To prepare monoclonal antibodies (mAbs) against structural proteins of severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), BALB/c mice were immunized using purified inactivated SFTSV virions as the antigens. Subsequently, hybridoma cell lines that secreted monoclonal antibodies against nucleoprotein (NP) and glycoproteins (GP) were obtained using a hybridoma technique. The antigen specificities of prepared mAbs were examined by indirect immunofluorescence and immunoprecipitation assays. Functional analyses were then performed,including the detection of IFA antibody titers,the levels of neutralizing activity and antibody affinities. After cell fusion and cloning,13 hybridoma cell lines secreted mAbs specifically against SFTSV-GP and 7 hybridoma cell lines secreted mAbs specifically against SFTSV-NP. Immunofluorescence and immunoprecipitation assays showed that the mAbs had high levels of antigen specificity. Among the 13 anti-SFTSV-GP mAbs,6 recognized Gn,whereas the others reacted with Gc. IFA titers of most anti-SFTSV-GP mAbs were between 1,280 and 20,480, and four anti-SFTSV-Gn mAbs showed neutralizing activity. Seven of the obtained anti-SFTSV-NP mAbs reacted specifically with NP,of which the IFA titers ranged from 5,120 to 20,480 with no observed neutralizing activity. Furthermore, two anti-SFTSV-GP mAbs, 1C8 and 1G8, showed high levels of affinity via a non-competitive ELISA. Our study lays the foundation for the development of further diagnostic assays and basic research into SFTSV.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antibody Specificity ; Bunyaviridae Infections ; immunology ; virology ; Female ; Humans ; Hybridomas ; immunology ; Mice ; Mice, Inbred BALB C ; Phlebovirus ; immunology ; Viral Structural Proteins ; immunology

OBJECTIVETo obtain recombinant human anti-EV71 antibodies from a EV71-associated hand-foot-and-mouth disease patient-derived antibody phage library.

METHODSA combinatorial human scFv library to enterovirus 71 (EV71) virus was constructed using antibody genes harvested from the blood of EV71 virus patients. The library was panned and selected by using purified VP1 protein of EV71 virus with phage display. After that the specific antibody was converted to full human IgG antibody with recombinant baculovirus/insect cell system.

RESULTSOne unique human scFv antibody specific for EV71 virus VP1 protein was obtained by ELISA, IFA and analysis of the antibody DNA sequence. The specific anti-VP1 human scFv antibody was converted to full human IgG antibody with recombinant baculovirus/insect cell system. The full human IgG antibody was tested in vitro for EV71 virus neutralization, resulting in no neutralizing activity with EV71 A type and EV71 C4 subtype.

CONCLUSIONThe obtained human anti-EV71 antibodies without neutralizing activity laid the foundation for diagnosis of human EV71-associated hand-foot-and-mouth disease.

Antibodies, Viral ; immunology ; Enterovirus ; immunology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G ; immunology ; Peptide Library

Antibodies, Viral ; blood ; Humans ; SARS Virus ; immunology ; Severe Acute Respiratory Syndrome ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

Objective To develop neutralizing monoclonal antibodies (MAbs) against H10N8 avian influenza virus hemagglutinin and to identify the binding sites. Methods MAbs against hemagglutinin of H10N8 avian influenza virus were developed by genetic engineering. Neutralizing MAbs were screened by microneutralization assay,and then tested by enzyme-linked immunosorbent assay and Western blot to identity the binding sites.The homology modeling process was performed using Discovery Studio 3.5 software,while the binding epitopes were analyzed by BioEdit software. Results One MAb that could neutralize the H10N8 pseudovirus was obtained and characterized. Analysis about epitopes suggested that the antibody could bind to the HA1 region of hemagglutinin,while the epitopes on antigen were conserved in H10 subtypes.Conclusions One neutralizing antibody was obtained by this research.The MAb may potentially be further developed as a pre-clinical candidate to treat avian influenza H10N8 virus infection.
Antibodies, Monoclonal ; immunology ; Antibodies, Neutralizing ; immunology ; Antibodies, Viral ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; immunology ; Hemagglutinin Glycoproteins, Influenza Virus ; immunology ; Influenza A Virus, H10N8 Subtype ; Neutralization Tests

With the development of bio-technological drugs, drug immunogenicity evaluation has become key factor of clarifying safety and efficacy of these drugs. It has become the focus to establish a stable and reliable evaluation system. Due to the advantages such as continuous real-time monitoring, surface plasmon resonance (SPR) technology has been widely used in bio-technological drugs immunogenicity assessments. Our study applied this technology to detect anti-drug antibody (ADA) of a recombinant human anti-rabies monoclonal antibody NM57 in the sera of 48 volunteers admitted in phase I clinical trials. This method could satisfy the basic requirements of detection of ADA.
Antibodies, Anti-Idiotypic ; analysis ; blood ; immunology ; Antibodies, Monoclonal ; analysis ; blood ; immunology ; Antibodies, Viral ; analysis ; blood ; immunology ; Humans ; Rabies virus ; immunology ; Recombinant Proteins ; analysis ; blood ; immunology ; Surface Plasmon Resonance

BACKGROUNDThe etiologic agent of severe acute respiratory syndrome (SARS) has been confirmed to be a novel coronavirus (CoV), namely SARS-CoV. Developing safe and effective SARS-CoV vaccines is essential for us to prevent the possible reemergence of its epidemic. Previous experiences indicate that inactivated vaccine is conventional and more hopeful to be successfully developed. Immunogenicity evaluation of an experimental inactivated SARS-CoV vaccine in rabbits was conducted and reported in this paper.

METHODSThe large-scale cultured SARS-CoV F69 strain was inactivated with 0.4% formaldehyde and purified, then used as the immunogen combined with Freund's adjuvant. Eight adult New Zealand rabbits were immunized four times with this experimental inactivated vaccine. Twelve sets of rabbit serum were sampled from the third day to the seventy-fourth day after the first vaccination. The titers of specific anti-SARS-CoV IgG antibody were determined by indirect enzyme-linked immunosorbent assay, and the neutralizing antibody titers were detected with micro-cytopathic effect neutralization test.

RESULTSRapid and potent humoral immune responses were induced by the inactivated SARS-CoV vaccine in all the eight test rabbits. Titers of both specific IgG antibody and neutralizing antibody peaked at about six weeks after first vaccination, with the maximum value of 1:81 920 and 1:20 480, respectively. After that, serum antibody levels remained at a plateau or had a slight decrease, though two boosters were given in the succedent 4 to 5 weeks. Cross neutralization response existed between SARS-CoV F69 strain and Z2-Y3 strain.

CONCLUSIONSThe inactivated SARS-CoV vaccine made from F69 strain owns strong immunogenicity, and the cross neutralization response between the two different SARS-CoV strains gives a hint of the similar neutralizing epitopes, which provide stable bases for the development of inactivated SARS-CoV vaccines.

Animals ; Antibodies, Viral ; blood ; Immunoglobulin G ; blood ; Neutralization Tests ; Rabbits ; SARS Virus ; immunology ; Vaccines, Inactivated ; immunology ; Viral Vaccines ; immunology

Antibodies, Viral ; blood ; Betacoronavirus ; Coronavirus ; immunology ; Coronavirus Infections ; immunology ; Humans ; Immunoglobulin G ; blood ; Pandemics ; Pneumonia, Viral ; immunology

In this study, three expression vectors encoding unmodified glycoproteins E1 and E2 from H77 (1a), Hebei (1b) and JFH1 (2a) strains were constructed to form pVRC-H77-E1E2, pVRC-HeBei-E1E2 and pVRC-JFH1-E1E2 expressing constructs. The protein expression was confirmed by immunofluorescene assay(IFA) and Western blot. The Lentiviral vector has the ability to package the cellular membrane into pseudo-particles. The plasmid expressing HCV E1-E2 glycoproteins in native form was co-transfected into 293FT cells with a lentiviral packaging plasmid (pHR'CMV delta R8.2)and a self-inactivated (SIN) transfer plasmid (pCS-CG) containing a reporter EGFP gene to produce infectious HCV pseudo-particles(pp). Flow cytometry assays showed that the HCVpp could infect Huh7 and Huh7-CD81, and the infectivity in Huh7-CD81 was about 2-3 times higher than that in Huh7 cells. Meanwhile, HCVpp could neither infect non-liver cells, for example, the 293 cells, nor HepG2 cell . Titration of HCVpp by p24 ELISA assay or infection assay showed that this HCVpp may contain 5-25 ng/mL p24 or 10(4)-10(5) TU (transducing unit)/ ml. An in vitro HCV neutralizing assays based on HCVpp (1a, 1b, 2a) were then established using AP33, a monoclone antibody with cross-neutralizing ability to different HCV strains. The neutralizing ability of the antibodies from HCV infected patients was further studied with this HCVpp system. In summary, three kinds of HCVpp (1a, 1b, 2a subtype) were successfully developed; In vitro HCV neutralizing assays based on HCVpp and SIN lentiviral system were established. This system paves a way for characterization of early steps of HCV infection (host tropisms, receptor binding, membrane fusion, et al. ) or screening anti-HCV drugs (such as inhibitor to virus entry). This system can be further applied to assess the human immune responses in HCV patients or evaluate HCV vaccine candidates.
Hepacivirus ; immunology ; Hepatitis C Antibodies ; immunology ; Hepatitis C, Chronic ; immunology ; Humans ; Neutralization Tests ; Viral Envelope Proteins ; immunology ; Virion ; immunology

Antibodies, Viral/immunology* ; COVID-19/immunology* ; Humans ; Immunoglobulin G/immunology* ; Immunoglobulin M/immunology* ; SARS-CoV-2/immunology*

7 strains of stable cell lines secreting monoclonal antibodies against AAV2 capsids were obtained by immunizing BALB/C mice with highly purified recombinant adeno-associated virus. Among them, the monoclonal antibodies B10 and G4 had neutralizing activity, and their subtypes were IgG1 and IgG2a, respectively. The binding characterizations of the two neutralizing antibodies were studied. Both B10 and G4 showed serotype specific binding activities to rAAV2 virus particles other than AAV1, AAV5, and AAV8, and the binding could not be blocked by heparin. After incubating with the two antibodies separately, rAAV2 viruses could still bind to sensitive cell line BHK-21, suggesting that the binding sites of the two antibodies to rAAV2 located at different positions on viral particle surface from the primary receptor binding sites of AAV2. Western blotting assay showed that B10 could bind to VP1, VP2 and VP3 of rAAV2. However, G4 bound none of them. The results suggested that B10 recognized a linear epitope of AAV2 capsid, whereas G4 probably recognized a conformational epitope on the surface of AAV2 virus particle. The two antibodies with different characteristics provided valuable tools for AAV2 virus particles detection and infection processes.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antibody Specificity ; Capsid ; immunology ; Cell Line ; Dependovirus ; genetics ; immunology ; Epitopes ; immunology ; Female ; Humans ; Immunoglobulin G ; immunology ; Mice ; Mice, Inbred BALB C