Background: A novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2. Aim: The cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed. Methods: The SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein. Results: An immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2infected cells and is suitable for use in a sandwich ELISA format. Conclusion: The cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.
Human papillomavirus (HPV) is the primary cause of human cervical cancer. The viral proteins E6 and E7 are essential to transform noncancerous epithelial cells into cancerous carcinomas by targeting key tumor suppressors p53 and retinoblastoma (Rb) proteins, respectively, but the cellular factors involved in E6 and E7 transcription themselves are incompletely understood. In this study, we defined a novel isoform of the mixed lineage leukemia 5 gene (MLL5b) as a specific and critical regulator of E6 and E7 transcription in cervical carcinoma cells. MLL5b is present in HPV16/18-positive cells including human primary cervical carcinoma specimens. Interaction of MLL5b with the AP-1-binding site at the distal region of the HPV18 long control region led to activation of E6/E7 transcription. Conversely, RNA interference-mediated knockdown of MLL5b downregulated both E6 and E7 expression. MLL5b downregulation was sufficient to restore p53 protein levels and reduce Rb phosphorylation, thereby reactivating apoptosis and cell-cycle checkpoints. By defining this novel MLL5b isoform and its specific critical role in activating E6/E7 gene transcription in HPV16/18-induced cervical cancers, our work highlights the potential of MLL5b as a biomarker and new therapeutic target in primary HPVinduced cervical cancers. Cancer Res; 71(21); 6696-707. Ó2011 AACR.
Keywords: envelope 2 protein, epitope, monoclonal antibody, virus pathogenesis Chikungunya virus (CHIKV) is a medically important human viral pathogen that causes Chikungunya fever accompanied with debilitating and persistent joint pain. Host-elicited or passively-transferred monoclonal antibodies (mAb) are essential mediators of CHIKV clearance. Therefore, this study aimed to generate and characterize a panel of mAbs for their neutralization efficacy against CHIKV infection in a cell-based and murine model.To evaluate their antigenicity and neutralization profile, indirect enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA) and a plaque reduction neutralization test were performed on mAbs of IgM isotype. CHIKV escape mutants against mAb 3E7b neutralization were generated, and reverse genetics techniques were then used to create an infectious CHIKV clone with a single mutation. 3E7b was also administered to neonate mice prior or after CHIKV infection. The survival rate, CHIKV burden in tissues and histopathology of the limb muscles were evaluated. Both IgM 3E7b and 8A2c bind strongly to native CHIKV surface and potently neutralize CHIKV replication. Further analyses of 3E7b binding and neutralization of CHIKV single-mutant clones revealed that N218 of CHIKV E2 protein is a potent neutralizing epitope. In a pre-binding neutralization assay, 3E7b blocks CHIKV attachment to permissive cells, possibly by binding to the surface-accessible E2-N218 residue. Prophylactic administration of 3E7b to neonate mice markedly reduced viremia and protected against CHIKV pathogenesis in various mice tissues. Given therapeutically at 4 h post-infection, 3E7b conferred 100% survival rate and similarly reduced CHIKV load in most mice tissues except the limb muscles. Collectively, these findings highlight the usefulness of 3E7b for future prophylactic or epitope-based vaccine design.
Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory diseases in humans and has a high mortality rate. During infection, MERS-CoV regulates several host cellular processes including antiviral response genes. In order to determine if the nucleocapsid protein of MERS-CoV (MERS-N) plays a role in viral–host interactions, a murine monoclonal antibody was generated so as to allow detection of the protein in infected cells as well as in overexpression system. Then, MERS-N was stably overexpressed in A549 cells, and a PCR array containing 84 genes was used to screen for genes transcriptionally regulated by it. Several up-regulated antiviral genes, namely TNF, IL6, IL8, and CXCL10, were selected for independent validation in transiently transfected 293FT cells. Out of these, the overexpression of MERS-N was found to up-regulate CXCL10 at both transcriptional and translational levels. Interestingly, CXCL10 has been reported to be up-regulated in MERS-CoV infected airway epithelial cells and lung fibroblast cells, as well as monocyte-derived macrophages and dendritic cells. High secretions and persistent increase of CXCL10 in MERS-CoV patients have been also associated with severity of disease. To our knowledge, this is the first report showing that the MERS-N protein is one of the contributing factors for CXCL10 up-regulation during infection. In addition, our results showed that a fragment consisting of residues 196–413 in MERS-N is sufficient to up-regulate CXCL10, while the N-terminal domain and serine-arginine (SR)-rich motif of MERS-N do not play a role in this up-regulation.
Monoclonal antibodies offer high specificity and this makes it an important tool for molecular biology, biochemistry and medicine. Typically, monoclonal antibodies are generated by fusing mouse spleen cells that have been immunized with the desired antigen with myeloma cells to create immortalized hybridomas. Here, we describe the generation of monoclonal antibodies that are specific to Chikungunya virus using ClonaCell-HY system.
We previously identified a novel MLL5 isoform, MLL5b, which was essential for E6 and E7 transcriptional activation in HPV16/18-associated cervical cancers. In this report, we investigated the potential of RNAimediated silencing of MLL5b through the use of MLL5b-siRNA as a novel therapeutic strategy for HPV16/18-positive cervical cancer. We observed concurrent downregulation of E6 and E7 after MLL5b silencing, leading to growth inhibition via the activation of apoptosis and senescence in the HeLa cell model. This corresponded with the enhanced antitumor effects of MLL5b-siRNA compared with E6-or E7-siRNA single treatments. Significant reduction in tumor size after MLLb-siRNA treatment in the HeLa xenograft tumor model further emphasized the importance of MLL5b in HPV16/18-associated tumor growth and the potential of RNAi therapeutics that target MLL5b. We also identified MLL5b as a modulator of gamma-irradiation (IR) sensitization properties of cisplatin. We observed that while MLL5b silencing alone was enough to evoke cisplatin-like IR sensitization in tumor cells in vitro, overexpression of MLL5b inhibited the ability of cisplatin to sensitize HeLa cells to IR-induced cytotoxicity. MLL5b-siRNA-IR cotreatment was also observed to enhance tumor growth inhibition in vivo. Taken together, our findings highlight the potential of targeted silencing of MLL5b via the use of MLL5b-siRNA as a novel therapeutic strategy and propose that MLL5b-siRNA could be a viable alternative for cisplatin in the current cisplatin-based chemotherapeutics for HPV16/18-associated cervical cancers. Mol Cancer Ther; 13(11); 2572-82. Ó2014 AACR.
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