RESUMO
Outer Membrane Vesicles (OMV) constitute a promising platform for the development of efficient vaccines. OMV can be decorated with heterologous antigens (proteins or polysaccharides), becoming attractive novel carriers for the development of multicomponent vaccines. Chemical conjugation represents a tool for linking antigens, also from phylogenetically distant pathogens, to OMV. Here we develop two simple and widely applicable conjugation chemistries targeting proteins or lipopolysaccharides on the surface of Generalized Modules for Membrane Antigens (GMMA), OMV spontaneously released from Gram-negative bacteria mutated to increase vesicle yield and reduce potential reactogenicity. A Design of Experiment approach was used to identify optimal conditions for GMMA activation before conjugation, resulting in consistent processes and ensuring conjugation efficiency. Conjugates produced by both chemistries induced strong humoral response against the heterologous antigen and GMMA. Additionally, the use of the two orthogonal chemistries allowed to control the linkage of two different antigens on the same GMMA particle. This work supports the further advancement of this novel platform with great potential for the design of effective vaccines.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Vesículas Extracelulares/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Vacinas Bacterianas/biossíntese , Feminino , Lipopolissacarídeos/imunologia , Camundongos , Neisseria meningitidis/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/química , Vacinas Protozoárias/biossíntese , Salmonella typhimurium/imunologia , Shigella sonnei/imunologiaRESUMO
Protein Glycan Coupling Technology (PGCT) uses purposely modified bacterial cells to produce recombinant glycoconjugate vaccines. This vaccine platform holds great potential in this context, namely due to its modular nature, the simplified production process in comparison to traditional chemical conjugation methods, and its amenability to scaled-up operations. As a result, a considerable reduction in production time and cost is expected, making PGCT-made vaccines a suitable vaccine technology for low-middle income countries, where vaccine coverage remains predominantly low and inconsistent. This work aims to develop an integrated whole-process automated platform for the screening of PGCT-made glycoconjugate vaccine candidates. The successful translation of a bench scale process for glycoconjugate production to a microscale automated setting was achieved. This was integrated with a numerical computational software that allowed hands-free operation and a platform adaptable to biological variation over the course of a production process. Platform robustness was proven with both technical and biological replicates and subsequently the platform was used to screen for the most favourable conditions for production of a pneumococcal serotype 4 vaccine candidate. This work establishes an effective automated platform that enabled the identification of the most suitable E. coli strain and genetic constructs to be used in ongoing early phase research and be further brought into preclinical trials.
Assuntos
ADP Ribose Transferases/metabolismo , Automação/métodos , Toxinas Bacterianas/metabolismo , Biotecnologia/métodos , Escherichia coli/metabolismo , Exotoxinas/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Polissacarídeos Bacterianos/metabolismo , Vacinas Conjugadas/biossíntese , Fatores de Virulência/metabolismo , Vacinas Bacterianas/biossíntese , Glicosilação , Humanos , Vacinas Pneumocócicas/biossíntese , Tecnologia Farmacêutica/métodos , Exotoxina A de Pseudomonas aeruginosaRESUMO
Carbohydrate-binding antibodies play diverse and critical roles in human health. Endogenous carbohydrate-binding antibodies that recognize bacterial, fungal, and other microbial carbohydrates prevent systemic infections and help maintain microbiome homeostasis. Anti-glycan antibodies can have both beneficial and detrimental effects. For example, alloantibodies to ABO blood group carbohydrates can help reduce the spread of some infectious diseases, but they also impose limitations for blood transfusions. Antibodies that recognize self-glycans can contribute to autoimmune diseases, such as Guillain-Barre syndrome. In addition to endogenous antibodies that arise through natural processes, a variety of vaccines induce anti-glycan antibodies as a primary mechanism of protection. Some examples of approved carbohydrate-based vaccines that have had a major impact on human health are against pneumococcus, Haemophilus influeanza type b, and Neisseria meningitidis. Monoclonal antibodies specifically targeting pathogen associated or tumor associated carbohydrate antigens (TACAs) are used clinically for both diagnostic and therapeutic purposes. This review aims to highlight some of the well-studied and critically important applications of anti-carbohydrate antibodies.
Assuntos
Síndrome de Guillain-Barré/imunologia , Infecções por Haemophilus/imunologia , Meningite Meningocócica/imunologia , Pneumonia Pneumocócica/imunologia , Polissacarídeos/imunologia , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/uso terapêutico , Autoanticorpos/biossíntese , Autoanticorpos/sangue , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/uso terapêutico , Sequência de Carboidratos , Síndrome de Guillain-Barré/patologia , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/prevenção & controle , Vacinas Anti-Haemophilus/biossíntese , Vacinas Anti-Haemophilus/uso terapêutico , Haemophilus influenzae/imunologia , Humanos , Meningite Meningocócica/microbiologia , Meningite Meningocócica/prevenção & controle , Neisseria meningitidis/imunologia , Vacinas Pneumocócicas/biossíntese , Vacinas Pneumocócicas/uso terapêutico , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/prevenção & controle , Polissacarídeos/antagonistas & inibidores , Polissacarídeos/química , Streptococcus pneumoniae/imunologiaRESUMO
In the present study, the importance of sodium bicarbonate antacid as an agent for an orally delivered attenuated Salmonella strain secreting Brucella antigens Cu-Zn superoxide dismutase (SodC) and outer membrane protein 19 (Omp19) as a live vaccine candidate against Brucella infection was investigated. First, Brucella antigens SodC and Omp19 were cloned into a prokaryotic constitutive expression vector, pJHL65. Then secretion of proteins was verified after transformation into an attenuated Salmonella typhimurium (ST) strain, JOL1800 (Δlon, ΔcpxR, Δasd, ΔrfaL), using western blot analysis. Mice were orally inoculated with phosphate-buffered saline (PBS) or with a co-mixture Salmonella secreting each antigens at a 1:1 ratio, each containing 1â¯×â¯108â¯CFU/mouse with and without sodium bicarbonate treatment. For antacid treatment, 1.3% w/v sodium bicarbonate was orally administered 30â¯min before and immediately after immunization with the Salmonella formulation. Humoral and cell-mediated immune responses were evaluated to investigate the efficacy of sodium bicarbonate in an oral formulation. The results indicated that addition of sodium bicarbonate to the vaccine significantly increased (Pâ¯<â¯0.05) levels of anti-Brucella-specific systemic IgG responses, lymphocyte proliferation, and CD4+ T cell responses, indicating induction of a mixed Th1-Th2 response. Immunohistochemical assays and bacterial enumeration in intestinal samples also indicated that administration of sodium bicarbonate enhanced colonization of Salmonella. These results indicate that ingestion of the Salmonella formulation with sodium bicarbonate can enhance colonization of Salmonella and induce a significant protective immune response against Brucella compared with a formulation without sodium bicarbonate. Thus, incorporation of sodium bicarbonate as an antacid buffer is highly recommended for this oral live vaccine.
Assuntos
Vacina contra Brucelose , Bicarbonato de Sódio , Vacinas Atenuadas , Administração Oral , Animais , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/química , Vacina contra Brucelose/administração & dosagem , Vacina contra Brucelose/biossíntese , Vacina contra Brucelose/química , Imunidade Celular , Imunidade Humoral , Intestinos/imunologia , Intestinos/microbiologia , Camundongos , Microrganismos Geneticamente Modificados , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Bicarbonato de Sódio/administração & dosagem , Transformação Bacteriana , Vacinação/métodos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/biossíntese , Vacinas Atenuadas/químicaRESUMO
OBJECTIVES.: To design and assess a multiepitopic protein as a candidate for a vaccine against Carrion disease. MATERIALS AND METHODS.: Using bioinformatics tools, epitopes of external membrane proteins were selected and a multiepitopic protein was designed. The multiepitopic protein gene was subcloned into the expression plasmid pET28b and transformed into E. coli BL21 pLys. The multiepitopic protein was expressed using isopropyl-ß-D-1-thiogalactopyranoside and purified using resin. This purified protein was used to immunize BALB/c mice obtaining polyclonal antibodies. In vitro invasion assays were conducted using a strain of Bartonella bacilliformis (B. bacilliformis) in human red blood cells. RESULTS.: The multiepitopic protein M1 presents preserved epitopes between isolates of B. bacilliformis with are non-toxic, and not homologous to human and surface proteins. Immunized mice presented IgG antibody levels capable of reducing in vitro the rate of invasion of B. bacilliformis into human red blood cells. CONCLUSIONS.: Multiepitopic protein M1 may serve as a candidate for a Carrion disease vaccine; however, more studies are needed to characterize the use of this antigen as a vaccine.
OBJETIVOS.: Diseñar y evaluar una proteína multiepítope como candidato a vacuna contra la enfermedad de Carrión. MATERIALES Y MÉTODOS.: Mediante herramientas bioinformáticas se seleccionó epítopes de proteínas de membrana externa y se diseñó una proteína multiepítope. El gen de la proteína multiepítope fue subclonado en el plásmido de expresión pET28b y transformado en E. coli BL21 pLys. La proteína multiepítope fue expresada usando isopropil-ß-D-1-tiogalactopiranósido y purificada usando resina. Esta proteína purificada fue utilizada para inmunizar ratones BALB/c y se obtuvo anticuerpos policlonales. Se realizaron ensayos de invasión in vitro usando una cepa de Bartonella bacilliformis (B. bacilliformis) a eritrocitos humanos. RESULTADOS.: La proteína multiepítope M1 presenta epítopes conservados entre aislamientos de B. bacilliformis, no tóxicos, no homólogos a proteínas humanas y superficiales. Los ratones inmunizados presentaron niveles de anticuerpos IgG capaces de reducir in vitro la tasa de invasión de B. bacilliformis a eritrocitos humanos. CONCLUSIONES.: La proteína multiepítope M1 podría servir como candidato a vacuna contra la enfermedad de Carrión; sin embargo, se requiere de más estudios para caracterizar el uso de este antígeno como vacuna.
Assuntos
Proteínas de Bactérias/biossíntese , Vacinas Bacterianas/biossíntese , Infecções por Bartonella/prevenção & controle , Desenho de Fármacos , Animais , Biologia Computacional , Epitopos , Feminino , Camundongos Endogâmicos BALB CAssuntos
Doenças Autoimunes/prevenção & controle , Controle de Doenças Transmissíveis/métodos , Hipersensibilidade/prevenção & controle , Imunoterapia/métodos , Neoplasias/prevenção & controle , Pesquisa Translacional Biomédica/tendências , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/uso terapêutico , Antígenos/química , Antígenos/genética , Antígenos/imunologia , Doenças Autoimunes/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/biossíntese , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/biossíntese , Doenças Transmissíveis/imunologia , Doenças Transmissíveis/patologia , Citocinas/agonistas , Citocinas/antagonistas & inibidores , Citocinas/genética , Citocinas/imunologia , Vacinas Fúngicas/administração & dosagem , Vacinas Fúngicas/biossíntese , Humanos , Hipersensibilidade/imunologia , Imunoensaio/métodos , Neoplasias/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/biossínteseRESUMO
RESUMEN Objetivos. Diseñar y evaluar una proteína multiepítope como candidato a vacuna contra la enfermedad de Carrión. Materiales y métodos. Mediante herramientas bioinformáticas se seleccionó epítopes de proteínas de membrana externa y se diseñó una proteína multiepítope. El gen de la proteína multiepítope fue subclonado en el plásmido de expresión pET28b y transformado en E. coli BL21 pLys. La proteína multiepítope fue expresada usando isopropil-β-D-1-tiogalactopiranósido y purificada usando resina. Esta proteína purificada fue utilizada para inmunizar ratones BALB/c y se obtuvo anticuerpos policlonales. Se realizaron ensayos de invasión in vitro usando una cepa de Bartonella bacilliformis (B. bacilliformis) a eritrocitos humanos. Resultados. La proteína multiepítope M1 presenta epítopes conservados entre aislamientos de B. bacilliformis, no tóxicos, no homólogos a proteínas humanas y superficiales. Los ratones inmunizados presentaron niveles de anticuerpos IgG capaces de reducir in vitro la tasa de invasión de B. bacilliformis a eritrocitos humanos. Conclusiones. La proteína multiepítope M1 podría servir como candidato a vacuna contra la enfermedad de Carrión; sin embargo, se requiere de más estudios para caracterizar el uso de este antígeno como vacuna.
ABSTRACT Objectives. To design and assess a multiepitopic protein as a candidate for a vaccine against Carrion disease. Materials and Methods. Using bioinformatics tools, epitopes of external membrane proteins were selected and a multiepitopic protein was designed. The multiepitopic protein gene was subcloned into the expression plasmid pET28b and transformed into E. coli BL21 pLys. The multiepitopic protein was expressed using isopropyl-β-D-1-thiogalactopyranoside and purified using resin. This purified protein was used to immunize BALB/c mice obtaining polyclonal antibodies. In vitro invasion assays were conducted using a strain of Bartonella bacilliformis (B. bacilliformis) in human red blood cells. Results. The multiepitopic protein M1 presents preserved epitopes between isolates of B. bacilliformis with are non-toxic, and not homologous to human and surface proteins. Immunized mice presented IgG antibody levels capable of reducing in vitro the rate of invasion of B. bacilliformis into human red blood cells. Conclusions. Multiepitopic protein M1 may serve as a candidate for a Carrion disease vaccine; however, more studies are needed to characterize the use of this antigen as a vaccine.
Assuntos
Animais , Feminino , Proteínas de Bactérias/biossíntese , Infecções por Bartonella/prevenção & controle , Vacinas Bacterianas/biossíntese , Desenho de Fármacos , Biologia Computacional , Camundongos Endogâmicos BALB C , EpitoposRESUMO
There is a growing public health interest in controlling sexually transmitted infections (STIs) through vaccination due to increasing recognition of the global disease burden of STIs and the role of STIs in women's reproductive health, adverse pregnancy outcomes, and the health and well-being of neonates. Neisseria gonorrhoeae has historically challenged vaccine development through the expression of phase and antigenically variable surface molecules and its capacity to cause repeated infections without inducing protective immunity. An estimated 78 million new N. gonorrhoeae infections occur annually and the greatest disease burden is carried by low- and middle-income countries (LMIC). Current control measures are clearly inadequate and threatened by the rapid emergence of antibiotic resistance. The gonococcus now holds the status of "super-bug" as there is currently no single reliable monotherapy for empirical treatment of gonorrhea. The problem of antibiotic resistance has elevated treatment costs and necessitated the establishment of large surveillance programs to track the spread of resistant strains. Here we review the need for a gonorrhea vaccine with respect to global disease burden and related socioeconomic and treatment costs, with an emphasis on the impact of gonorrhea on women and newborns. We also highlight the challenge of estimating the impact of a gonorrhea vaccine due to the need for more data on the burden of gonococcal pelvic inflammatory disease and related sequelae and of gonorrhea-associated adverse pregnancy outcomes and the problem of empirical diagnosis and treatment of STIs in LMIC. There is also a lack of clinical and basic science research in the area of gonococcal/chlamydia coinfection, which occurs in a high percentage of individuals with gonorrhea and should be considered when testing the efficacy of gonorrhea vaccines. Finally, we review recent research that suggests a gonorrhea vaccine is feasible and discuss challenges and research gaps in gonorrhea vaccine development.
Assuntos
Vacinas Bacterianas/biossíntese , Infecções por Chlamydia/prevenção & controle , Gonorreia/prevenção & controle , Doença Inflamatória Pélvica/prevenção & controle , Complicações Infecciosas na Gravidez/prevenção & controle , Antibacterianos/farmacologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/economia , Chlamydia/efeitos dos fármacos , Chlamydia/imunologia , Chlamydia/patogenicidade , Infecções por Chlamydia/epidemiologia , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Coinfecção , Farmacorresistência Bacteriana Múltipla , Feminino , Gonorreia/epidemiologia , Gonorreia/imunologia , Gonorreia/microbiologia , Humanos , Masculino , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/imunologia , Neisseria gonorrhoeae/patogenicidade , Doença Inflamatória Pélvica/epidemiologia , Doença Inflamatória Pélvica/imunologia , Doença Inflamatória Pélvica/microbiologia , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/microbiologia , Saúde Pública/economia , Saúde Pública/estatística & dados numéricos , Fatores SocioeconômicosRESUMO
Gastric adenocarcinoma is globally the third leading cause of death due to malignancy, with the bulk of this disease burden being suffered by low and middle income countries (LMIC), especially in Asia. The majority of these cancers develop as a result of a chronic gastritis that arises in response to infection with the stomach-dwelling bacterium, Helicobacter pylori. A vaccine against this pathogen would therefore be a powerful tool for preventing gastric adenocarcinoma. However, notwithstanding a proof-of-concept that vaccination can protect children from acquisition of H. pylori infection, there are currently no advanced vaccine candidates with only a single vaccine in Phase I clinical trial. Further, the development of a vaccine against H. pylori is not a current strategic priority of major pharmaceutical companies despite the large global disease burden. Given the involvement of such companies is likely to be critical for late stage development, there is therefore a need for an increased appreciation of the burden of this disease in LMIC and more investment to reinvigorate research in H. pylori vaccine Research and Development.
Assuntos
Adenocarcinoma/prevenção & controle , Vacinas Bacterianas/biossíntese , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/efeitos dos fármacos , Neoplasias Gástricas/prevenção & controle , Adenocarcinoma/etiologia , Adenocarcinoma/imunologia , Adenocarcinoma/microbiologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/genética , Pesquisa Biomédica/organização & administração , Criança , Ensaios Clínicos como Assunto , Países em Desenvolvimento/economia , Modelos Animais de Doenças , Indústria Farmacêutica/tendências , Infecções por Helicobacter/complicações , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/imunologia , Helicobacter pylori/patogenicidade , Humanos , Macaca mulatta , Camundongos , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/microbiologia , Vacinação/métodos , Vacinas de Subunidades AntigênicasRESUMO
According to the 2015 Global Burden of Disease Study, diarrhea ranked ninth among causes of death for all ages, and fourth among children under 5 years old, accounting for an estimated 499,000 deaths in this young age group. It was also the second most common cause of years lived with disability (2.39â¯billion YLDs). The goal of the WHO/UNICEF Integrated Global Action Plan for the Prevention and Control of Pneumonia and Diarrhea (GAPPD) is to reduce deaths from diarrhea in children under 5 years of age to less than 1 per 1000 live births, by 2025. Development of new and improved vaccines against diarrheal infections is a fundamental element of the strategy towards achieving this goal. Enterotoxigenic Escherichia coli (ETEC) and Shigella are enteropathogens that cause significant global mortality and morbidity, particularly in low- and middle-income countries. In 2016, WHO's Product Development for Vaccines Advisory Committee (PDVAC) recommended that the WHO's Initiative for Vaccine Research (IVR) engage in this area, based on PDVAC's criteria of prioritizing the development of vaccines against pathogens that will address a major unmet public health need, and for which clinical candidates with a good probability of technical success are in the pipeline. As a first step, WHO's IVR convened global subject matter experts to discuss the current global ETEC and Shigella disease burden estimates, including the current understanding of the long-term indirect effects of ETEC and Shigella infection, and how these data may affect future decision making on vaccine development for both pathogens. The available global burden estimates for ETEC and Shigella differ with respect to the relative importance of these two pathogens. The mortality estimates vary between iterations published by the same group, as well as between estimates of different groups, although the uncertainty intervals are broad and overlapping. These variances are attributable to differences in the data available and incorporated in the models; the methods used to detect the pathogens; the modelling methodologies; and, to actual changes in the total number of diarrheal deaths over time. The changes in the most recently reported mortality estimates for these pathogens, as compared to previous iterations, has led to debate as to whether investment in development of stand-alone vaccines, rather than combined vaccines, is warranted from cost-effectiveness and vaccine impact perspectives. Further work will be needed to understand better the variances and uncertainties in the reported mortality estimates to support investment decision making, and ultimately policy recommendations for vaccine use. In addition, a comprehensive assessment of the value proposition for vaccines against these pathogens is needed and will be strengthened if the long-term health consequences associated with diarrhea and dysentery due to these pathogens are better defined.
Assuntos
Diarreia/epidemiologia , Disenteria Bacilar/epidemiologia , Disenteria/epidemiologia , Escherichia coli Enterotoxigênica/patogenicidade , Infecções por Escherichia coli/epidemiologia , Shigella/patogenicidade , Vacinas Bacterianas/biossíntese , Pesquisa Biomédica/organização & administração , Ensaios Clínicos como Assunto , Congressos como Assunto , Diarreia/imunologia , Diarreia/microbiologia , Diarreia/prevenção & controle , Avaliação Pré-Clínica de Medicamentos , Disenteria/imunologia , Disenteria/microbiologia , Disenteria/prevenção & controle , Disenteria Bacilar/imunologia , Disenteria Bacilar/microbiologia , Disenteria Bacilar/prevenção & controle , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Humanos , Relatório de Pesquisa , Shigella/imunologia , Organização Mundial da SaúdeRESUMO
BACKGROUND: The sexual stages of Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas. The cysteine-rich Pfs48/45 protein, exposed on the surface of sexual stages, is one of the most advanced antigens for inclusion into a vaccine that will block transmission. However, clinical Pfs48/45 sub-unit vaccine development has been hampered by the inability to produce high yields of recombinant protein as the native structure is required for the induction of functional transmission-blocking (TB) antibodies. We have investigated a downstream purification process of a sub-unit (R0.6C) fragment representing the C-terminal 6-Cys domain of Pfs48/45 (6C) genetically fused to the R0 region (R0) of asexual stage Glutamate Rich Protein expressed in Lactococcus lactis. RESULTS: A series of R0.6C fusion proteins containing features, which aim to increase expression levels or to facilitate protein purification, were evaluated at small scale. None of these modifications affected the overall yield of recombinant protein. Consequently, R0.6C with a C-terminal his tag was used for upstream and downstream process development. A simple work-flow was developed consisting of batch fermentation followed by two purification steps. As such, the recombinant protein was purified to homogeneity. The composition of the final product was verified by HPLC, mass spectrometry, SDS-PAGE and Western blotting with conformation dependent antibodies against Pfs48/45. The recombinant protein induced high levels of functional TB antibodies in rats. CONCLUSIONS: The established production and purification process of the R0.6C fusion protein provide a strong basis for further clinical development of this candidate transmission blocking malaria vaccine.
Assuntos
Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/imunologia , Imunogenicidade da Vacina/imunologia , Lactococcus lactis/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Animais , Vacinas Bacterianas/isolamento & purificação , Reatores Biológicos , Lactococcus lactis/genética , Plasmodium falciparum/química , Plasmodium falciparum/imunologia , Subunidades Proteicas/biossíntese , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Ratos , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Clostridium difficile is a gram-positive intestine bacterium that causes a severe diarrhea and could eventually be lethal. The main virulence factor is related to the release of two major exotoxins, toxin A (TcdA) and toxin B (TcdB). Recent C. difficile-associated disease (CDAD) outbreaks have been caused by hypervirulent strains which secrete an additional binary toxin (CDTa/CDTb). Vaccination against these toxins is considered the best way to combat the CDAD. Recently, a novel tetravalent C. difficile vaccine candidate containing all four toxins produced from a baculovirus expression system has been developed. A dose assay to release this tetravalent C. difficile vaccine was developed using tandem ion-exchange HPLC chromatography. A sequential weak cation exchange (carboxyl group) and weak anion exchange (tertiary amine group) columns were employed. The four C. difficile vaccine antigen pIs range from 4.4 to 8.6. The final optimized separation employs salt gradient elution at two different pHs. The standard analytical parameters such as LOD, LOQ, linearity, accuracy, precision and repeatability were evaluated for this method and it was deemed acceptable as a quantitative assay for vaccine release. Furthermore, the developed method was utilized for monitoring the stability of the tetravalent C. difficile vaccine in final container.
Assuntos
Vacinas Bacterianas/análise , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/isolamento & purificação , Baculoviridae/genética , Clostridioides difficile/metabolismo , Enterocolite Pseudomembranosa/prevenção & controle , Enterotoxinas/genética , Enterotoxinas/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Current investigations have demonstrated that a multi-epitope peptide vaccine targeting multiple antigens could be considered as an ideal approach for prevention and treatment of brucellosis. According to the latest findings, the most effective immunogenic antigens of brucella to induce immune responses are included Omp31, BP26, BLS, DnaK and L7-L12. Therefore, in the present study, an in silico approach was used to design a novel multi-epitope vaccine to elicit a desirable immune response against brucellosis. First, five novel T-cell epitopes were selected from Omp31, BP26, BLS, DnaK and L7-L12 proteins using different servers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Selected epitopes were fused together by GPGPG linkers to facilitate the immune processing and epitope presentation. Moreover, cholera toxin B (CTB) was linked to N terminal of vaccine construct as an adjuvant by using EAAAK linker. A multi-epitope vaccine was designed based on predicted epitopes which was 377 amino acid residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this multi-epitope vaccine were assessed using immunoinformatics tools and servers. Based on obtained results, a soluble, and non-allergic protein with 40.59kDa molecular weight was constructed. Expasy ProtParam classified this chimeric protein as a stable protein and also 89.8% residues of constructed vaccine were located in favored regions of the Ramachandran plot. Furthermore, this multi-epitope peptide vaccine was able to strongly induce T cell and B-cell mediated immune responses. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide vaccine can be effectively expressed and potentially be used for prophylactic or therapeutic usages against brucellosis.
Assuntos
Antígenos de Bactérias/química , Vacinas Bacterianas/biossíntese , Brucella/imunologia , Brucelose/prevenção & controle , Proteínas Recombinantes de Fusão/química , Acetiltransferases/genética , Acetiltransferases/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/farmacologia , Brucella/química , Brucella/genética , Brucelose/imunologia , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Biologia Computacional , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/imunologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vacinas de Subunidades AntigênicasRESUMO
Helicobacter pylori have colonized the gastric mucosa of half of the population worldwide. This bacterium is classified as a definitive type I carcinogen by the World Health Organization and no effective vaccine has been found against it yet. Thus, a logical and rational vaccine design against H. pylori is necessary. Because of its tremendous complexity and elicited immune responses, the vaccine design should considered multiple antigens to enhance immune-protection, involved in the different stages of pathogenesis besides inducing a specific immune response by B- and T-cell multi-epitopes. In this study, emphasis was placed on the design of a new unique vaccine named CTB-multiHp. In silico techniques were used to design a chimeric construct consisting of cholera toxin B subunit fused to multi-epitope of urease B (residue 148-158, 188-198), cytotoxin-associated gene A (residue 584-602), neutrophil activating protein (residue 4-28), vacuolating cytotoxin gene A (residue 63-81), H. pylori adhesine A (residue77-99), heat shock protein A (residue 32-54) and gamma glutamyl transpeptidase (residue 271-293). The tertiary structure and features of the vaccine were analyzed. The chimeric protein was expressed in Escherichia coli BL21 and the serology analyses indicated that the CTB-multiHp protein produced exhibit immune-reactivity. The results showed that CTB-multiHp could be a good vaccine candidate against H. pylori. Ongoing studies will evaluate the effects of CTB-multiHp against H. pylori infection.
Assuntos
Vacinas Bacterianas/imunologia , Epitopos/imunologia , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/imunologia , Proteínas Recombinantes de Fusão/química , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/genética , Toxina da Cólera/química , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Clonagem Molecular , Desenho de Fármacos , Epitopos/química , Epitopos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Humanos , Interleucina-8/química , Interleucina-8/genética , Interleucina-8/imunologia , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Urease/química , Urease/genética , Urease/imunologia , gama-Glutamiltransferase/química , gama-Glutamiltransferase/genética , gama-Glutamiltransferase/imunologiaRESUMO
Clostridium botulinum group III strains are able to produce cytotoxins, C2 toxin and C3 exotoxin, along with botulinum neurotoxin types C and D. C2 toxin and C3 exotoxin produced by this organism are the most important members of bacterial ADP-ribosyltransferase superfamily. Both toxins have distinct pathophysiological functions in the avian and mammalian hosts. The members of this superfamily transfer an ADP-ribose moiety of NAD+ to specific eukaryotic target proteins. The present review describes the structure, function and evolution aspects of these toxins with a special emphasis to the development of veterinary vaccines. C2 toxin is a binary toxin that consists of a catalytic subunit (C2I) and a translocation subunit (C2II). C2I component is structurally and functionally similar to the VIP2 and iota A toxin whereas C2II component shows a significant homology with the protective antigen from anthrax toxin and iota B. Unlike C2 toxin, C3 toxin is devoid of translocation/binding subunit. Extensive studies on their sequence-structure-function link spawn additional efforts to understand the catalytic mechanisms and target recognition. Structural and functional relationships with them are often determined by using evolutionary constraints as valuable biological measures. Enzyme-deficient mutants derived from these toxins have been used as drug/protein delivery systems in eukaryotic cells. Thus, current knowledge on their molecular diversity is a well-known perspective to design immunotoxin or subunit vaccine for C. botulinum infection.
Assuntos
Toxinas Botulínicas/química , Botulismo/veterinária , Clostridium botulinum/patogenicidade , Evolução Molecular , Doenças das Aves Domésticas/microbiologia , Aves Domésticas/microbiologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/imunologia , Toxinas Botulínicas/genética , Toxinas Botulínicas/metabolismo , Botulismo/microbiologia , Botulismo/patologia , Botulismo/prevenção & controle , Domínio Catalítico , Clostridium botulinum/classificação , Clostridium botulinum/enzimologia , Clostridium botulinum/genética , Expressão Gênica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Filogenia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/prevenção & controle , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Ion-exchange (IEX) chromatography is one of many separation techniques that can be employed to analyze proteins. The separation mechanism is based on a reversible interaction between charged amino acids of a protein to the charged ligands attached to a column at a given pH. This interaction depends on both the pI and conformation of the protein being analyzed. The proteins are eluted by increasing the salt concentration or pH gradient. Here we describe the use of this technique to characterize the charge variant heterogeneities and to monitor stability of four protein antigen components of a Clostridium difficile vaccine. Furthermore, the IEX technique can be used to monitor reversion to toxicity for formaldehyde-treated Clostridium difficile toxins.
Assuntos
Vacinas Bacterianas/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Clostridioides difficile/imunologia , Enterocolite Pseudomembranosa/prevenção & controle , ADP Ribose Transferases/isolamento & purificação , ADP Ribose Transferases/toxicidade , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/toxicidade , Vacinas Bacterianas/biossíntese , Cromatografia Líquida de Alta Pressão , Clostridioides difficile/química , Enterocolite Pseudomembranosa/imunologia , Enterocolite Pseudomembranosa/microbiologia , Enterotoxinas/isolamento & purificação , Enterotoxinas/toxicidade , Formaldeído/química , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Cloreto de Sódio , Temperatura , Vacinas AtenuadasRESUMO
High-performance size-exclusion chromatography (HPSEC or SEC) is a method that can be applied to measure size distribution of proteins, including aggregates, monomers, and fragments. In the biopharmaceutical industry the quantitation of aggregates contained in biotherapeutics and protein-based vaccines is critical given the potential impact on safety, immunogenicity, and efficacy. Hence, aggregation analysis of therapeutic proteins or protein-based vaccine products is almost always a requirement of regulatory agencies. SEC, also referred to as gel-filtration chromatography, separates molecules by size through a porous resin stationary phase. Under isocratic flow small molecules are retained on the column longer than large molecules. Here we describe the use of this SEC technique to characterize aggregation levels for four different protein antigens for a Clostridium difficile vaccine.
Assuntos
Vacinas Bacterianas/isolamento & purificação , Cromatografia em Gel/métodos , Clostridioides difficile/imunologia , Enterocolite Pseudomembranosa/prevenção & controle , Potência de Vacina , ADP Ribose Transferases/química , ADP Ribose Transferases/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas/química , Toxinas Bacterianas/isolamento & purificação , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/genética , Baculoviridae/genética , Baculoviridae/metabolismo , Cromatografia Líquida de Alta Pressão , Clostridioides difficile/química , Enterocolite Pseudomembranosa/imunologia , Enterocolite Pseudomembranosa/microbiologia , Enterotoxinas/química , Enterotoxinas/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Formaldeído/química , Humanos , Agregados Proteicos , Vacinas Atenuadas , Vacinas SintéticasRESUMO
BACKGROUND: Chlamydia trachomatis is a human pathogen which causes a number of pathologies, including genital tract infections in women that can result in tubal infertility. Prevention of infection and disease control might be achieved through vaccination; however, a safe, efficacious and cost-effective vaccine against C. trachomatis infection remains an unmet medical need. C. trachomatis major outer membrane protein (MOMP), a ß-barrel integral outer membrane protein, is the most abundant antigen in the outer membrane of the bacterium and has been evaluated as a subunit vaccine candidate. Recombinant MOMP (rMOMP) expressed in E. coli cytoplasm forms inclusion bodies and rMOMP extracted from inclusion bodies results in a reduced level of protection compared to the native MOMP in a mouse challenge model. RESULTS: We sought to target the recombinant expression of MOMP to the E. coli outer membrane (OM). Successful surface expression was achieved with codon harmonization, utilization of low copy number vectors and promoters with moderate strength, suitable leader sequences and optimization of cell culture conditions. rMOMP was extracted from E. coli outer membrane, purified, and characterized biophysically. The OM expressed and purified rMOMP is immunogenic in mice and elicits antibodies that react to the native antigen, Chlamydia elementary body (EB). CONCLUSIONS: C. trachomatis MOMP was functionally expressed on the surface of E. coli outer membrane. The OM expressed and purified rMOMP elicits antibodies that react to the native antigen, Chlamydia EB, in a mouse immunogenicity model. Surface expression of MOMP could provide useful reagents for vaccine research, and the methodology could serve as a platform to produce other outer membrane proteins recombinantly.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Chlamydia trachomatis/genética , Escherichia coli/genética , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/biossíntese , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/química , Células Cultivadas , Infecções por Chlamydia/prevenção & controle , Clonagem Molecular , DNA Bacteriano/genética , Escherichia coli/metabolismo , Feminino , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Botulinum neurotoxin (BoNT) serotypes C and D are responsible for cattle botulism, a fatal paralytic disease that results in great economic losses in livestock production. Vaccination is the main approach to prevent cattle botulism. However, production of commercially available vaccines (toxoids) involves high risk and presents variation of BoNT production between batches. Such limitations can be attenuated by the development of novel nontoxic recombinant vaccines through a simple and reproducible process. The aim of this study was to evaluate the protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D. Bivalent vaccines containing 200 µg rHCC and rHCD each were formulated in three different ways: (1) purified antigens; (2) recombinant Escherichia coli bacterins; (3) recombinant E. coli cell lysates (supernatant and inclusion bodies). Guinea pigs immunized subcutaneously with recombinant formulations developed a protective immune response against the respective BoNTs as determined by a mouse neutralization bioassay with pooled sera. Purified recombinant antigens were capable of inducing 13 IU/mL antitoxin C and 21 IU/mL antitoxin D. Similarly, both the recombinant bacterins and the cell lysate formulations were capable of inducing 12 IU/mL antitoxin C and 20 IU/mL antitoxin D. These values are two times as high as compared to values induced by the commercial toxoid used as control, and two to ten times as high as the minimum amount required by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), respectively. Therefore, we used a practical, industry-friendly, and efficient vaccine production process that resulted in formulations capable of inducing protective immune response (neutralizing antitoxins) against botulism serotypes C and D.
Assuntos
Anticorpos Antibacterianos/sangue , Antitoxinas/sangue , Vacinas Bacterianas/administração & dosagem , Toxinas Botulínicas Tipo A/administração & dosagem , Toxinas Botulínicas/administração & dosagem , Botulismo/prevenção & controle , Animais , Anticorpos Antibacterianos/biossíntese , Antitoxinas/biossíntese , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/imunologia , Toxinas Botulínicas/biossíntese , Toxinas Botulínicas/imunologia , Toxinas Botulínicas Tipo A/biossíntese , Toxinas Botulínicas Tipo A/imunologia , Botulismo/sangue , Botulismo/imunologia , Clostridium botulinum/efeitos dos fármacos , Clostridium botulinum/genética , Clostridium botulinum/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cobaias , Imunidade Humoral/efeitos dos fármacos , Camundongos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Vacinação , Vacinas SintéticasRESUMO
Clostridium botulinum is a Gram-positive, spore-forming, anaerobic bacillus that produces a potent neurotoxin. Botulinum neurotoxins (BoNTs) are classified from serotypes A to H, and even though they have similar mechanisms of action, they show preferential hosts. In veterinary medicine, BoNT serotypes C and D are the most important, once several animal species are susceptible to them. Since BoNTs are the most potent toxins known in nature, the best way to control botulism in animals is through vaccination. However, current commercial vaccines are based on inactivated toxins (toxoids) and cells (bacterins) and present many drawbacks, such as a time-consuming production with variable antigen yield and biosafety risks. Recombinant vaccines, especially those produced by Escherichia coli expression system, have proved to be an interesting alternative to overcome these problems. E. coli is a very well-known microorganism that allows the production of large amounts of nontoxic recombinant antigens in a short period using simple culture medium reducing the production complexity and decreasing most of the biosafety risks involved in the process. We describe herein a method for the production of recombinant vaccines for veterinary medicine application, involving initial steps of gene design up to vaccine formulation and evaluation itself.