Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Several use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these novel, nucleic acid based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214mg/L in unoptimized, mammalian cell culture and in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100{degrees}C when lyophilized, upto 70{degrees}C in solution and stable for over four weeks at 37{degrees}C. In prime:boost guinea pig immunizations, when formulated with the MF59 like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

Green tea and Spirulina extracts inhibit SARS, MERS, and SARS-2 spike pseudotyped virus entry in vitro

Coronaviruses (CoVs) infect a wide range of animals and birds. Their tropism is primarily determined by the ability of the spike (S) protein to bind to a host cell surface receptor. The rapid outbreak of emerging novel coronavirus, SARS-CoV 2 in China inculcates the need for the development of hasty and effective intervention strategies. Medicinal plants and natural compounds have been traditionally used to treat viral infections. Here, we generated VSV based pseudotyped viruses (pvs) of SARS-, MERS-, and SARS-2 CoVs to screen entry inhibitors from natural products. In the first series of experiments, we demonstrated that pseudotyped viruses specifically bind on their receptors and enter into the cells. SARS and MERS polyclonal antibodies neutralize SARSpv and SARS-2pv, and MERSpv respectively. Incubation of soluble ACE2 inhibited entry of SARS and SARS-2 pvs but not MERSpv. In addition, expression of ACE2 and DPP4 in non-permissive BHK21 cells enabled infection by SARSpv, SARS-2pv, and MERSpv respectively. Next, we showed the antiviral properties of known enveloped virus entry inhibitors, Spirulina and Green tea extracts against CoVpvs. SARSpv, MERSpv, and SARS-2pv entry were blocked with higher efficiency when preincubated with either green tea or spirulina extracts. Green tea provided a better inhibitory effect than the spirulina extracts by binding to the S1 domain of spike and blocking the interaction of spike with its receptor. Further studies are required to understand the exact mechanism of viral inhibition. In summary, we demonstrate that pseudotyped virus is an ideal tool for screening viral entry inhibitors. Moreover, spirulina and green tea could be promising antiviral agents against emerging viruses.

Differential expression of olive flounder (Paralichthys olivaceus) transcriptome during viral hemorrhagic septicemia virus (VHSV) infection at warmer and colder temperature.

The data presented here are related to the research article entitled "Temperature-dependent immune response of olive flounder (Paralichthys olivaceus) infected with viral hemorrhagic septicemia virus (VHSV)" [1]. In the cited article, we sequenced the whole transcriptome of the olive flounder using Illumina RNA-Seq. Differentially expressed genes (DEG) analysis of VHSV infected head kidney samples showed perturbations in gene expression. Herein we made a comparison of DEGs at early stage of VHSV infection of olive flounder (4 h post infection) in colder (13 °C) and warmer (20 °C) temperatures. The analysis of signaling pathways showed that several major immune pathways were altered. The gene ontology terms associated with the genes differentially expressed are also presented.

The first report of siglec-3/CD33 gene in a teleost (rock bream, Oplegnathus fasciatus): An analysis of its spatial expression during stimulation to red seabream iridovirus (RSIV) and two bacterial pathogens.

Siglec-3/CD33 is a myeloid-specific inhibitory receptor that is expressed on cells of the immune system, where it is believed to play a regulatory role, modulating the inflammatory and immune responses. We characterized CD33 (RbCD33) in rock bream which is a transmembrane protein with two IG-like domains and a cytoplasmic tail. It has a deduced amino acid sequence of 390 residues and has tyrosine-based signaling motifs in the cytoplasmic tail. The RbCD33 mRNA was highly expressed in peripheral blood leukocytes and was also detected in the muscle, spleen, skin, head kidney, gills, trunk kidney, heart, stomach, brain, intestine and liver by quantitative real-time PCR. A temporal variation in expression of RbCD33 was observed in different tissues after stimulating with E. tarda, S. iniae and red seabream iridovirus (RSIV). In the head kidney tissue, E. tarda and S. iniae induced RbCD33, while a down regulation was observed with RSIV. In addition, in spleen tissue, S. iniae caused a very high induction of RbCD33 in comparison with an E. tarda and RSIV challenge. In the liver and gill tissues, all three pathogens induced a high expression of RbCD33. The expression pattern in various tissues and its high induction after pathogen stimulation suggests that RbCD33 plays an important role in initiating the immune response via the inhibition of signal transduction of the myeloid lineage cells.

Olive flounder CD276 (B7-H3) a coinhibitory molecule for T cells: Responses during viral hemorrhagic septicemia virus (VHSV) stimulation.

Coinhibitory pathways in the B7-CD28 family provide critical inhibitory signals that regulate immune homeostasis, defense and protect tissue integrity. CD276 (B7-H3) is an important immune checkpoint member of this family, which is induced on antigen-presenting cells (APCs), and plays an important role in the inhibition of T-cell function. We have characterized the CD276 gene of olive flounder, Paralichthys olivaceus. OfCD276 has an ORF of 912 bp that codes for 303 amino acids with a predicted molecular mass of 33 kDa. It is a type I transmembrane protein with a single extracellular V- and C-like Ig domains, a transmembrane region, and a highly diverse cytoplasmic tail. This gene was distinctly expressed in gill, spleen, and skin, and sparsely expressed in other tissues. Pathogen stimulation by VHSV revealed that transcription of OfCD276 was induced on early hours in liver and expressed late in head kidney, spleen, intestine and gill tissues. Flow cytometry analysis of leukocytes revealed the percentage of granulocytes and lymphocytes that expressed OfCD276 molecules on their cell surface was 85.1% and 3.1%, respectively. Our study shows a significant role played by this coinhibitory molecule that participate in the regulation of the cell mediated immune response.

Molecular characterization of a T cell co-stimulatory receptor, CD28 of rock bream (Oplegnathus fasciatus): Transcriptional expression during bacterial and viral stimulation.

CD28 is a co-stimulatory receptor that provides a critical second signal alongside T cell receptors for the activation of naive T cells. We characterized the CD28 gene of rock bream, which has a deduced amino acid sequence of 221 residues with an extracellular Ig-superfamily V domain, transmembrane region, and cytoplasmic tail. The conservation in domain structures and other motifs shows that it is highly likely that RbCD28 is a homologue of mammalian CD28 and may have related co-stimulatory functions. RbCD28 is constitutively expressed in most tissues that were analysed, with a relatively higher expression in teleost lymphoid organs, such as spleens, gills, trunk kidneys and skin. Unlike human CD28, RbCD28 is highly expressed in skin and gill-associated lymphoid organs. Although gills showed constitutive expression of RbCD28 in control animals, after a pathogen challenge, induction of CD28 was low, particularly in RSIV and E. tarda infection. Whereas induction of RbCD28 was observed in kidney during E. tarda and S. iniae infection, downregulation was observed during RSIV infection. In the case of the liver, E. tarda caused an initial upregulation of RbCD28. RbCD28 activation of T cells in the spleen was limited to S. iniae infection. Activation of RbCD28 observed in lymphoid organs during infection of various pathogens shows its key role as a co-stimulatory receptor of T cells.

Protective responses of two paralogs of granulocyte colony stimulating factor (GCSF) in rock bream, Oplegnathus fasciatus during bacterial and viral infection.

Granulocyte colony stimulating factor (GCSF) has a key role in the production of neutrophilic granulocytes during normal hematopoietic development and release of neutrophils into the blood circulation. In this study we have identified and characterized two paralogs of GCSF (RbGCSF) in rock bream. Although RbGCSF-1 and RbGCSF-2 share low sequence conservation, its domains and protein structure still share significant similarity. Basal levels of RbGCSF-1 gene expression was high in peripheral blood leukocytes (PBLs), spleen and intestine whereas the RbGCSF-2 was highly expressed in PBLs and kidney, of healthy animals. A significant induction of RbGCSFs were observed after the challenge with Streptococcus iniae in kidney, spleen and gills during initial hours of infection. Whereas Edwarsiella tarda infection caused a reasonable expression in kidney. Red seabream iridovirus caused induction of RbGCSF-1 transcription only in gills during initial hours. This higher expression of RbGCSF in early hours may be its response to induce emergency hematopoiesis, due to shortage of neutrophils to combat the surge in pathogens. The difference in induction of RbGCSF paralogs during infection may constitute to its different roles or overlapping functions.

White spot syndrome virus entry is dependent on multiple endocytic routes and strongly facilitated by Cq-GABARAP in a CME-dependent manner.

White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, including crayfish. However, the molecular mechanism underlying its cellular entry remains elusive due to the lack of shrimp cell lines for viral propagation. Crayfish hematopoietic tissue (Hpt) cell culture was recently established as a good model for WSSV infection study. Here, we showed that multiple endocytic routes, including clathrin-mediated endocytosis (CME), macropinocytosis and caveolae-mediated endocytosis, were indispensably employed for the viral entry into Hpt cell of the crayfish Cherax quadricarinatus. Intriguingly, cellular autophagic activity was positively correlated with efficient viral entry, in which a key autophagy-related protein, γ-aminobutyric acid receptor-associated protein (Cq-GABARAP), that not only localized but also co-localized with WSSV on the Hpt cell membrane, strongly facilitated WSSV entry by binding to the viral envelope VP28 in a CME-dependent manner that was negatively regulated by Cq-Rac1. Furthermore, cytoskeletal components, including Cq-ß-tubulin and Cq-ß-actin, bound to both recombinant rCq-GABARAP and WSSV envelope proteins, which likely led to viral entry promotion via cooperation with rCq-GABARAP. Even under conditions that promoted viral entry, rCq-GABARAP significantly reduced viral replication at an early stage of infection, which was probably caused by the formation of WSSV aggregates in the cytoplasm.

Proteomic analysis by iTRAQ in red claw crayfish, Cherax quadricarinatus, hematopoietic tissue cells post white spot syndrome virus infection.

To elucidate proteomic changes of Hpt cells from red claw crayfish, Cherax quadricarinatus, we have carried out isobaric tags for relative and absolute quantitation (iTRAQ) of cellular proteins at both early (1 hpi) and late stage (12 hpi) post white spot syndrome virus (WSSV) infection. Protein database search revealed 594 protein hits by Mascot, in which 17 and 30 proteins were present as differentially expressed proteins at early and late viral infection, respectively. Generally, these differentially expressed proteins include: 1) the metabolic process related proteins in glycolysis and glucogenesis, DNA replication, nucleotide/amino acid/fatty acid metabolism and protein biosynthesis; 2) the signal transduction related proteins like small GTPases, G-protein-alpha stimulatory subunit, proteins bearing PDZ- or 14-3-3-domains that help holding together and organize signaling complexes, casein kinase I and proteins of the MAP-kinase signal transduction pathway; 3) the immune defense related proteins such as α-2 macroglobulin, transglutaminase and trans-activation response RNA-binding protein 1. Taken together, these protein information shed new light on the host cellular response against WSSV infection in a crustacean cell culture.

Detrimental effect of CO2-driven seawater acidification on a crustacean brine shrimp, Artemia sinica.

The effects of the decline in ocean pH, termed as ocean acidification due to the elevated carbon dioxide in the atmosphere, on calcifying organisms such as marine crustacean are unclear. To understand the possible effects of ocean acidification on the physiological responses of a marine model crustacean brine shrimp, Artemia sinica, three groups of the cysts or animals were raised at different pH levels (8.2 as control; 7.8 and 7.6 as acidification stress according to the predictions for the end of this century and next century accordingly) for 24 h or two weeks, respectively, followed by examination of their hatching success, morphological appearance such as deformity and microstructure of animal body, growth (i.e. body length), survival rate, expression of selected genes (involved in development, immunity and cellular activity etc), and biological activity of several key enzymes (participated in antioxidant responses and physiological reactions etc). Our results clearly demonstrated that the cysts hatching rate, growth at late stage of acidification stress, and animal survival rate of brine shrimp were all reduced due to lower pH level (7.6 & 7.8) on comparison to the control group (pH 8.2), but no obvious change in deformity or microstructure of brine shrimp was present under these acidification stress by microscopy observation and section analysis. In addition, the animals subjected to a lower pH level of seawater underwent changes on their gene expressions, including Spätzle, MyD88, Notch, Gram-negative bacteria binding protein, prophenoloxidase, Apoptosis inhibitor 5, Trachealess, Caveolin-1 and Cyclin K. Meanwhile, several key enzyme activities, including superoxide dismutase, catalase, peroxidase, alkaline phosphatase and acid phosphatase, were also affected by acidified seawater stress. Taken together, our findings supports the idea that CO2-driven seawater acidification indeed has a detrimental effect, in case of hatching success, growth and survival, on a model crustacean brine shrimp, which will increase the risk of juvenile brine shrimp and possibly also other crustaceans, as important live feeds for aquaculture being introduced in the ecosystem especially the marine food webs.