Immunogenic profile of a plant-produced nonavalent African horse sickness viral protein 2 (VP2) vaccine in IFNAR-/- mice.

A safe, highly immunogenic multivalent vaccine to protect against all nine serotypes of African horse sickness virus (AHSV), will revolutionise the AHS vaccine industry in endemic countries and beyond. Plant-produced AHS virus-like particles (VLPs) and soluble viral protein 2 (VP2) vaccine candidates were developed that have the potential to protect against all nine serotypes but can equally well be formulated as mono- and bi-valent formulations for localised outbreaks of specific serotypes. In the first interferon α/ß receptor knock-out (IFNAR-/-) mice trial conducted, a nine-serotype (nonavalent) vaccine administered as two pentavalent (5 µg per serotype) vaccines (VLP/VP2 combination or exclusively VP2), were directly compared to the commercially available AHS live attenuated vaccine. In a follow up trial, mice were vaccinated with an adjuvanted nine-serotype multivalent VP2 vaccine in a prime boost strategy and resulted in the desired neutralising antibody titres of 1:320, previously demonstrated to confer protective immunity in IFNAR-/- mice. In addition, the plant-produced VP2 vaccine performed favourably when compared to the commercial vaccine. Here we provide compelling data for a nonavalent VP2-based vaccine candidate, with the VP2 from each serotype being antigenically distinguishable based on LC-MS/MS and ELISA data. This is the first preclinical trial demonstrating the ability of an adjuvanted nonavalent cocktail of soluble, plant-expressed AHS VP2 proteins administered in a prime-boost strategy eliciting high antibody titres against all 9 AHSV serotypes. Furthermore, elevated T helper cells 2 (Th2) and Th1, indicative of humoral and cell-mediated memory T cell immune responses, respectively, were detected in mouse serum collected 14 days after the multivalent prime-boost vaccination. Both Th2 and Th1 may play a role to confer protective immunity. These preclinical immunogenicity studies paved the way to test the safety and protective efficacy of the plant-produced nonavalent VP2 vaccine candidate in the target animals, horses.

Protective immunity of plant-produced African horse sickness virus serotype 5 chimaeric virus-like particles (VLPs) and viral protein 2 (VP2) vaccines in IFNAR-/- mice.

Next generation vaccines have the capability to contribute to and revolutionise the veterinary vaccine industry. African horse sickness (AHS) is caused by an arbovirus infection and is characterised by respiratory distress and/or cardiovascular failure and is lethal to horses. Mandatory annual vaccination in endemic areas curtails disease occurrence and severity. However, development of a next generation AHSV vaccine, which is both safe and efficacious, has been an objective globally for years. In this study, both AHSV serotype 5 chimaeric virus-like particles (VLPs) and soluble viral protein 2 (VP2) were successfully produced in Nicotiana benthamiana ΔXT/FT plants, partially purified and validated by gel electrophoresis, transmission electron microscopy and liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing before vaccine formulation. IFNAR-/- mice vaccinated with the adjuvanted VLPs or VP2 antigens in a 10 µg prime-boost regime resulted in high titres of antibodies confirmed by both serum neutralising tests (SNTs) and enzyme-linked immunosorbent assays (ELISA). Although previous studies reported high titres of antibodies in horses when vaccinated with plant-produced AHS homogenous VLPs, this is the first study demonstrating the protective efficacy of both AHSV serotype 5 chimaeric VLPs and soluble AHSV-5 VP2 as vaccine candidates. Complementary to this, coating ELISA plates with the soluble VP2 has the potential to underpin serotype-specific serological assays.

Biochemical Pulldown of mRNAs and Long Noncoding RNAs from Cellular Lysates Coupled with Mass Spectrometry to Identify Protein Binding Partners.

RNA binding proteins (RBPs) interact with cellular mRNAs, controlling various steps throughout the lifetime of these transcripts, including transcription, cellular transport, subcellular localization, translation and degradation. In addition to binding mRNA transcripts, a growing number of RBPs are shown to bind long noncoding RNAs (lncRNAs), controlling key cellular processes, including gene expression and translation of proteins. Current methodologies aimed at identifying and characterizing protein binding partners of specific RNAs of interest typically rely on tagging of the RNA with affinity aptamers, using in vitro transcribed RNA or immobilized oligonucleotides to capture RNA-protein complexes under native conditions. These assays are coupled with mass spectrometry or Western Blot analysis to identify or/and confirm interacting proteins. Here, we describe an alternative approach to identify protein binding partners of mRNAs and large long noncoding RNAs. This approach relies on biochemical pulldown of specific target RNAs and interacting protein partners from cellular lysates coupled with mass spectrometry to identify novel interacting proteins. By using 24-48 ~20 mer biotinylated DNA probes that hybridize to the target RNA, the method ensures high specificity and minimal off target binding. This approach is reproducible and fast and serves as a base for discovery studies to identify proteins that bind to RNAs of interest.

Plant-produced Bluetongue chimaeric VLP vaccine candidates elicit serotype-specific immunity in sheep.

Bluetongue (BT) is a hemorrhagic non-contagious, biting midge-transmitted disease of wild and domestic ruminants that is caused by bluetongue virus (BTV). Annual vaccination plays a pivotal role in BT disease control in endemic regions. Due to safety concerns of the current BTV multivalent live attenuated vaccine (LAV), a safe efficacious new generation subunit vaccine such as a plant-produced BT virus-like particle (VLP) vaccine is imperative. Previously, homogenous BTV serotype 8 (BTV-8) VLPs were successfully produced in Nicotiana benthamiana plants and provided protective immunity in sheep. In this study, combinations of BTV capsid proteins from more than one serotype were expressed and assembled to form chimaeric BTV-3 and BTV-4 VLPs in N. benthamiana plants. The assembled homogenous BTV-8, as well as chimaeric BTV-3 and chimaeric BTV-4 VLP serotypes, were confirmed by SDS-PAGE, Transmission Electron microscopy (TEM) and protein confirmation using liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing. As VP2 is the major determinant eliciting protective immunity, the percentage coverage and number of unique VP2 peptides detected in assembled chimaeric BT VLPs were used as a guide to assemble the most appropriate chimaeric combinations. Both plant-produced chimaeric BTV-3 and BTV-4 VLPs were able to induce long-lasting serotype-specific neutralizing antibodies equivalent to the monovalent LAV controls. Antibody levels remained high to the end of the trial. Combinations of homogenous and chimaeric BT VLPs have great potential as a safe, effective multivalent vaccine with the ability to distinguish between vaccinated and infected individuals (DIVA) due to the absence of non-structural proteins.

Conserved amino acid residues that are important for ligand binding in the type I gonadotropin-releasing hormone (GnRH) receptor are required for high potency of GnRH II at the type II GnRH receptor.

GnRH I regulates reproduction. A second form, designated GnRH II, selectively binds type II GnRH receptors. Amino acids of the type I GnRH receptor required for binding of GnRH I (Asp2.61(98), Asn2.65(102), and Lys3.32(121)) are conserved in the type II GnRH receptor, but their roles in receptor function are unknown. We have delineated their functions using mutagenesis, signaling and binding assays, immunoblotting, and computational modeling. Mutating Asp2.61(97) to Glu or Ala, Asn2.65(101) to Ala, or Lys3.32(120) to Gln decreased potency of GnRH II-stimulated inositol phosphate production. Consistent with proposed roles in ligand recognition, mutations eliminated measurable binding of GnRH II, whereas expression of mutant receptors was not decreased. In detailed analysis of how these residues affect ligand-dependent signaling, [Trp2]-GnRH I showed lesser decreases in potency than GnRH I at the Asp2.61(97)Glu mutant. In contrast, [Trp2]-GnRH II showed the same loss of potency as GnRH II at this mutant. This suggests that Asp2.61(97) contributes to recognition of His2 of GnRH I, but not of GnRH II. GnRH II showed a large decrease in potency at the Asn2.65(101)Ala mutant compared with analogs lacking the CO group of Gly10NH2. This suggests that Asn2.65(101) recognizes Gly10NH2 of GnRH II. GnRH agonists showed large decreases in potency at the Lys3.32(120)Gln mutant, but antagonist activity was unaffected. This suggests that Lys3.32(120) recognizes agonists, but not antagonists, as in the type I receptor. These data indicate that roles of conserved residues are similar, but not identical, in the type I and II GnRH receptors.

Expression, structure, function, and evolution of gonadotropin-releasing hormone (GnRH) receptors GnRH-R1SHS and GnRH-R2PEY in the teleost, Astatotilapia burtoni.

Multiple GnRH receptors are known to exist in nonmammalian species, but it is uncertain which receptor type regulates reproduction via the hypothalamic-pituitary-gonadal axis. The teleost fish, Astatotilapia burtoni, is useful for identifying the GnRH receptor responsible for reproduction, because only territorial males reproduce. We have cloned a second GnRH receptor in A. burtoni, GnRH-R1(SHS) (SHS is a peptide motif in extracellular loop 3), which is up-regulated in pituitaries of territorial males. We have shown that GnRH-R1(SHS) is expressed in many tissues and specifically colocalizes with LH in the pituitary. In A. burtoni brain, mRNA levels of both GnRH-R1(SHS) and a previously identified receptor, GnRH-R2(PEY), are highly correlated with mRNA levels of all three GnRH ligands. Despite its likely role in reproduction, we found that GnRH-R1(SHS) has the highest affinity for GnRH2 in vitro and low responsivity to GnRH1. Our phylogenetic analysis shows that GnRH-R1(SHS) is less closely related to mammalian reproductive GnRH receptors than GnRH-R2(PEY). We correlated vertebrate GnRH receptor amino acid sequences with receptor function and tissue distribution in many species and found that GnRH receptor sequences predict ligand responsiveness but not colocalization with pituitary gonadotropes. Based on sequence analysis, tissue localization, and physiological response we propose that the GnRH-R1(SHS) receptor controls reproduction in teleosts, including A. burtoni. We propose a GnRH receptor classification based on gene sequence that correlates with ligand selectivity but not with reproductive control. Our results suggest that different duplicated GnRH receptor genes have been selected to regulate reproduction in different vertebrate lineages.