Expression and in vitro activity of a neutralizing antibody against West Nile virus that reduces antibody-dependent enhancement / 中华微生物学和免疫学杂志

Objective:To establish an antibody expression system to reduce the antibody-dependent enhancement (ADE) effect of target antibody.Methods:Site-directed mutagenesis was used to mutate the 234 and 235 sites of the Fc region of the mammalian cell antibody expression vector-L234A and L235A to establish the antibody expression vector pFRT-IgG1κ-FcM. An antibody Wt-WNV with significant ADE effect obtained in previous work was selected and expressed by the pFRT-IgG1κ-FcM system to obtain mutant antibody FcM-WNV. The binding ability of FcM-WNV to target antigen West Nile virus envelope protein-DⅢ (WNV E-DⅢ) was detected by ELISA, and the its binding ability to human high-affinity IgG Fc receptor hFcγRⅠ (hCD64 ) was analyzed by flow cytometry. The neutralizing activity of FcM-WNV in vitro was detected by pseudovirus infection of host cells (BHK21 and K562). Results:The expression levels of FcM-WNV and Wt-WNV were comparable, and FcM-WNV could recognize and bind to WNVE-DIII in a concentration-dependent manner. Compared with Wt-WNV, the binding ability of FcM-WNV to hCD64 was significantly weakened, showing a significant decrease in fluorescence intensity. Consistent with the previous experimental results, Wt-WNV at a concentration of 5 μg/ml significantly enhanced the infection of K562 by WNV pseudovirus, while FcM-WNV at a concentration of 5 μg/ml could effectively block pseudovirus infection in both K562 and BHK21 cells.Conclusions:The established antibody expression system can effectively reduce the ADE effect of the target antibody.

Establishment of an in vivo neutralization model based on H5N1 pseudovirus / 中华微生物学和免疫学杂志

Objective:To establish an in vivo infection model of H5N1 pseudovirus and to detect the neutralizing activity of FHA3 antibody using this model. Methods:Based on the sequence information of hemagglutinin (HA) and neuraminidase (NA) of A/Anhui/1/2005/H5N1 strain, two recombinant plasmids of pcDNA3.1-HA5 and pcDNA3.1-NA1 were constructed. The two plasmids and plasmid pNL4-3.Luc.R-E- were co-transfected into 293T cells to prepare H5N1 pseudovirus supernatant. The morphology of pseudovirus particles in the supernatant was observed by electron microscopy. MDCK cells were infected with the pseudovirus supernatant and the virus titer was detected. BALB/c mice were injected with the pseudovirus supernatant by intraperitoneal injection and subjected to bioluminescence imaging at 2, 5, 8, and 12 d after infection to detect the pseudovirus infection in vivo. The functional activity of FHA3 antibody in vivo was evaluated using the established mouse infection model. Results:The recombinant plasmids pcDNA3.1-HA5 and pcDNA3.1-NA1 were correctly constructed and could be used to prepare pseudovirus supernatants of high titer by co-transfecting 293T cells with the plasmid pNL4-3.Luc.R-E-. The virus particles were round under electron microscope. H5N1 pseudovirus-infected mice exhibits strong fluorescence signals, which were attenuated by FHA3 treatment before challenge.Conclusions:The in vivo infection model of H5N1 pseudovirus was successfully constructed and FHA3 antibody was proved to be protective against the pseudovirus infection.

Research progress in optimization of therapeutic effect of antibodies against flavivirus / 中华微生物学和免疫学杂志

Flaviviruses are a class of positive-strand RNA viruses mainly transmitted by arthropods, which can cause high mortality and morbidity worldwide. At present, there is no specific therapy. Therapeutic antibodies bring hope for the treatment of flavivirus infection, but the antibody-dependent enhancement (ADE) effect induced by flavivirus infection can lead to disease progression. The ideal therapeutic antibodies against flaviviruses should not only treat flavivirus infection, but also avoid the harm caused by ADE. Therefore, researchers have optimized some of the antibodies to seek the best therapeutic antibodies. This review briefly described the research progress and mechanism of therapeutic antibodies against flaviviruses as well as some strategies to reduce the ADE effect induced by the therapeutic antibodies.

Progress in cross reactivity of neutralizing monoclonal antibodies against dengue virus / 中华微生物学和免疫学杂志

Neutralizing monoclonal antibodies against dengue virus cross-react with other flaviviruses due to the sequence homology between them, such as the antibodies targeting envelope protein and non-structural protein 1. Apart from exerting protective effects in infected animals, cross-neutralizing antibodies could also cause antibody-dependent enhancement (ADE) in vivo. This review summarized the progress in cross-reactivity of neutralizing antibodies against dengue virus.

Preparation and identification of a broad-spectrum antibody targeting hemagglutinin protein of influenza A virus / 中华微生物学和免疫学杂志

Objective:To prepare and identify a broad-spectrum antibody FHA3 targeting influenza A virus hemagglutinin (HA).Methods:According to the single-chain antibody fragment (scFv) sequence, the heavy chain (VH) and light chain (VL) variable regions of FHA3 were amplified by PCR and a recombinant plasmid pFRT-IgG1κ-FHA3 was constructed by linking the expression vector pFRT-IgG1κ. FHA3 was expressed in the ExpiCHO system and purified by affinity purification. The binding activity of FHA3 to influenza A virus HA was detected by ELISA. The neutralizing activity of FHA3 was detected in vitro by infecting host cells with pseudovirus. Results:SDS-PAGE showed that high-purity FHA3 was obtained. FHA3 could bind to H1N1 HA, H2N2 HA, H3N2 HA, H5N1 HA, H7N9 HA and H9N2 HA in a concentration-dependent manner. FHA3 had good neutralizing activity in vitro that was it could effectively block the invasion of H5N1 and H7N9 pseudoviruses into target cells at a low concentration of 5 μg/ml and H1N1 pseudovirus at 0.012 5 μg/ml. Conclusions:A broad spectrum antibody targeting HA protein of influenza A virus with neutralizing activity in vitro was obtained.

Preparation and identification of antibodies targeting influenza A virus N1 subtype neuraminidase / 中华微生物学和免疫学杂志

Objective:To prepare and identify a functional antibody FNA1 targeting the neuraminidase (NA) of influenza A virus N1 subtype.Methods:According to single-chain antibody fragment (scFv) sequence, the heavy chain and light chain variable region sequences of FNA1 were synthesized, and the recombinant expression plasmid pFRT-IgG1κ-FNA1 was constructed by linking the expression vector pFRT-IgG1κ. The FNA1 antibody was expressed in ExpiCHO cells and purified using affinity purification technique. The binding ability of FNA1 to the target proteins, influenza A virus N1 subtype NA antigens, was detected by ELISA. Flow cytometry was performed to analyze the binding ability of FNA1 to the NA antigens expressed on the surface of cell membrane. The in vitro activity of FNA1 against NA was evaluated by infecting 293T cells with pseudovirus. Results:Protein electrophoresis showed that FNA1 with high purity was obtained. FNA1 specifically recognized and bound to N1 subtype NA antigens in a concentration-dependent manner. FNA1 could effectively block NA activity by binding to N1 subtype NA protein expressed on the surface of cell membrane, thus inhibiting the release of packaged pseudovirus from cell surface and further inhibiting target cell infection.Conclusions:An antibody FNA1 targeting influenza A virus N1 subtype NA with in vitro functional activity was obtained.

TNFAIP8 gene silencing inhibits the migration of mouse RAW264. 7 macrophages / 中华微生物学和免疫学杂志

Objective To construct a shRNA lentiviral vector targeting the gene encoding tumor necrosis factor alpha-induced protein 8 (TNFAIP8) in RAW264. 7 cells, a mouse macrophage cell line, and to investigate the effects of TNFAIP8 gene silencing on the functions of mouse macrophages. Methods The shRNA sequence targeting TNFAIP8 gene was designed and DNA oligos containing small hairpin frame was synthesized. The double-stranded DNA was cloned into pLKO. 1-TRC vector after annealing. The recombi-nant vector was verified by using double enzyme digestion and gene sequencing. Lentiviruses were prepared by transfecting the constructed vector into 293T cells. Fluorescent quantitative RT-PCR and Western blot as-say were performed to detect the expression of TNFAIP8 at mRNA and protein levels after infecting the RAW264. 7 cells with lentiviruses. Flat dish adhesion experiment and wound-healing assay were used to evaluate the effects of TNFAIP8 gene silencing on the adhesion and migration of RAW264. 7 cells. Results The recombinant lentiviral vector was successfully constructed as indicated by double enzyme di-gestion and gene sequencing analysis. The expression of TNFAIP8 in RAW264. 7 cells at both mRNA and protein levels were significantly down-regulated after lentivirus infection (P<0. 05). Moreover, TNFAIP8 gene silencing significantly impaired the cell adhesion ability of RAW264. 7 cells after 15 min, 30 min or 2 hours of culture. Compared with the cells in control group, the RAW264. 7 cells harboring silenced TN-FAIP8 gene looked round with a smaller number of cellular extensions. The wound-healing assay showed that less TNFAIP8 gene-silenced RAW264. 7 cells migrated into the wounded area as compared with the cells in control group after 24 hours of culture (P<0. 05). The wound-healing rates of the experimental and control groups were 25% and 50%, respectively. Conclusion The recombinant lentiviral vector containing shRNA targeting the TNFAIP8 gene was successfully constructed. Transfecting the RAW264. 7 cells with the con-structed vector significantly silenced the expression of TNFAIP8 gene and inhibited the adhesion and migra-tion of these cells.

Osteoclast inhibitory lectin related protein 2 protects mice from endotoxemia / 中华微生物学和免疫学杂志

Objecitv e To investigate the effects of a recombinant protein osteoclast inhibitory lectin related protein 2( OCILRP2)-Fc on LPS-induced endotoxemia by blocking OCILRP 2 signaling pathway and to in-vestigate the roles of OCILRP2 during inflammation.Methods Real-time PCR was used to detect OCILRP2 ex-pression at mRNA level in RAW264.7cells before and after in vitro stimulation with LPS.A mouse model of en-dotoxemia was established by intraperitoneal injection of BALB /c mice with a median lethal dose of LPS .Two hours prior to LPS treatment, mice were intraperitoneally injected with OCILRP2-Fc, human IgG or PBS, re-spectively .Several parameters including the survival rate of BALB/c mice with and without LPS treatment , spleen weight for arterial hyperemia analyzing , histopathological changes of lung and liver by HE staining , serum levels of inflammatory cytokines (IL-6, IL-12, TNF-αand IFN-γ)by ELISA , NF-κB activity by Western blot, were analyzed .Results Real-time PCR showed that LPS elevated in vitro OCILRP2 expression at mRNA level in macrophages (P<0.05).Upon the treatment of OCILRP2-Fc, BALB/c mice suffered from endotoxemia showed obviously increased survival rate , decreased spleen hyperemia , attenuated pathological injury of lung and liver, reduced levels of IL-6, IL-12, TNF-αand IFN-γin serum samples (P <0.05) as compared with mice treated with human IgG and PBS .LPS induced NF-κB p65 phosphorylation and IκB degradation were inhibited by OCILRP2-Fc treatment.Conclusion OCILRP2-Fc protects mice from endotoxemia by blocking OCILRP 2 signaling, which suggests that OCILRP2 plays an important role in LPS induced inflammation.

Eukaryotic expression and preparation of polyclonal antibody for osteoclast inhibitory lectin related protein / 中国免疫学杂志

Objective:In order to get recombinant protein OCILRP 2-Fc and anti-OCILRP2 antibody for further study of OCILRP2.Methods:Eukaryotic expression vector pIg-CD5-OCILRP2 which fused with extracellular domain of OCILRP 2 and human IgG1 Fc fragment was constructed.G418 was used for stable expression cell strain after pIg-CD5-OCILRP2 transfected into CHO cells.Recombinant protein OCILRP 2-Fc purified from CHO cell supernatant was used to immunize rabbit and anti-OCILRP2 polyclonal antibody was purified from rabbit serum by using protein G column.Results: ELISA data showed that we got a high-titer anti-serum and anti-OCILRP2 antibody purified from the rabbit serum.Western blot indicated this antibody could specifically bind to OCILRP 2-Fc and OCILRP2 in NIH/3T3 lysate.OCILRP2 expression in murine bone marrow derived dendritic cells ( DC) was detected by this polyclonal antibody ,too.Compared to immature DC ,OCILRP2 expression was elevated in LPS induced-mature DC.Conclusion: This study has offered an available tool and provided a clue for further study of the roles of OCILRP 2 in immune response.

The mitochondrial-dependent molecular mechanisms for inducing apoptosis in Jurkat cells by a novel agonistic anti-human DR5 monoclonal antibody / 中国免疫学杂志

Objective:To investigate mitochondrial-dependent molecular mechanisms of a novel agonistic anti-human death receptor 5 (DR5) monoclonal antibody(mDRA-6) inducing apoptosis in Jurkat cell.Methods:The dose-dependent and time-dependent cell growth suppression of mDRA-6 in Jurkat cells was determined by MTT assay.The measurement of the mitochondrial transmembrane potential(ΔΨm) of Jurkat cells was detected by flow cytometry with JC-1 single staining.Caspase-8,9 as well as Bid,Bax,Bcl-2 and Cyto c of apoptotic Jurkat cells were analyzed by Western blot after mDRA-6 treatment.Results:The mDRA-6 induced cell growth suppression and cytotoxicity in dose-dependent manner and time-dependent manner.After mDRA-6 treatment at 2.0 μg/ml for15 min,30 min,60 min and 120 min,the change in ΔΨm were 20.14 %,19.34 %,21.11% and 30.90% respectively by JC-1 single staining.Western blot revealed that the level of active fragments of Caspase-8,9 and Bid,Bax,Bcl-2 and Cyto c respectively,and the amount of Cyto c was increased in cytosol concomitant with the related attenuation of Cyto c in mitochondria.Conclusion: Apoptotic pathway of Jurkat cells induced by mDRA-6 is initiated upon DR5 ligatian to mDRA-6 and exogenic Caspase-dependent cell apoptotic cascades is activated,and endogenic mitochondrial-dependent cell apoptosis pathway is activated.mDRA-6 may be a useful agent in investigating human leukemia therapy by using TRAIL/DR5.