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1.
Immunol Res ; 67(4-5): 398-407, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31773490

RESUMO

Limited protective effects of commercially available vaccines necessitate the development of novel pneumococcal vaccines. We recently reported a pneumococcal systemic vaccine containing two proteins, Pneumococcal surface protein A (PspA of family 1 and 2) and a bacterium-like particle-based pneumococcal mucosal vaccine containing PspA2 and PspA4 fragments, both eliciting broad protective immune responses. We had previously reported that subcutaneous (s.c.+s.c.+s.c.) immunization with the systemic vaccine induced more pronounced humoral serum IgG responses, while intranasal (i.n.+i.n.+i.n.) immunization with the mucosal vaccine elicited a more pronounced mucosal secretory IgA (sIgA) response. We hypothesized that a combinatorial administration of the two vaccines might elicit more pronounced and broader protective immune responses. Therefore, this study aimed to determine the efficacy of combinatorial prime-boost immunization using both systemic and mucosal vaccines for a pneumococcal infection. Combinatorial prime-boost immunization (s.c.+i.n. and i.n.+s.c.) induced not only IgG, but also mucosal sIgA production at high levels. Systemic priming and mucosal boosting immunization (s.c.+i.n.) provided markedly better protection than homologous prime-boost immunization (s.c.+s.c.+s.c. and i.n.+i.n.+i.n.). Moreover, it induced more robust Th1 and Th17 cell-mediated immune responses than mucosal priming and systemic boosting immunization (i.n.+s.c.). These results indicate that combinatorial prime-boost immunization potentially induces a robust systemic and mucosal immune response, making it an optimal alternative for maximum protection against lethal pneumococcal infections.

2.
Med Microbiol Immunol ; 208(2): 215-226, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30707297

RESUMO

Streptococcuspneumoniae, or pneumococcus, is a major respiratory-tract pathogen that causes high levels of mortality and morbidity in infants and elderly individuals. Despite the development of various capsular polysaccharide vaccines to prevent pneumococcal disease, it remains epidemic. Pneumococcal surface protein A (PspA) is a highly immunogenic surface protein existing in all strains of S. pneumoniae, and it can elicit immunizing protection against pneumococcal infection. In our previous studies, a fusion protein (PsaA-PspA23), consisting of PspA and pneumococcal surface antigen A (PsaA), displayed greater immunogenicity and provided better protection in mice against S. pneumoniae strains than either PsaA or PspA. In this study, the fusion protein PsaA-PspA23, together with PspA4, was formulated with four adjuvants Al(OH)3, MF59, AS03, and AS02, and subsequently subjected to dose optimization and immunological evaluation for determination of the antibody titers, bacterial burden, survival rates, and levels of cytokines in mice. All vaccines with high adjuvant doses displayed higher antigen-specific immunoglobulin G (IgG) titers. Bacterial burdens were notably decreased to different extents in the lungs and blood of mice immunized with the antigen and various adjuvants. Among these adjuvants, AS02 provided outstanding protection against challenge with pathogenic bacteria from different families and clades; it also induced high titers of IgG1 and IgG2a. Moreover, only AS02 elicited high levels of cytokines, such as TNF-α, IFN-γ, IL-2, and IL-4. These results suggest that PsaA-PspA23 and PspA4 formulated with AS02 may potentially be used as a subunit vaccine against deadly pneumococcal infection.


Assuntos
Adesinas Bacterianas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Proteínas de Bactérias/imunologia , Lipoproteínas/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Adesinas Bacterianas/genética , Animais , Anticorpos Antibacterianos/sangue , Carga Bacteriana , Proteínas de Bactérias/genética , Citocinas/análise , Modelos Animais de Doenças , Feminino , Lipoproteínas/genética , Camundongos Endogâmicos BALB C , Vacinas Pneumocócicas/administração & dosagem , Vacinas Pneumocócicas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Análise de Sobrevida , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
3.
Hum Vaccin Immunother ; 15(2): 371-380, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30235046

RESUMO

Streptococcus pneumoniae is an infectious pathogen mainly infecting host bodies through the respiratory system. An effective pneumococcal vaccine would be targeted to the mucosa and provide not only protection against invasive infection but also against colonization in the respiratory system. In the present work, we applied bacterium-like particles (BLPs) as an adjuvant for the development of a PspA mucosal vaccine, in which the PspA protein was displayed on the surface of BLPs. Intranasal immunization with the PspA-BLP pneumococcal vaccine, comprised of PspA2 from pneumococcal family 1 and PspA4 from pneumococcal family 2, not only induced a high level of serum IgG antibodies but also a high level of mucosal SIgA antibodies. Analysis of binding of serum antibodies to intact bacteria showed a broad coverage of binding to pneumococcal strains expressing PspA from clade 1 to 5. Immunization with the PspA-BLP vaccine conferred protection against fatal intranasal challenge with both PspA family 1 and family 2 pneumococcal strains regardless of serotype. Therefore, the PspA-BLP pneumococcal vaccine was demonstrated to be a promising strategy for mucosal immunization to enhance both systemic and mucosal immune responses.

4.
Immunol Res ; 66(4): 528-536, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30128745

RESUMO

Streptococcus pneumoniae is an important pathogen accounting for a large number of deaths worldwide. Despite the multitude of capsular polysaccharide vaccines used to guard against pneumococcal disease, fatal pneumococcal disease remains epidemic due to the narrow range of protection afforded by the capsular polysaccharide vaccines and rate of change in serotypes. The most promising solution is to develop an improved protein-based vaccine with broad protection. In this study, we tested a bivalent vaccine containing antigens mixed with the fusion protein PsaA-pneumococcal surface protein A (PspA)23 and single protein PspA4, including conserved PsaA and PspA from clades 2, 3, and 4 with coverage for families 1 and 2. The vaccine induced a significant increase of anti-PspA IgG, which demonstrated cross-reactivity with the 22 different S. pneumoniae strains from serotypes contained in PPV23 by Western blot. The wide ranging protection was determined by challenging mice with S. pneumoniae from PspA clades 1 to 5. Bacterial loads in the blood and lung and survival rate after challenge were measured. After immunization, the number of bacteria in mice was significantly reduced. The clearance rates with all strains were greater than 90% in the lung, and bacterial loads in the blood were decreased to lower than 10 CFU/ml. The survival rates in immunized animals also were greatly increased (all over 50%) compared with controls. Therefore, this bivalent PspA vaccine may be a good substitute for capsular polysaccharide vaccines.


Assuntos
Adesinas Bacterianas/imunologia , Proteínas de Bactérias/imunologia , Lipoproteínas/imunologia , Pulmão/imunologia , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/fisiologia , Animais , Anticorpos Antibacterianos/sangue , Carga Bacteriana , Reações Cruzadas , Feminino , Humanos , Imunoglobulina G/sangue , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Sorogrupo , Vacinação
5.
Microb Pathog ; 123: 115-119, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29959043

RESUMO

Streptococcus pneumoniae is a major respiratory tract pathogen causing high levels of mortality and morbidity in infants and the elderly. In spite of the multitude of capsular polysaccharide vaccines used to guard against pneumococcal disease, fatal pneumococcal disease remains epidemic. Immunization with pneumococcal surface protein A (PspA), a highly immunogenic surface protein present in all strains of S. pneumoniae, can elicit protection against deadly pneumococcal infection. We have previously evaluated PspA in systemic vaccination. However, the mucosal immune system, as a first line of defense against respiratory infection, plays the most important role against the invasion of S. pneumoniae. In this study, we employed bacterium-like particles (BLPs) as an adjuvant for a PspA mucosal vaccine. The BLPs served as a carrier for PspA proteins bound to their surface. Mice were immunized intranasally with the PspA-BLP pneumococcal vaccine consisting of PspA3 from pneumococcal family 2. Not only did the immunized mice show a high level of serum IgG antibodies but also a high level of SIgA antibodies in the respiratory tract. After immunization with the PspA3-BLP vaccine, the mice were broadly protected against fatal intranasal challenge with homologous and heterogenous pneumococcal strains of different PspA families regardless of serotype, and the colony count was notably decreased in the lungs. Therefore, the PspA3-BLP pneumococcal vaccine has the potential to serve as a novel mucosal vaccine to enhance both systemic and mucosal immune responses to this disease.


Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/imunologia , Vacinas Pneumocócicas/imunologia , Pneumonia Pneumocócica/imunologia , Pneumonia Pneumocócica/prevenção & controle , Streptococcus pneumoniae/imunologia , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Animais , Carga Bacteriana/imunologia , Proteínas de Bactérias/administração & dosagem , Proteção Cruzada/imunologia , Imunização/métodos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Pneumocócicas/administração & dosagem , Pneumonia Pneumocócica/microbiologia , Sistema Respiratório/imunologia
6.
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
7.
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
8.
Infect Immun ; 86(6)2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29610257

RESUMO

Streptococcus pneumoniae is a major cause of invasive pneumococcal disease, septicemia, and meningitis that can result in high morbidity rates in children under 5 years old. The current polysaccharide-based vaccines can provide type-specific immunity, but a broad-spectrum vaccine would provide greater coverage. Therefore, developing pneumococcal-protein-based vaccines that can extend to more serum types is highly important. In this study, we vaccinated mice via the subcutaneous (s.c.) route with a systemic vaccine that is a mixture of fusion protein PsaA-PspA23 and a single protein, PspA4, with aluminum hydroxide as an adjuvant. As a comparison, mice were immunized intranasally with a mucosal vaccine that is a mixture of PspA2-PA-BLP (where PA is protein anchor and BLP is bacterium-like particle) and PspA4-PA-BLP, via the intranasal (i.n.) route. The two immunization processes were followed by challenge with Streptococcus pneumoniae bacteria from two different PspA families. Specific IgG titers in the serum and specific IgA titers in the mucosa were determined following immunizations. Bacterial loads and survival rates after challenge were compared. Both the systemic vaccine and the mucosal vaccine induced a significant increase of IgG against PspAs. Only the mucosal vaccine also induced specific IgA in the mucosa. The two vaccines provided protection, but each vaccine showed an advantage. The systemic vaccine induced higher levels of serum antibodies, whereas the mucosal vaccine limited the bacterial load in the lung and blood. Therefore, coimmunizations with the two types of vaccines may be implemented in the future.


Assuntos
Proteínas de Bactérias/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/metabolismo , Animais , Anticorpos Antibacterianos/sangue , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Pneumocócicas/administração & dosagem , Proteínas Recombinantes
9.
Immunol Invest ; 47(4): 403-415, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29498560

RESUMO

BACKGROUND: Streptococcus pneumoniae is a major pathogen accounting for a large number of pneumococcal disease in worldwide. Due to the mucosal immune pathway induces both systemic and mucosal immune responses, the potential strategy to prevent pneumococcal disease may be to develop a mucosal vaccine. METHOD: In this study, we developed an intranasal pneumococcal protein vaccine based on a bacterium-like particle (BLP) delivery system. PspA is expressed and exposed on the surface of all pneumococcal strains, which confers the potential to induce immune responses to protect against pneumococcal infection. We fused one of the pneumococcal surface proteins (PspA, family2 clade4) with the protein anchor (PA) protein in order to display PspA on the surface of BLPs. RESULT: The current results showed that intranasal immunization with BLPs/PspA-PA efficiently induced both PspA-specific IgG in the serum and PspA-specific IgA in mucosal washes. And intranasal immunization of BLPs/PspA-PA could provide complete protection in a mouse challenge model with pneumococci of different two clades of both homologous and heterologous PspA families. DISCUSSION AND CONCLUSION: Thus, targeted delivery of multiple bacterial antigens via BLPs may prevent pneumococcal disease by inducing both systemic and mucosal immune responses.


Assuntos
Proteínas de Bactérias/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Pneumonia Pneumocócica/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/imunologia , Especificidade de Anticorpos , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Imunidade nas Mucosas , Imunização , Camundongos , Vacinas Pneumocócicas/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia
10.
AIDS ; 32(5): 555-563, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29239895

RESUMO

OBJECTIVE: Nonhuman primates (NHPs) are the only animal model that can be used to evaluate protection efficacy of HIV-1 envelope vaccines. However, whether broadly neutralizing antibodies (bnAbs) can be elicited in NHPs infected with simian/human immunodeficiency virus (SHIV) has not been fully understood. The objective of this study is to investigate whether broad neutralization activities were developed in SHIV-infected macaques after long-term infection as in humans. DESIGN: Neutralization breadth and specificities in plasmas from SHIV-infected macaques were determined by analyzing a panel of tier 2 viruses and their mutants. METHODS: Forty-four Chinese macaques infected with SHIV1157ipd3N4, SHIVSF162P3 or SHIVCHN19P4 were followed for 54-321 weeks. Archived plasmas from 19 macaques were used to determine neutralization breadth and specificities against 17 tier 2 envelope-pseudoviruses. RESULTS: Longitudinal plasma from three SHIVSF162P3-infected macaques and three SHIV1157ipd3N4-infected macaques rarely neutralized viruses (<25%) within 1 year of infection. The neutralization breadth in two SHIV1157ipd3N4-infected macaques significantly increased (≥65%) by year 6. Four of six SHIV1157ipd3N4-infected macaques could neutralize 50-75% viruses, whereas none of macaques infected with SHIVSF162P3 or SHIVCHN19P4 could neutralize more than 25% of viruses after 6 years of infection (P = 0.035). Neutralization specificity analysis showed mutations resistant to bnAbs in V2, V3 or CD4bs regions could abrogate neutralization by year-6 plasma from three SHIV1157ipd3N4-infected macaques. CONCLUSION: These results demonstrate that bnAbs targeting common HIV-1 epitopes can be elicited in SHIV1157ipd3N4-infected macaques as in humans after 4-6 years of infection, and SHIV/NHP can serve as an ideal model to study bnAb maturation.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Anti-HIV/sangue , HIV/imunologia , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Epitopos de Linfócito B/imunologia , Humanos , Estudos Longitudinais , Fatores de Tempo
11.
Immunol Lett ; 187: 41-46, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28487097

RESUMO

Pneumolysin (Ply) is an important virulence factor in pneumococcal infection and a conserved cholesterol-binding cytotoxin expressed by all serotypes of Streptococcus pneumoniae. We previously developed a highly detoxified Ply mutant designated Plym2 by replacement of two amino acids (C428G and W433F), which lost cytotoxicity but retained the ability to induce neutralizing antibodies. In the present work, we applied bacterium-like particles (BLPs) as a carrier and immunostimulant for the development of a Plym2 intranasal vaccine, in which the Plym2 protein was displayed on the surface of BLPs. Intranasal immunization of mice with BLP-Plym2 not only induced a high level of serum IgG antibodies but also a high level of mucosal SIgA antibodies in lung lavages. Antiserum induced by the BLP-Plym2 vaccine elicited high-titer neutralization activity which could inhibit the hemolysis of wild-type Ply. In conclusion, the BLP-Plym2 vaccine was demonstrated to be a promising strategy for intranasal immunization to enhance both systemic and mucosal immune responses.


Assuntos
Imunidade nas Mucosas/efeitos dos fármacos , Imunização , Imunoglobulina A/imunologia , Mutação de Sentido Incorreto , Vacinas Pneumocócicas , Estreptolisinas , Administração Intranasal , Substituição de Aminoácidos , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Pneumocócicas/genética , Vacinas Pneumocócicas/imunologia , Vacinas Pneumocócicas/farmacologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Estreptolisinas/genética , Estreptolisinas/imunologia
12.
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
13.
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
14.
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
15.
Appl Microbiol Biotechnol ; 100(3): 1231-1240, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26446387

RESUMO

Here, we describe a process for expression, purification, and characterization of truncated human papillomavirus type-6 (HPV-6) L1 virus-like particles (VLPs). The scalable cultivation process in a WAVE Bioreactor at the 10-L scale was optimized to express HPV-6 L1 VLPs using the baculovirus insect expression system. A hollow fiber membrane system was used for the integrated operation, including concentration, diafiltration, extraction, and clarification. The HPV-6 L1 protein was further purified by anion-exchange chromatography and hydrophobic chromatography. The HPV-6 L1 protein could self-assemble into VLPs with a diameter of approximately 50-60 nm after removal of the reductant dithiothreitol (DTT). The final purified HPV-6 L1 VLPs product was characterized to estimate yield and purity, and exceeds the requirements for pharmaceutical-grade VLP vaccine. Immunization of mice demonstrated that the vaccine could elicit high titer neutralizing antibodies in vivo. This study confirms the feasibility of producing pharmaceutical-grade HPV type-6 L1 VLPs on an industrial scale for clinical trials.


Assuntos
Proteínas do Capsídeo/metabolismo , Microbiologia Industrial/métodos , Infecções por Papillomavirus/virologia , Vacinas contra Papillomavirus/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Reatores Biológicos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Feminino , Humanos , Imunização , Microbiologia Industrial/instrumentação , Camundongos , Camundongos Endogâmicos BALB C , Vacinas contra Papillomavirus/química , Vacinas contra Papillomavirus/genética , Vacinas contra Papillomavirus/isolamento & purificação , Spodoptera
16.
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
17.
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
18.
Immunol Invest ; 44(5): 482-96, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26107747

RESUMO

Streptococcus pneumoniae is an important pathogen accounting for a large number of deaths worldwide. Due to drawbacks of the current polysaccharide-based vaccine, the most promising way to generate an improved vaccine may be to utilize protection-eliciting pneumococcal proteins. Pneumococcal surface adhesin A (PsaA) and pneumococcal surface protein A (PspA) are two vaccine candidates which have been evaluated against S. pneumoniae infection in animal models or human clinical trials with encouraging results. In this study, the efficacy of the fusion protein PsaA-PspA, which includes PsaA part and PspA part, in inducing immunoprotective effects against fatal pneumococcal challenge was evaluated in an animal model. PspA part of PsaA-PspA fusion protein contains both family1 N-terminal region and family 2 N-terminal clade-defining region of PspA. Immunization with the PsaA-PspA fusion protein induced high levels of antibodies against both PsaA and PspA, which could bind to intact S. pneumoniae strains bearing different PspAs. Ex vivo stimulation of splenocytes from mice immunized with PsaA-PspA induced IL-17A secretion. Mice immunized with PsaA-PspA showed reduced S. pneumoniae levels in the blood and lungs compared with the PBS group after intranasal infection. Finally, mice immunized with PsaA-PspA fusion proteins were protected against fatal challenge with pneumococcal strains expressing different PspAs regardless of the challenge route. These results support the PsaA-PspA fusion protein as a promising vaccine strategy, as demonstrated by its ability to enhance the immune response and stimulate production of high titer antibodies against S. pneumoniae strains bearing heterologous PspAs, as well as confer protection against fatal challenge with PspA family 1 and family 2 strains.


Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Lipoproteínas/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Adesinas Bacterianas/genética , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Avaliação Pré-Clínica de Medicamentos , Feminino , Interleucina-17/metabolismo , Lipoproteínas/genética , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Coelhos , Proteínas Recombinantes de Fusão/imunologia , Baço/citologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Vacinação , Vacinas Sintéticas/imunologia , Virulência
19.
Immunol Invest ; 43(7): 717-26, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25020076

RESUMO

Streptococcus pneumoniae is a major cause of infectious disease and complications worldwide, such as pneumonia, otitis media, bacteremia and meningitis. New generation protein-based pneumococcal vaccines are recognized as alternative vaccine candidates. Pneumolysin (Ply) is a cholesterol-dependent cytolysin produced by all clinical isolates of S. pneumoniae. Our research group previously developed a highly detoxified Ply mutant designated Plym2 by replacement of two animo acids (C428G and W433F). Exhibiting undetectable levels of cytotoxicity, Plym2 could still elicit high titer neutralizing antibodies against the native toxin. However, evaluation of the active immunoprotective effects of Plym2 by subcutaneous immunization and lethal challenge with S. pneumoniae in mice did not yield favorable results. In the present work, we confirmed the previous observations by using passive immunization and systemic challenge. Results of the passive immunization were consistent with those of active immunization. Further experiments were conducted to explain the inability of high titer neutralizing antibodies against Ply to protect mice from S. pneumoniae challenge. Pneumococcal Ply is known to be the major factor responsible for the induction of inflammation that benefits the host. Proinflammatory cytokines facilitate the clearance of invaders by the recruitment and activation of leukocytes at the early infection stage. We demonstrated that Plym2 could induce proinflammatory cytokines similarly to wild-type Ply. A systemic infection model was used to clarify that Plym2 lacking cytolytic activity could protect mice from intraperitoneal challenge directly, while antibodies to the mutant had no effect. Therefore, the protective function of Plym2 may be due to its induction of proinflammatory cytokines. When used in the systemic infection model, Plym2 antibodies may block the induction of proinflammatory cytokines by Ply. These findings demonstrate that a Ply-based vaccine would not be an effective primary vaccine component, but it may be beneficial as an adjuvant to stimulate cytokine production.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Infecções Pneumocócicas/prevenção & controle , Estreptolisinas/genética , Estreptolisinas/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunização Passiva , Interleucina-1beta/imunologia , Camundongos Endogâmicos BALB C , Mutação , Infecções Pneumocócicas/sangue , Infecções Pneumocócicas/imunologia , Coelhos , Streptococcus pneumoniae/imunologia , Fator de Necrose Tumoral alfa/imunologia
20.
Mol Immunol ; 59(2): 136-41, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24598312

RESUMO

Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57(VL85S) and FV57(VL84S). Furthermore, the disulfide bonds in the light chain of FV57, FV57(VL85S) and FV57(VL84S) were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies.


Assuntos
Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Glicoproteínas/imunologia , Profilaxia Pós-Exposição , Vacinas Antirrábicas/imunologia , Vírus da Raiva/imunologia , Raiva/prevenção & controle , Anticorpos de Cadeia Única/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Linhagem Celular , Cricetinae , Cisteína/genética , Feminino , Camundongos , Mutação , Raiva/imunologia , Vacinas Antirrábicas/administração & dosagem , Vacinas Antirrábicas/genética , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/genética
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