Identification and Full Genome Analysis of the First Putative Virus of Sea Buckthorn (Hippophae rhamnoides L.).

The agricultural importance of sea buckthorn (SBT; Hippophae rhamnoides L.) is rapidly increasing. Several bacterial and fungal pathogens infecting SBT have been identified and characterized; however, the viral pathogens are not yet known. In this study, we identified, isolated, and sequenced a virus from a wild plantation of SBT for the first time. Sequence analysis of the obtained viral genome revealed high similarity with several viruses belonging to the genus Marafivirus. The genome of the new virus is 6989 nucleotides (nt) in length according to 5', 3' RACE (without polyA-tail), with 5' and 3' 133 and 109 nt long untranslated regions, respectively. The viral genome encoded two open reading frames (ORFs). ORF1 encoded a polyprotein of 1954 amino acids with the characteristic marafivirus non-structural protein domains-methyltransferase, Salyut domain, papain-like cysteine protease, helicase, and RNA-dependent RNA polymerase. ORF1 was separated from ORF2 by 6 nt, encoding the coat protein (CP) with typical signatures of minor and major forms. Both CP forms were cloned and expressed in a bacterial expression system. Only the major CP was able to self-assemble into 30 nm virus-like particles that resembled the native virus, thus demonstrating that minor CP is not essential for virion assembly.

A scalable and highly immunogenic virus-like particle-based vaccine against SARS-CoV-2.

BACKGROUND: SARS-CoV-2 caused one of the most devastating pandemics in the recent history of mankind. Due to various countermeasures, including lock-downs, wearing masks, and increased hygiene, the virus has been controlled in some parts of the world. More recently, the availability of vaccines, based on RNA or adenoviruses, has greatly added to our ability to keep the virus at bay; again, however, in some parts of the world only. While available vaccines are effective, it would be desirable to also have more classical vaccines at hand for the future. Key feature of vaccines for long-term control of SARS-CoV-2 would be inexpensive production at large scale, ability to make multiple booster injections, and long-term stability at 4℃. METHODS: Here, we describe such a vaccine candidate, consisting of the SARS-CoV-2 receptor-binding motif (RBM) grafted genetically onto the surface of the immunologically optimized cucumber mosaic virus, called CuMVTT -RBM. RESULTS: Using bacterial fermentation and continuous flow centrifugation for purification, the yield of the production process is estimated to be >2.5 million doses per 1000-litre fermenter run. We demonstrate that the candidate vaccine is highly immunogenic in mice and rabbits and induces more high avidity antibodies compared to convalescent human sera. The induced antibodies are more cross-reactive to mutant RBDs of variants of concern (VoC). Furthermore, antibody responses are neutralizing and long-lived. In addition, the vaccine candidate was stable for at least 14 months at 4℃. CONCLUSION: Thus, the here presented VLP-based vaccine may be a good candidate for use as conventional vaccine in the long term.

Neutralization of MERS coronavirus through a scalable nanoparticle vaccine.

MERS-CoV continues to cause human outbreaks, so far in 27 countries worldwide following the first registered epidemic in Saudi Arabia in 2012. In this study, we produced a nanovaccine based on virus-like particles (VLPs). VLPs are safe vaccine platforms as they lack any replication-competent genetic material, and are used since many years against hepatitis B virus (HBV), hepatitis E virus (HEV) and human papilloma virus (HPV). In order to produce a vaccine that is readily scalable, we genetically fused the receptor-binding motif (RBM) of MERS-CoV spike protein into the surface of cucumber-mosaic virus VLPs. The employed CuMVTT-VLPs represent a new immunologically optimized vaccine platform incorporating a universal T cell epitope derived from tetanus toxin (TT). The resultant vaccine candidate (mCuMVTT-MERS) is a mosaic particle and consists of unmodified wild type monomers and genetically modified monomers displaying RBM, co-assembling within E. coli upon expression. mCuMVTT-MERS vaccine is self-adjuvanted with ssRNA, a TLR7/8 ligand which is spontaneously packaged during the bacterial expression process. The developed vaccine candidate induced high anti-RBD and anti-spike antibodies in a murine model, showing high binding avidity and an ability to completely neutralize MERS-CoV/EMC/2012 isolate, demonstrating the protective potential of the vaccine candidate for dromedaries and humans.

Recent Advances in the Use of Plant Virus-Like Particles as Vaccines.

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.

Correction to: Vaccination with nanoparticles combined with micro-adjuvants protects against cancer.

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.

Vaccination with nanoparticles combined with micro-adjuvants protects against cancer.

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.

Active immunisation targeting nerve growth factor attenuates chronic pain behaviour in murine osteoarthritis.

OBJECTIVES: Nerve growth factor (NGF) has emerged as a key driver of pain in osteoarthritis (OA) and antibodies to NGF are potent analgesics in human disease. Here, we validate a novel vaccine strategy to generate anti-NGF antibodies for reversal of pain behaviour in a surgical model of OA. METHODS: Virus-like particles were derived from the cucumber mosaic virus (CuMV) and coupled to expressed recombinant NGF to create the vaccine. 10-week-old male mice underwent partial meniscectomy to induce OA or sham-surgery. Spontaneous pain behaviour was measured by Linton incapacitance and OA severity was quantified using OARSI histological scoring. Mice (experimental and a sentinel cohort) were inoculated with CuMVttNGF (Vax) or CuMVttctrl (Mock) either before surgery or once pain was established. Efficacy of anti-NGF from the plasma of sentinel vaccinated mice was measured in vitro using a neurite outgrowth assay in PC12 cells. RESULTS: Anti-NGF titres were readily detectable in the vaccinated but not mock vaccinated mice. Regular boosting with fresh vaccine was required to maintain anti-NGF titres as measured in the sentinel cohort. Both prophylactic and therapeutic vaccination demonstrated a reversal of pain behaviour by incapacitance testing, and a meta-analysis of the two studies showing analgesia at peak anti-NGF titres was highly statistically significant. Serum anti-NGF was able to inhibit neurite outgrowth equivalent to around 150 ug/mL of recombinant monoclonal antibody. CONCLUSIONS: This study demonstrates therapeutic efficacy of a novel NGF vaccine strategy that reversibly alleviates spontaneous pain behaviour in surgically induced murine OA.

Use of plant viruses and virus-like particles for the creation of novel vaccines.

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.

Crystal structure of human gamma-butyrobetaine hydroxylase.

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 2.0A resolution. The GBBH monomer consists of a catalytic double-stranded beta-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.

The ryegrass mottle virus genome codes for a sobemovirus 3C-like serine protease and RNA-dependent RNA polymerase translated via -1 ribosomal frameshifting.

In the course of sobemovirus gene cloning the complete genome of Ryegrass mottle virus (RGMoV) was sequenced. Sequence analysis revealed differences including missing and extraneous nucleotides in comparison to the previously published sequence (Zhang, Toriyama, Takanashi, J. Gen. Plant Pathol. 67, 63 (2001)). A gene coding for a typical sobemovirus 3C-like serine protease was identified in ORF2a after multiple sequence alignment analysis. The newly identified 57-amino-acid stretch in ORF2a showed similarities ranging from 38.5 to 50.9% among sequenced genes of sobemovirus proteases. ORF analysis of the RGMoV polyprotein coding sequence demonstrated the arrangement of ORF2b coding for RNA-dependent RNA polymerase (RdRP) in the -1 frame in regard to ORF2a. The localization of conserved among sobemoviruses slippery sequence (UUUAAAC) at the 3'-end of ORF2a suggests the translation of RdRP via a -1 ribosomal frameshifting mechanism, allowing to include the RGMoV in the sobemovirus group with a Cocksfoot mottle virus-like (CfMV-like) genome organization.

The three-dimensional structure of cocksfoot mottle virus at 2.7 A resolution.

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 beta-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 maintained.