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In recent decades, the development of plant virology and genetic engineering techniques has resulted in the construction of plant virus-based vaccines for protection against different infectious agents, cancers and autoimmune diseases in both humans and animals. Interaction studies between plant viruses and mammalian organisms have suggested that plant viruses and virus-like particles (VLPs) are safe and noninfectious to humans and animals. Plant viruses with introduced antigens are powerful vaccine components due to their strongly organized, repetitive spatial structure; they can elicit strong immune responses similar to those observed with infectious mammalian viruses. The analysis of published data demonstrated that at least 73 experimental vaccines, including 61 prophylactic and 12 therapeutic vaccines, have been constructed using plant viruses as a carrier structure for presentation of different antigens. This information clearly demonstrates that noninfectious viruses are also applicable as vaccine carriers. Moreover, several plant viruses have been used for platform development, and corresponding vaccines are currently being tested in human and veterinary clinical trials. This review therefore discusses the main principles of plant VLP vaccine construction, emphasizing the physical, chemical, genetic and immunological aspects. Results of the latest studies suggest that several plant virus-based vaccines will join the list of approved human and animal vaccines in the near future. Unlabelled Image [ABSTRACT FROM AUTHOR]

Cocksfoot mottle virus is a plant virus that belongs to the genus Sobemovirus. The structure of the virus has been determined at 2.7 A˚ resolution. The icosahedral capsid has T = 3 quasisymmetry and 180 copies of the coat protein. Except for a couple of stacked bases, the viral RNA is not visible in the electron density map. The coat protein has a jelly-roll β-sandwich fold and its conformation is very similar to that of other sobemoviruses and tobacco necrosis virus. The N-terminal arm of one of the three quasiequivalent subunits is partly ordered and follows the same path in the capsid as the arm in rice yellow mottle virus, another sobemovirus. In other sobemoviruses, the ordered arm follows a different path, but in both cases the arms from three subunits meet and form a similar structure at a threefold axis. A comparison of the structures and sequences of viruses in this family shows that the only conserved parts of the protein–protein interfaces are those that form binding sites for calcium ions. Still, the relative orientations and position of the subunits are mantained. [Copyright &y& Elsevier]

Streptomyces olivaceoviridis secretes a so far unique protein of 18.7 kDa (CHB1) which lacks catalytic activity. It interacts highly specifically with α-chitin, but not with β-chitin, chitosan, or cellulose. Each of the five codons for tryptophan (Trp) in the chb1 gene was replaced by those for leucine (Leu) or tyrosine (Tyr). Eight corresponding mutant proteins and the wild-type protein were purified to homogeneity and their binding capacity to α-chitin was determined. The relative affinities to anti-CHB1 antibodies, the kinetics of binding, the dissociation constants, circular dichroism, and fluorescence emission spectra for three mutant types were compared to the characteristics of CHB1. The presented data lead to the following conclusions. (a) CHB1 presents a highly flexible protein lacking α-helices. (b) Replacement of each of the buried Trp residues (Trp134 and Trp184) leads to conformational alterations and, in due course, to a considerably reduced binding affinity of the protein. (c) The exchange of the exposed Trp 57 by either Leu or Tyr results in relatively slight topological changes, but entails a loss of binding capacity of about 90%. (d) The dissociation constant was highest for the mutant protein [L57]CHB1 (2.17 µM), followed by [LI34]CHB1 (0.91 µM) and [LI84]CHB1 (0.26 µM), and lowest for the progenitor CHB1 (0.11 µM), indicating its strong affinity to the unsoiuble substrate. (e) The data suggest that the exposed Trp57 contributes directly and significantly to the interaction of CHB1 with α-chitin. [ABSTRACT FROM AUTHOR]

Vaccination is one of the most effective public health interventions of the 20th century. All vaccines can be classified into different types, such as vaccines against infectious diseases, anticancer vaccines and vaccines against autoimmune diseases. In recent decades, recombinant technologies have enabled the design of experimental vaccines against a wide range of diseases using plant viruses and virus-like particles as central elements to stimulate protective and long-lasting immune responses. The analysis of recent publications shows that at least 97 experimental vaccines have been constructed based on plant viruses, including 71 vaccines against infectious agents, 16 anticancer vaccines and 10 therapeutic vaccines against autoimmune disorders. Several plant viruses have already been used for the development of vaccine platforms and have been tested in human and veterinary studies, suggesting that plant virus-based vaccines will be introduced into clinical and veterinary practice in the near future. [ABSTRACT FROM AUTHOR]

Background: Virus‐like particle (VLP) Peanut is a novel immunotherapeutic vaccine candidate for the treatment of peanut allergy. The active pharmaceutical ingredient represents cucumber mosaic VLPs (CuMVTT‐VLPs) that are genetically fused with one of the major peanut allergens, Ara h 2 (CuMVTT‐Ara h 2). We previously demonstrated the immunogenicity and the protective capacity of VLP Peanut‐based immunization in a murine model for peanut allergy. Moreover, a Phase I clinical trial has been initiated using VLP Peanut material manufactured following a GMP‐compliant manufacturing process. Key product characterization studies were undertaken here to understand the role and contribution of critical quality attributes that translate as predictive markers of immunogenicity and protective efficacy for clinical vaccine development. Method: The role of prokaryotic RNA encapsulated within VLP Peanut on vaccine immunogenicity was assessed by producing a VLP Peanut batch with a reduced RNA content (VLP Peanut low RNA). Immunogenicity and peanut allergen challenge studies were conducted with VLP Peanut low RNA, as well as with VLP Peanut in WT and TLR 7 KO mice. Furthermore, mass spectrometry and SDS‐PAGE based methods were used to determine Ara h 2 antigen density on the surface of VLP Peanut particles. This methodology was subsequently applied to investigate the relationship between Ara h 2 antigen density and immunogenicity of VLP Peanut. Results: A TLR 7 dependent formation of Ara h 2 specific high‐avidity IgG antibodies, as well as a TLR 7 dependent change in the dominant IgG subclass, was observed following VLP Peanut vaccination, while total allergen‐specific IgG remained relatively unaffected. Consistently, a missing TLR 7 signal caused only a weak decrease in allergen tolerability after vaccination. In contrast, a reduced RNA content for VLP Peanut resulted in diminished total Ara h 2 specific IgG responses, followed by a significant impairment in peanut allergen tolerability. The discrepant effect on allergen tolerance caused by an absent TLR 7 signal versus a reduced RNA content is explained by the observation that VLP Peanut‐derived RNA not only stimulates TLR 7 but also TLR 3. Additionally, a strong correlation was observed between the number of Ara h 2 antigens displayed on the surface of VLP Peanut particles and the vaccine's immunogenicity and protective capacity. Conclusions: Our findings demonstrate that prokaryotic RNA encapsulated within VLP Peanut, including antigen density of Ara h 2 on viral particles, are key contributors to the immunogenicity and protective capacity of the vaccine. Thus, antigenicity and RNA content are two critical quality attributes that need to be determined at the stage of manufacturing, providing robust information regarding the immunogenicity and protective capacity of VLP Peanut in the mouse which has translational relevance to the human setting. [ABSTRACT FROM AUTHOR]

Ryegrass mottle virus (RGMoV; genus: Sobemovirus) is a single-stranded positive RNA virus with a 30 nm viral particle size. It exhibits T = 3 symmetry with 180 coat protein (CP) subunits forming a viral structure. The RGMoV genome comprises five open reading frames that encode P1, Px, a membrane-anchored 3C-like serine protease, a viral genome-linked protein, P16, an RNA-dependent RNA polymerase, and CP. The RGMoV genome size varies, ranging from 4175 nt (MW411579.1) to 4253 nt (MW411579.1) in the deposited sequences. An earlier deposited RGMoV complete genome sequence of 4212 nt length (EF091714.1) was used to develop an infectious complementary DNA (icDNA) construct for in vitro gRNA transcription from the T7 promoter. However, viral infection was not induced when the transcribed gRNA was introduced into oat plants, indicating the potential absence of certain sequences in either the 5' or 3' untranslated regions (UTR) or both. The complete sequence of the 3' UTR was determined through 3' end RACE, while the 5' UTR was identified using high-throughput sequencing (HTS)-RNA-Seq to resolve the potential absences. Only the icDNA vector containing the newly identified UTR sequences proved infectious, resulting in typical viral infection symptoms and subsequent propagation of progeny viruses, exhibiting the ability to cause repeated infections in oat plants after at least one passage. The successful generation of icDNA highlighted the synergistic potential of utilizing both methods when a single approach failed. Furthermore, this study demonstrated the reliability of HTS as a method for determining the complete genome sequence of viral genomes. [ABSTRACT FROM AUTHOR]

Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its cis and trans activity is mediated by the naturally unfolded virus-genome-linked protein (VPg). Nuclear magnetic resonance studies show a Pro–VPg complex interaction and VPg tertiary structure; however, information regarding structural changes of the Pro–VPg complex during interaction is lacking. Here, we solved a full Pro–VPg 3D structure of ryegrass mottle virus (RGMoV) that demonstrates the structural changes in three different conformations due to VPg interaction with Pro. We identified a unique site of VPg interaction with Pro that was not observed in other sobemoviruses, and observed different conformations of the Pro β2 barrel. This is the first report of a full plant Pro crystal structure with its VPg cofactor. We also confirmed the existence of an unusual previously unmapped cleavage site for sobemovirus Pro in the transmembrane domain: E/A. We demonstrated that RGMoV Pro in cis activity is not regulated by VPg and that in trans, VPg can also mediate Pro in free form. Additionally, we observed Ca2+ and Zn2+ inhibitory effects on the Pro cleavage activity. [ABSTRACT FROM AUTHOR]

Background: The highly contagious SARS‐CoV‐2 is mainly transmitted by respiratory droplets and aerosols. Consequently, people are required to wear masks and maintain a social distance to avoid spreading of the virus. Despite the success of the commercially available vaccines, the virus is still uncontained globally. Given the tropism of SARS‐CoV‐2, a mucosal immune reaction would help to reduce viral shedding and transmission locally. Only seven out of hundreds of ongoing clinical trials are testing the intranasal delivery of a vaccine against COVID‐19. Methods: In the current study, we evaluated the immunogenicity of a traditional vaccine platform based on virus‐like particles (VLPs) displaying RBD of SARS‐CoV‐2 for intranasal administration in a murine model. The candidate vaccine platform, CuMVTT‐RBD, has been optimized to incorporate a universal T helper cell epitope derived from tetanus‐toxin and is self‐adjuvanted with TLR7/8 ligands. Results: CuMVTT‐RBD vaccine elicited a strong systemic RBD‐ and spike‐IgG and IgA antibodies of high avidity. Local immune response was assessed, and our results demonstrate a strong mucosal antibody and plasma cell production in lung tissue. Furthermore, the induced systemic antibodies could efficiently recognize and neutralize different variants of concern (VOCs). Conclusion: Our data demonstrate that intranasal administration of CuMVTT‐RBD induces a protective systemic and local specific antibody response against SARS‐CoV‐2 and its VOCs. [ABSTRACT FROM AUTHOR]

Abstract: Gamma-butyrobetaine hydroxylase (GBBH) is a 2-ketoglutarate-dependent dioxygenase that catalyzes the biosynthesis of l-carnitine by hydroxylation of gamma-butyrobetaine (GBB). l-carnitine is required for the transport of long-chain fatty acids into mitochondria for generating metabolic energy. The only known synthetic inhibitor of GBBH is mildronate (3-(2,2,2-trimethylhydrazinium) propionate dihydrate), which is a non-hydroxylatable analog of GBB. To aid in the discovery of novel GBBH inhibitors by rational drug design, we have solved the three-dimensional structure of recombinant human GBBH at 2.0Å resolution. The GBBH monomer consists of a catalytic double-stranded β-helix (DBSH) domain, which is found in all 2KG oxygenases, and a smaller N-terminal domain. Extensive interactions between two monomers confirm earlier observations that GBBH is dimeric in its biological state. Although many 2KG oxygenases are multimeric, the dimerization interface of GBBH is very different from that of related enzymes. The N-terminal domain of GBBH has a similar fold to the DUF971 superfamily, which consists of several short bacterial proteins with unknown function. The N-terminal domain has a bound Zn ion, which is coordinated by three cysteines and one histidine. Although several other 2KG oxygenases with known structures have more than one domain, none of them resemble the N-terminal domain of GBBH. The N-terminal domain may facilitate dimer formation, but its precise biological role remains to be discovered. The active site of the catalytic domain of GBBH is similar to that of other 2KG oxygenases, and Fe(II)-binding residues form a conserved His–X–Asp–X n –His triad, which is found in all related enzymes. [ABSTRACT FROM AUTHOR]

Abstract: The crystal structure of the sobemovirus Ryegrass mottle virus (RGMoV) has been determined at 2.9 Å resolution. The coat protein has a canonical jellyroll β-sandwich fold. In comparison to other sobemoviruses the RGMoV coat protein is missing several residues in two of the loop regions. The first loop contributes to contacts between subunits around the quasi-threefold symmetry axis. The altered contact interface results in tilting of the subunits towards the quasi-threefold axis. The assembly of the T=3 capsid of sobemoviruses is controlled by the N-termini of C subunits forming a so-called β-annulus. The other loop that is smaller in the RGMoV structure contains a helix that participates in stabilization of the β-annulus in other sobemoviruses. The loss of interaction between the RGMoV loop and the β-annulus has been compensated for by additional interactions between the N-terminal arms. As a consequence of these differences, the diameter of the RGMoV particle is 8 Å smaller than that of the other sobemoviruses. The interactions of coat proteins in sobemovirus capsids involve calcium ions. Depletion of calcium ions results in particle swelling, which is considered a first step in disassembly. We could not identify any density for metal ions in the proximity of the conserved residues normally involved in calcium binding, but the RGMoV structure does not show any signs of swelling. A likely reason is the low pH (3.0) of the crystallization buffer in which the groups interacting with the calcium ions are not charged. [Copyright &y& Elsevier]

Vaccine-induced immune response can be greatly enhanced by mimicking pathogen properties. The size and the repetitive geometric shape of virus-like particles (VLPs) influence their immunogenicity by facilitating drainage to secondary lymphoid organs and enhancing interaction with and activation of B cells and innate humoral immune components. VLPs derived from the plant Bromovirus genus, specifically cowpea chlorotic mottle virus (CCMV), are T = 3 icosahedral particles. (T) is the triangulation number that refers to the number and arrangements of the subunits (pentamers and hexamers) of the VLPs. CCMV-VLPs can be easily expressed in an E. coli host system and package ssRNA during the expression process. Recently, we have engineered CCMV-VLPs by incorporating the universal tetanus toxin (TT) epitope at the N-terminus. The modified CCMV TT -VLPs successfully form icosahedral particles T = 3 , with a diameter of ~30 nm analogous to the parental VLPs. Interestingly, incorporating TT epitope at the C-terminus of CCMV TT -VLPs results in the formation of Rod-shaped VLPs, ~1 μm in length and ~ 30 nm in width. In this study, we have investigated the draining kinetics and immunogenicity of both engineered forms (termed as Round-shaped CCMV TT -VLPs and Rod-shaped CCMV TT -VLPs) as potential B cell immunogens using different in vitro and in vivo assays. Our results reveal that Round-shaped CCMV TT -VLPs are more efficient in draining to secondary lymphoid organs to charge professional antigen-presenting cells as well as B cells. Furthermore, compared to Rod-shaped CCMV TT -VLPs, Round-shaped CCMV TT -VLPs led to more than 100-fold increased systemic IgG and IgA responses accompanied by prominent formation of splenic germinal centers. Round-shaped CCMV TT -VLPs could also polarize the induced T cell response toward Th1. To our knowledge, this is the first study investigating and comparing the draining kinetics and immunogenicity of one and the same VLP monomer forming nano-sized icosahedra or rods in the micrometer size. Unlabelled Image [ABSTRACT FROM AUTHOR]

Background: Peanut allergy is the most prevalent and dangerous food allergy. Peanuts consist of a large number of different allergens and peanut-allergic patients are frequently sensitized to multiple allergens. Hence, conventional desensitization approaches aim at targeting as many allergens as possible. Methods: The monoclonal anti-Ara h 2 antibody (mAb) was produced by hybridoma cells derived from WT BALB/c mice after immunization with a vaccine based on virus-like particles coupled to Ara h 2. BALB/c mice were sensitized intraperitoneally with peanut extract absorbed to alum and mAbs were applied i.v. Challenge was performed the next day with the whole peanut extract intravenously and via skin prick test. Results: Here we show in peanut-allergic mice that a single high-affinity mAb specific for Ara h 2 is able to block systemic and local allergic reactions induced by the complex peanut extract. We confirm in vitro binding of the mAb to the inhibitory low-affinity FcγRIIb receptor using a sensitive biosensor and demonstrate in vivo that protection was dependent on FcγRIIb. Conclusion: A single mAb specific for Ara h 2 is able to improve local and systemic allergic symptoms induced by the whole allergen mixture. [ABSTRACT FROM AUTHOR]

Background: Induction of strong T cell responses, in particular cytotoxic T cells, is a key for the generation of efficacious therapeutic cancer vaccines which yet, remains a major challenge for the vaccine developing world. Here we demonstrate that it is possible to harness the physiological properties of the lymphatic system to optimize the induction of a protective T cell response. Indeed, the lymphatic system sharply distinguishes between nanoscale and microscale particles. The former reaches the fenestrated lymphatic system via diffusion, while the latter either need to be transported by dendritic cells or form a local depot. Methods: Our previously developed cucumber-mosaic virus-derived nanoparticles termed (CuMVTT-VLPs) incorporating a universal Tetanus toxoid epitope TT830-843 were assessed for their draining kinetics using stereomicroscopic imaging. A nano-vaccine has been generated by coupling p33 epitope as a model antigen to CuMVTT-VLPs using bio-orthogonal Cu-free click chemistry. The CuMVTT-p33 nano-sized vaccine has been next formulated with the micron-sized microcrystalline tyrosine (MCT) adjuvant and the formed depot effect was studied using confocal microscopy and trafficking experiments. The immunogenicity of the nanoparticles combined with the micron-sized adjuvant was next assessed in an aggressive transplanted murine melanoma model. The obtained results were compared to other commonly used adjuvants such as B type CpGs and Alum. Results: Our results showed that CuMVTT-VLPs can efficiently and rapidly drain into the lymphatic system due to their nano-size of ~ 30 nm. However, formulating the nanoparticles with the micron-sized MCT adjuvant of ~ 5 μM resulted in a local depot for the nanoparticles and a longer exposure time for the immune system. The preclinical nano-vaccine CuMVTT-p33 formulated with the micron-sized MCT adjuvant has enhanced the specific T cell response in the stringent B16F10p33 murine melanoma model. Furthermore, the micron-sized MCT adjuvant was as potent as B type CpGs and clearly superior to the commonly used Alum adjuvant when total CD8+, specific p33 T cell response or tumour protection were assessed. Conclusion: The combination of nano- and micro-particles may optimally harness the physiological properties of the lymphatic system. Since the nanoparticles are well defined virus-like particles and the micron-sized adjuvant MCT has been used for decades in allergen-specific desensitization, this approach may readily be translated to the clinic. [ABSTRACT FROM AUTHOR]

Background: Insect‐bite hypersensitivity (IBH) in horses is a chronic allergic dermatitis caused by insect bites. Horses suffer from pruritic skin lesions, caused by type‐I/type‐IV allergic reactions accompanied by prominent eosinophil infiltration into the skin. Interleukin‐5 (IL‐5) is the key cytokine for eosinophils and we have previously shown that targeting IL‐5 by vaccination reduces disease symptoms in horses. Objective: Here, we analyzed the potential for long‐term therapy by assessing a second follow‐up year of the previously published study. Methods: The vaccine consisted of equine IL‐5 (eIL‐5) covalently linked to a cucumber mosaic virus‐like particle (VLP) containing a universal T cell epitope (CuMVTT) using a semi‐crossover design to follow vaccinated horses during a second treatment season. Thirty Icelandic horses were immunized with 300 μg of eIL‐5‐CuMVTT without adjuvant. Results: The vaccine was well tolerated and did not reveal any safety concerns throughout the study. Upon vaccination, all horses developed reversible anti‐eIL‐5 auto‐antibody titers. The mean course of eosinophil levels was reduced compared to placebo treatment leading to significant reduction of clinical lesion scores. Horses in their second vaccination year showed a more pronounced improvement of disease symptoms when compared to first treatment year, most likely due to more stable antibody titers induced by a single booster injection. Hence, responses could be maintained over two seasons and the horses remained protected against disease symptoms. Conclusion: Yearly vaccination against IL‐5 may be a long‐term solution for the treatment of IBH and other eosinophil‐mediated diseases in horses and other species including humans. Basic vaccination regimen consisting of three vaccine injections in the first treatment year induces sufficient antibody titers leading to significant improvement of insect bite hypersensitivity (IBH) lesion scores. A single booster vaccine injection in the second treatment year re‐induces anti‐IL‐5 antibody titers leading to enhanced improvement of IBH lesion scores. Anti‐IL‐5 vaccination in horses significantly reduces eosinophil levels in blood. [ABSTRACT FROM AUTHOR]

Following publication of the original article [1], the author reported an author's family name has been misspelled. Paul Engroff should be replace Paul Engeroff. Furthermore, there are two mistake in two affiliations: 9) Department of dermatology, University of Zurich, Bern, Switzerland and 10) Department of Oncology, University of Lausanne, Bern,Switzerland should be replace with 9) Department of dermatology, University of Zurich, Zurich, Switzerland. 10) Department of Oncology, University of Lausanne, Lausanne, Switzerland. [ABSTRACT FROM AUTHOR]

Background: Induction of strong T cell responses, in particular cytotoxic T cells, is a key for the generation of efficacious therapeutic cancer vaccines which yet, remains a major challenge for the vaccine developing world. Here we demonstrate that it is possible to harness the physiological properties of the lymphatic system to optimize the induction of a protective T cell response. Indeed, the lymphatic system sharply distinguishes between nanoscale and microscale particles. The former reaches the fenestrated lymphatic system via diffusion, while the latter either need to be transported by dendritic cells or form a local depot. Methods: Our previously developed cucumber-mosaic virus-derived nanoparticles termed (CuMVTT-VLPs) incorporating a universal Tetanus toxoid epitope TT830–843 were assessed for their draining kinetics using stereomicroscopic imaging. A nano-vaccine has been generated by coupling p33 epitope as a model antigen to CuMVTT-VLPs using bio-orthogonal Cu-free click chemistry. The CuMVTT-p33 nano-sized vaccine has been next formulated with the micron-sized microcrystalline tyrosine (MCT) adjuvant and the formed depot effect was studied using confocal microscopy and trafficking experiments. The immunogenicity of the nanoparticles combined with the micron-sized adjuvant was next assessed in an aggressive transplanted murine melanoma model. The obtained results were compared to other commonly used adjuvants such as B type CpGs and Alum. Results: Our results showed that CuMVTT-VLPs can efficiently and rapidly drain into the lymphatic system due to their nano-size of ~ 30 nm. However, formulating the nanoparticles with the micron-sized MCT adjuvant of ~ 5 μM resulted in a local depot for the nanoparticles and a longer exposure time for the immune system. The preclinical nano-vaccine CuMVTT-p33 formulated with the micron-sized MCT adjuvant has enhanced the specific T cell response in the stringent B16F10p33 murine melanoma model. Furthermore, the micron-sized MCT adjuvant was as potent as B type CpGs and clearly superior to the commonly used Alum adjuvant when total CD8+, specific p33 T cell response or tumour protection were assessed. Conclusion: The combination of nano- and micro-particles may optimally harness the physiological properties of the lymphatic system. Since the nanoparticles are well defined virus-like particles and the micron-sized adjuvant MCT has been used for decades in allergen-specific desensitization, this approach may readily be translated to the clinic. [ABSTRACT FROM AUTHOR]