RESUMO
Live vaccines are ideal for inducing immunity but suffer from the need to attenuate their pathogenicity or replication to preclude the possibility of escape. Unnatural amino acids (UAAs) provide a strategy to engineer stringent auxotrophies, yielding conditionally replication incompetent live bacteria with excellent safety profiles. Here, we engineer Pseudomonas aeruginosa to maintain auxotrophy for the UAA p-benzoyl-L-phenylalanine (BzF) through its incorporation into the essential protein DnaN. In vivo evolution using an Escherichia coli-based two-hybrid selection system enabled engineering of a mutant DnaN homodimeric interface completely dependent on a BzF-specific interaction. This engineered strain, Pa Vaccine, exhibits undetectable escape frequency (<10-11) and shows excellent safety in naïve mice. Animals vaccinated via intranasal or intraperitoneal routes are protected from lethal challenge with pathogenic P. aeruginosa PA14. These results establish UAA-auxotrophic bacteria as promising candidates for bacterial vaccine therapy and outline a platform for expanding this technology to diverse bacterial pathogens.
Assuntos
Infecções por Pseudomonas , Vacinas contra Pseudomonas , Pseudomonas aeruginosa , Animais , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/genética , Infecções por Pseudomonas/prevenção & controle , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Camundongos , Feminino , Vacinas contra Pseudomonas/imunologia , Vacinas contra Pseudomonas/genética , Aminoácidos , Fenilalanina/análogos & derivados , Escherichia coli/imunologia , Escherichia coli/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/genética , Camundongos Endogâmicos BALB CRESUMO
Pseudomonas aeruginosa is a complex nosocomial infectious agent responsible for numerous illnesses, with its growing resistance variations complicating treatment development. Studies have emphasized the importance of virulence factors OprE and OprF in pathogenesis, highlighting their potential as vaccine candidates. In this study, B-cell, MHC-I, and MHC-II epitopes were identified, and molecular linkers were active to join these epitopes with an appropriate adjuvant to construct a vaccine. Computational tools were employed to forecast the tertiary framework, characteristics, and also to confirm the vaccine's composition. The potency was weighed through population coverage analysis and immune simulation. This project aims to create a multi-epitope vaccine to reduce P. aeruginosa-related illness and mortality using immunoinformatics resources. The ultimate complex has been determined to be stable, soluble, antigenic, and non-allergenic upon inspection of its physicochemical and immunological properties. Additionally, the protein exhibited acidic and hydrophilic characteristics. The Ramachandran plot, ProSA-web, ERRAT, and Verify3D were employed to ensure the final model's authenticity once the protein's three-dimensional structure had been established and refined. The vaccine model showed a significant binding score and stability when interacting with MHC receptors. Population coverage analysis indicated a global coverage rate of 83.40%, with the USA having the highest coverage rate, exceeding 90%. Moreover, the vaccine sequence underwent codon optimization before being cloned into the Escherichia coli plasmid vector pET-28a (+) at the EcoRI and EcoRV restriction sites. Our research has developed a vaccine against P. aeruginosa that has strong binding affinity and worldwide coverage, offering an acceptable way to mitigate nosocomial infections.
Assuntos
Biologia Computacional , Infecções por Pseudomonas , Pseudomonas aeruginosa , Sepse , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/genética , Humanos , Infecções por Pseudomonas/prevenção & controle , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Sepse/prevenção & controle , Sepse/imunologia , Sepse/microbiologia , Biologia Computacional/métodos , Epitopos/imunologia , Epitopos/química , Pneumonia/prevenção & controle , Pneumonia/imunologia , Pneumonia/microbiologia , Vacinas contra Pseudomonas/imunologia , Vacinas Bacterianas/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/genéticaRESUMO
Extensively drug-resistant Pseudomonas aeruginosa infections are emerging as a significant threat associated with adverse patient outcomes. Due to this organism's inherent properties of developing antibiotic resistance, we sought to investigate alternative strategies such as identifying "high value" antigens for immunotherapy-based purposes. Through extensive database mining, we discovered that numerous Gram-negative bacterial (GNB) genomes, many of which are known multidrug-resistant (MDR) pathogens, including P. aeruginosa, horizontally acquired the evolutionarily conserved gene encoding Zonula occludens toxin (Zot) with a substantial degree of homology. The toxin's genomic footprint among so many different GNB stresses its evolutionary importance. By employing in silico techniques such as proteomic-based phylogenetic tracing, in conjunction with comparative structural modeling, we discovered a highly conserved intermembrane associated stretch of 70 amino acids shared among all the GNB strains analyzed. The characterization of our newly identified antigen reveals it to be a "high value" vaccine candidate specific for P. aeruginosa. This newly identified antigen harbors multiple non-overlapping B- and T-cell epitopes exhibiting very high binding affinities and can adopt identical tertiary structures among the least genetically homologous P. aeruginosa strains. Taken together, using proteomic-driven reverse vaccinology techniques, we identified multiple "high value" vaccine candidates capable of eliciting a polarized immune response against all the P. aeruginosa genetic variants tested.
Assuntos
Filogenia , Infecções por Pseudomonas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/genética , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Humanos , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/química , Vacinas contra Pseudomonas/imunologia , Vacinas contra Pseudomonas/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/genéticaRESUMO
This study marks the first utilization of reverse vaccinology to develop recombinant subunit vaccines against Pseudomonas koreensis infection in Empurau (Tor tambroides). The proteome (5538 proteins) was screened against various filters to prioritize proteins based on features that are associated with virulence, subcellular localization, transmembrane helical structure, antigenicity, essentiality, non-homology with the host proteome, molecular weight, and stability, which led to the identification of eight potential vaccine candidates. These potential vaccine candidates were cloned and expressed, with six achieving successful expression and purification. The antigens were formulated into two distinct vaccine mixtures, Vac A and Vac B, and their protective efficacy was assessed through in vivo challenge experiments. Vac A and Vac B demonstrated high protective efficacies of 100 % and 81.2 %, respectively. Histological analyses revealed reduced tissue damage in vaccinated fish after experimental infection, with Vac A showing no adverse effects, whereas Vac B exhibited mild degenerative changes. Quantitative real-time PCR results showed a significant upregulation of TNF-α and downregulation of IL-1ß in the kidneys, spleen, gills, and intestine in both Vac A- and Vac B-immunized fish after challenged with P. koreensis. Additionally, IL-8 exhibits tissue-specific differential expression, with significant upregulation in the kidney, gills, and intestine, and downregulation in the spleen, particularly notable in Vac A-immunized fish. The research underscores the effectiveness of the reverse vaccinology approach in fish and demonstrates the promising potential of Vac A and Vac B as recombinant subunit vaccines.
Assuntos
Doenças dos Peixes , Infecções por Pseudomonas , Pseudomonas , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/prevenção & controle , Pseudomonas/imunologia , Infecções por Pseudomonas/veterinária , Infecções por Pseudomonas/prevenção & controle , Infecções por Pseudomonas/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinologia , Vacinas Sintéticas/imunologia , Cyprinidae/imunologia , Vacinas contra Pseudomonas/imunologia , Proteoma/imunologiaRESUMO
Pseudomonas aeruginosa (Pa) is an opportunistic bacterial pathogen responsible for severe hospital acquired infections in immunocompromised and elderly individuals. Emergence of increasingly drug resistant strains and the absence of a broad-spectrum prophylactic vaccine against both T3SA+ (type III secretion apparatus) and ExlA+/T3SA- Pa strains worsen the situation in a post-pandemic world. Thus, we formulated a candidate subunit vaccine (called ExlA/L-PaF/BECC/ME) against both Pa types. This bivalent vaccine was generated by combining the C-terminal active moiety of exolysin A (ExlA) produced by non-T3SA Pa strains with our T3SA-based vaccine platform, L-PaF, in an oil-in-water emulsion. The ExlA/L-PaF in ME (MedImmune emulsion) was then mixed with BECC438b, an engineered lipid A analogue and a TLR4 agonist. This formulation was administered intranasally (IN) to young and elderly mice to determine its potency across a diverse age-range. The elderly mice were used to mimic the infection seen in elderly humans, who are more susceptible to serious Pa disease compared to their young adult counterparts. After Pa infection, mice immunized with ExlA/L-PaF/BECC/ME displayed a T cell-mediated adaptive response while PBS-vaccinated mice experienced a rapid onset inflammatory response. Important genes and pathways were observed, which give rise to an anti-Pa immune response. Thus, this vaccine has the potential to protect aged individuals in our population from serious Pa infection.
Assuntos
Emulsões , Infecções por Pseudomonas , Vacinas contra Pseudomonas , Pseudomonas aeruginosa , Vacinas de Subunidades Antigênicas , Animais , Pseudomonas aeruginosa/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Camundongos , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Vacinas contra Pseudomonas/administração & dosagem , Feminino , Desenvolvimento de Vacinas , Humanos , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/sangue , Modelos Animais de Doenças , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/genéticaRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen considered a common cause of nosocomial infection with high morbidity and mortality in burn patients. Immunoprophylaxis techniques may lower the mortality rate of patients with burn wounds infected by P. aeruginosa; consequently, this may be an efficient strategy to manage infections caused by this bacterium. Several pathogenic Gram-negative bacteria like P. aeruginosa release outer membrane vesicles (OMVs), and structurally OMV consists of several antigenic components capable of generating a wide range of immune responses. Here, we evaluated the immunogenicity and efficacy of P. aeruginosa PA-OMVs (PA-OMVs) conjugated with the diphtheria toxoid (DT) formulated with alum adjuvant (PA-OMVs-DT + adj) in a mice model of burn wound infection. ELISA results showed that in the group of mice immunized with PA-OMVs-DT + adj conjugated, there was a significant increase in specific antibodies titer compared to non-conjugated PA-OMVs or control groups. In addition, the vaccination of mice with PA-OMVs-DT + adj conjugated generated greater protective effectiveness, as seen by lower bacterial loads, and eightfold decreased inflammatory cell infiltration with less tissue damage in the mice burn model compared to the control group. The opsonophagocytic killing results confirmed that humoral immune response might be critical for PA-OMVs mediated protection. These findings suggest that PA-OMV-DT conjugated might be used as a new vaccine against P. aeruginosa in burn wound infection.
Assuntos
Queimaduras , Toxoide Diftérico , Vacinas contra Pseudomonas , Pseudomonas aeruginosa , Infecção dos Ferimentos , Animais , Camundongos , Proteínas da Membrana Bacteriana Externa/imunologia , Queimaduras/microbiologia , Toxoide Diftérico/imunologia , Pseudomonas aeruginosa/imunologia , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/prevenção & controle , Vacinas contra Pseudomonas/imunologiaRESUMO
Pseudomonas aeruginosa infection continues to be a major threat to global public health, and new safe and efficacious vaccines are needed for prevention of infections caused by P. aeruginosa. X-ray irradiation has been used to prepare whole-cell inactivated vaccines against P. aeruginosa infection. However, the immunological mechanisms of X-ray-inactivated vaccines are still unclear and require further investigation. Our previous study found that an X-ray-inactivated whole-cell vaccine could provide protection against P. aeruginosa by boosting T cells. The aim of the present study was to further explore the immunological mechanisms of the vaccine. Herein, P. aeruginosa PAO1, a widely used laboratory strain, was utilized to prepare the vaccine, and we found nucleic acids and 8-hydroxyguanosine in the supernatant of X-ray-inactivated PAO1 (XPa). By detecting CD86, CD80, and MHCII expression, we found that XPa fostered dentritic cell (DC) maturation by detecting. XPa stimulated the cGAS-STING pathway as well as Toll-like receptors in DCs in vitro, and DC finally underwent apoptosis and pyroptosis after XPa stimulation. In addition, DC stimulated by XPa induced CD8+ T-cell proliferation in vitro and generated immunologic memory in vivo. Moreover, XPa vaccination induced both Th1 and Th2 cytokine responses in mice and reduced the level of inflammatory factors during infection. XPa protected mice in pneumonia models from infection with PAO1 or multidrug-resistant clinical isolate W9. Chronic obstructive pulmonary disease (COPD) mice immunized with XPa could resist PAO1 infection. Therefore, a new mechanism of an X-ray-inactivated whole-cell vaccine against P. aeruginosa infection was discovered in this study.
Assuntos
Proteínas de Membrana/imunologia , Nucleotidiltransferases/imunologia , Infecções por Pseudomonas/imunologia , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Transdução de Sinais/imunologia , Animais , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Nucleotidiltransferases/genética , Infecções por Pseudomonas/genética , Vacinas contra Pseudomonas/farmacologia , Células RAW 264.7 , Transdução de Sinais/genéticaRESUMO
In a dairy cow herd consisted of herd of 200 lactating Holstein-Friesian cows and heifers, clinical signs of mastitis in 40 out of 170 animals were observed. Treatments with antibiotics were proved ineffective. Milk bacterial cultures from 15 affected animals revealed Pseudomonas aeruginosa. An autogenous vaccine was administered subcutaneously, twice in a month period, to all adults. Cases of clinical mastitis declined significantly (p⟨0.0001) during next 3 months.
Assuntos
Surtos de Doenças/veterinária , Mastite Bovina/microbiologia , Infecções por Pseudomonas/terapia , Infecções por Pseudomonas/veterinária , Vacinas contra Pseudomonas/imunologia , Animais , Bovinos , Feminino , Mastite Bovina/prevenção & controle , Mastite Bovina/terapia , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/administração & dosagem , Pseudomonas aeruginosaRESUMO
Pseudomonas aeruginosa (P. aeruginosa) displays high drug resistance and biofilm-mediated adaptability, which makes its infections difficult to treat. Alternative intervention methods and targets have made such infections treatment manageable. One of the biofilm components, functional amyloids of Pseudomonas (Fap) is correlated positively with virulence and mucoidy phenotype found in infection in cystic fibrosis (CF) patients. Extracellular accessibility, conservation across P. aeruginosa isolates and linkage with lung infections phenotype in CF patients, makes Fap a promising intervention target. Furthermore, the reported effect of bacterial amyloid on neuronal function and immune response makes it a targetable candidate. In the current study, Fap C protein and its immediate interactions were explored to extract antigenic T-cell and B-cell epitopes. A combination of epitopes and peptide adjuvants has been linked to derive vaccine candidate structures. The vaccine candidates were validated for antigenicity, allergenicity, physiochemical properties, stability and interactions with TLRs and MHC alleles. Immunosimulation studies have demonstrated that vaccines elicit Th1 dominated response, which can assist in good prognosis of infection in CF patients.
Assuntos
Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Desenvolvimento de Vacinas , Biologia ComputacionalRESUMO
Pseudomonas aeruginosa is a Gram-negative pathogen that causes severe pulmonary infections associated with high morbidity and mortality in immunocompromised patients. The development of a vaccine against P. aeruginosa could help prevent infections caused by this highly antibiotic-resistant microorganism. We propose that identifying the vaccine-induced correlates of protection against P. aeruginosa will facilitate the development of a vaccine against this pathogen. In this study, we investigated the mechanistic correlates of protection of a curdlan-adjuvanted P. aeruginosa whole-cell vaccine (WCV) delivered intranasally. The WCV significantly decreased bacterial loads in the respiratory tract after intranasal P. aeruginosa challenge and raised antigen-specific antibody titers. To study the role of B and T cells during vaccination, anti-CD4, -CD8, and -CD20 depletions were performed prior to WCV vaccination and boosting. The depletion of CD4+, CD8+, or CD20+ cells had no impact on the bacterial burden in mock-vaccinated animals. However, depletion of CD20+ B cells, but not CD8+ or CD4+ T cells, led to the loss of vaccine-mediated bacterial clearance. Also, passive immunization with serum from WCV group mice alone protected naive mice against P. aeruginosa, supporting the role of antibodies in clearing P. aeruginosa We observed that in the absence of T cell-dependent antibody production, mice vaccinated with the WCV were still able to reduce bacterial loads. Our results collectively highlight the importance of the humoral immune response for protection against P. aeruginosa and suggest that the production of T cell-independent antibodies may be sufficient for bacterial clearance induced by whole-cell P. aeruginosa vaccination.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Anticorpos Antibacterianos/imunologia , Pneumonia Bacteriana/prevenção & controle , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/administração & dosagem , Vacinas contra Pseudomonas/imunologia , Animais , Humanos , Imunização , Camundongos , Modelos Animais , Pneumonia Bacteriana/fisiopatologia , Infecções por Pseudomonas/fisiopatologia , VacinaçãoRESUMO
Pseudomonas aeruginosa is a leading cause of chronic respiratory infections in people with cystic fibrosis (CF), bronchiectasis or chronic obstructive pulmonary disease (COPD), and acute infections in immunocompromised individuals. The adaptability of this opportunistic pathogen has hampered the development of antimicrobial therapies, and consequently, it remains a major threat to public health. Due to its antimicrobial resistance, vaccines represent an alternative strategy to tackle the pathogen, yet despite over 50 years of research on anti-Pseudomonas vaccines, no vaccine has been licensed. Nevertheless, there have been many advances in this field, including a better understanding of the host immune response and the biology of P. aeruginosa. Multiple antigens and adjuvants have been investigated with varying results. Although the most effective protective response remains to be established, it is clear that a polarised Th2 response is sub-optimal, and a mixed Th1/Th2 or Th1/Th17 response appears beneficial. This comprehensive review collates the current understanding of the complexities of P. aeruginosa-host interactions and its implication in vaccine design, with a view to understanding the current state of Pseudomonal vaccine development and the direction of future efforts. It highlights the importance of the incorporation of appropriate adjuvants to the protective antigen to yield optimal protection.
Assuntos
Anticorpos Antibacterianos , Fibrose Cística/microbiologia , Infecções por Pseudomonas/imunologia , Vacinas contra Pseudomonas/imunologia , Infecções Respiratórias/microbiologia , Adjuvantes Imunológicos , Alginatos/química , Animais , Antígenos/metabolismo , Fibrose Cística/imunologia , Exotoxinas/metabolismo , Flagelos/metabolismo , Humanos , Imunidade Inata , Lipopolissacarídeos , Estudos Longitudinais , Pulmão/imunologia , Pulmão/virologia , Camundongos , Pseudomonas aeruginosa , Infecções Respiratórias/imunologia , Células Th1/virologia , Células Th17/virologia , Células Th2/virologia , Vacinas de DNA/metabolismoRESUMO
Hospital-acquired infections with Pseudomonas aeruginosa have become a great challenge in caring for critically ill and immunocompromised patients. The cause of high mortality is the presence of multi-drug resistant (MDR) strains, which confers a pressing need for vaccines. Although vaccines against P. aeruginosa have been in development for more than several decades, there is no vaccine for patients at present. In this study, we purified genomic DNA of P. aeruginosa from sera of patients affected, constructed genome-wide library with random recombinants, and screened candidate protein antigens by evaluating their protective effects in vivo. After 13-round of screening, 115 reactive recombinants were obtained, among which 13 antigens showed strong immunoreactivity (more than 10% reaction to PcrV, a well-characterized V-antigen of P. aeruginosa). These 13 antigens were: PpiA, PtsP, OprP, CAZ10_34235, HmuU_2, PcaK, CarAd, RecG, YjiR_5, LigD, KinB, RtcA, and PscF. In vivo studies showed that vaccination with PscF protected against lethal P. aeruginosa challenge, and decreased lung inflammation and injury. A genomic library of P. aeruginosa could be constructed in this way for the first time, which could not only screen candidate antigens but also in a high-throughput way. PscF was considered as an ideal promising vaccine candidate for combating P. aeruginosa infection and was supported for further evaluation of its safety and efficacy.
Assuntos
Antígenos de Bactérias/genética , Infecção Hospitalar/prevenção & controle , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Infecção Hospitalar/sangue , Infecção Hospitalar/imunologia , Infecção Hospitalar/microbiologia , DNA Bacteriano/genética , Modelos Animais de Doenças , Feminino , Biblioteca Genômica , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunogenicidade da Vacina , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Infecções por Pseudomonas/sangue , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Vacinas contra Pseudomonas/administração & dosagem , Vacinas contra Pseudomonas/genética , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Several vaccine candidates have been introduced for immunization against Pseudomonas aeruginosa strains. Despite extensive efforts in recent decades, there is no accurate immunogenic candidate against this pathogen in the market yet. Due to the rapid increase in several drug-resistant strains, P. aeruginosa has caused various health concerns worldwide. It encodes many specific virulence features, which can be used as an appropriate vaccine candidate. The primary stage of the pathogenesis of P. aeruginosa is the expression of many dynamic adhesive molecules, such as type IV pili (T4P), which acts as a principal colonization factor. It has been confirmed that three different subtypes of T4P, including type IVa (T4aP), type IVb (T4bP) and tight adherence (Tad) pili are expressed by P. aeruginosa. The IVa fimbriae type is almost the main cause of challenges to design an effective pili based-immunotherapy method. Nevertheless, in terms of heterogeneity, variability and hidden conserved binding site of T4aP, this attitude has been remained controversial and there is no permitted human study based on IVa pilin commercially. The engineered synthetic peptide-based vaccines are highly talented to mimic the target. In this research, for the first time, some dominant immunogenic features of the Flp protein, such as both B- and T-cell-associated epitopes, presence of IgE-associated epitopes, solvent-accessible surface area were evaluated by analytical immunoinformatics methods. In addition, we designed the engineered Flp pilin as an effective immunogenic substance against several clinically important P. aeruginosa strains. Moreover, by practical active immunization approaches, the humoral and cellular immune response against the extremely conserved region of the engineered synthetic Flp (EFlp) formulated in Montanide ISA 266 compared to the control group. The results of active immunization against EFlp significantly signified that EFlp-Montanide ISA 266 (EFLP-M) strongly could induce both humoral and cellular immune responses. We concluded that Flp pilin has therapeutic potential against numerous clinically significant P. aeruginosa strains and can be served as a novel immunogen for further investigations for development of effective immunotherapy methods against P. aeruginosa as a dexterous pathogen.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Biologia Computacional , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Pseudomonas/prevenção & controle , Vacinação , Vacinas Sintéticas/imunologiaRESUMO
IC43, a truncate form of outer membrane proteins OprF190-342 and OprI21-83 from Pseudomonas aeruginosa, is a promising candidate antigen and exists as monomer in solution. In this study, we generated the heptamer of IC43 by carrier protein aided oligomerization, which was confirmed by gel-filtration and chemical cross-linking analysis. The carrier protein naturally exists as a homo-heptamer, and IC43 was displayed on the surface of the carrier protein in the fusion protein. Immunization with this fusion protein resulted in increased level of antigen specific IgG antibodies and higher survival rate after infection. The improved efficacy was correlated with lower bacteria burden, inflammation and tissue damage in the lungs of immunized mice. Further studies revealed that immunization with this fusion protein resulted in increased levels of IL-4 and antigen specific IgG1, suggesting a stronger Th2 immune response was induced. The improved immunogenicity may be attributed to the exposure of more epitopes on the antigen, which was confirmed by results from immune-dominant peptide mapping and passive immunization. These results demonstrated a possible strategy to improve the immunogenicity of an antigen by carrier protein aided oligomerization.
Assuntos
Imunogenicidade da Vacina , Fragmentos de Peptídeos/imunologia , Pneumonia Bacteriana/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Proteínas de Bactérias/imunologia , Feminino , Imunoglobulina G/imunologia , Interleucina-4/metabolismo , Lipoproteínas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/química , Multimerização Proteica , Vacinas contra Pseudomonas/químicaRESUMO
BACKGROUND: Pseudomonas aeruginosa has an important role in nosocomial infections. OBJECTIVE: To evaluate biological activity of the detoxified LPS (D-LPS) entrapped into Poly lactic-co-glycolic acid (PLGA) nanoparticles. MATERIALS: LPS was extracted and detoxified from the P. aeruginosa strain PAO1. The D-LPS, conjugated to the PLGA nanoparticles with 1-ethyl-3-dimethyl aminopropyl carbodiimide (EDAC) and N-hydroxy-succinimide (NHS). The connection was evaluated by FTIR (Fourier transform infrared), Zetasizer, and Atomic Force Microscope (AFM). The BALB/c mice injected intramuscularly with the D-LPS-PLGA with two-week intervals and then challenged two weeks after the last immunization. The bioactivity of the induced specific antisera and cytokines responses against D-LPS-PLGA antigen was assessed by ELISA. RESULTS: D-LPS-PLGA conjugation was confirmed by FTIR, Zetasizer, and AFM. The ELISA results showed that D-LPS was successful in the stimulation of the humoral immune response. The immune responses raised against the D-LPS-PLGA, significantly decreased bacterial titer in the spleen of the immunized mice after challenge with PAO1 strain in comparison with the control groups. CONCLUSION: The conjugation of the bacterial LPS to the PLGA nanoparticle increased their functional activity by decrease in bacterial dissemination and increase the killing of opsonized bacteria.
Assuntos
Antígenos de Bactérias , Lipopolissacarídeos , Nanopartículas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Vacinas contra Pseudomonas , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/farmacologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosaRESUMO
Pulmonary infection caused by Pseudomonas aeruginosa (PA) has created an urgent need for an efficient vaccine, but the protection induced by current candidates is limited, partially because of the high variability of the PA genome. Antigens targeting pulmonary Th17 responses are able to provide antibody-independent and broad-spectrum protection; however, little information about Th17-stimulating antigens in PA is available. Herein, we identified two novel PA antigens that effectively induce Th17-dependent protection, namely, PcrV (PA1706) and AmpC (PA4110). Compared to intramuscular immunization, intranasal immunization enhanced the protection of rePcrV due to activation of a Th17 response. The Th17-stimulating epitopes of PcrV and AmpC were identified, and the recombinant protein PVAC was designed and generated by combining these Th17-stimulating epitopes. PVAC was successfully produced in soluble form and elicited broad protective immunity against PA. Our results provide an alternative strategy for the development of Th17-based vaccines against PA and other pathogens.
Assuntos
Antígenos de Bactérias , Proteínas de Bactérias , Toxinas Bacterianas , Epitopos de Linfócito T , Proteínas Citotóxicas Formadoras de Poros , Vacinas contra Pseudomonas , Pseudomonas aeruginosa , Células Th17/imunologia , beta-Lactamases , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Camundongos , Camundongos Knockout , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/imunologia , Vacinas contra Pseudomonas/genética , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , beta-Lactamases/genética , beta-Lactamases/imunologiaRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen causing acute and chronic respiratory infections associated with morbidity and mortality, especially in patients with cystic fibrosis. Vaccination against P. aeruginosa before colonization may be a solution against these infections and improve the quality of life of at-risk patients. To develop a vaccine against P. aeruginosa, we formulated a novel peptide-based P. aeruginosa subunit vaccine based on the extracellular regions of one of its major siderophore receptors, FpvA. We evaluated the effectiveness and immunogenicity of the FpvA peptides conjugated to keyhole limpet hemocyanin (KLH) with the adjuvant curdlan in a murine vaccination and challenge model. Immunization with the FpvA-KLH vaccine decreased the bacterial burden and lung edema after P. aeruginosa challenge. Vaccination with FpvA-KLH lead to antigen-specific IgG and IgM antibodies in sera, and IgA antibodies in lung supernatant. FpvA-KLH immunized mice had an increase in recruitment of CD11b+ dendritic cells as well as resident memory CD4+ T cells in the lungs compared to non-vaccinated challenged mice. Splenocytes isolated from vaccinated animals showed that the FpvA-KLH vaccine with the adjuvant curdlan induces antigen-specific IL-17 production and leads to a Th17 type of immune response. These results indicate that the intranasal FpvA-KLH conjugate vaccine can elicit both mucosal and systemic immune responses. These observations suggest that the intranasal peptide-based FpvA-KLH conjugate vaccine with curdlan is a potential vaccine candidate against P. aeruginosa pneumonia.
Assuntos
Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/prevenção & controle , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Vacinas Conjugadas/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Hemocianinas/química , Hemocianinas/imunologia , Humanos , Imunidade nas Mucosas , Imunização , Memória de Curto Prazo , Camundongos , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/patologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Vacinas contra Pseudomonas/administração & dosagem , Proteínas Recombinantes , Vacinas Conjugadas/administração & dosagem , Vacinas de Subunidades Antigênicas/administração & dosagemRESUMO
Objective To construct and identify Bifidobacterium bifidum-vectored outer membrane protein F-I[rBb(pGEX-OprF-I)] vaccine of Pseudomonas aeruginosa and observe its protection against Pseudomonas aeruginosa infection in mice. Methods OprF and OprI genes were amplified by PCR, then the OprF-I fusion gene obtained by gene SOEing was digested and ligated into the vector pGEX-1λT to construct the recombinant plasmid pGEX-OprF-I. The plasmid was transformed into Bifidobacterium bifidum (Bb) by electroporation, and the rBb(pGEX-OprF-I) vaccine was constructed and identified by double enzyme digestion and PCR. Expression products of the vaccine induced by IPTG were analyzed and identified by SDS-PAGE and Western blot analysis. Twenty-one BALB/c mice were randomly divided into rBb(pGEX-OprF-I) vaccine group, Bb-pGEX-1λT empty vector group and Bb control group. The 5×108 CFUs vaccine was intragastrically administered for 3 consecutive days per week for 3 weeks. All mice were challenged intranasally with 5×107 CFUs of PA01 strain at the 4th week after the first immunization. At the 2nd week after the challenge, all mice were sacrificed to count the lung bacteria loads. IgG levels in sera from the mice before immunization, 4th week after the first immunization and 2nd week after the challenge were detected by routine ELISA. Results A total of 1289 bp OprF-I fusion gene was amplified by PCR. Double enzyme digestion and PCR identification confirmed that the gene was ligated into pGEX-1λT and transformed into Bb, and the rBb(pGEX-OprF-I) vaccine was successfully constructed. SDS-PAGE showed that the fusion protein with a relative molecular mass (Mr) of about 68 000 could be expressed by IPTG-induced vaccine. Western blot analysis indicated that the protein could be specifically recognized by the sera of Pseudomonas aeruginosa-infected mice. The number of bacteria colonies in the lung of the mice immunized with rBb(pGEX-OprF-I) vaccine was significantly lower than that of the control group. The IgG levels in the sera of the immunized mice increased successively at 4th week after the first immunization and 2nd week after the challenge, and higher than that in the other control groups at the same time point. Conclusion The rBb(pGEX-OprF-I) vaccine has been successfully constructed, and it may take a certain protective effect on the mice against Pseudomonas aeruginosa infection.
Assuntos
Proteínas de Bactérias/imunologia , Bifidobacterium bifidum , Lipoproteínas/imunologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Animais , Camundongos , Camundongos Endogâmicos BALB C , Pseudomonas aeruginosa , Distribuição Aleatória , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Pseudomonas aeruginosa (PA) is a major cause of nosocomial infections, which remain an unsolved problem in the clinic despite conventional antibiotic treatment. A PA vaccine could be both an effective and economical strategy to address this issue. Many studies have shown that PcrV, a structural protein of the type 3 secretion system (T3SS) from PA, is an ideal target for immune prevention and therapy. However, difficulties in the production of high-quality PcrV likely hinder its further application in the vaccine industry. Thus, we hypothesized that an optimized PcrV derivative with a rational design could be produced. In this study, the full-length PcrV was divided into four domains with the guidance of its structure, and the Nter domain (Met1-Lys127) and H12 domain (Leu251-Ile294) were found to be immunodominant. Subsequently, Nter and H12 were combined with a flexible linker to generate an artificial PcrV derivative (PcrVNH). PcrVNH was successfully produced in E. coli and behaved as a homogenous monomer. Moreover, immunization with PcrVNH elicited a multifactorial immune response and conferred broad protection in an acute PA pneumonia model and was equally effective to full-length PcrV. In addition, passive immunization with anti-PcrVNH antibodies alone also showed significant protection, at least based on inhibition of the T3SS and mediation of opsonophagocytic killing activities. These results provide an additional example for the rational design of antigens and suggest that PcrVNH is a promising vaccine candidate for the control of PA infection.
Assuntos
Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/prevenção & controle , Vacinas contra Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Epitopos Imunodominantes/imunologia , Camundongos , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/imunologia , Infecções por Pseudomonas/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Respiratory infections are a leading cause of morbidity and mortality globally. This is partially due to a lack of effective vaccines and a clear understanding of how vaccination route and formulation influence protective immunity in mucosal tissues such as the lung. Pseudomonas aeruginosa is an opportunistic pathogen capable of causing acute pulmonary infections and is a leading cause of hospital-acquired and ventilator-associated pneumonia. With multidrug-resistant P. aeruginosa infections on the rise, the need for a vaccine against this pathogen is critical. Growing evidence suggests that a successful P. aeruginosa vaccine may require mucosal antibody and Th1- and Th17-type CD4+ T cells to prevent pulmonary infection. Intradermal immunization with adjuvants, such as the bacterial ADP-Ribosylating Enterotoxin Adjuvant (BARE) double mutant of E. coli heat-labile toxin (dmLT), can direct protective immune responses to mucosal tissues, including the lungs. We reasoned that intradermal immunization with P. aeruginosa outer membrane proteins (OMPs) adjuvanted with dmLT could drive neutralizing antibodies and migration of CD4+ T cells to the lungs and protect against P. aeruginosa pneumonia in a murine model. Here we show that mice immunized with OMPs and dmLT had significantly more antigen-specific IgG and Th1- and Th17-type CD4+ memory T cells in the pulmonary environment compared to control groups of mice. Furthermore, OMPs and dmLT immunized mice were significantly protected against an otherwise lethal lung infection. Protection was associated with early IFN-γ and IL-17 production in the lungs of immunized mice. These results indicate that intradermal immunization with dmLT can drive protective immunity to the lung mucosa and may be a viable vaccination strategy for a multitude of respiratory pathogens.