Plant crude extracts containing oligomeric hemagglutinins protect chickens against highly Pathogenic Avian Influenza Virus after one dose of immunization.

Highly pathogenic avian influenza viruses (HPAIV) have been responsible for causing several severe outbreaks across the world. To protect poultry farms and to prevent the possible spread of new influenza pandemics, vaccines that are both efficacious and low-cost are in high demand. We produced stable, large hemagglutinin H5 oligomers in planta by the specific interaction between S•Tag and S•Protein. H5 oligomers combined via S•Tag::S•Protein interaction in plant crude extracts induced strong humoral immune responses, strong neutralizing antibody responses, and resistance in chickens after challenge with a wild type HPAIV H5 virus strain. In all three parameters, plant crude extracts with H5 oligomers induced better responses than crude extracts containing trimers. The neutralizing antibodies induced by by two-dose and one dose immunization with an adjuvanted crude extract containing H5 oligomer protected vaccinated chickens from two lethal H5N1 virus strains with the efficiency of 92% and 100%, respectively. Following housing vaccinated chickens together with ten non-immunized chickens, only one of these chickens had detectable levels of the H5N1 virus. To facilitate the easy storage of a candidate vaccine, the H5 oligomer crude extracts were mixed with adjuvants and stored for 3.5 and 5.5 months at 4 °C, and chickens were immunized with these crude extracts. All these vaccinated chickens survived after a lethal H5N1 virus challenge. H5 oligomer crude extracts are comparable to commercial vaccines as they also induce strong virus-neutralizing immune responses following the administration of a single dose. The cost-effective production of plant crude extract vaccine candidates and the high stability after long-term storage will enable and encourage the further exploration of this technology for veterinary vaccine development.

The immunogenicity of plant-based COE-GCN4pII protein in pigs against the highly virulent porcine epidemic diarrhea virus strain from genotype 2.

Porcine epidemic diarrhea virus (PEDV) is a serious infectious causative agent in swine, especially in neonatal piglets. PEDV genotype 2 (G2) strains, particularly G2a, were the primary causes of porcine epidemic diarrhea (PED) outbreaks in Vietnam. Here, we produced a plant-based CO-26K-equivalent epitope (COE) variant from a Vietnamese highly virulent PEDV strain belonging to genotype 2a (COE/G2a) and evaluated the protective efficacy of COE/G2a-GCN4pII protein (COE/G2a-pII) in piglets against the highly virulent PEDV G2a strain following passive immunity. The 5-day-old piglets had high levels of PEDV-specific IgG antibodies, COE-IgA specific antibodies, neutralizing antibodies, and IFN-γ responses. After virulent challenge experiments, all of these piglets survived and had normal clinical symptoms, no watery diarrhea in feces, and an increase in their body weight, while all of the negative control piglets died. These results suggest that the COE/G2a-pII protein produced in plants can be developed as a promising vaccine candidate to protect piglets against PEDV G2a infection in Vietnam.

Effects of Size and Surface Properties of Nanodiamonds on the Immunogenicity of Plant-Based H5 Protein of A/H5N1 Virus in Mice.

Nanodiamond (ND) has recently emerged as a potential nanomaterial for nanovaccine development. Here, a plant-based haemagglutinin protein (H5.c2) of A/H5N1 virus was conjugated with detonation NDs (DND) of 3.7 nm in diameter (ND4), and high-pressure and high-temperature (HPHT) oxidative NDs of ~40-70 nm (ND40) and ~100-250 nm (ND100) in diameter. Our results revealed that the surface charge, but not the size of NDs, is crucial to the protein conjugation, as well as the in vitro and in vivo behaviors of H5.c2:ND conjugates. Positively charged ND4 does not effectively form stable conjugates with H5.c2, and has no impact on the immunogenicity of the protein both in vitro and in vivo. In contrast, the negatively oxidized NDs (ND40 and ND100) are excellent protein antigen carriers. When compared to free H5.c2, H5.c2:ND40, and H5.c2:ND100 conjugates are highly immunogenic with hemagglutination titers that are both 16 times higher than that of the free H5.c2 protein. Notably, H5.c2:ND40 and H5.c2:ND100 conjugates induce over 3-folds stronger production of both H5.c2-specific-IgG and neutralizing antibodies against A/H5N1 than free H5.c2 in mice. These findings support the innovative strategy of using negatively oxidized ND particles as novel antigen carriers for vaccine development, while also highlighting the importance of particle characterization before use.

Plant-Derived Trimeric CO-26K-Equivalent Epitope Induced Neutralizing Antibodies Against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) is a causative agent of a highly infectious disease with a high mortality rate, especially in newborn piglets in Asian countries resulting in serious economic loss. The development of a rapid, safe, effective and cost-efficient vaccine is crucial to protect pigs against PEDV infection. The COE antigen is regarded to be a major target for subunit vaccine development against PEDV infection. The naturally assembled COE protein forms a homotrimeric structure. In the present study, we successfully produced a trimeric COE protein as a native structure by fusion with the C-terminal isoleucine zipper trimerization (GCN4pII) motif in Nicotiana benthamiana, with a high expression level shown via semi-quantified Western blots. Trimeric COE protein was purified via immobilized metal affinity chromatography (IMAC), and its trimeric structure was successfully demonstrated by a cross-linking reaction, and a native PAGE gel. A crude extract containing the COE trimer was used for evaluating immunogenicity in mice. After 1 and 2 booster immunizations, the crude extract containing trimeric COE elicited elevated PEDV-specific humoral responses, as demonstrated by ELISA and Western blot analyses. Notably, a virus-neutralizing antibody assay indicated that the neutralization activities of sera of mice vaccinated with the crude extract containing COE-GCN4pII were similar to those of mice vaccinated with a commercial vaccine. These results suggest that crude extract containing trimeric COE is a promising plant-based subunit vaccine candidate for PEDV prevention.

Immunization with Plant-Derived Multimeric H5 Hemagglutinins Protect Chicken against Highly Pathogenic Avian Influenza Virus H5N1.

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.

A novel p.A191D matrilin-3 variant in a Vietnamese family with multiple epiphyseal dysplasia: a case report.

BACKGROUND: Multiple epiphyseal dysplasia (MED) is a common skeletal dysplasia that is characterized by variable degrees of epiphyseal abnormality primarily involving the hip and knee joints. Mutations in a gene encoding matrilin-3 (MATN3) have been reported as disease causing of autosomal dominant MED. The current study identified a novel c.572 C > A variant (p.A191D) in exon 2 of MATN3 in a Vietnamese family with MED. CASE PRESENTATION: A standard clinical tests and radiological examination were performed in an 8-year-old Vietnamese girl patient. The clinical examination showed that patient height was under average, with bent lower limbs, limited mobility and dislocation of the joints at both knees. Radiological documentation revealed abnormal cartilage development at the epiphysis of the femur and patella. The patient has a varus deformity of the lower limbs. The patient was diagnosed with autosomal dominant MED using molecular testing in the order of the coding sequences and flanking sequences of five genes: COMP (exons 8-19), MATN3 (exon 2), COL9A2 (exon 3), COL9A3 (exon 3), COL9A1 (exon 8) by Sanger sequencing. A novel heterozygous missense variant (c.572 C > A, p.A191D) in MATN3 was identified in this family, which were not inherited from parents. The p.A191D was predicted and classified as a pathogenic variant. When the two predicted structures of the wild type and mutant matrilin-3 were compared, the p.A191D substitution caused conformational changes near the substitution site, resulting in deformity of the ß-sheet of the single A domain of matrilin- 3. CONCLUSIONS: This is the first Vietnamese MED family attributed to p.A191D matrilin-3 variant, and our clinical, radiological and molecular data suggest that the novel de novo missense variant in MATN3 contributed to MED.

Detection of G338R FGFR2 mutation in a Vietnamese patient with Crouzon syndrome.

Crouzon syndrome is a rare autosomal dominant genetic disorder, which causes the premature fusion of the cranial suture. Fibroblast growth factor receptor 2 (FGFR2) mutations are well-known causatives of Crouzon syndrome. The current study aimed to assess the FGFR2 gene associated with Crouzon syndrome in a Vietnamese family of three generations and to characterize their associated clinical features. The family included in the present study underwent complete clinical examination. A patient was clinically examined and presented with typical features of Crouzon syndrome including craniosynostosis, shallow orbits, ocular proptosis and midface hypoplasia. However the patient had normal hands and feet, a normal hearing ability and normal intelligence. Genomic DNA collected from all family members (except from a 16 week-old-foetus) and 200 unrelated control subjects from the same population was extracted from leukocytes obtained from peripheral blood samples. Genomic DNA was extracted from the 16-week-old foetus via the amniotic fluid of the mother. All coding sequences of FGFR2 were amplified via polymerase chain reaction and directly sequenced. A heterozygous FGFR2 missense mutation (c.1012G>C, p.G338R) in exon 10 was identified in the patient with Crouzon but not in other family members, the 16 week-old-foetus or the controls. This mutation was therefore determined to be the causative agent of Crouzon syndrome. In addition, a novel heterozygous silent mutation (c.1164C>T, p.I388I) in exon 11 of the FGFR2 gene was identified in the patient with Crouzon, his mother and the 16-week-old fetus, but not in other family members. The mutation in exon 10 of FGRF2 was confirmed via restriction-enzyme digestion. The gain of the BsoBI site confirmed the FGFR2 mutation in exon 10 of the patient with Crouzon. This molecular finding may provide useful information to aid clinicians in the diagnosis of Crouzon syndrome and may also aid early prenatal diagnoses.

Nanodiamond enhances immune responses in mice against recombinant HA/H7N9 protein.

BACKGROUND: 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. RESULTS: 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). CONCLUSIONS: These results indicated a potential effect inherent to nanodiamond towards modulating immune systems, which should be further evaluated and broadly applied in nanovaccine development.

Neutralizing immune responses induced by oligomeric H5N1-hemagglutinins from plants.

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.