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1.
Biologicals ; 47: 11-17, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28404459

RESUMEN

Mucoid strains of Pseudomonas aeruginosa are closely associated with chronic pulmonary infections. In this report we describe a straightforward approach to conjugate high molecular weight alginate to type b-flagellin (FLB) and investigation of its bioactivity. The conjugation process was performed by using ADH and EDAC. The endotoxin was eliminated from the candidate vaccine by LPS removal resin followed by LAL test. The bioconjugate molecules were verified by simultaneously determination of polysaccharide/protein content followed by gel filtration chromatography and FTIR spectroscopy. Groups of eight BALB/c mice were injected intranasally with 5 µg (per each nostril) of purified alginate, FLB and conjugated alginate-FLB with two week intervals. The functional activity of the vaccine was evaluated by ELISA and opsonophagocytosis tests. Vaccination with the alginate-FLB conjugate induced a significant (P = 0.0033) rise in alginate specific IgG in mice. At all dilution ranges, the opsonic activity of the conjugate vaccine antisera was significantly higher than alginate alone (61.9% vs. 17.3% at 1:4 dilution; P = 0.0067). The alginate-FLB conjugate could elicit high specific antibodies titer against alginate by improving its immunogenicity. In addition, the antisera raised against conjugate vaccine act as a suitable opsonin for phagocytosis of the mucoid strains of P. aeruginosa.


Asunto(s)
Flagelina , Inmunoconjugados , Infecciones por Pseudomonas/prevención & control , Vacunas contra la Infección por Pseudomonas , Pseudomonas aeruginosa , Animales , Femenino , Flagelina/química , Flagelina/inmunología , Flagelina/farmacología , Inmunoconjugados/química , Inmunoconjugados/inmunología , Inmunoconjugados/farmacología , Ratones , Ratones Endogámicos BALB C , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/patología , Vacunas contra la Infección por Pseudomonas/química , Vacunas contra la Infección por Pseudomonas/inmunología , Vacunas contra la Infección por Pseudomonas/farmacología , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/inmunología
2.
Appl Microbiol Biotechnol ; 99(2): 667-80, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25381907

RESUMEN

Pseudomonas aeruginosa is an opportunistic pathogen that localizes to and colonizes mucosal tissue. Thus, vaccines that elicit a strong mucosal response against P. aeruginosa should be superior to other vaccination strategies. In this study, to stimulate rapid and enhanced mucosal immune responses, mannose-modified chitosan microspheres loaded with the recombinant outer membrane protein OprF190-342-OprI21-83 (FI) (FI-MCS-MPs) of P. aeruginosa were developed as a potent subunit vaccine for mucosal delivery. FI-MCS-MPs were successfully obtained via the tripolyphosphate ionic crosslinking method. Confocal and immunohistochemical analyses indicated that FI-MCS-MPs exhibited the ability to bind the macrophage mannose receptor (MMR, CD206) in vitro and in vivo. After intranasal immunization of mice with FI-MCS-MPs, FI-specific humoral immune responses were detected, measured as local IgM antibody titers in lung tissue slurry; IgA antibody titers in nasal washes, bronchoalveolar lavage (BAL), and intestinal lavage; and systemic IgA and IgG antibody titers in serum. FI-MCS-MPs induced early and high mucosal and systemic humoral antibody responses comparable to those in the group vaccinated with unmodified mannose. High levels of IFN-γ and IL-4 in addition to T lymphocyte subsets induced a mixed Th1/Th2 response in mice immunized with FI-MCS-MPs, resulting in the establishment of cellular immunity. Additionally, when immunized mice were challenged with P. aeruginosa via the nasal cavity, FI-MCS-MPs demonstrated 75 % protective efficacy. Together, these data indicate that mannose-modified chitosan microspheres are a promising subunit delivery system for vaccines against P. aeruginosa infection.


Asunto(s)
Quitosano/farmacología , Inmunidad Mucosa , Manosa/farmacología , Infecciones por Pseudomonas/inmunología , Vacunas contra la Infección por Pseudomonas/inmunología , Pseudomonas aeruginosa , Administración Intranasal , Animales , Anticuerpos Antibacterianos/sangre , Formación de Anticuerpos , Proteínas Bacterianas/inmunología , Secuencia de Bases , Línea Celular , Quitosano/química , Femenino , Inmunoglobulina A/sangre , Inmunoglobulina A/química , Inmunoglobulina G/sangre , Interferón gamma/inmunología , Interleucina-4/inmunología , Lipoproteínas/inmunología , Macrófagos/química , Macrófagos/inmunología , Manosa/química , Ratones , Ratones Endogámicos BALB C , Microesferas , Datos de Secuencia Molecular , Infecciones por Pseudomonas/prevención & control , Vacunas contra la Infección por Pseudomonas/química , Proteínas Recombinantes/inmunología , Linfocitos T/inmunología
3.
Int J Biol Macromol ; 159: 174-182, 2020 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-32413471

RESUMEN

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.


Asunto(s)
Inmunogenicidad Vacunal , Fragmentos de Péptidos/inmunología , Neumonía Bacteriana/prevención & control , Vacunas contra la Infección por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Proteínas Bacterianas/inmunología , Femenino , Inmunoglobulina G/inmunología , Interleucina-4/metabolismo , Lipoproteínas/inmunología , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/química , Multimerización de Proteína , Vacunas contra la Infección por Pseudomonas/química
4.
Vaccine ; 37(38): 5762-5769, 2019 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-30262247

RESUMEN

Efforts to develop a vaccine for the elimination of malaria include the use of carrier proteins to assemble monomeric antigens into nanoparticles to maximize immunogenicity. Recombinant ExoProtein A (EPA) is a detoxified form of Pseudomonas aeruginosa Exotoxin A which has been used as a carrier in the conjugate vaccine field. A pilot-scale process developed for purification of EPA yielded product that consistently approached a preset upper limit for host cell protein (HCP) content per human dose. To minimize the risk of bulk material exceeding the specification, the purification process was redeveloped using mixed-mode chromatography resins. Purified EPA derived from the primary and redeveloped processes were comparable following full biochemical and biophysical characterization. However, using a process specific immunoassay, the HCP content was shown to decrease from a range of 0.14-0.24% w/w of total protein to below the level of detection with the revised process. The improved process reproducibly yields EPA with highly similar quality characteristics as the original process but with an improved profile for the HCP content.


Asunto(s)
ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/inmunología , Toxinas Bacterianas/química , Toxinas Bacterianas/inmunología , Fenómenos Químicos , Exotoxinas/química , Exotoxinas/inmunología , Vacunas contra la Infección por Pseudomonas/química , Vacunas contra la Infección por Pseudomonas/inmunología , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunología , Factores de Virulencia/química , Factores de Virulencia/inmunología , ADP Ribosa Transferasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Toxinas Bacterianas/aislamiento & purificación , Epítopos/inmunología , Exotoxinas/aislamiento & purificación , Humanos , Inmunogenicidad Vacunal , Ratones , Péptidos/inmunología , Procesamiento Proteico-Postraduccional , Infecciones por Pseudomonas/prevención & control , Vacunas contra la Infección por Pseudomonas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Análisis Espectral , Vacunas Sintéticas/aislamiento & purificación , Factores de Virulencia/aislamiento & purificación , Exotoxina A de Pseudomonas aeruginosa
5.
Chem Biol Drug Des ; 76(4): 293-304, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20807222

RESUMEN

One of the main challenges of Pseudomonas aeruginosa vaccine development is the design of an antigen that elicits cross-reactive antibodies against multiple virulent strains. Using a rational design approach, we have developed a single 17-residue peptide immunogen that generates antibodies that target the receptor-binding domain of the type IV pilus of more than one strain of P. aeruginosa. Using the receptor-binding domain sequence, of native strain PAO as a template, we have systematically changed up to five residues in the PAO sequence of the peptide immunogen into that of the PAK sequence. We show by indirect and competitive ELISA that the mutant peptide immunogens elicit the development of polyclonal sera that is cross-reactive to both native strain PAO and PAK pilin. We further show that there are at least two separate antibody populations in the polyclonal sera that possess closely related epitopes but which are each strain specific. Moreover, part of the epitope for the PAO-specific antibodies consists of several residues outside the disulfide loop of the receptor-binding domain. This allows us to create two unique epitopes within the same receptor-binding domain sequence.


Asunto(s)
Anticuerpos Antibacterianos/inmunología , Epítopos/inmunología , Proteínas Fimbrias/inmunología , Vacunas contra la Infección por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Anticuerpos Antibacterianos/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Datos de Secuencia Molecular , Mutación , Péptidos/síntesis química , Péptidos/química , Péptidos/inmunología , Unión Proteica , Estructura Terciaria de Proteína , Vacunas contra la Infección por Pseudomonas/química , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunología
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