Cochleates derived from Vibrio cholerae O1 proteoliposomes : The impact of structure transformation on mucosal immunisation

Cochleates are phospholipid-calcium precipitates derived from the interaction of anionic lipid vesicles with divalent cations. Proteoliposomes from bacteria may also be used as a source of negatively charged components, to induce calcium-cochleate formation. In this study, proteoliposomes from V. cholerae O1 (PLc) (sized 160.7±1.6 nm) were transformed into larger (16.3±4.6 µm) cochleate-like structures (named Adjuvant Finlay Cochleate 2, AFCo2) and evaluated by electron microscopy (EM). Measurements from transmission EM (TEM) showed the structures had a similar size to that previously reported using light microscopy, while observations from scanning electron microscopy (SEM) indicated that the structures were multilayered and of cochleate-like formation. The edges of the AFCo2 structures appeared to have spaces that allowed penetration of negative stain or Ovalbumin labeled with Texas Red (OVA-TR) observed by epi-fluorescence microscopy. In addition, freeze fracture electron microscopy confirmed that the AFCo2 structures consisted of multiple overlapping layers, which corresponds to previous descriptions of cochleates. TEM also showed that small vesicles co-existed with the larger cochleate structures, and in vitro treatment with a calcium chelator caused the AFCo2 to unfold and reassemble into small proteoliposome-like structures. Using OVA as a model antigen, we demonstrated the potential loading capacity of a heterologous antigen and in vivo studies showed that with simple admixing and administration via intragastric and intranasal routes AFCo2 provided enhanced adjuvant properties compared with PLc

Immune adjuvant effect of V. cholerae O1 derived Proteoliposome coadministered by intranasal route with Vi polysaccharide from Salmonella Typhi

Proteoliposome derived from Vibrio cholerae O1 (PLc) is a nanoscaled structure obtained by detergent extraction process. Intranasal (i.n) administration of PLc was immunogenic at mucosal and systemic level vs. V. cholerae; however the adjuvant potential of this structure for non-cholera antigens has not been proven yet. The aim of this work was to evaluate the effect of coadministering PLc with the Vi polysaccharide antigen (Poli Vi) of S. Typhi by i.n route. The results showed that Poli Vi coadministered with PLc (PLc+Poli Vi) induce higher IgA response in saliva (p0.05) to that induced in a group of mice immunised by parenteral route with the Cuban anti-typhoid vaccine vax-TyVi®, although this vaccine did not induce mucosal response. In conclusion, this work demonstrates that PLc can be used as mucosal adjuvant to potentiate the immune response against a polysaccharide antigen like Poli Vi

Consideraciones sobre el empleo de antisueros somáticos en la clasificación por serotipos de cepas de Pseudomonas aeruginosa

El lipopolisacárido (LPS) de bacterias gramnegativas se emplea en el desarrollo de candidatos vacunales, como antígeno expuesto en la preparación o formando parte de esta para aumentar su poder inmunogénico. Sin embargo, esta estructura química de la membrana externa puede ser también utilizada para clasificar bacterias. Según el LPS, presente en la membrana externa bacteriana, Pseudomonas aeruginosa se puede clasificar en 20 tipos somáticos diferentes, clasificación muy útil para estudios epidemiológicos. P. aeruginosa expone sus LPS en la membrana de forma estable, pero ocurren cambios en el fenotipo del LPS bacteriano durante la infección, sucesos que dificultan su clasificación. En estos momentos, los estudios que se desarrollan en el Instituto Finlay necesitan del empleo de tecnologías modernas de clasificación y del diagnóstico molecular para poder establecer patrones más precisos de expresión de los LPS por métodos que junto a los ya existentes permitan diferenciar cepas bacterianas de la misma especie en cualquier tipo de infección

Evaluación en animales del efecto protector de una inmunoglobulina anti Pseudomonas aeruginosa para uso terapéutico

Pseudomonas aeruginosa constituye uno de los agentes patógenos oportunistas de mayor frecuencia de aislamiento en los diversos procesos infecciosos, por lo que es reconocido como un gran problema de salud a nivel mundial. Al no existir un fármaco de alta efectividad ni vacunas disponibles contra esta bacteria, se emplea una terapia con inmunoglobulinas polivalentes comerciales que de forma combinada con los antibióticos contribuyen a eliminar la infección, aunque los preparados disponibles en el mercado no contienen concentraciones suficientemente elevadas de anticuerpos específicos contra este microorganismo. En este trabajo se llevó a cabo la evaluación en un modelo animal de una inmunoglobulina anti- Pseudomonas aeruginosa para uso terapéutico mediante un ensayo de reto con una cepa virulenta. Se evaluó dosis y vía de administración de la misma, así como el valor profiláctico o terapéutico de los anticuerpos. Esta gammaglobulina resultó ser protectora en animales mostrando una sobreviviencia cercana a un 75% en comparación con el grupo control no protegido y además se logra eliminar el estado de portador en los individuos infectados

Labeling AFCo2 and PLc with OVA-TR.

<p>(A) AFCo2 admixed with OVA-TR. The images were obtained with a coupled-device camera linked to an epi-fluorescence microscope. After sonication and ultrafiltration AFCo2+OVA-TR was analyzed by light microscopy as shown in (B). The broken structure is a consequence of the sonication process. (C) The same AFCo2 structure observed by epi-fluorescence showing the distribution of the Ova-TR in AFCo2. (D) PLc+OVA-TR observed by epi-fluorescence showing a lack of signal.</p

AFCo2 formation.

<p>(A) White milky suspension (AFCo2) obtained after calcium interaction with a solution of PLc, (B) AFCo2 observed by light microscopy with an Opton Standard 25 microscope (magnification ×400), (C) distribution percentage (82.6%) of AFCo2 with a length of 16.3±4.6 µm measured using a graduated scale on the ocular lens of the microscope.</p

Scanning electron micrographs of AFCo2.

<p>(A) Tubular and multilamellar structures observed at ×1400 [multilamellarity is indicated by white arrows], (B) the open end of the structure magnified ×3300.</p

Systemic and mucosal anti-OVA IgA and IgG response induced by intranasal immunization of AFCo2+ OVA or PLc+OVA in BALB/c mice.

<p>AFCo2 or PLc (100 µg) was admixed with 20 µg of OVA and administered by i.n route to BALB/c mice. Results are expressed as the logarithm of the mean ± SEM. Statistical significance of the variance between multiple groups of experiments were calculated with one-way ANOVA, followed by a Tukey's multiple comparison test.</p>a,b,c<p>Different letters on the same column mean that values are significantly different (p<0.05); - no response detected.</p

Systemic and mucosal immune responses induced by mucosal immunization of AFCo2 or PLc in BALB/c mice.

<p>AFCo2 or PLc (100 µg) was administered by i.n or i.g route in BALB/c mice. Results of anti-PLc IgA in saliva and faeces and anti-PLc IgG in sera are expressed as the logarithm of the mean ± SEM. The ratio of IgG subclasses IgG2a/IgG1 is also reported. Vibriocidal activity of sera from immunized mice is also expressed as logarithm of the mean ± SEM. Statistical significance of the variance between multiple groups of experiments were calculated with one-way ANOVA, followed by a Tukey's multiple comparison test.</p>a,b,c<p>Different letters on the same column mean that values are significantly different (p<0.05); - no response detected.</p

AFCo2 unfolding with EDTA.

<p>The micrographs represent stills of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046461#pone.0046461.s001" target="_blank">Video S1</a> (supporting information) that shows the unfolding process of AFCo2 under the action of EDTA chelating agent (25 mM). AFCo2 starts to shrinks in the first 15 seconds and completely disappears at 25 seconds of exposure with EDTA.</p