Recombinant neutralizing secretory IgA antibodies for preventing mucosal acquisition and transmission of SARS-CoV-2.

Passive delivery of antibodies to mucosal sites may be a valuable adjunct to COVID-19 vaccination to prevent infection, treat viral carriage, or block transmission. Neutralizing monoclonal IgG antibodies are already approved for systemic delivery, and several clinical trials have been reported for delivery to mucosal sites where SARS-CoV-2 resides and replicates in early infection. However, secretory IgA may be preferred because the polymeric complex is adapted for the harsh, unstable external mucosal environment. Here, we investigated the feasibility of producing neutralizing monoclonal IgA antibodies against SARS-CoV-2. We engineered two class-switched mAbs that express well as monomeric and secretory IgA (SIgA) variants with high antigen-binding affinities and increased stability in mucosal secretions compared to their IgG counterparts. SIgAs had stronger virus neutralization activities than IgG mAbs and were protective against SARS-CoV-2 infection in an in vivo murine model. Furthermore, SIgA1 can be aerosolized for topical delivery using a mesh nebulizer. Our findings provide a persuasive case for developing recombinant SIgAs for mucosal application as a new tool in the fight against COVID-19.

ß1,3-galactosyltransferase on chromosome 6 is essential for the formation of Lewis a structure on N-glycan in Oryza sativa.

ß1,3-galactose is the component of outer-chain elongation of complex N-glycans that, together with α1,4-fucose, forms Lewis a structures in plants. Previous studies have revealed that N-glycan maturation is mediated by sequential attachment of ß1,3-galactose and α1,4-fucose by individual ß1,3-galactosyltransferase (GalT) and α1,4-fucosyltransferase (1,4-FucT), respectively. Although GalT from several species has been studied, little information about GalT from rice is available. I therefore characterized three GalT candidate genes on different chromosomes in Oryza sativa. Seeds of rice lines that had T-DNA insertions in regions corresponding to individual putative GalT genes were obtained from a Rice Functional Genomic Express Database and plants grown until maturity. Homozygotes were selected from the next generation by genotyping PCR, and used for callus induction. Callus extracts of two independent T-DNA mutant rice which have T-DNA insertions at the same gene on chromosome 6 but in different exons showed highly reduced band intensity on a western blots using an anti-Lewis a antibody. Cell extracts and cultured media from suspension culture of the one of these mutant rice were further analysed by N-glycan profiling using matrix-associated laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). Identified N-glycan species containing ß1,3-galactose from both cell extracts and cultured media of knock-out mutant were less than 0.5% of total N-glycans while that of WT cells were 9.8% and 49.1%, respectively. This suggests that GalT located on rice chromosome 6 plays a major role in N-glycan galactosylation, and mutations within it lead to blockage of Lewis a epitope formation.

Production of Plant-Derived Japanese Encephalitis Virus Multi-Epitope Peptide in Nicotiana benthamiana and Immunological Response in Mice.

The current production of the Japanese encephalitis virus (JEV) vaccine is based on animal cells, where various risk factors for human health should be resolved. This study used a transient expression system to express the chimeric protein composed of antigenic epitopes from the JEV envelope (E) protein in Nicotiana benthamiana. JEV multi-epitope peptide (MEP) sequences fused with FLAG-tag or 6× His-tag at the C- or N-terminus for the purification were introduced into plant expression vectors and used for transient expression. Among the constructs, vector pSK480, which expresses MEP fused with a FLAG-tag at the C-terminus, showed the highest level of expression and yield in purification. Optimization of transient expression procedures further improved the target protein yield. The purified MEP protein was applied to an ICR mouse and successfully induced an antibody against JEV, which demonstrates the potential of the plant-produced JEV MEP as an alternative vaccine candidate.

Plant-based expression and characterization of SARS-CoV-2 virus-like particles presenting a native spike protein.

We have investigated the use of transient expression to produce virus-like particles (VLPs) of severe acute respiratory syndrome coronavirus 2, the causative agent of COVID-19, in Nicotiana benthamiana. Expression of a native form of the spike (S) protein, either alone or in combination with the envelope (E) and membrane (M) proteins, all of which were directed to the plant membranes via their native sequences, was assessed. The full-length S protein, together with degradation products, could be detected in total protein extracts from infiltrated leaves in both cases. Particles with a characteristic 'crown-shaped' or 'spiky' structure could be purified by density gradient centrifugation. Enzyme-linked immunosorbent assays using anti-S antibodies showed that threefold higher levels of VLPs containing the full-length S protein were obtained by infiltration with S alone, compared to co-infiltration of S with M and E. The S protein within the VLPs could be cleaved by furin in vitro and the particles showed reactivity with serum from recovering COVID-19 patients, but not with human serum taken before the pandemic. These studies show that the native S protein expressed in plants has biological properties similar to those of the parent virus. We show that the approach undertaken is suitable for the production of VLPs from emerging strains and we anticipate that the material will be suitable for functional studies of the S protein, including the assessment of the effects of specific mutations. As the plant-made material is noninfectious, it does not have to be handled under conditions of high containment.

Investigation of the N-Glycosylation of the SARS-CoV-2 S Protein Contained in VLPs Produced in Nicotiana benthamiana.

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the fast development of efficient therapeutics. Of the major structural proteins encoded by the SARS-CoV-2 genome, the SPIKE (S) protein has attracted considerable research interest because of the central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein is heavily glycosylated with 22 predicted N-glycosylation consensus sites as well as numerous mucin-type O-glycosylation sites. As a consequence, O- and N-glycosylations of this viral protein have received particular attention. Glycans N-linked to the S protein are mainly exposed at the surface and form a shield-masking specific epitope to escape the virus antigenic recognition. In this work, the N-glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana benthamiana (N. benthamiana) was investigated using a glycoproteomic approach. We show that 20 among the 22 predicted N-glycosylation sites are dominated by complex plant N-glycans and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana adopts an overall 3D structure similar to that of recombinant homologues produced in mammalian cells.

Salinomycin suppresses TGF-ß1-induced EMT by down-regulating MMP-2 and MMP-9 via the AMPK/SIRT1 pathway in non-small cell lung cancer.

Salinomycin (Sal) is a recently identified anti-tumor drug for treating several types of solid tumor; however, its effects on the migratory and invasive properties of non-small cell lung cancer (NSCLC) remain unclear. This study investigated the inhibitory effect underlying mechanisms of Salon transforming growth factor-ß1 (TGF-ß1)-induced epithelial-to-mesenchymal transition (EMT) and cell migration. Sal solidly blocked cell migration and invasion enhancement by TGF-ß1-induced EMT, through recovering E-cadherin loss and suppressing mesenchymal markers induction, as well as TGF-ß1-mediated AMPK/SIRT signaling activity upregulation. The pharmacologic inhibition or knockdown of AMPK or SIRT1 can act synergistically with Sal to inhibit TGF-ß1-induced MMP-2 and MMP-9. In contrast, AMPK or SIRT1 upregulation can protect against TGF-ß1-induced MMP-2 and MMP-9 inhibition by Sal. Next we demonstrated that the MMP-2 and MMP-9 knockdown can act synergistically with Sal to inhibit TGF-ß1-induced EMT. Moreover, treatment of PMA of MMP activator increased TGF-ß1-induced MMP-2 and MMP-9, even with Sal. Our results demonstrate that Sal suppresses TGF-ß1-induced EMT by downregulating MMP-2 and MMP-9 through the AMPK/SIRT pathway, thereby inhibiting lung cancer cell migration and invasion.

Inactivation of the ß (1, 2)-xylosyltransferase and the α (1, 3)-fucosyltransferase gene in rice (Oryza sativa) by multiplex CRISPR/Cas9 strategy.

KEY MESSAGE: CRISPR/Cas9-mediated OsXylT and OsFucT mutation caused the elimination of plant-specific ß1,2-xylose and α1,3-fucose residues on glycoproteins in rice, which is the first report of OsXylT/OsFucT double KO mutation in rice. N-glycosylation pathway is the one of post-translational mechanism and is known as highly conserved in eukaryotes. However, the process for complex-N-glycan modification is different between mammals and plants. In plant-specific manner, ß1,2-xylose and α1,3-fucose residues are transferred to N-glycan core structure on glycoproteins by ß1,2-xylosyltransferase (ß1,2-XylT) and α1,3-fucosyltransferase (α1,3-FucT), respectively. As an effort to use plants as a platform to produce biopharmaceuticals, the plant-specific N-glycan genes of rice (Oryza sativa), ß1,2-xylT (OsXylT) and α1,3-FucT (OsFucT), were knocked out using multiplex CRISPR/Cas9 technology. The double knock-out lines were found to have frameshift mutations by INDELs. Both ß1,2-xylose and α1,3-fucose residues in the lines were not detected in Western blot analysis. Consistently, there was no peak corresponding to the N-glycans in MALDI-TOF/MS analysis. Although α1,3-fucose and ß1,2-xylose residues were not detected in the line, other plant-specific residues of ß1,3-galactose and α1,4-fucose were detected. Thus, we suggest that each enzymes working on the process for complex N-glycan biosynthesis might independently act in rice, hence the double knock-out of both OsXylT and OsFucT might be not enough to humanize N-glycan structure in rice.

Production of functional recombinant cyclic citrullinated peptide monoclonal antibody in transgenic rice cell suspension culture.

Cyclic citrullinated peptide (CCP) antibody has been shown recently to be a promising marker for early detection and diagnosis of rheumatoid arthritis (RA). In order to exploit newly developed therapies for RA, early intervention is crucial in preventing irreversible joint damage. Here, we describe use of a plant expression system to produce a CCP antibody that could be used in the early diagnosis of RA. Heavy and light chain gene sequences of a CCP monoclonal antibody (CCP mAb) were cloned from the hybridoma cell (12G1) and introduced into two separate plant expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter system. The vectors were introduced into rice calli (Oryza sativa L. cv. Dongjin) using Agrobacterium tumefaciens mediated transformation. Integration of the CCP mAb genes into rice chromosomes was confirmed by a genomic DNA polymerase chain reaction and expression was verified by northern blot analysis of mRNA. The in vivo assembly and secretion of CCP mAb occurred in transgenic rice cell suspension culture under the RAmy3D expression system; accumulated CCP mAbs in the medium were purified by protein G affinity chromatography. Immunoblot assays and ELISA showed these plant-produced CCP mAbs successfully bound to a synthetic CCP antigen. Taken together, our results suggest that CCP mAb produced in a transgenic rice suspension culture were easily purified and biologically active against their antigen in the RA, and thus may be used a specific serological marker, which is present very early in the RA.

Production of recombinant human acid ß-glucosidase with high mannose-type N-glycans in rice gnt1 mutant for potential treatment of Gaucher disease.

Gaucher disease is an inherited metabolic disease caused by genetic acid ß -glucosidase (GBA) deficiency and is currently treated by enzyme replacement therapy. For uptake into macrophages, GBA needs to carry terminal mannose residues on their N-glycans. Knockout mutant rice of N-acetylglucosaminyltransferase-I (gnt1) have a disrupted N-glycan processing pathway and produce only glycoproteins with high mannose residues. In this study, we introduced a gene encoding recombinant human GBA into both wild-type rice (WT) and rice gnt1 calli. Target gene integration and mRNA expression were confirmed by genomic DNA PCR and Northern blotting, respectively. Secreted rhGBAs in culture media from cell lines originating from both WT (WT-GBA) and rice gnt1 (gnt1-GBA) were detected by Western blotting. Each rhGBA was purified by affinity and ion exchange chromatography. In vitro catalytic activity of purified rhGBA was comparable to commercial Chinese hamster ovary cell-derived rhGBA. N-glycans were isolated from WT-GBA and gnt1-GBA and analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The amounts of high mannose-type N-glycans were highly elevated in gnt1-GBA (100%) compared to WT-GBA (1%).

Delayed solitary mediastinal lymph node metastasis from a benign meningothelial meningioma six years after surgical resection of an intracranial tumor.

Meningiomas are common intracranial neoplasms with benign features, and extracranial metastases are very rare. There have been no previous reports of solitary mediastinal lymph node metastasis from benign meningiomas without pulmonary lesions. Here, we present a case of an 82-year-old female who visited our department for mediastinal lymphadenopathy with a history of meningioma treated with total surgical resection six years prior. Endobronchial ultrasound-guided transbronchial needle aspiration of the left lower paratracheal lymph node revealed a benign meningothelial meningioma. In patients with a history of meningioma, extracranial metastasis should be considered in the differential diagnosis of mediastinal lymphadenopathy.

Anesthetic management during whole-lung lavage using lung ultrasound in a patient with pulmonary alveolar proteinosis: a case report.

Pulmonary alveolar proteinosis (PAP) is an uncommon disease characterized by progressive accumulation of lipoprotein material in the lungs due to impaired surfactant clearance. Whole-lung lavage (WLL) is the current standard treatment and consists of sequential lavage of each lung to mechanically remove the residual material from the alveoli. Although WLL is considered safe, unexpected complications can occur. Moreover, due to the rarity of the disease itself, this procedure is unknown to many physicians, and management of intraoperative complications can be challenging for anesthesiologists. Lung ultrasound (LUS) provides reliable and valuable information for detecting perioperative pulmonary complications and, in particular, quantitation of lung water content. There have been reports on monitoring the different stages of controlled deaeration of the non-ventilated lung during WLL using LUS. However, it has been limited to non-ventilated lungs. Therefore, we report the use of LUS in WLL to proactively detect pulmonary edema in the ventilated lung and implement a safe and effective anesthesia strategy. Given the limited diagnostic tools available to anesthesiologists in the operating room, LUS is a reliable, fast, and noninvasive method for identifying perioperative pulmonary complications in patients with PAP undergoing WLL.

Mobile computed tomography for the efficient allocation of medical resources in patients with COVID-19 pneumonia: A case report.

RATIONALE: The highly contagious Coronavirus 2019 (COVID-19) infection raise social and economic burden. Medical staff and resources are being diverted for the care of patients with COVID-19. There are problems for healthcare systems, including burnout syndrome for the medical staff and exhaustion of medical resources. PATIENT CONCERNS: The patient was a 65-year-old woman presenting with fever, cough, and dyspnea due to COVID-19 pneumonia. She received antiviral agents, broad-spectrum antibiotics, and conservative treatment. Although her clinical condition improved, there was no significant improvement in portable chest X-ray results. DIAGNOSES: Due to concerns over the propagation of infection when transferring to patients for scanning and the need for excessive medical personnel to move patients, we moved a mobile chest computed tomography (CT) machine to an isolation ward for CT scanning. INTERVENTIONS: We report our experience using mobile chest CT to effectively allocate medical resources and assess treatment response in patient with COVID-19 pneumonia. OUTCOMES: Follow-up mobile CT scans disclosed progressive resolution of the multifocal ground-glass opacities and mixed consolidations distributed peripheral to subpleural spaces. During the mobile chest CT scan, there were no adverse or unforeseen events. Three medical personnel were required to performed mobile chest CT, including a clinician, a nurse, and a radiologist. LESSONS: As a result of using mobile chest CT on COVID-19 patients, the number of medical personnel required for CT scanning decreased by about 83%, rapid, and safe compared with a patient who performed conventional CT.

Producing Vaccines against Enveloped Viruses in Plants: Making the Impossible, Difficult.

The past 30 years have seen the growth of plant molecular farming as an approach to the production of recombinant proteins for pharmaceutical and biotechnological uses. Much of this effort has focused on producing vaccine candidates against viral diseases, including those caused by enveloped viruses. These represent a particular challenge given the difficulties associated with expressing and purifying membrane-bound proteins and achieving correct assembly. Despite this, there have been notable successes both from a biochemical and a clinical perspective, with a number of clinical trials showing great promise. This review will explore the history and current status of plant-produced vaccine candidates against enveloped viruses to date, with a particular focus on virus-like particles (VLPs), which mimic authentic virus structures but do not contain infectious genetic material.

Immunoproteomics profiling of blood stage Plasmodium vivax infection by high-throughput screening assays.

Completed genome sequences and stage-specific transcriptomes of the intraerythrocytic developmental cycle of Plasmodium vivax offers the opportunity to profile immune responses against P. vivax infection using innovative screening approaches. To detect the immune responses to blood stage-specific proteins, we applied a protein array technology to screen the sera of vivax malaria patients. Herein, a set of genes from the P. vivax blood stage was cloned using the In-Fusion cloning method and expressed by a wheat germ cell-free system. A total of 94 open reading frames (ORFs) were cloned and 89 (95%, 89/94) proteins were expressed, which were screened with sera from P. vivax-infected patients and healthy individuals using protein arrays. A total of 18 (19.1%, 18/94) highly immunoreactive proteins were identified, including 7 well-characterized vivax vaccine candidates. The remaining 11 ORFs have not been previously described as immunologically reactive. These novel immunoproteomes of the vivax malaria blood stage will be further studied as potential vaccine candidates. In this first report, high-throughput screening assays have been applied to investigate blood stage-specific immunoproteomes from vivax malaria. These methods may be used to determine immunodominant candidate antigens from the P. vivax genome.

Serodiagnostic antigens of Clonorchis sinensis identified and evaluated by high-throughput proteogenomics.

Clonorchiasis caused by Clonorchis sinensis is endemic in East Asia; approximately 15 million people have been infected thus far. To diagnose the infection, serodiagnostic tests with excellent functionality should be performed. First, 607 expressed sequence tags encoding polypeptides with a secretory signal were expressed into recombinant proteins using an in vitro translation system. By protein array-based screening using C. sinensis-infected sera, 18 antigen candidate proteins were selected and assayed for cross-reactivity against Opisthorchis viverrini-infected sera. Of the six antigenic proteins selected, four were synthesized on large scale in vitro and evaluated for antigenicity against the flukes-infected human sera using ELISA. CsAg17 antigen showed the highest sensitivity (77.1%) and specificity (71.2%). The sensitivity and specificity of the bacterially produced CsAg17-28GST fusion antigen was similar to those of CsAg17 antigen. CsAg17 antigen can be used to develop point-of-care serodiagnostic tests for clonorchiasis.

A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells.

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis andinflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation between spots was 2.6% (n=3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n=3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.

Spontaneous pepsin C-catalyzed activation of human pepsinogen C in transgenic rice cell suspension culture: Production and characterization of human pepsin C.

A human pepsinogen C (hPGC) gene was synthesized with rice-optimized codon usage and cloned into a rice expression vector containing the promoter, signal peptide, and terminator derived from the rice α-amylase 3D (Ramy3D) gene. In addition, a 6-His tag was added to the 3' end of the synthetic hPGC gene for easy purification. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. The integration of the hPGC gene into the chromosome of the transgenic rice callus and hPGC expression in transgenic rice cell suspensions was verified via genomic DNA polymerase chain reaction amplification and Northern blot analysis. Western blot analysis indicated both hPGC and its mature form, human pepsin C, with masses of 42- and 36-kDa in the culture medium under sugar starvation conditions. Human pepsin C was purified from the culture medium using a Ni-NTA agarose column and the NH2-terminal 5-residue sequences were verified by amino acid sequencing. The hydrolyzing activity of human pepsin C was confirmed using bovine hemoglobin as a substrate. The optimum pH and temperature for pepsin activity were 2.0 and 40°C, respectively.

Scalable Solvothermal Synthesis of Superparamagnetic Fe3O4 Nanoclusters for Bioseparation and Theragnostic Probes.

Magnetic nanoparticles have had a significant impact on a wide range of advanced applications in the academic and industrial fields. In particular, in nanomedicine, the nanoparticles require specific properties, including hydrophilic behavior, uniform and tunable dimensions, and good magnetic properties, which are still challenging to achieve by industrial-scale synthesis. Here, we report a gram-scale synthesis of hydrophilic magnetic nanoclusters based on a one-pot solvothermal system. Using this approach, we achieved the nanoclusters with controlled size composed of magnetite nanocrystals in close-packed superstructures that exhibited hydrophilicity, superparamagnetism, high magnetization, and colloidal stability. The proposed solvothermal method is found to be highly suitable for synthesizing industrial quantities (gram-per-batch level) of magnetic spheres with unchanged structural and magnetic properties. Furthermore, coating the magnetic spheres with an additional silica layer provided further stability and specific functionalities favorable for biological applications. Using in vitro and in vivo studies, we successfully demonstrated both positive and negative separation and the use of the magnetic nanoclusters as a theragnostic nanoprobe. This scalable synthetic procedure is expected to be highly suitable for widespread use in biomedical, energy storage, photonics, and catalysis fields, among others.

Production of recombinant human acid α-glucosidase with high-mannose glycans in gnt1 rice for the treatment of Pompe disease.

Lysosomal storage diseases are a group of inherited metabolic disorders. Patients are treated with enzyme replacement therapy (ERT), in which the replacement enzymes are required to carry terminal mannose or mannose 6-phosphate residues to allow efficient uptake into target cells and tissues. N-acetylglucosaminyltransferase-I (GnTI) mediates N-glycosylation in the cis cisternae of the Golgi apparatus by adding N-acetylglucosamine to the exposed terminal mannose residue of core N-glycan structures for further processing. Mutant rice lacking GnTI produces only high mannosylated glycoproteins. In this study, we introduced a gene encoding recombinant human acid α-glucosidase (rhGAA), which is used in ERT for Pompe disease, into gnt1 rice callus by particle bombardment. Integration of the target gene into the genome of the gnt1 rice line and its mRNA expression were confirmed by PCR and Northern blot, respectively. Western blot analysis was performed to confirm secretion of the target proteins into the culture media. Using an indirect enzyme linked immunosorbent assay, we determined the maximum expression of rhGAA to be approximately 45mg/L, 13days after induction. To assay the enzymatic activity and determine the N-glycan profile of rhGAA, we purified the protein using a 6×histidine tag. The in vitro α-glucosidase activity of rhGAA from gnt1 rice callus (gnt1-GAA) was 3.092U/mg, similar to the activity of the Chinese hamster ovary cell-derived GAA (3.154U/mg). N-glycan analysis revealed the presence of high-mannose N-glycans on gnt1-GAA. In addition, the production of high-mannose GAA using gnt1 rice calli as an expression host was characterized, which may aid the future development of therapeutic enzymes for the treatment of Pompe disease.

Analysis of protein interactions on protein arrays by a novel spectral surface plasmon resonance imaging.

We presented a novel surface plasmon resonance (SPR) imaging method for analysis of protein arrays based on a wavelength interrogation-based SPR biosensor. The spectral imaging was performed by the combination of position control and resonance wavelengths calculated from SPR reflectivity spectra. The imaging method was evaluated by analyzing interactions of glutathione S-transferase-fusion proteins with their antibodies. Antigen-antibody interactions were successfully analyzed on glutathione S-transferase-fusion protein arrays by using the spectral imaging method, and the results were confirmed by a parallel analysis using a previously used spectral SPR biosensor based on wavelength interrogation. Specific binding of anti-Rac1 and anti-RhoA to Rac1 and RhoA on the protein arrays was qualitatively and quantitatively analyzed by the spectral SPR imaging. Thus, it was suggested that the novel spectral SPR imaging was a useful tool for the high-throughput analysis of protein-protein interactions on protein arrays.