RESUMO
Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study, we developed a new indirect ELISA (rE2-ELISA) for serologic detection of BVDV. The assay considers three recombinant E2 protein subtypes as antigens, allowing serologic diagnosis of BVDV-1b (high prevalence worldwide), BVDV-1d and 1e (high prevalence in southern Chile) sub-genotypes. Recombinant E2 (rE2) proteins were successfully expressed in stably transfected CHO cells. Conditions for rE2 ELISAs were established after determining appropriate concentrations of antigen, blocking agent, secondary antibody, and serum dilutions to achieve maximum discrimination between positive and negative serum samples. The developed rE2-ELISA showed a sensitivity of 92.86% and a specificity of 98.33%. Clinical testing of 180 serum samples from herds in southern Chile showed high accuracy (kappa > 0.8) compared to the commercial BVDV Total Ab kit (IDEXX), with 95.37% positive and 87.5% negative predictive value. In addition, the rE2 ELISA has shown the capability to detect anti-BVDV antibodies from naturally infected animals with sub-genotypes 1b, 1e, or undetermined. These results indicate that the developed indirect ELISA could serve as a valid, and efficient alternative for identifying BVDV-infected animals, thus contributing to the success of disease control and eradication programs.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina , Ensaio de Imunoadsorção Enzimática , Sensibilidade e Especificidade , Animais , Ensaio de Imunoadsorção Enzimática/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Bovinos , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Doença das Mucosas por Vírus da Diarreia Viral Bovina/sangue , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Chile , Genótipo , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Vírus da Diarreia Viral Bovina Tipo 1/isolamento & purificação , Vírus da Diarreia Viral Bovina/imunologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , Antígenos Virais/imunologia , Cricetulus , Células CHO , Anticorpos Antivirais/sangue , Proteínas Recombinantes/imunologiaRESUMO
Background: Transmission-blocking vaccines (TBVs) can effectively prevent the community's spread of malaria by targeting the antigens of mosquito sexual stage parasites. At present, only a few candidate antigens have demonstrated transmission-blocking activity (TBA) potential in P. vivax. Quiescin-sulfhydryl oxidase (QSOX) is a sexual stage protein in the rodent malaria parasite Plasmodium berghei and is associated with a critical role in protein folding by introducing disulfides into unfolded reduced proteins. Here, we reported the immunogenicity and transmission-blocking potency of the PvQSOX in P. vivax. Methods and findings: The full-length recombinant PvQSOX protein (rPvQSOX) was expressed in the Escherichia coli expression system. The anti-rPvQSOX antibodies were generated following immunization with the rPvQSOX in rabbits. A parasite integration of the pvqsox gene into the P. berghei pbqsox gene knockout genome was developed to express full-length PvQSOX protein in P. berghei (Pv-Tr-PbQSOX). In western blot, the anti-rPvQSOX antibodies recognized the native PvQSOX protein expressed in transgenic P. berghei gametocyte and ookinete. In indirect immunofluorescence assays, the fluorescence signal was detected in the sexual stages, including gametocyte, gamete, zygote, and ookinete. Anti-rPvQSOX IgGs obviously inhibited the ookinetes and oocysts development both in vivo and in vitro using transgenic parasites. Direct membrane feeding assays of anti-rPvQSOX antibodies were conducted using four field P. vivax isolates (named isolates #1-4) in Thailand. Oocyst density in mosquitoes was significantly reduced by 32.00, 85.96, 43.52, and 66.03% with rabbit anti-rPvQSOX antibodies, respectively. The anti-rPvQSOX antibodies also showed a modest reduction of infection prevalence by 15, 15, 20, and 22.22%, respectively, as compared to the control, while the effect was insignificant. The variation in the DMFA results may be unrelated to the genetic polymorphisms. Compared to the P.vivax Salvador (Sal) I strain sequences, the pvqsox in isolate #1 showed no amino acid substitution, whereas isolates #2, #3, and #4 all had the M361I substitution. Conclusions: Our results suggest that PvQSOX could serve as a potential P. vivax TBVs candidate, which warrants further evaluation and optimization.
Assuntos
Anticorpos Antiprotozoários , Vacinas Antimaláricas , Malária Vivax , Plasmodium berghei , Plasmodium vivax , Proteínas Recombinantes , Plasmodium vivax/imunologia , Plasmodium vivax/genética , Plasmodium vivax/enzimologia , Animais , Coelhos , Vacinas Antimaláricas/imunologia , Anticorpos Antiprotozoários/imunologia , Malária Vivax/prevenção & controle , Malária Vivax/transmissão , Malária Vivax/imunologia , Plasmodium berghei/imunologia , Plasmodium berghei/genética , Plasmodium berghei/enzimologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Camundongos , Escherichia coli/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Feminino , Humanos , Imunogenicidade da Vacina , Camundongos Endogâmicos BALB CRESUMO
Transgenic chicken bioreactors can efficiently produce egg whites containing large quantities of recombinant proteins. We previously developed transgenic chickens that produce recombinant monoclonal antibodies (mAbs) against epidermal growth factor receptor 2 (HER2). However, the practical applications of mAbs derived from transgenic eggs have not yet been examined. Therefore, we aimed to evaluate whether these recombinant mAbs can be used in enzyme-linked immunosorbent assay (ELISA). Recombinant HER2 mAbs from transgenic eggs were dissolved in phosphate-buffered saline and applied directly to 96-well microplates as immobilized antibodies without purification. The performance of ELISA using the unpurified recombinant HER2 mAbs from transgenic eggs was comparable to that of ELISA using commercially available purified recombinant HER2 mAbs. Moreover, ELISA using unpurified recombinant HER2 mAbs from transgenic eggs demonstrated high antigen specificity and was successfully applied to samples from cultured cell lysates derived from HER2-positive and HER2-negative cell lines. The unpurified recombinant HER2 mAbs from transgenic eggs were also efficiently used as immobilized antibodies in paper-based ELISA. In conclusion, our findings suggest that recombinant mAbs from transgenic eggs have the potential to be used to develop economic ELISA devices. To the best of our knowledge, this study is the first to use recombinant HER2 mAbs from transgenic eggs in ELISA.
Assuntos
Animais Geneticamente Modificados , Anticorpos Monoclonais , Reatores Biológicos , Galinhas , Ensaio de Imunoadsorção Enzimática , Receptor ErbB-2 , Proteínas Recombinantes , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Receptor ErbB-2/imunologia , Receptor ErbB-2/genética , Humanos , Linhagem Celular TumoralRESUMO
Predicting hepatitis B surface antigen (HBsAg) clearance is important for chronic hepatitis B (CHB) patients receiving pegylated interferon-alfa (Peg-IFN) therapy. We aimed to determine the predictive value of serum hepatitis B core antibody (anti-HBc) for HBsAg clearance. A total of 189 HBeAg-negative CHB patients who received Peg-IFN based therapy were retrospectively included and classified into two groups: nucleos(t)ide analogues (NAs) add-on Peg-IFN group (add-on group, n = 94) and Peg-IFN combined with NAs or Peg-IFN monotherapy group (combination or monotherapy group, n = 95). After 48 weeks of treatment, 27.5% (52/189) and 15.9% (30/189) of patients achieved HBsAg clearance and seroconversion, respectively. Patients in the combination or monotherapy group tended to achieve relatively higher HBsAg clearance (31.6% vs. 23.4%, p = 0.208) and seroconversion (21.1% vs. 10.6%, p = 0.050) rates than those in the add-on group. In combination or monotherapy group, anti-HBc levels at week 12 were lower in patients with HBsAg clearance (9.0 S/CO vs. 9.9 S/CO, p < 0.001) and seroconversion (8.8 S/CO vs. 9.8 S/CO, p < 0.001) than those without. Anti-HBc level at week 12 was an independent predictor of HBsAg clearance and seroconversion. Patients with lower anti-HBc levels at week 12 showed a more significant decline in HBsAg levels during treatment. Combination of anti-HBc at week 12 and baseline HBsAg could identify over 70% of patients who achieved HBsAg clearance after 48 weeks of treatment. In addition to HBsAg, anti-HBc level could be used as a promising marker for selecting HBeAg-negative CHB patients who are more likely to respond to Peg-IFN-based therapy.
Assuntos
Antivirais , Anticorpos Anti-Hepatite B , Antígenos de Superfície da Hepatite B , Antígenos E da Hepatite B , Hepatite B Crônica , Interferon-alfa , Humanos , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Hepatite B Crônica/sangue , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/imunologia , Masculino , Feminino , Adulto , Estudos Retrospectivos , Anticorpos Anti-Hepatite B/sangue , Antivirais/uso terapêutico , Pessoa de Meia-Idade , Antígenos E da Hepatite B/sangue , Interferon-alfa/uso terapêutico , Vírus da Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/sangue , Resultado do Tratamento , Quimioterapia Combinada , Soroconversão , Adulto Jovem , Polietilenoglicóis/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Proteínas Recombinantes/imunologiaRESUMO
P32 protein serves as a crucial structural component of Goat pox virus (GTPV), which causes a highly virulent infectious disease in sheep and goats. Despite the fact that P32 has been widely expressed in the previous studies, it is difficult to obtain recombinant P32 efficiently. This study aimed to achieve soluble expression of P32 recombinant protein and to develop its specific monoclonal antibody. The gene fragment of P32Δ (GP32Δ) was synthesized by optimizing the coding sequence of amino acids 1-246 of the known goatpox P32 protein. Subsequently, GP32Δ was cloned into a prokaryotic expression vector for expression and purification, resulting in the successful production of soluble recombinant protein rP32Δ. Utilizing rP32Δ, an indirect ELISA method was established by immunizing 6-week-old BALB/c mice with inactivated GTPV as the antigen. Through hybridoma technology, three monoclonal antibody hybridoma cell lines secreting anti-goat pox virus rP32Δ were screened, designated as 2F3, 3E8, and 4H5, respectively. These monoclonal antibodies, classified as IgG1, IgG2a, and IgG2b, respectively, with κappa light chains, were characterized following ascites preparation and purification. Indirect ELISA results demonstrated that the ELISA potency of the three monoclonal antibodies exceeded 1:12800. Furthermore, Western blot analysis revealed specific reactivity of both 3E8 and 4H5 with rP32Δ, while immunofluorescence assays confirmed 3E8's ability to specifically recognize GTPV in cells. The preceding findings demonstrate the successful acquisition of the soluble expressed recombinant P32 protein and its specific monoclonal antibody 3E8 in this study, thereby laying a foundational material basis for the establishment of a GTPV detection method.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Capripoxvirus , Ensaio de Imunoadsorção Enzimática , Cabras , Camundongos Endogâmicos BALB C , Proteínas Recombinantes , Animais , Anticorpos Monoclonais/imunologia , Capripoxvirus/genética , Capripoxvirus/imunologia , Anticorpos Antivirais/imunologia , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Hibridomas , Imunoglobulina G , Expressão Gênica , Proteínas Virais/genética , Proteínas Virais/imunologia , Infecções por Poxviridae/imunologia , Feminino , Doenças das Cabras/virologia , Clonagem MolecularRESUMO
BACKGROUND: The development of therapies and vaccines for various diseases often necessitates the analysis of cellular immunity. However, unlike other rodents, the limited availability of reagents for Syrian hamsters restricts immunological analysis, particularly in the determination of serum effector molecules such as cytokines. In this study, we aim to produce and characterize the cytokines IFN-γ, TGF-ß, IL-6, IL-10, and TNF-α from Syrian hamsters in recombinant form and to generate polyclonal antibodies against them in rats. METHODS AND RESULTS: Cytokine transcript sequences were cloned into expression vectors in E. coli. Recombinant proteins were produced, purified through affinity chromatography, and characterized by Western blot using an anti-6xHis monoclonal antibody. Rats were immunized with the recombinant proteins to generate polyclonal antibodies (pAbs). These pAbs were characterized by Western blot and titrated by indirect ELISA. The recombinant cytokines rTNF-α, rIL-10, rIFN-γ, rTGF-ß, and rIL-6 were produced and specifically recognized at their expected molecular weights of 22.3 kDa, 19.8 kDa, 18.9 kDa, 11.8 kDa, and 22.9 kDa. pAbs were produced and demonstrated the ability to specifically recognize their target proteins with titers of 409,600 (rIL-10), 204,800 (rTNF-α), 102,400 (rIL-10), 51,200 (rTGF-ß), and 25,600 (rIFN-É£). CONCLUSIONS: The reagents produced represent a starting point for developing immunoassays to detect hamster cytokines, facilitating the analysis of cellular immunity in this biomodel.
Assuntos
Citocinas , Imunidade Celular , Mesocricetus , Proteínas Recombinantes , Animais , Citocinas/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Cricetinae , Ratos , Anticorpos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
Canine Visceral Leishmaniasis (CVL) is the most fatal form of Leishmania infection in dogs and is caused by L. infantum in the Americas. This parasite follows a zoonotic life cycle, raising concerns within domestic households, where dogs act as the primary reservoir of the parasite. Accurately detecting infected dogs is vital for effective epidemiological control in both canine and human populations. However, existing diagnostic methods in Brazil have limitations, particularly in detecting asymptomatic and oligosymptomatic dogs, leading to ineffective disease control. To address this challenge, we evaluated a novel recombinant antigen from L. infantum, the rLiNTPDase2. Previous studies have confirmed its high performance via ELISA, leading us to assess its suitability for a Lateral Flow Immunochromatographic Assay (LFIA), which is ideal for point-of-care testing. Standardization of the assay involved testing two nitrocellulose membranes (HF135 and HF120, Millipore), three blocking protocols, and five sample dilutions (1:10, 1:20, 1:40, 1:80, and 1:160). Following the chosen conditions (HF120 membrane, 1-minute blocking protocol, and 1:80 sample dilution), we validated our assay with a sample size of 78 dogs, comprising 32 negatives and 46 positives, including symptomatic (n=23), oligosymptomatic (n=17), and asymptomatic (n=6) cases. The results revealed a sensitivity of 86.9â¯%, specificity of 62.5â¯%, and accuracy of 76.9â¯%, which is consistent with ELISA performance for the same samples. Compared to DPP-LVC, our assay demonstrated promising results in detecting asymptomatic and oligosymptomatic cases. This study underscores the suitability of the rLiNTPDase2 antigen for the LFIA format, suggesting its potential as a novel point-of-care diagnostic test for CVL.
Assuntos
Antígenos de Protozoários , Doenças do Cão , Leishmaniose Visceral , Sensibilidade e Especificidade , Animais , Cães , Leishmaniose Visceral/veterinária , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/análise , Cromatografia de Afinidade/veterinária , Cromatografia de Afinidade/métodos , Leishmania infantum/enzimologia , Leishmania infantum/imunologia , Proteínas Recombinantes/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Ensaio de Imunoadsorção Enzimática/métodosRESUMO
In the landscape of infectious diseases, human coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2 pose significant threats, characterized by severe respiratory illnesses and notable resistance to conventional treatments due to their rapid evolution and the emergence of diverse variants, particularly within SARS-CoV-2. This study investigated the development of broad-spectrum coronavirus vaccines using heterodimeric RBD-Fc proteins engineered through the "Knob-into-Hole" technique. We constructed various recombinant proteins incorporating the receptor-binding domains (RBDs) of different coronaviruses. Heterodimers combining RBDs from SARS-CoV-2 with those of SARS-CoV or MERS-CoV elicited superior neutralizing responses compared to homodimeric proteins in murine models. Additionally, heterotetrameric proteins, specifically D614G_Delta/BA.1_XBB.1.5-RBD and MERS_D614G/BA.1_XBB.1.5-RBD, elicited remarkable breadth and potency in neutralizing all known SARS-CoV-2 variants, SARS-CoV, related sarbecoviruses like GD-Pangolin and WIV1, and even MERS-CoV pseudoviruses. Furthermore, these heterotetrameric proteins also demonstrated enhanced cellular immune responses. These findings underscore the potential of recombinant hetero proteins as a universal vaccine strategy against current and future coronavirus threats.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , Coronavírus da Síndrome Respiratória do Oriente Médio , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Camundongos , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Vacinas contra COVID-19/imunologia , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/química , COVID-19/prevenção & controle , COVID-19/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Camundongos Endogâmicos BALB C , Feminino , Domínios Proteicos , Testes de Neutralização , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genéticaRESUMO
Introduction: Tularemia, caused by the bacterium Francisella tularensis, poses health risks to humans and can spread through a variety of routes. It has also been classified as a Tier 1 Select agent by the CDC, highlighting its potential as a bioterrorism agent. Moreover, it is difficult to diagnose in a timely fashion, owing to the non-specific nature of tularemia infections. Rapid, sensitive, and accurate detection methods are required to reduce mortality rates. We aimed to develop antibodies directed against the outer membrane protein A of F. tularensis (FopA) for rapid and accurate diagnosis of tularemia. Methods: We used a baculovirus insect cell expression vector system to produce the FopA antigen and generate anti-FopA antibodies through immunization of BALB/c mice. We then employed hybridoma and phage display technologies to screen for antibodies that could recognize unique epitopes on FopA. Result: Two monoclonal antibodies, 6B12 and 3C1, identified through phage display screening specifically bound to recombinant FopA in a dose-dependent manner. The binding affinity of the anti-FopA 6B12 and 3C1 antibodies was observed to have an equilibrium dissociation constant of 1.76 × 10-10 M and 1.32 × 10-9 M, respectively. These antibodies were used to develop a sandwich ELISA system for the diagnosis of tularemia. This assay was found to be highly specific and sensitive, with detection limits ranging from 0.062 ng/mL in PBS to 0.064 ng/mL in skim milk matrices. Discussion: Our findings demonstrate the feasibility of a novel diagnostic approach for detecting F. tularensis based on targeting FopA, as opposed to existing tests that target the bacterial lipopolysaccharide.
Assuntos
Anticorpos Antibacterianos , Anticorpos Monoclonais , Proteínas da Membrana Bacteriana Externa , Francisella tularensis , Camundongos Endogâmicos BALB C , Proteínas Recombinantes , Tularemia , Tularemia/diagnóstico , Animais , Francisella tularensis/imunologia , Francisella tularensis/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Anticorpos Monoclonais/imunologia , Camundongos , Imunoensaio/métodos , Sensibilidade e Especificidade , Feminino , Técnicas de Visualização da Superfície Celular , Epitopos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/genética , Hibridomas , Baculoviridae/genéticaRESUMO
Tropical theileriosis is a lymphoproliferative disease caused by Theileria annulata and is transmitted by Ixodid ticks of the genus Hyalomma. It causes significant losses in livestock, especially in exotic cattle. The existing methods for controlling it, chemotherapeutic agents and a vaccine based on an attenuated schizont stage parasite, have several limitations. A promising solution to control this disease is the use of molecular vaccines based on potential immunogenic proteins of T. annulata. For this purpose, we selected five antigenic sequences of T. annulata, i.e. SPAG-1, Tams, TaSP, spm2, and Ta9. These were subjected to epitope prediction for cytotoxic T lymphocytes, B-cells, and helper T lymphocytes. CTL and B-cell epitopes with a higher score whereas those of HTL with a lower score, were selected for the construct. A single protein was constructed using specific linkers and evaluated for high antigenicity and low allergenicity. The construct was acidic, hydrophobic, and thermostable in nature. Secondary and tertiary structures of this construct were drawn using the PSIPRED and RaptorX servers, respectively. A Ramachandran plot showed a high percentage of residues in this construct in favorable, allowed, and general regions. Molecular docking studies suggested that the complex was stable and our construct could potentially be a good candidate for immunization trials. Furthermore, we successfully cloned it into the pET-28a plasmid and transformed it into the BL21 strain. A restriction analysis was performed to confirm the transformation of our plasmid. After expression and purification, recombinant protein of 49 kDa was confirmed by western blotting. An ELISA detected increased specific antibody levels in the sera of the immunized animals compared with the control group, and flow cytometric analysis showed a stronger cell-mediated immune response. We believe our multi-epitope recombinant protein has the potential for the large-scale application for disease prevention globally in the bovine population. This study will act as a model for similar parasitic challenges.
Assuntos
Imunidade Celular , Imunidade Humoral , Proteínas Recombinantes , Theileria annulata , Theileriose , Theileria annulata/imunologia , Theileria annulata/genética , Animais , Bovinos , Theileriose/imunologia , Theileriose/parasitologia , Theileriose/prevenção & controle , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito B/imunologia , Vacinas Protozoárias/imunologia , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/genética , Simulação por Computador , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/genética , Anticorpos Antiprotozoários/imunologia , Anticorpos Antiprotozoários/sangueRESUMO
The recombinant Staphylococcal protein A (SpA) is widely used in biotechnology to purify polyclonal and monoclonal IgG antibodies. At very low concentrations, the highly-purified form of the protein A can down-regulate the activation of human B-lymphocytes and macrophages which are the key cells in determining autoimmune diseases. In the present study, the efficiency of three different forms of protein A, including native full-length SpA, the recombinant full-length SpA, and a recombinant truncated form of SpA on the reduction of 4 inflammatory cytokines, including IL-8, IL-1ß, TNF-α, and IL-6 by peripheral blood mononuclear cell (PBMCs) were studied and compared to an anti-rheumatoid arthritis commercial drug, Enbrel. The recombinant proteins were expressed in E. coli and the native form of SpA was commercially provided. PBMCs were obtained from adult patients with active rheumatoid arthritis (RA) and healthy control donors. Then, the effect of different doses of the three pure forms of SpA in comparison with Enbrel was investigated by analyzing the expression of selected cytokines using ELISA. The results showed that the truncated form of recombinant SpA significantly reduced the expression of cytokines more effectively than the other full-length formulations as well as the commercial drug Enbrel. In silico analysis shows that in the truncated protein, as the radius of gyration increases, the structure of IgG-binding domains become more open and more exposed to IgG. To summarize, our findings indicate that the truncated form of protein A is the most efficient form of SpA as it significantly decreases the secretion of evaluated cytokines from PBMCs in vitro.
Assuntos
Citocinas , Leucócitos Mononucleares , Proteína Estafilocócica A , Staphylococcus aureus , Humanos , Proteína Estafilocócica A/imunologia , Proteína Estafilocócica A/metabolismo , Citocinas/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Staphylococcus aureus/imunologia , Adulto , Proteínas Recombinantes/imunologia , Artrite Reumatoide/imunologia , Feminino , Masculino , Pessoa de Meia-Idade , Doenças Autoimunes/imunologiaRESUMO
INTRODUCTION: Producing commercial bacterins/toxoids against Clostridium spp. is laborious and hazardous. Conversely, developing prototype vaccines using purified recombinant toxoids, though safe and effective, is both laborious and costly for application in production animals. OBJECTIVE: Considering that inactivated recombinant Escherichiacoli (bacterin) is a simple, cost-effective, and to be safe solution, we evaluated, for the first time, a pentavalent formulation of recombinant bacterins containing the alpha, beta, and epsilon toxins of Clostridiumperfringens and C and D neurotoxins of Clostridiumbotulinum in sheep. METHODS: Subcutaneously, 18 Texel sheep received two doses (200 µg of each antigen) of recombinant bacterin (n = 7) or purified recombinant antigens (n = 6) on days 0 and 28, while the control group (n = 5) did not receive an immunization. Sera samples from days 0 (before the 1st dose), 28 (before the 2nd dose), and 56, 84, and 112 were used for measuring IgG (indirect ELISA) and neutralizing antibodies (mouse serum neutralization). RESULTS: Both formulations induced significant levels of IgG against all five toxins (p < 0.05) up to day 112, with peaks at days 28 and 56 post-immunization. The expected booster effect occurred only for the botulinum toxins. The neutralizing antibody titers were satisfactory against ETX (≥2 IU/ml for both formulations) and BoNT-D [5 IU/ml (bacterin) and 10 IU/ml (purified)]. CONCLUSION: While adjustments are required, the recombinant bacterin platform holds great potential for polyvalent vaccines due to its straightforward, safe, and cost-effective production, establishing it as a user-friendly technology for the veterinary immunobiological industry.
Assuntos
Anticorpos Antibacterianos , Anticorpos Neutralizantes , Vacinas Bacterianas , Botulismo , Enterotoxemia , Animais , Botulismo/prevenção & controle , Botulismo/veterinária , Botulismo/imunologia , Ovinos , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Anticorpos Antibacterianos/sangue , Enterotoxemia/prevenção & controle , Enterotoxemia/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Doenças dos Ovinos/prevenção & controle , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Imunoglobulina G/sangue , Escherichia coli/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , FemininoRESUMO
SARS-CoV-2 is the causative virus of COVID-19, which has been responsible for millions of deaths worldwide since its discovery. After its emergence, several variants have been identified that challenge the efficacy of the available vaccines. Previously, we generated and evaluated a vaccine based on a recombinant Bacillus Calmette-Guérin (rBCG) expressing the nucleoprotein (N) of SARS-CoV-2 (rBCG-N-SARS-CoV-2). This protein is a highly immunogenic antigen and well conserved among variants. Here, we tested the administration of this vaccine with recombinant N and viral Spike proteins (S), or Receptor Binding Domain (RBD-Omicron variant), plus a booster with the recombinant proteins only, as a novel and effective strategy to protect against SARS-CoV-2 variants. METHODS: BALB/c mice were immunized with rBCG-N-SARS-CoV-2 and recombinant SARS-CoV-2 proteins in Alum adjuvant, followed by a booster with recombinant proteins to assess the safety and virus-specific cellular and humoral immune responses against SARS-CoV-2 antigens. RESULTS: Immunization with rBCG-N-SARS-CoV-2 + recombinant proteins as a vaccine was safe and promoted the activation of CD4+ and CD8+ T cells that recognize SARS-CoV-2 N, S, and RBD antigens. These cells were able to secrete cytokines with an antiviral profile. This immunization strategy also induced robust titers of specific antibodies against N, S, and RBD and neutralizing antibodies of SARS-CoV-2. CONCLUSIONS: Co-administration of the rBCG-N-SARS-CoV-2 vaccine with recombinant SARS-CoV-2 proteins could be an effective alternative to control particular SARS-CoV-2 variants. Due to its safety and capacity to induce virus-specific immune responses, we believe the rBCG-N-SARS-CoV-2 + Proteins vaccine could be an attractive candidate to protect against this virus, especially in newborns.
Assuntos
Anticorpos Antivirais , Vacina BCG , Vacinas contra COVID-19 , COVID-19 , Camundongos Endogâmicos BALB C , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Camundongos , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/prevenção & controle , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacina BCG/imunologia , Vacina BCG/administração & dosagem , Vacina BCG/genética , Feminino , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Imunização Secundária , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagem , Imunidade Humoral , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , Linfócitos T CD8-Positivos/imunologia , Fosfoproteínas/imunologia , Fosfoproteínas/genética , Adjuvantes Imunológicos/administração & dosagem , Imunidade CelularRESUMO
The COVID-19 pandemic continues to cause infections and deaths, which are attributable to the SARS-CoV-2 Omicron variant of concern (VOC). Moderna's response to the declining protective efficacies of current SARS-CoV-2 vaccines against Omicron was to develop a bivalent booster vaccine based on the Spike (S) protein from the Wuhan and Omicron BA.4/BA.5 strains. This approach, while commendable, is unfeasible in light of rapidly emerging mutated viral strains. PubMed and Google Scholar were systematically reviewed for peer-reviewed papers up to January 2024. Articles included focused on specific themes such as the clinical history of recombinant protein vaccine development against different diseases, including COVID-19, the production of recombinant protein vaccines using different host expression systems, aspects to consider in recombinant protein vaccine development, and overcoming problems associated with large-scale recombinant protein vaccine production. In silico approaches to identify conserved and immunogenic epitopes could provide broad protection against SARS-CoV-2 VOCs but require validation in animal models. The recombinant protein vaccine development platform has shown a successful history in clinical development. Recombinant protein vaccines incorporating conserved epitopes may utilize a number of expression systems, such as yeast (Saccharomyces cerevisiae), baculovirus-insect cells (Sf9 cells), and Escherichia coli (E. coli). Current multi-epitope subunit vaccines against SARS-CoV-2 utilizing synthetic peptides are unfeasible for large-scale immunizations. Recombinant protein vaccines based on conserved and immunogenic proteins produced using E. coli offer high production yields, convenient purification, and cost-effective production of large-scale vaccine quantities capable of protecting against the SARS-CoV-2 D614G strain and its VOCs.
Assuntos
Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Vacinas Sintéticas , Humanos , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Vacinas Sintéticas/imunologia , Animais , Proteínas Recombinantes/imunologia , Desenvolvimento de Vacinas , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Epitopos/imunologia , Vacinas de Subunidades ProteicasAssuntos
Anticorpos Neutralizantes , Anticorpos Neutralizantes/imunologia , Humanos , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/genética , Imunização Secundária , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Animais , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/prevenção & controleRESUMO
BACKGROUND: Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease that presents with severe hemorrhagic manifestations and is associated with significant fatality rates. The causative agent, Crimean-Congo Hemorrhagic Fever Virus (CCHFV), is a high-priority pathogen identified by the World Health Organization with no approved vaccine or specific treatment available. In addition, there is a critical need for enhanced diagnostic tools to improve public health awareness, prevention measures, and disease control strategies. METHODS: We designed plasmids to enable the purification of soluble CCHFV glycoprotein Gc expressed in mammalian 293 F cells, followed by purification using affinity and size exclusion chromatography. The purified antigen was analyzed by SDS-PAGE and Western blotting to confirm its reactivity to antibodies from CCHF survivors. Additionally, an in-house indirect ELISA was developed using the purified Gc as a coating antigen. RESULTS: The optimized expression system successfully produced soluble and pure Gc antigen after affinity chromatography. The protein showed specific reactivity with CCHFV-positive serum antibodies in Western blot analysis. The indirect ELISA assay demonstrated high efficacy in distinguishing between CCHFV-positive and -negative serum samples, indicating its potential as a valuable diagnostic tool. Size exclusion chromatography further confirmed the presence of aggregates in our protein preparation. CONCLUSIONS: The purified Gc antigen shows promise for developing direct diagnostic assays for CCHFV. The antigen's suitability for subunit vaccine development and its application as bait for monoclonal antibody isolation from survivors could be investigated further. This work lays the foundation for future research into the development of rapid diagnostic tests for field deployment.
Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo , Proteínas Recombinantes , Humanos , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Células HEK293 , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/isolamento & purificação , Febre Hemorrágica da Crimeia/diagnóstico , Febre Hemorrágica da Crimeia/virologia , Ensaio de Imunoadsorção Enzimática , Animais , Cromatografia de Afinidade/métodos , Cromatografia em Gel , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangueRESUMO
We compared the immunogenicity of recombinant S. pneumoniae pneumolysin (rPly) when administered with and without Al(OH)3 adjuvant, and evaluated the protective properties of recombinant protein in the active defense experiment. It was shown that double immunization with rPly+Al(OH)3 increases the levels of IgG antibodies in comparison with the control (p<0.01), while triple immunization results in a more significant increase in IgG antibody levels (p<0.001). Double immunization with rPly without Al(OH)3 does not induce a significant increase in antibody levels in comparison with the control, while triple immunization results in a slight but significant increase in antibody levels (p<0.05). The active defense test proved the protective activity of rPly against S. pneumoniae serotype 3 at intranasal infection.
Assuntos
Anticorpos Antibacterianos , Proteínas de Bactérias , Imunoglobulina G , Proteínas Recombinantes , Streptococcus pneumoniae , Estreptolisinas , Estreptolisinas/imunologia , Estreptolisinas/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/genética , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/genética , Animais , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/sangue , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Camundongos , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Infecções Pneumocócicas/microbiologia , Adjuvantes Imunológicos , Hidróxido de Alumínio/imunologia , Hidróxido de Alumínio/administração & dosagem , Vacinas Pneumocócicas/imunologia , Vacinas Pneumocócicas/administração & dosagem , FemininoRESUMO
Toxoplasma gondii (T. gondii) is widely spread around the world, which can cause serious harm to immunosuppressed patients. Currently, the commercial test kits are poor at assessing T. gondii infection and vaccine effectiveness, making an urgent need to exploit effective enzyme-linked immunosorbent assay with great performance to compensate for this deficiency. Here, the TgIMP1 recombinant protein was expressed in E. coli BL(21) cells. The TgIMP1 was purified with affinity chromatography and the reactivity was retained with anti-TgIMP1 antibodies. The TgIMP1 was then used to develop an indirect ELISA (IMP1-iELISA) and the reaction conditions of IMP1-iELISA were optimized. As a result, the cut-off value was determined to be 0.2833 by analyzing the OD450nm values of forty T. gondii-negative sera. The coefficient of variation of 6 T. gondii-positive sera within and between runs were both less than 10%. The IMP1-iELISA was non-cross-reactive with the sera of cytomegalovirus, herpes virus, rubella virus, Cryptosporidium spp., Theileria spp., Neospora spp. and Plasmodium spp.. Furthermore, the sensitivity and specificity of IMP1-iELISA were 98.9% and 96.7%, respectively, based on testing 150 serum samples. The results suggest that this IMP1-iELISA is specific, sensitive, repeatable and can be applied to the detection of T. gondii infections in the medical and health industries.
Assuntos
Anticorpos Antiprotozoários , Ensaio de Imunoadsorção Enzimática , Toxoplasma , Toxoplasmose , Animais , Humanos , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/genética , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Imunoglobulina G/sangue , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Sensibilidade e Especificidade , Toxoplasma/imunologia , Toxoplasma/genética , Toxoplasmose/diagnóstico , Toxoplasmose/imunologia , Toxoplasmose/parasitologiaRESUMO
Loxoscelism is the pathological condition triggered by a brown spider bite. The venom of these spiders is rich in phospholipases D (PLDs), which can induce virtually all local and systemic manifestations. Recombinant mutated PLDs from clinically relevant Loxosceles species in South America have been investigated as potential antigens to develop novel therapeutic strategies for loxoscelism. However, certain gaps need to be addressed before a clinical approach can be implemented. In this study, we examined the potential of these recombinant mutated PLDs as antigens by testing some variations in the immunization scheme. Furthermore, we evaluated the efficacy of the produced antibodies in neutralizing the nephrotoxicity and sphingomyelinase activity of brown spider venoms. Our findings indicate that the number of immunizations has a greater impact on the effectiveness of neutralization compared to the amount of antigen. Specifically, two or three doses were equally effective in reducing dermonecrosis and edema. Additionally, three immunizations proved to be more effective in neutralizing mice lethality than one or two. Moreover, immunizations mitigated the signs of kidney injury, a crucial aspect given that acute renal failure is a serious systemic complication. In vitro inhibition of the sphingomyelinase activity of Loxosceles venoms, a key factor in vivo toxicity, was nearly complete after incubation with antibodies raised against these antigens. These findings underscore the importance of implementing an effective immunization scheme with multiple immunizations, without the need for high antigen doses, and enhances the spectrum of neutralization exhibited by antibodies generated with these antigens. In summary, these results highlight the strong potential of these antigens for the development of new therapeutic strategies against cutaneous and systemic manifestations of loxoscelism.
Assuntos
Fosfolipase D , Proteínas Recombinantes , Venenos de Aranha , Animais , Fosfolipase D/imunologia , Fosfolipase D/genética , Venenos de Aranha/imunologia , Camundongos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Picada de Aranha/imunologia , Aranha Marrom Reclusa/imunologia , Feminino , Antígenos/imunologia , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/imunologia , Anticorpos Neutralizantes , Antivenenos/imunologia , Antivenenos/administração & dosagem , Modelos Animais de Doenças , Imunização , Diester Fosfórico HidrolasesRESUMO
The development of cross-reactive vaccines is one of the central aims of modern vaccinology. Continuous mutation and the emergence of new SARS-CoV-2 variants and subvariants create the problem of universal coronavirus vaccine design. Previously, the authors devised three recombinant coronavirus antigens, which were based on the sequence collected in 2019 (the Wuhan variant) and produced in an E. coli bacterial expression system. The present work has shown, for the first time, that these recombinant antigens induce the production of antibodies that clearly interact with produced in CHO full-length S-protein of the Omicron variant. The immunogenicity of these recombinant antigens was studied in formulations with different adjuvants: Freund's adjuvant, Al(OH)3 and an adjuvant based on spherical particles (SPs), which are structurally modified plant virus. All adjuvanted formulations effectively stimulated Omicron-specific IgG production in mice. These universal coronavirus antigens could be considered the main component for the further development of broad-spectrum coronavirus vaccines for the prevention of SARS-CoV-2 infection. The present work also provides evidence that the synthetic biology approach is a promising strategy for the development of highly cross-reactive vaccines. Moreover, it is important to note that the bacterial expression system might be appropriate for the production of antigenically active universal antigens.