High-affinity monoclonal antibodies against the porcine epidemic diarrhea virus S1 protein.

The porcine epidemic diarrhea virus (PEDV) infection inflicted substantial economic losses upon the global pig-breeding industry. This pathogen can infect all pigs and poses a particularly high fatality risk for suckling piglets. The S1 subunit of spike protein is a crucial target protein for inducing the particularly neutralizing antibodies that can intercept the virus-host interaction and neutralize virus infectivity. In the present study, the HEK293F eukaryotic expression system was successfully utilized to express and produce recombinant S1 protein. Through quantitative analysis, five monoclonal antibodies (mAbs) specifically targeting the recombinant S1 protein of PEDV were developed and subsequently evaluated using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and flow cytometry assay (FCA). The results indicate that all five mAbs belong to the IgG1 isotype, and their half-maximal effective concentration (EC50) values measured at 84.77, 7.42, 0.89, 14.64, and 7.86 pM. All these five mAbs can be utilized in ELISA, FCA, and IFA for the detection of PEDV infection. MAb 5-F9 exhibits the highest sensitivity to detect as low as 0.3125 ng/mL of recombinant PEDV-S1 protein in ELISA, while only 0.096 ng/mL of mAb 5-F9 is required to detect PEDV in FCA. The results from antigen epitope analysis indicated that mAb 8-G2 is the sole antibody capable of recognizing linear epitopes. In conclusion, this study has yielded a highly immunogenic S1 protein and five high-affinity mAbs specifically targeting the S1 protein. These findings have significant implications for early detection of PEDV infection and provide a solid foundation for further investigation into studying virus-host interactions.

Characterization of monoclonal antibodies against porcine epidemic diarrhea virus S1/S2 junction protein.

Pig producers have faced considerable economic losses due to porcine epidemic diarrhea virus (PEDV) infection, emphasizing the need for PEDV antibody development. The S1/S2 junction (S1S2J) cleavage site of the S protein of PEDV is one of the major determinants of coronavirus infection success. In this study, we specifically selected the S1S2J protein of PEDV-AJ1102 (a representative strain of the G2 type) as a target protein to immunize mice and generated monoclonal antibodies (mAbs) using hybridoma technology. Three mAbs with high-binding activities to the S1S2J protein and were obtained and further analyzed. To reveal the characterization of these mAbs, variable region genes of antibodies were studied by using DNA sequencing, thereby revealing differences in their CDR3 amino acid sequences. We then developed a new method to identify the isotypes of these three mAbs. Results showed that these three antibodies were of the IgM type. As for the functions of these three mAbs, indirect immunofluorescence assay confirmed their good binding ability to Vero E6 cells infected with the PEDV-SP-C strain (G1 type). Epitope analysis showed linear epitopes for all three mAbs. These antibodies were also used to detect infected cells via flow cytometry analysis. In summary, we prepared and examined three mAbs against PEDV-S1S2J. These mAbs can be employed as detection antibodies for diagnostic reagents and further developed for other applications. We also designed a novel technique for easy and cost-saving identification of isotypes of mouse mAbs. Our results lay a good foundation for the development of research on PEDV.

Discovery and characterization of SARS-CoV-2 reactive and neutralizing antibodies from humanized CAMouseHG mice through rapid hybridoma screening and high-throughput single-cell V(D)J sequencing.

The coronavirus disease 2019 pandemic has caused more than 532 million infections and 6.3 million deaths to date. The reactive and neutralizing fully human antibodies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are effective detection tools and therapeutic measures. During SARS-CoV-2 infection, a large number of SARS-CoV-2 reactive and neutralizing antibodies will be produced. Most SARS-CoV-2 reactive and neutralizing fully human antibodies are isolated from human and frequently encoded by convergent heavy-chain variable genes. However, SARS-CoV-2 viruses can mutate rapidly during replication and the resistant variants of neutralizing antibodies easily survive and evade the immune response, especially in the face of such focused antibody responses in humans. Therefore, additional tools are needed to develop different kinds of fully human antibodies to compensate for current deficiency. In this study, we utilized antibody humanized CAMouseHG mice to develop a rapid antibody discovery method and examine the antibody repertoire of SARS-CoV-2 RBD-reactive hybridoma cells derived from CAMouseHG mice by using high-throughput single-cell V(D)J sequencing analysis. CAMouseHG mice were immunized by 28-day rapid immunization method. After electrofusion and semi-solid medium screening on day 12 post-electrofusion, 171 hybridoma clones were generated based on the results of SARS-CoV-2 RBD binding activity assay. A rather obvious preferential usage of IGHV6-1 family was found in these hybridoma clones derived from CAMouseHG mice, which was significantly different from the antibodies found in patients with COVID-19. After further virus neutralization screening and antibody competition assays, we generated a noncompeting two-antibody cocktail, which showed a potent prophylactic protective efficacy against SARS-CoV-2 in cynomolgus macaques. These results indicate that humanized CAMouseHG mice not only provide a valuable platform to obtain fully human reactive and neutralizing antibodies but also have a different antibody repertoire from humans. Thus, humanized CAMouseHG mice can be used as a good complementary tool in discovery of fully human therapeutic and diagnostic antibodies.

[Development of an improved virus plaque assay based on avicel].

Avicel is made of a mixture of microcrystalline cellulose (MCC) and carboxymethyl cellulose (CMC), and used for virus plaque assay. The avicel in common use is produced by FMC Biopolymer. Due to the relatively fixed proportion of MCC and CMC, avicel in common use is not suitable for plaque determination experiment of all types of viruses. In this study, we evaluated the effect of avicel made of different proportions of MCC and CMC on virus plaque assay, and developed an improved avicel virus plaque assay featured with simple and convenient operation, good practicability and high stability. To generate avicel overlays with different proportions of MCC and CMC, twelve different 2×avicel solutions were prepared. Their overall viscosity and bottom viscosity were measured to evaluate the ease of operation. The results showed that most of the 2×avicel solutions (except the 4.8% MCC+1.4% CMC and 4.8% MCC+1.0% CMC group) were easy to absorb and prepare nutrient overlap than 2×CMC solution. In order to find the best scheme to detect the titer of porcine epidemic diarrhea virus (PEDV), these avicel overlay solutions with different proportion of MCC and CMC were used as a replacement in the standard plaque assay. By comparing the size, clarity, stability and titer accuracy of virus plaque, we identified that 0.6% MCC and 0.7% CMC was the most preferable composition of avicel overlay for PEDV plaque assay. In conclusion, we developed an improved virus plaque assay based on avicel, which may facilitate the research of virus etiology, antiviral drugs and vaccines.

Development of a Tetravalent T-Cell Engaging Bispecific Antibody Against Glypican-3 for Hepatocellular Carcinoma.

Cancer therapies benefit from accelerated development of biotechnology, and many immunotherapeutic strategies spring up including vaccines, the immune checkpoint blockade, chimeric antigen receptor T cells, and bispecific antibodies (BsAbs). Glypican-3 (GPC3) is a member of the heparan sulfate proteoglycan family of proteins and is highly expressed in hepatocellular carcinoma (HCC) cell membranes. Here, the authors describe a new tetravalent BsAb h8B-BsAb targeting GPC3 and CD3 antigens and studied its antitumor activities against HCC. h8B-BsAb was designed based on immunoglobulin G with a fragment variable fused to the light chain, whose biophysical stabilities including degradation resistance and thermostability were improved by introducing disulfide bonds. In vitro activity of h8B-BsAb showed potent T-cell recruitment and activation for HCC cell lysis by the presence of peripheral blood mononuclear cells, but no specific killing in GPC3-negative cells. In HCC xenograft mouse studies, h8B-BsAb induced robust regression of tumors. In summary, we engineered a highly stable and efficacious BsAb as a potential candidate for HCC treatment.

Generation of fully human anti-GPC3 antibodies with high-affinity recognition of GPC3 positive tumors.

The acceleration of therapeutic antibody development has been motivated by the benefit to and their demand for human health. In particular, humanized transgenic antibody discovery platforms, combined with immunization, hybridoma fusion and/or single cell DNA sequencing are the most reliable and rapid methods for mining the human monoclonal antibodies. Human GPC3 protein is an oncofetal antigen, and it is highly expressed in most hepatocellular carcinomas and some types of squamous cell carcinomas. Currently, no fully human anti-GPC3 therapeutic antibodies have been reported and evaluated in extensive tumor tissues. Here, we utilized a new humanized transgenic mouse antibody discovery platform (CAMouse) that contains large V(D)J -regions and human gamma-constant regions of human immunoglobulin in authentic configurations to generate fully human anti-GPC3 antibodies. Our experiments resulted in four anti-GPC3 antibodies with high-specific binding and cytotoxicity to GPC3 positive cancer cells, and the antibody affinities are in the nanomolar range. Immunohistochemistry analysis demonstrated that these antibodies can recognize GPC3 protein on many types of solid tumors. In summary, the human anti-human GPC3 monoclonal antibodies described here are leading candidates for further preclinical studies of cancer therapy, further, the CAMouse platform is a robust tool for human therapeutic antibody discovery.

A novel targeted GPC3/CD3 bispecific antibody for the treatment hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer but has shown limited success to date in the treatment of advanced stage. Recruitment of T cells for cancer treatment is a rapidly growing strategy in immunotherapy such as chimeric antigen receptor T cells and bispecific antibodies. However, unwanted aggregations, structural instability or short serum half-life are major challenges of bispecific antibodies. Here, we developed a new format of T cell-redirecting antibody that is bispecific for membrane proteoglycans GPC3 of HCC and the T-cell-specific antigen CD3, which demonstrated to be favorable stability and productivity. Cross-linking of T cells with GPC3 positive tumor cells by the anti-GPC3/CD3 bispecific antibody-mediated potent GPC3-dependent and concentration-dependent cytotoxicity in vitro. Administration of the bispecific antibody with different concentrations in murine xenograft models of human HCC significantly inhibited tumor growth. In addition, no effects on tumor growth were observed in the absence of human effector cells or the bispecific antibody. Taken together, the anti-GPC3/CD3 bispecific antibody might be a potential therapeutic treatment for HCC.

Screening high-quality fetal bovine serum for porcine oocyte maturation in vitro.

Fetal bovine serum (FBS) is widely used in cell cultures due to its high stability and easy access. It was also used as a substitute for porcine follicular fluid (PFF) in previous studies. However, FBS components are unclear, and the presence of FBS in culture media may introduce a variation from batch to batch. This study aimed to establish an effective method to screen FBS in place of PFF in the culture media for porcine oocytes in vitro. We screened FBS from different sources by using porcine fetal fibroblast cells. The effects of six FBS samples on porcine fetal fibroblast cell growth were tested via frozen cell survival assay, cell clone formation assay, cell growth curve, and cell passage activity assay. The best serum that we called GFBS (heat-inactivated FBS, cat. no. 10500-64; Gibco) showed a similar effect on the maturation and development of porcine oocytes to that of PFF and can be used as a good substitute for PFF. These results suggested that the porcine fetal fibroblast cell culture test can be used as a valuable method to screen FBS for porcine oocyte maturation and embryonic development in vitro.

[MicroRNA Expression Profiles of Porcine Kidney 15 Cell Line Infected with Porcine Epidemic Diahorrea Virus].

To explore the effect of porcine epidemic diarrhea virus(PEDV)on microRNA expression profiles of porcine kidney 15cell(PK-15),total RNA was isolated from PK-15 cells with or without PEDV infection. Then, we obtained the miRNAs by using solexa sequencing technology and analyzed these differentially expressed miRNAs. Heatmap cluster analysis and GO (ontology, GO) (Gene function, MF, Molecular) analysis was performed on the significant differences in expression of the miRNA, and 10 significant differences in expression of miRNA were selected by RT-qPCR. The result showed 214 kinds of microRNAs (miRNAs) expression levels were significantly different in PEDV-infected cells, compared with normal PK-15 cells. Among of them,175 kinds of miRNAs were significantly up-regulated while 39 kinds of miRNAs were significantly down-regulated. Furthermore, qPCR results of the expression trends of miRNA were similar with that of solexa sequencing. Heatmap cluster analysis showed that the vast majority of the viral groups were differentially expressed in miRNA compared with the control group, Go analysis showed that miRNAs are widely involved in combination, binding protein, protein kinase activity, transfer enzyme activity, phosphorus containing radicals transfer, phosphotransferase enzyme activity and other biological effects. The expression trends of RT-qPCR verified by miRNA were consistent with the high throughput sequencing results. The results showed that the swine epidemic diarrhea virus infection had a significant impact on the level of miRNA expression in PK-15 cells, thus providing a new idea for the further study of the miRNA preparation for the treatment of PEDV.

Differential expression of micrornas in porcine parvovirus infected porcine cell line.

BACKGROUND: Porcine parvovirus (PPV), a member of the Parvoviridae family, causes great economic loss in the swine industry worldwide. MicroRNAs (miRNAs) are a class of non-protein-coding genes that play many diverse and complex roles in viral infections. FINDING: Aiming to determine the impact of PPV infections on the cellular miRNAome, we used high-throughput sequencing to sequence two miRNA libraries prepared from porcine kidney 15 (PK-15) cells under normal conditions and during PPV infection. There was differential miRNA expression between the uninfected and infected cells: 65 miRNAs were upregulated and 128 miRNAs were downregulated. We detected the expression of miR-10b, miR-20a, miR-19b, miR-181a, miR-146b, miR-18a, and other previously identified immune-related miRNAs. Gene Ontology analysis and KEGG function annotations of the host target genes suggested that the miRNAs are involved in complex cellular pathways, including cellular metabolic processes, immune system processes, and gene expression. CONCLUSIONS: These data suggest that a large group of miRNAs is expressed in PK-15 cells and that some miRNAs were altered in PPV-infected PK-15 cells. A number of microRNAs play an important role in regulating immune-related gene expression. Our findings should help with the development of new control strategies to prevent or treat PPV infections in swine.

Identification and analysis of differentially-expressed microRNAs in Japanese encephalitis virus-infected PK-15 cells with deep sequencing.

Japanese encephalitis virus (JEV), a mosquito-borne Flavivirus, causes acute viral encephalitis with high morbidity and mortality in humans and animals. MicroRNAs (miRNAs) are small noncoding RNAs that are important modulators of the intricate host-pathogen interaction networks. However, our knowledge of the changes that occur in miRNAs in host cells after JEV infection is still limited. To understand the molecular pathogenesis of JEV at the level of posttranscriptional regulation, we used Illumina deep sequencing to sequence two small RNA libraries prepared from PK-15 cells before and after JEV infection. We identified 522 and 427 miRNAs in the infected and uninfected cells, respectively. Overall, 132 miRNAs were expressed significantly differently after challenge with JEV: 78 were upregulated and 54 downregulated. The sequencing results for selected miRNAs were confirmed with RT-qPCR. GO analysis of the host target genes revealed that these dysregulated miRNAs are involved in complex cellular pathways, including the metabolic pathway, inflammatory response and immune response. To our knowledge, this is the first report of the comparative expression of miRNAs in PK-15 cells after JEV infection. Our findings will underpin further studies of miRNAs' roles in JEV replication and identify potential candidates for antiviral therapies against JEV.