.

Developing new vaccine candidates is considered the best strategy for protecting poultry against artificial haemagglutinin (A/H5N1) strains. The transient expression system in plants has been a very efficient method for rapidly producing haemagglutinin-based recombinant vaccines. In this study, two novel artificial trimeric haemagglutinin constructs representing A/H5N1 strains that were detected in poultry from 2005 to 2015 in Vietnam, H5.c1 (representing all of the subclades 1.1, 1.1.1, and 1.1.2) and H5.c2 (representing all of the subclades 2.3.2.1, 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c), were designed for transient expression in Nicotiana benthamiana via agroinfiltration. However, only the H5.c1 protein, which showed the best expression and biofunction via the haemagglutination test, was selected for purification by immobilized metal ion affinity chromatography (IMAC). The trimeric structure of the IMAC-purified H5.c1 protein was well characterized by cross-linking reaction and size exclusion chromatography. An indirect ELISA and Western blot analysis of vaccinated mouse sera demonstrated that the H5.c1 protein strongly induced HA-specific Immunoglobulin G (IgG) immune responses. Notably, the H5.c1 protein induced strongly neutralizing antibodies against homologous H5.c1 protein and that of three heterologous native strains of clade, 1, 1.1, and 2.3.2.1c, in haemagglutination inhibition assays. Therefore, the plant-based artificial H5.c1 protein can be a promising vaccine candidate for conferring poultry resistance against A/H5N1 viruses in Vietnam.

The continuing spread of the newly emerged H7N9 virus among poultry in China, as well as the possibility of human-to-human transmission, has attracted numerous efforts to develop an effective vaccine against H7N9. The use of nanoparticles in vaccinology is inspired by the fact that most pathogens have a dimension within the nano-size range and therefore can be processed efficiently by the immune system, which leads to a potent immune response. Herein, we report a facile approach to increase antigen size to achieve not only fast but also effective responses against the recombinant HA/H7N9 protein via a simple conjugation of the protein onto the surface of nanodiamond particles. In this study, trimeric Haemagglutinin (H7) that is transiently expressed in N. benthamiana was purified using affinity chromatography, and its trimeric state was revealed successfully by the cross-linking reaction. The trimeric H7 solution was subsequently mixed with a nanodiamond suspension in different ratios. The successful conjugation of the trimeric H7 onto the surface of nanodiamond particles was demonstrated by the changes in size and Zeta-potential of the particles before and after protein coating, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western-blot analysis. Next, biofunction of the protein-nanodiamond conjugates was screened using a haemagglutination assay. A mixture containing 5 µg of trimeric H7 and 60 µg of nanodiamond corresponds to a ratio of 1:12 (w/w) of agglutinated chicken red blood cells at HA titer of 1024, which is 512-fold higher than the HA titer of free trimeric H7. After the 2nd and 3rd immunization in mice, ELISA and Western blot analyses demonstrated that the physical mixture of trimeric H7 protein and nanodiamond (1:12, w/w) elicited statistically significant stronger H7-specific-IgG response demonstrated by higher amounts of H7N9-specific IgG (over 15.4-fold with P < 0.05 after the second immunization). These results indicated a potential effect inherent to nanodiamond towards modulating immune systems, which should be further evaluated and broadly applied in nanovaccine development.

Plant-based transient expression is an alternative platform to produce hemagglutinin-based subunit vaccines. This production system provides not only fast and effective response in the context of a pandemic but also enables the supply of big volume vaccines at low cost. Crude plant extracts containing influenza hemagglutinin are considered to use as vaccine sources because of avoidance of related purification steps resulting in low cost production allowing veterinary applications. Highly immunogenic influenza hemagglutinins are urgently required to meet these pre-conditions. Here, we present a new and innovative way to generate functional H5 oligomers from avian flu hemagglutinin in planta by the specific interaction of S·Tag and S·Protein. A S·Tag was fused to H5 trimers and this construct was transiently co-expressed in planta with S·Protein-TPs which was multimerized by disulfide bonds via cysteine residues in tailpiece sequences (TP) of IgM antibody. Multimerized S·Protein-TPs serve as bridges/molecular docks to combine S·Tag-fused hemagglutinin trimers to form very large hemagglutinin H5 oligomers. H5 oligomers in the plant crude extract were highly active in hemagglutination resulting in high titers. Immunization of mice with two doses of plant crude extracts containing H5 oligomers after storage for 1 week at 4 °C caused strong immune responses and induced neutralizing specific humoral immune responses in mice. These results allow for the development of cheap influenza vaccines for veterinary application in future.