A Novel Conserved Linear Neutralizing Epitope on the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein.

The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made it challenging to develop broad-spectrum prophylactic vaccines and therapeutic antibodies. Here, we have identified a broad-spectrum neutralizing antibody and its highly conserved epitope in the receptor-binding domain (RBD) of the spike protein (S) S1 subunit of SARS-CoV-2. First, nine monoclonal antibodies (MAbs) against the RBD or S1 were generated; of these, one RBD-specific MAb, 22.9-1, was selected for its broad RBD-binding abilities and neutralizing activities against SARS-CoV-2 variants. An epitope of 22.9-1 was fine-mapped with overlapping and truncated peptide fusion proteins. The core sequence of the epitope, 405D(N)EVR(S)QIAPGQ414, was identified on the internal surface of the up-state RBD. The epitope was conserved in nearly all variants of concern of SARS-CoV-2. MAb 22.9-1 and its novel epitope could be beneficial for research on broad-spectrum prophylactic vaccines and therapeutic antibody drugs. IMPORTANCE The continuous emergence of new variants of SARS-CoV-2 has caused great challenge in vaccine design and therapeutic antibody development. In this study, we selected a broad-spectrum neutralizing mouse monoclonal antibody which recognized a conserved linear B-cell epitope located on the internal surface of RBD. This MAb could neutralize all variants until now. The epitope was conserved in all variants. This work provides new insights in developing broad-spectrum prophylactic vaccines and therapeutic antibodies.

Lentogenic NDV V protein inhibits IFN responses and represses cell apoptosis.

The V protein of Newcastle disease virus (NDV) has been shown to inhibit the secretion of interferon (IFN) during infection, which is responsible for the promotion of NDV pathogenicity. However, the ability of the V protein to suppress host innate immunity is not well understood. In this study, we explored the function of V protein and its relationship with virulence by generating V protein-inserted recombinant (r) NDVs. Using rNDVs as a model, we examined the efficiency of infection, IFN responses, and apoptosis of host cells during infection. We found that viral propagation occurred smoothly when V protein from lentogenic NDV is inserted instead of the V protein from the velogenic strain. The infection of lentogenic V protein-inserted rNDV induced less expression of IFNs and downstream antiviral proteins via efficient degradation of p-STAT1 and MDA5. Moreover, velogenic V protein triggered a higher apoptosis rate during infection thereby restricting the replication of NDV. Conversely, lentogenic V protein inhibits IFN responses efficiently and induces less apoptosis compared to the velogenic strain. Our findings provide a novel understanding of the role of V protein in NDV pathogenicity.

Newcastle Disease Virus Inhibits the Proliferation of T Cells Induced by Dendritic Cells In Vitro and In Vivo.

Newcastle disease virus (NDV) infects poultry and antagonizes host immunity via several mechanisms. Dendritic cells (DCs) are characterized as specialized antigen presenting cells, bridging innate and adaptive immunity and regulating host resistance to viral invasion. However, there is little specific knowledge of the role of DCs in NDV infection. In this study, the representative NDV lentogenic strain LaSota was used to explore whether murine bone marrow derived DCs mature following infection. We examined surface molecule expression and cytokine release from DCs as well as proliferation and activation of T cells in vivo and in vitro in the context of NDV. The results demonstrated that infection with lentogenic strain LaSota induced a phenotypic maturation of immature DCs (imDCs), which actually led to curtailed T cell responses. Upon infection, the phenotypic maturation of DCs was reflected by markedly enhanced MHC and costimulatory molecule expression and secretion of proinflammatory cytokines. Nevertheless, NDV-infected DCs produced the anti-inflammatory cytokine IL-10 and attenuated T cell proliferation, inducing Th2-biased responses. Therefore, our study reveals a novel understanding that DCs are phenotypically mature but dysfunctional in priming T cell responses during NDV infection.

Newcastle disease virus-based MERS-CoV candidate vaccine elicits high-level and lasting neutralizing antibodies in Bactrian camels.

Middle East respiratory syndrome coronavirus (MERS-CoV), a member of the Coronaviridae family, is the causative pathogen for MERS that is characterized by high fever, pneumonia, acute respiratory distress syndrome (ARDS), as well as extrapulmonary manifestations. Currently, there are no approved treatment regimens or vaccines for MERS. Here, we generated recombinant nonvirulent Newcastle disease virus (NDV) LaSota strain expressing MERS-CoV S protein (designated as rLa-MERS-S), and evaluated its immunogenicity in mice and Bactrian camels. The results revealed that rLa-MERS-S showed similar growth properties to those of LaSota in embryonated chicken eggs, while animal immunization studies showed that rLa-MERS-S induced MERS-CoV neutralizing antibodies in mice and camels. Our findings suggest that recombinant rLa-MERS-S may be a potential MERS-CoV veterinary vaccine candidate for camels and other animals affected by MERS.

Chimeric rabies glycoprotein with a transmembrane domain and cytoplasmic tail from Newcastle disease virus fusion protein incorporates into the Newcastle disease virion at reduced levels.

Rabies remains an important worldwide health problem. Newcastle disease virus (NDV) was developed as a vaccine vector in animals by using a reverse genetics approach. Previously, our group generated a recombinant NDV (LaSota strain) expressing the complete rabies virus G protein (RVG), named rL-RVG. In this study, we constructed the variant rL-RVGTM, which expresses a chimeric rabies virus G protein (RVGTM) containing the ectodomain of RVG and the transmembrane domain (TM) and a cytoplasmic tail (CT) from the NDV fusion glycoprotein to study the function of RVG's TM and CT. The RVGTM did not detectably incorporate into NDV virions, though it was abundantly expressed at the surface of infected BHK-21 cells. Both rL-RVG and rL-RVGTM induced similar levels of NDV virus-neutralizing antibody (VNA) after initial and secondary vaccination in mice, whereas rabies VNA induction by rL-RVGTM was markedly lower than that induced by rL-RVG. Though rL-RVG could spread from cell to cell like that in rabies virus, rL-RVGTM lost this ability and spread in a manner similar to the parental NDV. Our data suggest that the TM and CT of RVG are essential for its incorporation into NDV virions and for spreading of the recombinant virus from the initially infected cells to surrounding cells.

Lens proteomics: analysis of rat crystallins when lenses are exposed to dexamethasone.

To identify glucocorticoid induced cataract (GIC)-specific modified crystallins and related changes, we analyzed rat crystallins and related changes in lenses exposed to dexamethasone (Dex). To carry out proteomics analyses, we separated soluble lens proteins with two-dimensional electrophoresis (2-DE) and modified crystallins were analyzed with matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Related changes in mRNA, protein levels and morphological and functional changes of modified crystallins were also determined. Measured masses (except for γD-crystallin as the larger and cross-link form), the isoelectric points (PIs; except for ßB3-crystallin as the alkalinization form) and amino acid sequences of all known rat crystallins matched previously reported data. Analysis by 2-DE indicated that αA, αB, ßB3 and γD increased when lenses were exposed to 5 µM Dex; ßA4 increased when lenses were exposed to 1 µM Dex and the five proteins that had the highest expressional trend were identical with the results of Q-PCR. ßA3/A1 crystallin (expressional trend identical with results of Q-PCR) and the serum albumin precursor gradually disappeared when exposed to 1-50 µM Dex. Results of Western blotting, immunohistochemistry or fluorescence analysis showed that αA and αB increased most when exposed to 5 µM Dex and ßA1/A3 and KI-67 decreased obviously when exposed to 1-50 µM Dex. Electron microscopy showed that the condition of the lens was better when lenses were exposed to 5 µM Dex than at other levels and cracks between the fiber cells became larger when lenses were exposed to 1-50 µM Dex. A chaperone role of α-crystallin protecting heated catalase (CAT) and the activity of superoxide dismutase (SOD), glutathione (GSH), and caspase-3 were highest when exposed to 5 µM Dex. Moreover, αA-crystallins were associated with increased phosphorylation (PI decreased). In conclusion, the proteomics analysis and related changes of rat crystallins when lenses were exposed to Dex in this study will be useful for comparison with normal lens proteins and GIC. We also provided a mechanism for GIC from a proteomics aspect based on the in vitro model.

Comparison of the antiangiogenic activity of modified RGDRGD-endostatin to endostatin delivered by gene transfer in vivo rabbit neovascularization model.

PURPOSE: Endostatin plays an important role in inhibiting corneal neovascularization (CNV). The aim of this study was to evaluate the antiangiogenic activities of lipid-mediated subconjunctival injection of the modified RGDRGD (arginine- glycin- aspartic- arginine- glycin- aspartic- endostatin gene in a rabbit model of neovascularization in vivo. METHODS: A modified human endostatin gene containing an RGDRGD motif was obtained by rapid site-directed mutagenesis. Forty New Zealand white rabbits underwent alkaline burn and developed CNV, which were randomly divided into four groups: an experimental control group, a PCI empty vector group, a PCI-endostatin group, and a PCI-RGDRGD-endostatin group. The vector, endostatin, and RGDRGD-endostatin groups received injections into the superior bulbar conjunctiva after the burn. An injection of 5 µg was given twice at 1-week intervals. Four eyes of two rabbits received neither treatment nor alkaline burn and served as absolute normal controls. The areas of CNV were monitored after 7 and 14 days. Corneas were examined by histology, and VEGF (vascular endothelial growth factor) and CD31 (platelet endothelial cell adhesion molecule-1) expression was detected by immunohistochemistry after 7 and 14 days. Retina, liver, and kidney were examined by histology, and CD38 expression in the inflammatory cells was detected by immunohistochemistry at 90 days. RESULTS: Subconjunctival injection of both native endostatin and modified RGDRGD-endostatin genes resulted in a significant suppression of CNV in vivo, with modified RGDRGD-endostatin being more effective than native endostatin. The mean concentration of VEGF in the PCI-RGDRGD-endostatin group significantly decreased compared to the means in the other groups. Upon histological examination, the endostatin-treated and RGDRGD-endostatin-treated eyes showed significantly less neovascular area and fewer vessels than the control and vector-injected groups. Retinal, hepatic, and renal tissue sections were normal, and there was no inflammatory cell infiltration observed. CONCLUSIONS: Native and modified endostatin can significantly inhibit CNV by suppressing the expression of VEGF. However, modified endostatin with the RGDRGD motif is far more effective than the endostatin gene in antiangiogenic activity.

Identification and characterization of a virus-specific continuous B-cell epitope on the PrM/M protein of Japanese Encephalitis Virus: potential application in the detection of antibodies to distinguish Japanese Encephalitis Virus infection from West Nile Virus and Dengue Virus infections.

BACKGROUND: Differential diagnose of Japanese encephalitis virus (JEV) infection from other flavivirus especially West Nile virus (WNV) and Dengue virus (DV) infection was greatly hindered for the serological cross-reactive. Virus specific epitopes could benefit for developing JEV specific antibodies detection methods. To identify the JEV specific epitopes, we fully mapped and characterized the continuous B-cell epitope of the PrM/M protein of JEV. RESULTS: To map the epitopes on the PrM/M protein, we designed a set of 20 partially overlapping fragments spanning the whole PrM, fused them with GST, and expressed them in an expression vector. Linear epitope M14 (105VNKKEAWLDSTKATRY120) was detected by enzyme-linked immunosorbent assay (ELISA). By removing amino acid residues individually from the carboxy and amino terminal of peptide M14, we confirmed that the minimal unit of the linear epitope of PrM/M was M14-13 (108KEAWLDSTKAT118). This epitope was highly conserved across different JEV strains. Moreover, this epitope did not cross-react with WNV-positive and DENV-positive sera. CONCLUSION: Epitope M14-13 was a JEV specific lineal B-cell epitpe. The results may provide a useful basis for the development of epitope-based virus specific diagnostic clinical techniques.

[Study on the DNA immunogenicity of fusion and attachment glycoproteins of Nipah virus].

The two mammalian codon optimized genes, F and G genes of Nipah virus, were generated by assembly PCR, and inserted into mammalian expression vector pCAGGS under chicken beta-actin promoter to construct pCAGG-NiV-F and pCAGG-NiV-G. Syncytium formation was induced in BHK cells by plasmid pCAGG-NiV-F and pCAGG-NiV-G transfection, which indicate recombination proteins F and G were expressed in BHK cell and possessed good biologic activity. Six-week-old female BALB/c mice were intramuscularly primed with 100 microg pCAGG-NiV-F, pCAGG-NiV-G or pCAGG-NiV-F+ pCAGG-NiV-G respectively, and boosted with same dose after 4 weeks. The sera were collected at 3 weeks post second boost. The serum IgG against Nipah virus F and G proteins was detected by indirect ELISA using recombinant Baculovirus expressed Nipah F and G glycoproteins. The results showed that specific antibodies possessed good sensitivity and specificity. Furthermore, the G and F proteins' specific antibodies could neutralize the infectivity of VSVdeltaG* F/G (the NiV F and G envelope glycoproteins psudotyped recombinant vesicular stomatitis virus expressing green fluorescence protein). And, pCAGG-NiV-G also induced higher titer of neutralizing antibody response than pCAGG-NiV-F did. The result indicates that DAN immunization is an efficient vaccine strategy against Nipah virus.

[Study on DNA immune of envelope protein gene of Rift Valley Fever Virus].

DNA vaccines have successfully induced effective antibody and cellular immune response to many viral pathogens. The antibody response of DNA immunization induction in mouse model with envelope glycoproteins of Rift Valley Fever Virus (RVFV), G (N + C), GN and GC was investigated. For this purpose, three codon G (N + C), GN and GC gene were insert into mammalian expression vector pCAGGS under chicken beta-actin promoter to construct pCAGG-RVFV-GN, pCAGG-RVFV-GC and pCAGG-RVFV-G (N + C). The expression of recommbinant GN or / and GC protein in BHK cells transfected with pCAGG-RVFV-GC or pCAGG-RVFV-G (N + C) DNA were confirmed by immunoprecipitation. Six-week-old female BALB/c mice were intramuscularly primed with 100 (g pCAGG-RVFV-GN + pCAGG-RVFV-GC + pCAGG-RVFV-G (N + C), and boosted with same dose after 4 weeks. The serums were collected at 3 weeks post final boost. The serum IgG against Rift Valley Fever Virus G (N + C) protein were detect by indirect ELISA using recombinant Baculovirus expressed Rift Valley Fever Virus GN and GC glycoprotein. The mixture of pCAGG-RVFV-GN, pCAGG-RVFV-GC and pCAGG-RVFV-G (N + C) elicited much strong IgG response. For serum neutralization antibody assay, a recombinant Vesicular Stomatitis Virus pseudotype, in which the VSV envelope protein G gene was replaced with the green fluorescent protein gene (VSVdeltaG x G, Whitt M A) and complemented with Rift Valley Fever Virus G (N + C) glycoprotein expressed in transient (VSVdeltaG x RVFV-G), was use to replace the authentic Rift Valley Fever Virus. The mixture of pCAGG-RVFV-GN, pCAGG-RVFV-GC and pCAGG-RVFV-G (N + C) also induced high titer of neutralization antibody response. These result indicates that DNA immunization is an efficient vaccine strategy against Rift Valley Fever Virus.

[Expression of porcine gamma-interferon in recombinant baculovirus and determination of its antiviral activity].

The full-length porcine interferon gamma(PoIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into honor plasmid pFastBac 1 of Bac-To-Bac Baculovirus Expression System. These recombinant plasmids, pFastBac -PoIFN-gamma, were transformed into DH(10Bac) host bacteria to get recombinant shuttle plasmids, rBacmid-PoIFN-gamma. Recombinant baculovirus, rBac-PoIFN-gamma, was generated for expressing PoIFN-gamma, by transfecting rBacmid-PoIFN-gamma with Cellfectin Reagent into sf9 insect cells. The expression of PoIFN-gamma in insect cells was confirmed by Western Blot, indirect immunofluorescence assay and indirect ELISA. The antiviral activity assay shows that PoIFN-gamma expressed by the rBac-PoIFN-gamma can efficiently inhibit the replication of the recombinant Vesicular Stomatitis Virus expressing green fluorescence protein in PK-15 cells. The antiviral activity of PoIFN-gamma can be specifically blocked by anti-PoIFN-gamma mouse serum. The antiviral titer of culture supernatant of insect cells infected by rBac-PoIFN-gamma is 2 x 10(4) IU/mL. The results demonstrat that the rBac-PoIFN-gamma can express rPoIFN-gamma efficiently and rPoIFN-gamma has high antiviral activity.

[Rescue of a recombinant Newcastle disease virus expressing the green fluorescent protein].

A recombinant Newcastle disease virus (NDV) expressing the green fluorescent protein (GFP) was generated by applying reverse genetics techniques. The GFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the phosphoprotein (P) and matrix protein (M) in a full-length cDNA clone of NDV Lasota vaccine strain. This plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into cells stably expressing T7 RNA polymerase. The rescued virus was first propagated in 10-day-old embryonated eggs and the allantoic fluid was used to infect primary chicken embryo fibroblasts (CEF) cells. The appearance of GFP in live infected cells confirmed further the recovery of a recombinant NDV (rNDV-GFP) expressing this reporter gene. Nine successive passages in embryonated chicken eggs were performed. Allantoic fluid samples were then titrated by a microtiter plate HA test. HA positive ailantoic fluid were used for further egg passages. All the allantoic fluid samples were titrated by end point dilutions and infected cells were examined for the presence of GFP expression. To analyze virus growth, 10-day-old embryonated SPF chicken eggs were inoculated with 1 x 10(4) EID50 rNDV or rNDV-GFP. At 24,48,72 and 96 h p.i. the allantoic fluid of inoculated eggs containing live embryos was harvested and clarified by centrifugation. Supernatants were used for titration of EID50 in 10-day-old embryonated SPF chicken eggs. rNDV and rNDV-GFP grew to similar titers (10(9) EID50/mL). In order to test the virulence of rNDV-GFP, infectious allantoic fluid of rNDV-GFP were inoculated into embryonated SPF chicken eggs at 1 x 10(6) EID50. No dead embryonated egg was found within 96 hours. The replication kinetics and pathogenicity in SPF embryonated eggs of rNDV-GFP did not differ significantly from that of the parent virus. LaSota is a widely used NDV live vaccine strain. The reverse genetic system established for this LaSota vaccine strain provided a useful platform for development of novel live viral vector vaccines in future.

[Generation of recombinant vaccinia virus expressing attachment glycoprotein of Nipah virus].

The mammalian condon optimized G gene was synthesized by over-lapping PCR and used to generate recombinant vaccinia virus, rWR-NiV-G. The expression of Nipah virus G protein in rWR-NiV-G infected HeLa cells was confirmed by western-blot with NiV G protein specific mouse antiserum generated by DNA immunization.The recombinant G protein showed sensitive and specific antigenic reaction to rabbit serum anti-Nipah virus in indirect florescence. Syncytium formation was induced in BHK cells by rWR-NiV-G infection following NiV F protein expressing plasmid pCAGG-NiV-F transfection. Immunization with rWR-NiV-G elicited G protein specific antibody responses in mice. The prokaryotic expressing G protein fragment showed sensitive and specific antigenic reaction to NiV G protein specific antibody from rWR-NiV-G immunized mice serum in indirect ELISA. Furthermore, the G protein specific antibodies could neutralize the infectivity of the recombinant Vesicular Stomatitis Virus pseudotype VSVAG * F/G, in which the VSV envelope protein G gene was replaced with the green fluorescent protein gene (VSVAG * G, Whitt MA) and complemented with Nipah virus F and G glycoprotein expressed in transient (VSVAG * F/G).The results here demonstrated the G protein expressed by rWR-NiV-G keeps native immunogenicity and biological activity. The recombinant virus could be promising vaccine strategy for the prevention of Nipah virus.

[Study of fusion protein and attachment glycoprotein of Nipah virus expressed in recombinant baculovirus].

In this study, Recombinant baculoviruses rBac-NF and rBac-NG were generated for expressing F and G proteins Nipah virus (NiV) . The expression of recommbinant G (rNG) and F (rNF) protein in rBac-NF and rBac-NG infected cells were confirmed by western-blot. Both rNG and rNF showed sensitive and specific antigenic reaction to rabbit serum anti-Nipah virus in indirect immunofluorescence detection and indirect ELISA. Immunization with rBac-NF and rBac-NG infected insect cells elicited G ad F protein specific antibody responses in mice. Furthermore, the G ad F specific antibodies could neutralize the infectivity of the VSVdeltaG* F/G, the NiV F and G envelope glycoproteins psudotyped recombinant Vesicular Stomatitis Virus expressing green fluorescence protein. The results demonstrated F and G protein expressed by the recombinant baculoviruses could be safe economic diagnostic antigens for the surveillance and monitoring of NiV and promising subunit vaccines for the prevention of NiV.

Identification and antigenic epitope mapping of immunodominant region amino residues 510 to 672 on the spike protein of the severe acute respiratory syndrome coronavirus.

The severe acute respiratory syndrome (SARS) is a newly emerging human infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. In this study, the S1 domain of the spike protein and three truncated fragments were expressed by fusion with GST in a pGEX-6p-1 vector. Western blot results demonstrated that the 510-672 fragment of the S1 domain is a linear epitope dominant region. To map the antigenic epitope of this linear epitope dominant region, a set of 16 partially overlapping fragments spanning the fragment were fused with GST and expressed. Four antigenic epitopes S1C3 (539-559), S1C4 (548-567), S1C7/8 (583-606), and S1C10/11 (607-630) were identified. Immunization of mice with each of the four antigenic epitope-fused proteins revealed that all four proteins could elicit spike protein specific antisera. All of them were able to bind to the surface domain of the whole spike protein expressed by recombinant baculovirus in insect cells. Identification of antigenic epitopes of the spike protein of SARS-CoV may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for the severe acute respiratory syndrome.