scholarly journals Expression and Purification of a PEDV-Neutralizing Antibody and Its Functional Verification

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 472
Author(s):  
Wenshu Shi ◽  
Haiyang Hao ◽  
Mengran Li ◽  
Jianqin Niu ◽  
Yaning Hu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Neutralizing Antibodies ◽  
Hek293 Cell ◽  
Neutralizing Antibody ◽  
Functional Verification ◽  
High Morbidity ◽  
Rapid Variation ◽  
Diarrhea Virus ◽  
Pathogenic Virus

Porcine epidemic diarrhea virus (PEDV) is a highly infectious and pathogenic virus causing high morbidity and mortality, especially in newborn piglets. There remain problems with contemporary PEDV vaccines, in part because of the rapid variation of PEDV, poor conferred immunity, and numerous side effects. The ability to produce PEDV-neutralizing antibodies suggests that we may be able to increase the success rate of PEDV prevention in piglets using these antibodies. In this study, we produced an anti-PEDV S protein monoclonal antibody (anti-PEDV mAb-2) that neutralized PEDV-CV777 (a G1 strain), PEDV-SDSX16 and PEDV-Aj1102 (two G2 strains). In vivo challenge experiments demonstrated that anti-PEDV mAb-2 inhibited the PEDV infection in piglets. We also produced three HEK293 cell lines that expressed anti-PEDV mAb-2. Overall, our study showed that anti-PEDV mAb-2 produced from hybridoma supernatants effectively inhibited PEDV infection in piglets, and the recombinant HEK293 cell lines expressed anti-PEDV mAb-2 genes.

scholarly journals Long non-coding RNA CCDC183-AS1 acts AS a miR-589-5p sponge to promote the progression of hepatocellular carcinoma through regulating SKP1 expression

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
He Zhu ◽  
Hongwei Zhang ◽  
Youliang Pei ◽  
Zhibin Liao ◽  
Furong Liu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Human Cancer ◽  
Quantitative Polymerase Chain Reaction ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
High Morbidity ◽  
Regulatory Relationship

Abstract Background Hepatocellular carcinoma (HCC) is a common type of malignant human cancer with high morbidity and poor prognosis, causing numerous deaths per year worldwide. Growing evidence has been demonstrated that long non-coding RNAs (lncRNAs) are closely associated with hepatocarcinogenesis and metastasis. However, the roles, functions, and working mechanisms of most lncRNAs in HCC remain poorly defined. Methods Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of CCDC183-AS1 in HCC tissues and cell lines. Cell proliferation, migration and invasion ability were evaluated by CCK-8 and transwell assay, respectively. Animal experiments were used to explore the role of CCDC183-AS1 and miR-589-5p in vivo. Bioinformatic analysis, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to confirm the regulatory relationship between CCDC183-AS1, miR-589-5p and SKP1. Results Significantly upregulated expression of CCDC183-AS1 was observed in both HCC tissues and cell lines. HCC patients with higher expression of CCDC183-AS1 had a poorer overall survival rate. Functionally, overexpression of CCDC183-AS1 markedly promoted HCC cell proliferation, migration and invasion in vitro and tumor growth and metastasis in vivo, whereas the downregulation of CCDC183-AS1 exerted opposite effects. MiR-589-5p inhibitor counteracted the proliferation, migration and invasion inhibitory effects induced by CCDC183-AS1 silencing. Mechanistically, CCDC183-AS1 acted as a ceRNA through sponging miR-589-5p to offset its inhibitory effect on the target gene SKP1, then promoted the tumorigenesis of HCC. Conclusions CCDC183-AS1 functions as an oncogene to promote HCC progression through the CCDC183-AS1/miR-589-5p/SKP1 axis. Our study provided a novel potential therapeutic target for HCC patients.

scholarly journals CircTP63 promotes hepatocellular carcinoma progression by sponging miR-155-5p and upregulating ZBTB18

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jiantao Wang ◽  
Jinbiao Che
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Circular Rnas ◽  
Epithelial Cell Line ◽  
High Morbidity ◽  
Mrna Levels ◽  
Promoting Effect ◽  
Tumor Stages ◽  
Hcc Cells

Abstract Background Hepatocellular carcinoma (HCC) is the leading cause of tumor-related death worldwide due to high morbidity and mortality, yet lacking effective biomarkers and therapies. Circular RNAs (circRNAs) are a group of non-coding RNAs that regulate gene expression through interacting with miRNAs, implicating in the tumorigenesis and progression. A novel circRNA, circTP63, was reported to be an oncogene in HCC. However, its role in HCC remains unclear. Methods qRT-PCR was used to assess the mRNA levels of CircTP63 in 90 pairs of tumor and adjacent normal tissues from HCC patients, one human normal hepatic epithelial cell line and HCC cell lines. CCK-8, colony formation, transwell, and flow cytometry assays were performed to detect the cellular function of circTP63/miR-155-5p/ZBTB18 in HCC cells. HCC xenograft mice models were established to assess the in vivo effect of circTP63. Bioinformatic analysis, RNA pull-down and luciferase assays were used to determine the interaction among circTP63/miR-155-5p/ZBTB18. Results circTP63 was significantly upregulated in HCC tissues and cell lines. High circTP63 expression is closely associated with the tumor stages, lymph node metastasis, and poor prognosis of HCC patients. Functionally, knockdown of circTP63 inhibited cell proliferation, migration, invasion, and promoted cell apoptosis of HCC. Meanwhile, overexpression of circTP63 enhanced HCC progression. Mechanically, circTP63 was a sponge of miR-155-5p to facilitate the ZBTB18 expression, and the ZBTB18 expression in HCC tissues was negatively associated with the survival rate of HCC patients. Furthermore, rescued assays revealed that the reduced tumor-promoting effect on HCC cells induced by knockdown of circTP63 can be reversed by miR-155-5p inhibitor or ZBTB18 overexpression. Conclusion Our data highlight a critical circTP63-miR-155-5p-ZBTB18 regulatory network involved in the HCC progression, gaining mechanistic insights into the function of circRNAs in HCC progression, and providing effective biomarkers and therapeutic targets for HCC treatment.

scholarly journals Screening of potent neutralizing antibodies against SARS-CoV-2 using convalescent patients-derived phage-display libraries

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yongbing Pan ◽  
Jianhui Du ◽  
Jia Liu ◽  
Hai Wu ◽  
Fang Gui ◽  
...  
Keyword(s):  
Phage Display ◽  
Neutralizing Antibodies ◽  
Variable Region ◽  
Neutralizing Antibody ◽  
Chain Gene ◽  
Prophylactic Efficacy ◽  
Heavy Chains ◽  
Effective Interventions ◽  
Phage Display Libraries

AbstractAs the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health worldwide, the development of effective interventions is urgently needed. Neutralizing antibodies (nAbs) have great potential for the prevention and treatment of SARS-CoV-2 infection. In this study, ten nAbs were isolated from two phage-display immune libraries constructed from the pooled PBMCs of eight COVID-19 convalescent patients. Eight of them, consisting of heavy chains encoded by the immunoglobulin heavy-chain gene-variable region (IGHV)3-66 or IGHV3-53 genes, recognized the same epitope on the receptor-binding domain (RBD), while the remaining two bound to different epitopes. Among the ten antibodies, 2B11 exhibited the highest affinity and neutralization potency against the original wild-type (WT) SARS-CoV-2 virus (KD = 4.76 nM for the S1 protein, IC50 = 6 ng/mL for pseudoviruses, and IC50 = 1 ng/mL for authentic viruses), and potent neutralizing ability against B.1.1.7 pseudoviruses. Furthermore, 1E10, targeting a distinct epitope on RBD, exhibited different neutralization efficiency against WT SARS-CoV-2 and its variants B.1.1.7, B.1.351, and P.1. The crystal structure of the 2B11–RBD complexes revealed that the epitope of 2B11 highly overlaps with the ACE2-binding site. The in vivo experiment of 2B11 using AdV5-hACE2-transduced mice showed encouraging therapeutic and prophylactic efficacy against SARS-CoV-2. Taken together, our results suggest that the highly potent SARS-CoV-2-neutralizing antibody, 2B11, could be used against the WT SARS-CoV-2 and B.1.1.7 variant, or in combination with a different epitope-targeted neutralizing antibody, such as 1E10, against SARS-CoV-2 variants.

scholarly journals CircTP63 promotes hepatocellular carcinoma progression by sponging miR-155-5p and upregulating ZBTB18

2020 ◽  
Author(s):  
Jiantao Wang ◽  
Jinbiao Che
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Circular Rnas ◽  
Epithelial Cell Line ◽  
High Morbidity ◽  
Mrna Levels ◽  
Promoting Effect ◽  
Tumor Stages ◽  
Hcc Cells

Abstract Background: Hepatocellular carcinoma (HCC) is the leading cause of tumor-related death worldwide due to high morbidity and mortality, yet lacking effective biomarkers and therapies. Circular RNAs (circRNAs) are a group of non-coding RNAs that regulate gene expression through interacting with miRNAs, implicating in the tumorigenesis and progression. A novel circRNA, circTP63, was reported to be an oncogene in HCC. However, its role in HCC remains unclear.Methods: qRT-PCR was used to assess the mRNA levels of CircTP63 in 90 pairs of tumor and adjacent normal tissues from HCC patients, one human normal hepatic epithelial cell line and HCC cell lines. CCK-8, colony formation, transwell, and flow cytometry assays were performed to detect the cellular function of circTP63/miR-155-5p/ZBTB18 in HCC cells. HCC xenograft mice models were established to assess the in vivo effect of circTP63. Bioinformatic analysis, RNA pull-down and luciferase assays were used to determine the interaction among circTP63/miR-155-5p/ZBTB18.Results: circTP63 was significantly upregulated in HCC tissues and cell lines. High circTP63 expression is closely associated with the tumor stages, lymph node metastasis, and poor prognosis of HCC patients. Functionally, knockdown of circTP63 inhibited cell proliferation, migration, invasion, and promoted cell apoptosis of HCC. Meanwhile, overexpression of circTP63 enhanced HCC progression. Mechanically, circTP63 was a sponge of miR-155-5p to facilitate the ZBTB18 expression, and the ZBTB18 expression in HCC tissues was negatively associated with the survival rate of HCC patients. Furthermore, rescued assays revealed that the reduced tumor-promoting effect on HCC cells induced by knockdown of circTP63 can be reversed by miR-155-5p inhibitor or ZBTB18 overexpression.Conclusion: Our data highlight a critical circTP63-miR-155-5p-ZBTB18 regulatory network involved in the HCC progression, gaining mechanistic insights into the function of circRNAs in HCC progression, and providing effective biomarkers and therapeutic targets for HCC treatment.

scholarly journals Role of TGF-alpha in the progression of diabetic kidney disease

2017 ◽  
Vol 312 (6) ◽  
pp. F951-F962 ◽  
Author(s):  
Josef G. Heuer ◽  
Shannon M. Harlan ◽  
Derek D. Yang ◽  
Dianna L. Jaqua ◽  
Jeffrey S. Boyles ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Renal Disease ◽  
Neutralizing Antibodies ◽  
Diabetic Kidney Disease ◽  
Transforming Growth Factor ◽  
Renin Angiotensin System ◽  
Neutralizing Antibody ◽  
Diabetic Kidney

Transforming growth factor-alpha (TGFA) has been shown to play a role in experimental chronic kidney disease associated with nephron reduction, while its role in diabetic kidney disease (DKD) is unknown. We show here that intrarenal TGFA mRNA expression, as well as urine and serum TGFA, are increased in human DKD. We used a TGFA neutralizing antibody to determine the role of TGFA in two models of renal disease, the remnant surgical reduction model and the uninephrectomized (uniNx) db/db DKD model. In addition, the contribution of TGFA to DKD progression was examined using an adeno-associated virus approach to increase circulating TGFA in experimental DKD. In vivo blockade of TGFA attenuated kidney disease progression in both nondiabetic 129S6 nephron reduction and Type 2 diabetic uniNx db/db models, whereas overexpression of TGFA in uniNx db/db model accelerated renal disease. Therapeutic activity of the TGFA antibody was enhanced with renin angiotensin system inhibition with further improvement in renal parameters. These findings suggest a pathologic contribution of TGFA in DKD and support the possibility that therapeutic administration of neutralizing antibodies could provide a novel treatment for the disease.

scholarly journals Propagation of SARS-CoV-2 in Calu-3 Cells to Eliminate Mutations in the Furin Cleavage Site of Spike

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2434
Author(s):  
John James Baczenas ◽  
Hanne Andersen ◽  
Sujatha Rashid ◽  
David Yarmosh ◽  
Nikhita Puthuveetil ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Cleavage Site ◽  
Furin Cleavage ◽  
Model Studies ◽  
Furin Cleavage Site ◽  
Pathogenic Virus ◽  
Epithelial Cell Lines ◽  
Propagation Methods

SARS-CoV-2 pathogenesis, vaccine, and therapeutic studies rely on the use of animals challenged with highly pathogenic virus stocks produced in cell cultures. Ideally, these virus stocks should be genetically and functionally similar to the original clinical isolate, retaining wild-type properties to be reliably used in animal model studies. It is well-established that SARS-CoV-2 isolates serially passaged on Vero cell lines accumulate mutations and deletions in the furin cleavage site; however, these can be eliminated when passaged on Calu-3 lung epithelial cell lines, as presented in this study. As numerous stocks of SARS-CoV-2 variants of concern are being grown in cell cultures with the intent for use in animal models, it is essential that propagation methods generate virus stocks that are pathogenic in vivo. Here, we found that the propagation of a B.1.351 SARS-CoV-2 stock on Calu-3 cells eliminated viruses that previously accumulated mutations in the furin cleavage site. Notably, there were alternative variants that accumulated at the same nucleotide positions in virus populations grown on Calu-3 cells at multiple independent facilities. When a Calu-3-derived B.1.351 virus stock was used to infect hamsters, the virus remained pathogenic and the Calu-3-specific variants persisted in the population. These results suggest that Calu-3-derived virus stocks are pathogenic but care should still be taken to evaluate virus stocks for newly arising mutations during propagation.

scholarly journals Kinetics of Nucleocapsid, Spike and Neutralizing Antibodies, and Viral Load in Patients with Severe COVID-19 Treated with Convalescent Plasma

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1844
Author(s):  
Thomas P. Thomopoulos ◽  
Margherita Rosati ◽  
Evangelos Terpos ◽  
Dimitris Stellas ◽  
Xintao Hu ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Rna Binding ◽  
Neutralizing Antibody ◽  
Binding Domain ◽  
High Morbidity ◽  
Viral Loads ◽  
Effective Treatment Modality ◽  
Meta Analyses ◽  
Humoral Responses ◽  
Kinetics Of

COVID-19 is an ongoing pandemic with high morbidity and mortality. Despite meticulous research, only dexamethasone has shown consistent mortality reduction. Convalescent plasma (CP) infusion might also develop into a safe and effective treatment modality on the basis of recent studies and meta-analyses; however, little is known regarding the kinetics of antibodies in CP recipients. To evaluate the kinetics, we followed 31 CP recipients longitudinally enrolled at a median of 3 days post symptom onset for changes in binding and neutralizing antibody titers and viral loads. Antibodies against the complete trimeric Spike protein and the receptor-binding domain (Spike-RBD), as well as against the complete Nucleocapsid protein and the RNA binding domain (N-RBD) were determined at baseline and weekly following CP infusion. Neutralizing antibody (pseudotype NAb) titers were determined at the same time points. Viral loads were determined semi-quantitatively by SARS-CoV-2 PCR. Patients with low humoral responses at entry showed a robust increase of antibodies to all SARS-CoV-2 proteins and Nab, reaching peak levels within 2 weeks. The rapid increase in binding and neutralizing antibodies was paralleled by a concomitant clearance of the virus within the same timeframe. Patients with high humoral responses at entry demonstrated low or no further increases; however, virus clearance followed the same trajectory as in patients with low antibody response at baseline. Together, the sequential immunological and virological analysis of this well-defined cohort of patients early in infection shows the presence of high levels of binding and neutralizing antibodies and potent clearance of the virus.

scholarly journals LACK OF IDENTITY IN NEUTRALIZING AND HEMAGGLUTINATION-INHIBITING ANTIBODIES AGAINST INFLUENZA VIRUSES

1950 ◽  
Vol 91 (1) ◽  
pp. 65-86 ◽  
Author(s):  
Duard L. Walker ◽  
Frank L. Horsfall
Keyword(s):  
Influenza Virus ◽  
Hemagglutination Inhibition ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Immune Serum ◽  
Influenza Viruses ◽  
In Ovo ◽  
Neutralization Line

There is an exponential linear relationship between the quantity of influenza virus neutralized and the quantity of immune serum employed in in ovo neutralization. The slope of the neutralization line is extremely steep. The concentration of neutralizing antibody can be measured with considerable precision in ovo if the constant virus-varying serum technique is utilized. The amounts of hemagglutination-inhibiting and neutralizing antibodies which are absorbed by a given quantity of influenza virus (PR8) were found to be predictable and the degree of reactivity of these two antibodies was shown to be directly related to the extent of immunization. It was demonstrated that there are marked discrepancies in correlation between antibody titers obtained by in vitro hemagglutination-inhibition and in vivo neutralization techniques and that neutralizing antibody is preferentially absorbed by a given quantity of virus. Inasmuch as the results were found not to be attributable to peculiarities of the techniques employed, it appears that the antibodies measured by hemagglutination-inhibition in vitro and by neutralization in vivo are not identical.

scholarly journals Intranasal administration of a monoclonal neutralizing antibody protects mice against SARS-CoV-2 infection

2021 ◽  
Author(s):  
Sandro Halwe ◽  
Alexandra Kupke ◽  
Kanika Vanshylla ◽  
Falk Liberta ◽  
Henning Gruell ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Pharmacokinetic Profile ◽  
Lung Pathology ◽  
Therapeutic Drugs ◽  
Intranasal Application ◽  
Important Drug

Despite recent availability of vaccines against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there is an urgent need for specific anti-SARS-CoV-2 drugs. Monoclonal neutralizing antibodies are an important drug class in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection and their potential to be used as both, prophylactic and therapeutic drugs. Clinically used neutralizing antibodies against respiratory viruses are currently injected intravenously, which can lead to suboptimal pulmonary bioavailability and thus to a lower effectiveness. Here we describe DZIF-10c, a fully human monoclonal neutralizing antibody that binds the receptor-binding domain of SARS-CoV-2 spike protein. DZIF-10c displays an exceptionally high neutralizing potency against SARS-CoV-2 and retains activity against the variants of concern B.1.1.7 and B.1.351. Importantly, not only systemic but also intranasal application of DZIF-10c abolished presence of infectious particles in the lungs of SARS-CoV-2 infected mice and mitigated lung pathology. Along with a favorable pharmacokinetic profile, these results highlight DZIF-10c as a novel human SARS-CoV-2 neutralizing antibody with high in vitro and in vivo antiviral potency. The successful intranasal application of DZIF-10c paves the way for clinical trials investigating topical delivery of anti-SARS-CoV-2 antibodies.

Targeting FGFR3 in t(4;14) Mutiple Myeloma: Pre-Clinical Studies of PRO-001, a Novel Anti-FGFR3 Neutralizing Antibody.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2479-2479
Author(s):  
Suzanne Trudel ◽  
Ellen Wei ◽  
Zhi Hua Li ◽  
Eran Rom ◽  
Ira Chumakov ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Lines ◽  
Neutralizing Antibody ◽  
Myeloma Cell ◽  
Chromosomal Translocations ◽  
Minimal Growth ◽  
Erk Phosphorylation ◽  
Human Myeloma Cell

Abstract As with other B-cell malignancies, chromosomal translocations to the immunoglobulin heavy-chain (IgH) locus on chromosome 14q32 are believed to be a hallmark of multiple myeloma (MM), occurring in approximately 50% of patients. Identification of these chromosomal translocations has resulted in the discovery of powerful prognostic tools and novel molecular targets that promise to revolutionize the treatment of this malignancy. Five recurrent translocation partners have been defined, resulting in the dysregulation of the genes encoding cyclin D1 and D3, c-maf, mafB and Fibroblast Growth Factor Receptor 3 (FGFR3) together with MMSET. Genetic analysis of 14q32 translocations in MM has identified distinct groups of patients with separate clinical outcomes supporting a biological correlation of these genes in MM. In particular, the t(4;14) translocation portends a particularly bad prognosis. The association of FGFR3 expression with t(4;14) myeloma and the demonstration of the transforming potential of this receptor tyrosine kinase (RTK), make this a particularly attractive target for drug development for this poor prognosis group. We report here the development of a novel and highly specific anti-FGFR3 neutralizing antibody (PRO-001) isolated from a phage display human combinatorial antibody library. PRO-001 binds with high affinity (Kd=1.3 nM) to FGFR3 in in vitro binding assays and blocks ligand-dependent and independent FGFR3 phosphorylation and signal transduction in cell-based assays. Furthermore, PRO-001 potently inhibits FGFR3-dependent solid tumor growth in mouse xenograft models. We found that PRO-001 bound to, and competed with FGF binding to the surface of FGFR3 on human myeloma cell lines. PRO-001 inhibited FGF-induced phosphorylation of wild-type FGFR3 and downstream ERK phosphorylation in stable B9 cell transfectants (B9-WT) and FGFR3 expressing human myeloma cell lines. The antibody inhibited FGF-mediated growth of B9-WT with an IC50 of 3 μg/ml as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of PRO-001 for FGFR3. PRO-001 inhibited the viability of the FGFR3 expressing, human myeloma cell line, UTMC2. Inhibition of viability was still observed when cells were co-cultured with stroma or in the presence of IL-6, a potent growth factor for MM cells. Several myeloma cell lines lacking FGFR3, showed minimal growth inhibition demonstrating selectivity and lack of non-specific toxic at effective dose concentrations. Finally, PRO-001 bound to FGFR3 on the cell surface, inhibited ERK phosphorylation, and induced cytotoxic responses in primary MM samples derived from t(4;14) positive patients. A xenograft mouse model has been established and studies assessing in vivo activity of PRO-001 are planned and will be reported. Taken together, the data demonstrate that PRO-001 is a specific and potent inhibitor of FGFR3 and that it deserves further study for targeted therapy in MM.

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