RESUMO
Tetanus toxoid (TTxd), developed over 100 years ago, is a clinically effective, legacy vaccine against tetanus. Due to the extreme potency of native tetanus toxin, manufacturing and regulatory efforts often focus on TTxd production, standardization, and safety, rather than product modernization. Recently, a genetically detoxified, full-length tetanus toxin protein (8MTT) was reported as a tetanus vaccine alternative to TTxd (Przedpelski et al. mBio, 2020). Here we describe the production of 8MTT in Gor/MetTM E. coli, a strain engineered to have an oxidative cytoplasm, allowing for the expression of soluble, disulfide-bonded proteins. The strain was also designed to efficiently cleave N-terminal methionine, the obligatory start amino acid for E. coli expressed proteins. 8MTT was purified as a soluble protein from the cytoplasm in a two-column protocol to > 99 % purity, yielding 0.5 g of purified 8MTT/liter of fermentation broth with low endotoxin contamination, and antigenic purity of 3500 Lf/mg protein nitrogen. Mouse immunizations showed 8MTT to be an immunogenic vaccine and effective as a carrier protein for peptide and polysaccharide conjugates. These studies validate 8MTT as commercially viable and, unlike the heterogenous tetanus toxoid, a uniform carrier protein for conjugate vaccines. The development of a recombinant, genetically detoxified toxin produced in E. coli aligns the tetanus vaccine with modern manufacturing, regulatory, standardization, and safety requirements.
Assuntos
Toxina Tetânica , Tétano , Animais , Anticorpos Antibacterianos , Proteínas de Transporte , Escherichia coli/metabolismo , Camundongos , Tétano/prevenção & controle , Toxina Tetânica/efeitos adversos , Toxina Tetânica/genética , Toxoide Tetânico/efeitos adversos , Toxoide Tetânico/genética , Vacinas ConjugadasRESUMO
Lyme disease (LD) accounts for over 70% of tick-borne disease reported in the United States. The disease in humans is characterized by skin rash, arthritis, cardiac and neurological signs. Vaccination is the most efficient preventive measure that could be taken to reduce the incidence of the LD worldwide; however, at present no vaccine is available. In this study, evaluation of the Borrelia burgdorferi BB0172-derived peptide (PepB) in conjugated formulations was investigated as a vaccine candidate in murine model of LD. In brief, PepB was conjugated to the Cross-Reacting Material 197 (CRM197) and to Tetanus Toxoid heavy chain (TTHc) molecules, and subsequently used to immunize C3H/HeN mice. Following the challenge with 105 spirochetes/mouse via subcutaneous inoculation, TTHc:PepB construct showed protection in 66% of the immunized animals. Hence, to further evaluate the efficacy of TTHc:PepB, immunized mice were challenged with B. burgdorferi using the tick model of infection. The outcome of this experiment revealed that serum from TTHc:PepB immunized mice was borrelicidal. After tick infection, bacterial burden was significantly reduced (over 70%) in vaccinated animals when compared with the control groups regardless of whether the mice were infested 8 or 12-weeks post-priming. Therefore, we conclude that PepB conjugated antigens can serve as an alternative to prevent LD; nevertheless, further studies will be needed to dissect the mechanisms by which anti-PepB IgG antibodies are able to kill B. burgdorferi in vitro and in vivo to further advance in the development of formulations and delivery alternative to generate a safe anti-LD vaccine.
Assuntos
Proteínas de Bactérias/imunologia , Borrelia burgdorferi/imunologia , Vacinas contra Doença de Lyme/imunologia , Doença de Lyme/prevenção & controle , Vacinas Conjugadas/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imunização , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Vacinas contra Doença de Lyme/administração & dosagem , Camundongos , Peptídeos/imunologia , Carrapatos/microbiologia , Vacinas Conjugadas/administração & dosagem , Vacinas de Subunidades Antigênicas/administração & dosagemRESUMO
Klebsiella pneumoniae (KP) and Pseudomonas aeruginosa (PA) are important human pathogens that are associated with a range of infection types, including wound and disseminated infections. Treatment has been complicated by rising rates of antimicrobial resistance. Immunoprophylactic strategies are not constrained by antimicrobial resistance mechanisms. Vaccines against these organisms would be important public health tools, yet they are not available. KP surface O polysaccharides (OPS) are protective antigens in animal models of infection. Similarly, PA flagellin (Fla), the major subunit of the flagellar filament, is required for virulence and is a target of protective antibodies in animal models. We report herein the development of a combined KP and PA glycoconjugate vaccine comprised of the four most common KP OPS types associated with human infections (O1, O2, O3, O5), chemically linked to the two Fla types of PA (FlaA, FlaB). Conjugation of KP OPS to PA Fla enhanced anti-polysaccharide immune responses and produced a formulation that generated antibody titers to the four KP OPS types and both PA Fla antigens in rabbits. Passive transfer of vaccine-induced rabbit antisera reduced the bacterial burden and protected mice against fatal intravenous KP infection. Mice passively transferred with conjugate-induced antisera were also protected against PA infection after thermal injury with a FlaB-expressing isolate, but not a FlaA isolate. Taken together, these promising preclinical results provide important proof-of-concept for a broad spectrum human vaccine to prevent KP and PA infections.
Assuntos
Vacinas Bacterianas , Infecções por Klebsiella/prevenção & controle , Infecções por Pseudomonas/prevenção & controle , Infecção dos Ferimentos/prevenção & controle , Animais , Anticorpos Antibacterianos/metabolismo , Proteínas de Bactérias/imunologia , Feminino , Glicoconjugados/imunologia , Humanos , Imunidade Humoral , Imunização , Klebsiella pneumoniae/imunologia , Camundongos , Estudo de Prova de Conceito , Pseudomonas aeruginosa/imunologia , CoelhosRESUMO
Surface adhered bacterial colonies or biofilms are an important problem in medical and food industries. Bacteria use a chemical language to monitor their quorum and to express virulence factors, which eventually help them in colonization and manifestation of an infection. The LasR-LasI and RhlR-RhlI quorum-sensing (QS) systems of Pseudomonas aeruginosa control expression of virulence factors in a population density-dependent fashion. In this study we investigated the role of synthetic analogs to RhlR-RhlI system of P. aeruginosa strains (PAO-1; wild-type and mutants JP-1, PDO-100, and JP-2) responsible for production of acyl-homoserine lactones-2; butanol homoserine lactone (AHL-2; C(4)-HSL). We synthesized double (QS1207) and single (QS0108) sulfur analogs against (C(4)-HSL; AHL-2), an autoinducer of Pseudomonas QS system. Extensive biological investigation of these analogs suggested a growth promoting activity for these analogs in Pseudomonas controlling biofilm production and exo-protease secretion. We hypothesized that these thiolactone analogs could be potentially utilized as potent drug-delivery vehicles against biofilm-producing pathogens. As a proof of principle we conjugated the single sulfur analog QS0108 with the broad-spectrum antibiotic, ciprofloxacin (QS0108-Cip). The QS analog-antibiotic conjugate was significantly more effective at disrupting both the nascent and mature biofilms of P. aeruginosa than the free antibiotic.
Assuntos
Sistemas de Liberação de Medicamentos , Percepção de Quorum/efeitos dos fármacos , Biofilmes , Espectrometria de Massas , Veículos Farmacêuticos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Piocianina/genética , RNA Mensageiro/genéticaRESUMO
A rival to native peroxidase! An existing binding site for glutathione was combined with the catalytic residue tellurocysteine by using an auxotrophic expression system to create an engineered enzyme that functions as a glutathione peroxidase from the scaffold of a glutathione transferase (see picture). The catalytic activity of the telluroenzyme in the reduction of hydroperoxides by glutathione is comparable to that of native glutathione peroxidase.
Assuntos
Cisteína/análogos & derivados , Glutationa Peroxidase/metabolismo , Glutationa Transferase/química , Catálise , Cisteína/síntese química , Cisteína/química , Glutationa Transferase/metabolismo , Peróxido de Hidrogênio/química , OxirreduçãoRESUMO
Stable isotope-labeled N-acyl selones have been constructed in fewer than four steps from readily available starting materials. Site-specific labeling was achieved using the following synthons: bromo[2-(13)C]acetic acid, [(13)C]formic acid, and elemental (77)Se. These labeled selones have been found to provide unique insights into enolate structure and may be useful in the detection and quantitation of remotely disposed chiral centers in compounds in short supply.
Assuntos
Compostos de Selênio/química , Isótopos de Carbono , Isótopos , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
Our group has used Ti-promoted aldol additions with an oxazolidineselone as the chiral auxiliary with much success. In these reactions, the Se atom in the auxiliary both promotes stereospecific addition as well as reports on, through the use of 77Se NMR spectroscopy, the ratio of diastereomers produced and the geometry of intermediates as the reaction proceeds. Through stable isotope labeling and NMR spectroscopy, we are able to experimentally observe a Ti enolate in solution and gain insight into its structure and reactivity. Results from molecular modeling calculations are also presented for comparison with NMR data.