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1.
Mol Immunol ; 101: 197-202, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30007229

RESUMO

Monoclonal antibodies (MAbs) are a unique and attractive class of biologics and are potential substitutes for post-exposure rabies prophylaxis. The safety, tolerance, and broad neutralization efficiency of a MAb cocktail called CL184, composed of the antibodies CR4098 and CR57, was confirmed in a phase I clinical trial. We have prepared a series of single-chain Fv fragments (scFvs) and leucine zipper Fv fragments (zipFvs) from CR57 and CR4098. In this study, we selected and formed scFv and zipFv cocktails and compared their protective effects against the rabies virus. Mice and hamster challenge models demonstrated the improved protection of the zipFv cocktail compared with scFv cocktail, because of its stronger affinity. The results indicate that zipFv production is a promising novel method for the genetic engineering of antibody fragments and improving affinity through systematic screening may be important when designing small molecule antibodies against RV.


Assuntos
Anticorpos Monoclonais/imunologia , Zíper de Leucina , Vírus da Raiva/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Afinidade de Anticorpos , Cricetinae , Modelos Animais de Doenças , Humanos , Camundongos , Testes de Neutralização
2.
Protein Expr Purif ; 151: 56-61, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29908315

RESUMO

Streptococcus pneumoniae is a major pathogen that causes life-threatening diseases, such as pneumonia, otitis media, bacteremia, and meningitis, worldwide and especially in young children and the elderly. Pneumococcal surface protein A (PspA) is a widely studied candidate protein vaccine that represents a promising replacement for current polysaccharide and polysaccharide-conjugate vaccines. In this study, we describe a simple method to produce PspA of clade 4 from an Escherichia coli expression system using hydroxylapatite and ion-exchange chromatography. Using this method, we successfully expressed soluble PspA4 in 10 L of autoinducing culture medium, with a wet-cell yield of 19 g/L and a final PspA4 concentration of 22.8 mg/L. Additionally, we improved PspA4 purity from 17% to 70% in a single step through the use of hydroxylapatite, resulting in acquisition of recombinant PspA4 (>95% purity) at a final yield of 43% from the starting cell-lysis solution. We subsequently verified the secondary structure molecular weight of recombinant PspA4 by circular dichroism and mass spectrometry, respectively. These results demonstrated a highly efficient method for mass producing PspA4 protein and that can also be applied for purification of PspA proteins from other clades.


Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Durapatita/química , Escherichia coli/metabolismo , Cromatografia por Troca Iônica , Escherichia coli/genética , Fermentação , Expressão Gênica , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação
3.
Immunol Lett ; 186: 9-14, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28389318

RESUMO

Rabies is an acute zoonotic infectious disease with a high fatality rate but is preventable with vaccination and rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv), a small engineered antigen-binding protein derived from antibody variable heavy (VH) and light (VL) chains connected by a peptide linker, can potentially be used to replace RIG. Here, we produced two peptides VH-JUN-HIS and VL-FOS-HA separately in Escherichia coli and assembled them to form zipFv successfully in vitro. The new zipFv utilizes FOS and JUN leucine zippers to form an antibody structure similar to the IgG counterpart with two free N-terminal ends of VH and VL. The zipFv protein showed notable improvement in binding ability and affinity over its corresponding scFv. The zipFv also demonstrated greater stability in serum and the same protective rate as RIG against challenge with a standard rabies virus (CVS-24) in mice. Our results indicated zipFv as a novel and efficient antibody form with enhanced neutralizing potency.


Assuntos
Antígenos Virais/imunologia , Glicoproteínas/imunologia , Região Variável de Imunoglobulina/genética , Zíper de Leucina/genética , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Raiva/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/genética , Afinidade de Anticorpos , Escherichia coli/genética , Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos , Engenharia de Proteínas , Estabilidade Proteica , Vacinas Antirrábicas/genética , Anticorpos de Cadeia Única/genética , Vacinação
4.
J Microbiol Biotechnol ; 27(4): 718-724, 2017 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-28068664

RESUMO

The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and halflife. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Antivirais/imunologia , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/imunologia , Potência de Vacina , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Afinidade de Anticorpos , Combinação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Glicoproteínas/imunologia , Meia-Vida , Humanos , Camundongos , Modelos Animais , Testes de Neutralização , Engenharia de Proteínas , Redobramento de Proteína , Raiva/imunologia , Vírus da Raiva/patogenicidade , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/farmacologia , Proteínas do Envelope Viral/imunologia , alfa-Sinucleína/química
5.
Protein Expr Purif ; 126: 26-32, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27157441

RESUMO

An anti-rabies virus single-chain antibody fragment of an anti-glycoprotein with the VL-linker-VH orientation, designated scFv57RN, was successfully and conveniently prepared in this study. The scFv57RN protein was mainly expressed in inclusion bodies in Escherichia coli. After washing and purification, the inclusion bodies were finally obtained with an on-column refolding procedure. Further purification by gel exclusion chromatography was performed to remove inactive multimers. About 360 mg of final product was recovered from 1 L of bacterial culture. The final product showed a high neutralizing titer of 950 IU/mg to the CVS-11 strain as measured using the rapid fluorescent focus inhibition test. Our study demonstrated a highly efficient method to mass produce scFV57RN with activity from inclusion bodies, which may be applied in the purification of other insoluble proteins.


Assuntos
Expressão Gênica , Glicoproteínas/análise , Redobramento de Proteína , Vírus da Raiva/química , Anticorpos de Cadeia Única , Proteínas Virais/antagonistas & inibidores , Escherichia coli , Glicoproteínas/química , Corpos de Inclusão/química , Corpos de Inclusão/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/isolamento & purificação , Proteínas Virais/química
6.
Protein Pept Lett ; 23(1): 24-32, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26497316

RESUMO

Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.


Assuntos
Glicoproteínas/imunologia , Vírus da Raiva/metabolismo , Raiva/prevenção & controle , Anticorpos de Cadeia Única/genética , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Camundongos , Peptídeos/genética , Peptídeos/metabolismo , Vírus da Raiva/genética , Vírus da Raiva/imunologia , Anticorpos de Cadeia Única/metabolismo , Proteínas Virais/imunologia , Vacinas Virais/administração & dosagem
7.
Mol Immunol ; 68(2 Pt A): 168-75, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26325475

RESUMO

Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency.


Assuntos
Anticorpos Neutralizantes/imunologia , Zíper de Leucina/genética , Vacinas Antirrábicas/imunologia , Raiva/prevenção & controle , Anticorpos de Cadeia Única/imunologia , Proteínas do Envelope Viral/antagonistas & inibidores , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/genética , Cricetulus , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Células HEK293 , Humanos , Imunização , Imunoglobulina G/administração & dosagem , Zíper de Leucina/imunologia , Camundongos , Testes de Neutralização , Plasmídeos/química , Plasmídeos/metabolismo , Multimerização Proteica , Raiva/imunologia , Raiva/virologia , Vacinas Antirrábicas/administração & dosagem , Vacinas Antirrábicas/genética , Vírus da Raiva/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/genética , Análise de Sobrevida , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
8.
Appl Microbiol Biotechnol ; 98(4): 1547-55, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24241896

RESUMO

Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Glicoproteínas/imunologia , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Vírus da Raiva/metabolismo , Anticorpos de Cadeia Única/biossíntese , Proteínas Virais/imunologia
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