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Many pathogens, including Plasmodium spp., exploit the interaction of programmed death-1 (PD-1) with PD-1-ligand-1 (PD-L1) to "deactivate" T cell functions, but the role of PD-L2 remains unclear. We studied malarial infections to understand the contribution of PD-L2 to immunity. Here we have shown that higher PD-L2 expression on blood dendritic cells, from Plasmodium falciparum-infected individuals, correlated with lower parasitemia. Mechanistic studies in mice showed that PD-L2 was indispensable for establishing effective CD4(+) T cell immunity against malaria, because it not only inhibited PD-L1 to PD-1 activity but also increased CD3 and inducible co-stimulator (ICOS) expression on T cells. Importantly, administration of soluble multimeric PD-L2 to mice with lethal malaria was sufficient to dramatically improve immunity and survival. These studies show immuno-regulation by PD-L2, which has the potential to be translated into an effective treatment for malaria and other diseases where T cell immunity is ineffective or short-lived due to PD-1-mediated signaling.
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Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Adamantano/análogos & derivados , Adamantano/uso terapêutico , Adulto , Animais , Antimaláricos/uso terapêutico , Antígeno B7-H1/genética , Células Cultivadas , Ensaios Clínicos como Assunto , Células Dendríticas/parasitologia , Feminino , Humanos , Imunidade Celular , Ativação Linfocitária , Malária Falciparum/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Parasitemia/imunologia , Peróxidos/uso terapêutico , Proteína 2 Ligante de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/genética , Pirimidinas/uso terapêutico , Triazóis/uso terapêutico , Adulto JovemRESUMO
Antibody with high affinity and specificity to antigen has widely used as a tool to combat various diseases. The variable domain of immunoglobulin new antigen receptor (VNAR) naturally found in shark contains autonomous function as single-domain antibody. Due to its excellent characteristics, the small, non-complex, and highly stable have made shark VNAR can acquires the antigen-binding capability that might not be reached by conventional antibody. Phage display technology enables shark VNAR to be presented on the surface of phage, allowing the exploration of shark VNAR as an alternative antibody format to target antigens from various infectious diseases. The application of phage-displayed shark VNAR in antibody library and biopanning eventually leads to the discovery and isolation of antigen-specific VNARs with diagnostic and therapeutic potential towards infectious diseases. This review provides an overview of the shark VNAR antibody, the types of phage display technology with comparison to the other types of display system, as well as the application and case studies of phage-displayed shark VNAR antibodies against infectious diseases.
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Bacteriófagos , Doenças Transmissíveis , Tubarões , Animais , Anticorpos , Antígenos , Biblioteca de PeptídeosRESUMO
AIMS: Schistosomes infect approximately 250 million people worldwide. To date, there is no effective vaccine available for the prevention of schistosome infection in endemic regions. There remains a need to develop means to confer long-term protection of individuals against reinfection. In this study, an annexin, namely annexin B30, which is highly expressed in the tegument of Schistosoma mansoni was selected to evaluate its immunogenicity and protective efficacy in a mouse model. METHODS AND RESULTS: Bioinformatics analysis showed that there were three potential linear B-cell epitopes and four conformational B-cell epitopes predicted from annexin B30, respectively. Full-length annexin B30 was cloned and expressed in Escherichia coli BL21(DE3). In the presence of adjuvants, the soluble recombinant protein was evaluated for its protective efficacy in two independent vaccine trials. Immunization of CBA mice with recombinant annexin B30 formulated either in alum only or alum/CpG induced a mixed Th1/Th2 cytokine profile but no significant protection against schistosome infection was detected. CONCLUSION: Recombinant annexin B30 did not confer significant protection against the parasite. The molecule may not be suitable for vaccine development. However, it could be an ideal biomarker recommended for immunodiagnostics development.
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Anexinas/imunologia , Antígenos de Helmintos/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Adjuvantes Imunológicos , Animais , Anexinas/administração & dosagem , Anexinas/análise , Anticorpos Anti-Helmínticos/imunologia , Formação de Anticorpos , Feminino , Camundongos , Camundongos Endogâmicos CBA , Proteínas Recombinantes/imunologia , Schistosoma mansoni/química , Esquistossomose mansoni/diagnóstico , Vacinas/imunologiaRESUMO
Anti-salbutamol antibodies remain as important tools for the detection of salbutamol abuse in athletic doping. This study evaluated the feasibility and efficiency of the chicken (Gallus gallus domesticus) as an immunization host to generate anti-salbutamol scFv antibodies by phage display. A phage display antibody library was constructed from a single chicken immunized against salbutamol-KLH conjugate. After a stringent biopanning strategy, a novel scFv clone which was inhibited by free salbutamol recorded the highest affinity. This scFv was expressed as soluble and functional protein in Escherichia coli T7 SHuffle Express B (DE3) strain. Cross-reactivity studies of the scFv towards other relevant ß2-agonists revealed that the scFv cross-reacted significantly towards clenbuterol. The determined IC50 of the scFv towards the two ß2-agonists were; IC50 salbutamolâ¯=â¯â¼0.310⯵g/ml, IC50 clenbuterolâ¯=â¯â¼0.076⯵g/ml. The generated scFv demonstrated poor stability based on accelerated stability studies. The scFv was used to develop an competitive indirect ELISA (LODâ¯=â¯0.125⯵g/ml) for detection of parent salbutamol in spiked human urine (nâ¯=â¯18) with â¼83.4% reliability at the cut-off of 1⯵g/ml currently implemented by WADA and may be of potential use in human doping urinalysis.
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Albuterol/urina , Proteínas Aviárias/química , Clembuterol/urina , Dopagem Esportivo , Anticorpos de Cadeia Única/química , Animais , Especificidade de Anticorpos/genética , Proteínas Aviárias/genética , Galinhas , Humanos , Anticorpos de Cadeia Única/genética , UrináliseRESUMO
BACKGROUND: Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform. Unfortunately, conventional monoclonal antibodies are subject to degradation shortening shelf lives of RDTs. The variable region of the receptor (VNAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability. METHODS: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken. RESULTS: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13. CONCLUSIONS: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.
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Anticorpos Antiprotozoários/metabolismo , Testes Diagnósticos de Rotina , Malária/diagnóstico , Proteínas de Protozoários/metabolismo , Tubarões , Animais , Anticorpos Monoclonais/metabolismo , Biomarcadores , Escherichia coli/metabolismo , Camundongos , Biblioteca de Peptídeos , Proteínas Recombinantes/metabolismo , Tubarões/imunologiaRESUMO
BACKGROUND: Increased expression of voltage-gated sodium channels (VGSCs) have been implicated with strong metastatic potential of human breast cancer in vitro and in vivo where the main culprits are cardiac isoform Nav1.5 and its 'neonatal' splice variant, nNav1.5. Several factors have been associated with Nav1.5 and nNav1.5 gain of expression in breast cancer mainly hormones, and growth factors. AIM: This study aimed to investigate the role of epigenetics via transcription repressor, repressor element silencing transcription factor (REST) and histone deacetylases (HDACs) in enhancing Nav1.5 and nNav1.5 expression in human breast cancer by assessing the effect of HDAC inhibitor, trichostatin A (TSA). METHODS: The less aggressive human breast cancer cell line, MCF-7 cells which lack Nav1.5 and nNav1.5 expression was treated with TSA at a concentration range 10-10,000 ng/ml for 24 h whilst the aggressive MDA-MB-231 cells was used as control. The effect of TSA on Nav1.5, nNav1.5, REST, HDAC1, HDAC2, HDAC3, MMP2 and N-cadherin gene expression level was analysed by real-time PCR. Cell growth (MTT assay) and metastatic behaviors (lateral motility and migration assays) were also measured. RESULTS: mRNA expression level of Nav1.5 and nNav1.5 were initially very low in MCF-7 compared to MDA-MB-231 cells. Inversely, mRNA expression level of REST, HDAC1, HDAC2, and HDAC3 were all greater in MCF-7 compared to MDA-MB-231 cells. Treatment with TSA significantly increased the mRNA expression level of Nav1.5 and nNav1.5 in MCF-7 cells. On the contrary, TSA significantly reduced the mRNA expression level of REST and HDAC2 in this cell line. Remarkably, despite cell growth inhibition by TSA, motility and migration of MCF-7 cells were enhanced after TSA treatment, confirmed with the up-regulation of metastatic markers, MMP2 and N-cadherin. CONCLUSIONS: This study identified epigenetics as another factor that regulate the expression level of Nav1.5 and nNav1.5 in breast cancer where REST and HDAC2 play important role as epigenetic regulators that when lacking enhances the expression of Nav1.5 and nNav1.5 thus promotes motility and migration of breast cancer. Elucidation of the regulatory mechanisms for gain of Nav1.5 and nNav1.5 expression may be helpful for seeking effective strategies for the management of metastatic diseases.
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Structural vaccinology is pivotal in expediting vaccine design through high-throughput screening of immunogenic antigens. Leveraging the structural and functional characteristics of antigens and immune cell receptors, this approach employs protein structural comparison to identify conserved patterns in key pathogenic components. Molecular modeling techniques, including homology modeling and molecular docking, analyze specific three-dimensional (3D) structures and protein interactions and offer valuable insights into the 3D interactions and binding affinity between vaccine candidates and target proteins. In this review, we delve into the utilization of various immunoinformatics and molecular modeling tools to streamline the development of broad-protective vaccines against coronavirus disease 2019 variants. Structural vaccinology significantly enhances our understanding of molecular interactions between hosts and pathogens. By accelerating the pace of developing effective and targeted vaccines, particularly against the rapidly mutating severe acute respiratory syndrome coronavirus 2 and other prevalent infectious diseases, this approach stands at the forefront of advancing immunization strategies. The combination of computational techniques and structural insights not only facilitates the identification of potential vaccine candidates but also contributes to the rational design of vaccines, fostering a more efficient and targeted approach to combatting infectious diseases.
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BACKGROUND: The scale-up of zoonoses prevention control and eradication in China, coupled with numerous academic articles in Chinese journals has led to the development of new tools and strategies aimed at further consolidating parasite control goals. As a result, there is a growing need for an up-to-date understanding of the research progress and prevention and control experience of parasitic diseases in China. METHODS: To understand the research status of schistosomiasis and toxoplasmosis in China, academic articles published in Chinese journals from 1980 to 2021 were retrieved from China National Knowledge Infrastructure (CNKI) and Wanfang databases. The Bibliographic Items Co-occurrence Matrix Builder (BICOMB) software was used to extract and analyze the keyword frequencies. The 'K/A ratio' as the frequency of a keyword that occurred in all the articles within a certain time stage was calculated to compare the popularity of the same keyword in different time stages. Keyword co-occurrence network maps were constructed by VOSviewer software. RESULTS: A total of 18,508 articles in the research field of Schistosoma and 13,289 articles in the field of Toxoplasma gondii were included. Results in both fields showed some similarities: the annual number of articles presented an increasing trend before entering the 21st century and decreased rapidly in recent years. Two opposite changing trends of keyword frequency could be observed in the K/A ratio analysis: the K/A ratios of 'Surveillance' and 'Infection' continuously increased over time, while those of 'Schistosoma mansoni' and 'Mesenteric lymph nodes' decreased. The diversification of keyword co-occurrence networks could be observed in the co-occurrence network maps. CONCLUSIONS: This bibliometric analysis reveals trends in research themes in the fields of Schistosoma and Toxoplasma gondii from 1980 to 2021, presenting China's experience such as a high degree of government involvement and multidisciplinary participation in schistosomiasis and toxoplasmosis control and elimination.
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Publicações Periódicas como Assunto , Esquistossomose , Toxoplasma , Toxoplasmose , Animais , Humanos , População do Leste Asiático , Bibliometria , China , Schistosoma mansoniRESUMO
The transmission of dengue virus (DENV) from an infected Aedes mosquito to a human, causes illness ranging from mild dengue fever to fatal dengue shock syndrome. The similar conserved structure and sequence among distinct DENV serotypes or different flaviviruses has resulted in the occurrence of cross reaction followed by antibody-dependent enhancement (ADE). Thus far, the vaccine which can provide effective protection against infection by different DENV serotypes remains the biggest hurdle to overcome. Therefore, deep investigation is crucial for the potent and effective therapeutic drugs development. In addition, the cross-reactivity of flaviviruses that leads to false diagnosis in clinical settings could result to delay proper intervention management. Thus, the accurate diagnostic with high specificity and sensitivity is highly required to provide prompt diagnosis in respect to render early treatment for DENV infected individuals. In this review, the recent development of neutralizing antibodies, antiviral agents, and vaccine candidates in therapeutic platform for DENV infection will be discussed. Moreover, the discovery of antigenic cryptic epitopes, principle of molecular mimicry, and application of single-chain or single-domain antibodies towards DENV will also be presented.
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Vacinas contra Dengue , Vírus da Dengue , Dengue , Vacinas , Animais , Humanos , Dengue/diagnóstico , Dengue/prevenção & controle , Anticorpos Antivirais/uso terapêutico , Anticorpos NeutralizantesRESUMO
The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally devastated public health, the economies of many countries and quality of life universally. The recent emergence of immune-escaped variants and scenario of vaccinated individuals being infected has raised the global concerns about the effectiveness of the current available vaccines in transmission control and disease prevention. Given the high rate mutation of SARS-CoV-2, an efficacious vaccine targeting against multiple variants that contains virus-specific epitopes is desperately needed. An immunoinformatics approach is gaining traction in vaccine design and development due to the significant reduction in time and cost of immunogenicity studies and increasing reliability of the generated results. It can underpin the development of novel therapeutic methods and accelerate the design and production of peptide vaccines for infectious diseases. Structural proteins, particularly spike protein (S), along with other proteins have been studied intensively as promising coronavirus vaccine targets. Numbers of promising online immunological databases, tools and web servers have widely been employed for the design and development of next generation COVID-19 vaccines. This review highlights the role of immunoinformatics in identifying immunogenic peptides as potential vaccine targets, involving databases, and prediction and characterization of epitopes which can be harnessed for designing future coronavirus vaccines.
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COVID-19 , Vacinas Virais , Humanos , Vacinas contra COVID-19 , SARS-CoV-2 , Vacinas Virais/química , Vacinas Virais/genética , Qualidade de Vida , Reprodutibilidade dos Testes , Epitopos de Linfócito B , Epitopos de Linfócito T , Simulação de Acoplamento MolecularRESUMO
The coronavirus 2019 (COVID-19) pandemic was caused by a positive sense single-stranded RNA (ssRNA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other human coronaviruses (hCoVs) exist. Historical pandemics include smallpox and influenza, with efficacious therapeutics utilized to reduce overall disease burden through effectively targeting a competent host immune system response. The immune system is composed of primary/secondary lymphoid structures with initially eight types of immune cell types, and many other subtypes, traversing cell membranes utilizing cell signaling cascades that contribute towards clearance of pathogenic proteins. Other proteins discussed include cluster of differentiation (CD) markers, major histocompatibility complexes (MHC), pleiotropic interleukins (IL), and chemokines (CXC). The historical concepts of host immunity are the innate and adaptive immune systems. The adaptive immune system is represented by T cells, B cells, and antibodies. The innate immune system is represented by macrophages, neutrophils, dendritic cells, and the complement system. Other viruses can affect and regulate cell cycle progression for example, in cancers that include human papillomavirus (HPV: cervical carcinoma), Epstein-Barr virus (EBV: lymphoma), Hepatitis B and C (HB/HC: hepatocellular carcinoma) and human T cell Leukemia Virus-1 (T cell leukemia). Bacterial infections also increase the risk of developing cancer (e.g., Helicobacter pylori). Viral and bacterial factors can cause both morbidity and mortality alongside being transmitted within clinical and community settings through affecting a host immune response. Therefore, it is appropriate to contextualize advances in single cell sequencing in conjunction with other laboratory techniques allowing insights into immune cell characterization. These developments offer improved clarity and understanding that overlap with autoimmune conditions that could be affected by innate B cells (B1+ or marginal zone cells) or adaptive T cell responses to SARS-CoV-2 infection and other pathologies. Thus, this review starts with an introduction into host respiratory infection before examining invaluable cellular messenger proteins and then individual immune cell markers.
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In the last few years, annexins have been discovered in several nematodes and other parasites, and distinct differences between the parasite annexins and those of the hosts make them potentially attractive targets for anti-parasite therapeutics. Annexins are ubiquitous proteins found in almost all organisms across all kingdoms.Here, we present an overview of novel annexins from parasitic organisms, and summarize their phylogenetic and biochemical properties, with a view to using them as drug or vaccine targets. Building on structural and biological information that has been accumulated for mammalian and plant annexins, we describe a predicted additional secondary structure element found in many parasite annexins that may confer unique functional properties, and present a specific antigenic epitope for use as a vaccine.
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Anexinas/antagonistas & inibidores , Descoberta de Drogas , Parasitos/metabolismo , Vacinas/biossíntese , Sequência de Aminoácidos , Animais , Anexinas/química , Anti-Infecciosos/farmacologia , Doença , Humanos , Dados de Sequência Molecular , Parasitos/efeitos dos fármacosRESUMO
Reverse vaccinology (RV) was first introduced by Rappuoli for the development of an effective vaccine against serogroup B Neisseria meningitidis (MenB). With the advances in next generation sequencing technologies, the amount of genomic data has risen exponentially. Since then, the RV approach has widely been used to discover potential vaccine protein targets by screening whole genome sequences of pathogens using a combination of sophisticated computational algorithms and bioinformatic tools. In contrast to conventional vaccine development strategies, RV offers a novel method to facilitate rapid vaccine design and reduces reliance on the traditional, relatively tedious, and labor-intensive approach based on Pasteur"s principles of isolating, inactivating, and injecting the causative agent of an infectious disease. Advances in biocomputational techniques have remarkably increased the significance for the rapid identification of the proteins that are secreted or expressed on the surface of pathogens. Immunogenic proteins which are able to induce the immune response in the hosts can be predicted based on the immune epitopes present within the protein sequence. To date, RV has successfully been applied to develop vaccines against a variety of infectious pathogens. In this chapter, we apply a pipeline of bioinformatic programs for identification of Shigella flexneri potential vaccine candidates as an illustration immunoinformatic tools available for RV.
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Neisseria meningitidis Sorogrupo B , Shigella flexneri , Vacinas Bacterianas , Biologia Computacional , Neisseria meningitidis Sorogrupo B/imunologia , Shigella flexneri/genética , VacinologiaRESUMO
The current COVID-19 pandemic, an infectious disease caused by the novel coronavirus (SARS-CoV-2), poses a threat to global health because of its high rate of spread and death. Currently, vaccination is the most effective method to prevent the spread of this disease. In the present study, we developed a novel multiepitope vaccine against SARS-CoV-2 containing Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (BA.1) variants. To this end, we performed a robust immunoinformatics approach based on multiple epitopes of the four structural proteins of SARS-CoV-2 (S, M, N, and E) from 475 SARS-CoV-2 genomes sequenced from the regions with the highest number of registered cases, namely the United States, India, Brazil, France, Germany, and the United Kingdom. To investigate the best immunogenic epitopes for linear B cells, cytotoxic T lymphocytes (CTL), and helper T lymphocytes (HTL), we evaluated antigenicity, allergenicity, conservation, immunogenicity, toxicity, human population coverage, IFN-inducing, post-translational modifications, and physicochemical properties. The tertiary structure of a vaccine prototype was predicted, refined, and validated. Through docking experiments, we evaluated its molecular coupling to the key immune receptor Toll-Like Receptor 3 (TLR3). To improve the quality of docking calculations, quantum mechanics/molecular mechanics calculations (QM/MM) were used, with the QM part of the simulations performed using the density functional theory formalism (DFT). Cloning and codon optimization were performed for the successful expression of the vaccine in E. coli. Finally, we investigated the immunogenic properties and immune response of our SARS-CoV-2 multiepitope vaccine. The results of the simulations show that administering our prototype three times significantly increases the antibody response and decreases the amount of antigens. The proposed vaccine candidate should therefore be tested in clinical trials for its efficacy in neutralizing SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Vacinas contra COVID-19 , Pandemias/prevenção & controle , Vacinologia , COVID-19/prevenção & controle , Escherichia coli , Epitopos de Linfócito B , Epitopos de Linfócito T , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Vacinas de Subunidades Antigênicas/químicaRESUMO
Shigella is a Gram-negative bacteria that cause shigellosis. Treatment with antibiotics cannot be sustained to control the bacterial infection due to the risk of antibiotic resistance. Vaccine development against the highly prevalent Shigella serotypes could provide a generous benefit in reducing the occurrence of shigellosis. The present study is aimed to identify the peptides that could be the ideal candidates for the Shigella vaccine development. THP-1 human macrophage cell lines were infected with clinical strains of Shigella flexneri 2a. The bacterial peptides bound on HLA class II molecules of infected THP-1 were analyzed and identified using the immunopeptidomics approach. Following mass spectrometry identification, a total of 14 proteins were predicted by PSORTb, CELLO, and Gneg-mPLoc as outer membrane proteins (OMPs) of Shigella. Of which, 12 OMPs were found to be conserved among Shigella species and had no significance with human proteomes. Outer membrane receptor FepA and TonB-dependent receptor were among the OMPs predicted to possess the high number of immunogenic B- and T-cell epitopes. The epitopes with high antigenicity from FepA and TonB were identified as potential peptide candidates for Shigella vaccine development. The immunoreactivity of the constructed recombinant proteins were determined using the Shigella-infected human and rabbit sera, respectively. Their protective efficacy and immune responses in controlling the Shigella infection will further be investigated in experimental animal models.
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Disenteria Bacilar , Shigella , Animais , Epitopos de Linfócito T , Humanos , Espectrometria de Massas , Peptídeos , Coelhos , VacinologiaRESUMO
Co-existence of Japanese Encephalitis virus (JEV) with highly homologous antigenic epitopes results in antibody-based serodiagnosis being inaccurate at detecting and distinguishing JEV from other flaviviruses. This often causes misdiagnosis and inefficient treatments of flavivirus infection. Generation of JEV NS1 protein remains a challenge as it is notably expressed in the form of inactive aggregates known as inclusion bodies using bacterial expression systems. This study evaluated two trxB and gor E. coli strains in producing soluble JEV NS1 via a cold-shock expression system. High yield of JEV NS1 inclusion bodies was produced using cold-shocked expression system. Subsequently, a simplified yet successful approach in generating soluble, active JEV NS1 protein through solubilization, purification and in vitro refolding of JEV NS1 protein from inclusion bodies was developed. A step-wise dialysis refolding approach was used to facilitate JEV NS1 refolding. The authenticity of the refolded JEV NS1 was confirmed by specific antibody binding on indirect ELISA commercial anti-NS1 antibodies which showed that the refolded JEV NS1 was highly immunoreactive. This presented approach is cost-effective, and negates the need for mammalian or insect cell expression systems in order to synthesize this JEV NS1 protein of important diagnostic and therapeutic relevance in Japanese Encephalitis disease.
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Anticorpos Antivirais/metabolismo , Vírus da Encefalite Japonesa (Espécie)/isolamento & purificação , Escherichia coli/crescimento & desenvolvimento , Proteínas não Estruturais Virais/genética , Dissulfetos/química , Vírus da Encefalite Japonesa (Espécie)/imunologia , Epitopos/imunologia , Escherichia coli/classificação , Escherichia coli/genética , Corpos de Inclusão Viral/imunologia , Corpos de Inclusão Viral/metabolismo , Engenharia de Proteínas , Redobramento de Proteína , Solubilidade , Transformação Bacteriana , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismoRESUMO
Conventional monoclonal antibodies (mAbs) have been widely used in research and diagnostic applications due to their high affinity and specificity. However, multiple limitations, such as large size, complex structure and sensitivity to extreme ambient temperature potentially weaken the performance of mAbs in certain applications. To address this problem, the exploration of new antigen binders is extensively required in relation to improve the quality of current diagnostic platforms. In recent years, a new immunoglobulin-based protein, namely variable domain of new antigen receptor (VNAR) was discovered in sharks. Unlike conventional mAbs, several advantages of VNARs, include small size, better thermostability and peculiar paratope structure have attracted interest of researchers to further explore on it. This article aims to first present an overview of the shark VNARs and outline the characteristics as an outstanding new reagent for diagnostic and therapeutic applications.
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Anticorpos Monoclonais , Proteínas de Peixes , Receptores de Antígenos , Tubarões/imunologia , Anticorpos de Cadeia Única , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Proteínas de Peixes/imunologia , Proteínas de Peixes/uso terapêutico , Receptores de Antígenos/imunologia , Receptores de Antígenos/uso terapêutico , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/uso terapêuticoRESUMO
Shigella is an intracellular bacterial pathogen that causes bacterial dysentery called shigellosis. The assessment of pro- and anti-inflammatory mediators produced by immune cells against this bacteria are vital in identifying the effectiveness of the immune reaction in protecting the host. In Malaysia, Shigella is ranked as the third most common bacteria causing diarrheal disease among children below 5 years old. In the present study, we aim to examine the differential cytokine gene expressions of macrophages in response to two types of clinical strains of Shigella flexneri 2a (S. flexneri 2a) isolated from patients admitted in Hospital Universiti Sains Malaysia, Kelantan, Malaysia. THP-1-derived macrophages, as the model of human macrophages, were infected separately with S. flexneri 2a mild (SH062) and virulence (SH057) strains for 6, 12, and 24 h, respectively. The gene expression level of inflammatory mediators was identified using real-time quantitative polymerase chain reaction (RT-qPCR). The production of nitric oxide (NO) by the macrophages was measured by using a commercialized NO assay kit. The ability of macrophages to kill the intracellular bacteria was assessed by intracellular killing assay. Induction of tumor necrosis factor-alpha (TNFα), interleukin (IL)-1ß, IL-6, IL-12, inducible NO synthase (iNOS), and NO, confirmed the pro-inflammatory reaction of the THP-1-derived macrophages in response to S. flexneri 2a, especially against the SH507 strain. The SH057 also induced a marked increase in the expression levels of the anti-inflammatory cytokine mRNAs at 12 h and 24 h post-infection. In the intracellular killing assay, both strains showed less viable, indicating the generation of pro-inflammatory cytokines in the presence of iNOS and NO was crucial in the stimulation of macrophages for the host defense against shigellosis. Transcription analysis of THP-1-derived macrophages in this study identifies differentially expressed cytokine genes that correlated with the virulence factor of S. flexneri 2a.
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Citocinas/genética , Citocinas/metabolismo , Disenteria Bacilar/genética , Disenteria Bacilar/fisiopatologia , Macrófagos/microbiologia , Shigella flexneri/genética , Fatores de Virulência/genética , Virulência/genética , Animais , Pré-Escolar , Modelos Animais de Doenças , Disenteria Bacilar/epidemiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Variação Genética , Genótipo , Humanos , Lactente , Recém-Nascido , Malásia/epidemiologia , Masculino , Shigella flexneri/patogenicidadeRESUMO
Shigellosis is one of the most common diseases found in the developing countries, especially those countries that are prone flood. The causative agent for this disease is the Shigella species. This organism is one of the third most common enteropathogens responsible for childhood diarrhea. Since Shigella can survive gastric acidity and is an intracellular pathogen, it becomes difficult to treat. Also, uncontrolled use of antibiotics has led to development of resistant strains which poses a threat to public health. Therefore, there is a need for long term control of Shigella infection which can be achieved by designing a proper and effective vaccine. In this study, emphasis was made on designing a candidate that could elicit both B-cell and T-cell immune response. Hence B- and T-cell epitopes of outer membrane channel protein (OM) and putative lipoprotein (PL) from S. flexneri 2a were computationally predicted using immunoinformatics approach and a chimeric construct (chimeric-OP) containing the immunogenic epitopes selected from OM and PL was designed, cloned and expressed in E. coli system. The immunogenicity of the recombinant chimeric-OP was assessed using Shigella antigen infected rabbit antibody. The result showed that the chimeric-OP was a synthetic peptide candidate suitable for the development of vaccine and immunodiagnostics against Shigella infection.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Engenharia de Proteínas , Shigella flexneri/imunologia , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/química , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteínas Recombinantes de Fusão , Shigella flexneri/genética , Relação Estrutura-AtividadeRESUMO
PURPOSE: In the developing world, bacillary dysentery is one of the most common communicable diarrheal infections. There are approximately 169 million cases of shigellosis reported worldwide. The disease is transmitted by a group of Gram-negative intracellular enterobacteria known as Shigella flexneri, S. sonnei, S. dysenteriae, and S. boydii. Conventional treatment regimens for Shigella have been less effective due to the development of resistant strains against antibiotics. Therefore, an effective vaccine for the long term control of Shigella transmission is urgently needed. MATERIALS AND METHODS: In this study, a reverse vaccinology approach was employed to identify most conserved and immunogenic outer membrane proteins (OMPs) of S. flexneri 2a. RESULTS: Five OMPs including fepA, ompC, nlpD_1, tolC, and nlpD_2 were identified as potential vaccine candidates. Protein-protein interactions analysis using STRING software (https://string-db.org/) revealed that five of these OMPs may potentially interact with other intracellular proteins which are involved in beta-lactam resistance pathway. B- and T-cell epitopes of the selected OMPs were predicted using BCPred as well as Propred I and Propred (http://crdd.osdd.net/raghava/propred/), respectively. Each of these OMPs contains regions which are capable to induce B- and T-cell immune responses. CONCLUSION: Analysis acquired from this study showed that five selected OMPs have great potential for vaccine development against S. flexneri infection. The predicted immunogenic epitopes can also be used for development of peptide vaccines or multi-epitope vaccines against human shigellosis. Reverse vaccinology is a promising strategy for the discovery of potential vaccine candidates which can be used for future vaccine development against global persistent infections.