Ana I. Camacho

Mucosal immunization with Shigella flexneri outer membrane vesicles induced protection in mice

Vaccination appears to be the only rational prophylactic approach to control shigellosis. Unfortunately, there is still no safe and efficacious vaccine available. We investigated the protection conferred by a new vaccine containing outer membrane vesicles (OMVs) from Shigella flexneri with an adjuvant based on nanoparticles in an experimental model of shigellosis in mice. OMVs were encapsulated in poly(anhydride) nanoparticles prepared by a solvent displacement method with the copolymer PMV/MA. OMVs loaded into NPs (NP-OMVs) were homogeneous and spherical in shape, with a size of 197nm (PdI=0.06). BALB/c mice (females, 9-week-old, 20±1g) were immunized by intradermal, nasal, ocular (20μg) or oral route (100μg) with free or encapsulated OMV. Thirty-five days after administration, mice were infected intranasally with a lethal dose of S. flexneri (1×10(7)CFU). The new vaccine was able to protect fully against infection when it was administered via mucosa. By intradermal route the NP-OMVs formulation increased the protection from 20%, obtained with free extract, to 100%. Interestingly, both OMVs and OMV-NP induced full protection when administered by the nasal and conjuntival route. A strong association between the ratio of IL-12p40/IL-10 and protection was found. Moreover, low levels of IFN-γ correlate with protection. Under the experimental conditions used, the adjuvant did not induce any adverse effects. These results place OMVs among promising candidates to be used for vaccination against Shigellosis.

Poly(methyl vinyl ether-co-maleic anhydride) nanoparticles as innate immune system activators

Adjuvant research is being oriented to TLR-agonists, but complement activation has been relatively unexplored. In previous studies it was demonstrated that poly(methyl vinyl ether-co-maleic anhydride) nanoparticles (PVMA NPs) used as adjuvant differentially activate dendritic cells through toll like receptors (TLR) stimulation, however, a high dose of these NPs was used. Now, we demonstrated a dose-response effect, with a concentration as low as 20μg/mL able to stimulate TLR2 and TLR4 transfected dendritic cells. In addition, we investigated whether PVMA NPs are able to exploit also the immunomodulatory benefits of complement activation. Results indicated that the hydroxylated surface of these NPs highly activated the complement cascade, as measured by adsorption studies and a complement fixation bioassay. Stable binding of C3b to NPs was confirmed as indicated by lability to SDS treatment after washing resistance. Complement consumption was confirmed as the lytic capacity of complement exposed to NPs was abolished against antibody-sensitized sheep erythrocytes, with a minimal inhibitory concentration of 50μg NPs, equivalent to a surface of 1cm(2). On the contrary, nanoparticles prepared with poly(lactic-co-glycolic acid) (PLGA), used as a reference, did not consume complement at a concentration ≥3mg NPs (≥40cm(2)). Complement consumption was inhibited when PVMA NPs were cross-linked with diamino groups (1,3-diaminopropane), indicating the role of hydroxyl groups as responsible of the phenomenon. These results favour a model whereby PVMA NPs adjuvant activate complement on site to attract immature antigen presenting cells that are activated through TLR2 and TLR4.

Evaluation of a Novel Enzyme-Linked Immunosorbent Assay To Detect Immunoglobulin G Antibody to Enolase for Serodiagnosis of Invasive Candidiasis

ABSTRACT The performance of a new test to detect antibodies to Candida albicans recombinant enolase was investigated in 47 immunocompromised and 51 immunocompetent patients. The sensitivity, specificity, and positive and negative predictive values of the test for the diagnosis of invasive candidiasis were 81.0, 83.9, 79.1, and 85.5%, respectively.

Recent progress towards development of a Shigella vaccine

The burden of dysentery due to shigellosis among children in the developing world is still a major concern. A safe and efficacious vaccine against this disease is a priority, since no licensed vaccine is available. This review provides an update of vaccine achievements focusing on subunit vaccine strategies and the forthcoming strategies surrounding this approach. In particular, this review explores several aspects of the pathogenesis of shigellosis and the elicited immune response as being the basis of vaccine requirements. The use of appropriate Shigella antigens, together with the right adjuvants, may offer safety, efficacy and more convenient delivery methods for massive worldwide vaccination campaigns.

Development of poly(anhydride) nanoparticles loaded with peanut proteins: the influence of preparation method on the immunogenic properties.

Allergen-specific immunotherapy is based on the administration of allergens with the main disadvantage of inducing an allergic reaction. Within this context, we report the generation of an adjuvant and allergen-delivery system for peanut allergen immunotherapy with reduced IgE induction. Therefore, we prepared and characterized poly(anhydride) nanoparticles loaded with peanut proteins using the solvent displacement method, with some modifications in the manufacturing process. The precipitation of polymer was performed with either a mixture of ethanol and water or water. The resultant nanoparticles were dried by either freeze-drying or spray-drying, respectively. Poly(anhydride) nanoparticles loaded with peanut proteins were successfully developed, achieving both high encapsulation efficiency (70-80%) and manufacturing yield (60-80%). After intradermal immunization of mice (C57Bl/6) with peanut proteins incorporated into poly(anhydride) nanoparticles, a strong mixed T(H)1/T(H)2-type immune response was observed. Furthermore, we also provide, to our knowledge for the first time, clear evidence of the influence of formulation design on the immunostimulatory properties of nanoparticles. Taken together, our findings indicate that poly(anhydride) nanoparticles are efficient stimulators of immune responses and promising adjuvants and allergen-delivery systems applied for immunotherapy.

Nanoparticle-based vaccine for mucosal protection against Shigella flexneri in mice

Shigellosis is one of the leading causes of diarrhea worldwide with more than 130 million cases annually. Hence, the research of an effective vaccine is still a priority. Unfortunately, a safe and efficacious vaccine is not available yet. We have previously demonstrated the capacity of outer membrane vesicles (OMVs) to protect mice against an experimental infection with Shigella flexneri. Now, we present results on the capacity of this antigenic complex to confer a longer-term protection by oral or nasal routes when encapsulated into nanoparticles. OMVs were encapsulated in poly(anhydride) nanoparticles (NP) prepared by a solvent displacement method with the copolymer poly methyl vinyl ether/maleic anhydride. OMVs loaded into nanoparticles (NP-OMVs) were homogeneous and spherical in shape, with a size of 148nm (PdI=0.2). BALB/c mice were immunized with OMVs either free or encapsulated in nanoparticles by nasal (20μg or 10μg of OMVs) or oral route (100μg or 50μg of OMVs). All immunized animals remained in good health after administration. Challenge infection was performed intranasally on week 8th with a lethal dose of 5×10(7)CFU/mouse of S. flexneri 2a. The number of dead mice after challenge was recorded daily. Results confirmed the value of OMVs as a vaccine. By oral route, the OMV-vaccine was able to protect independently either the dose or the formulation. When vaccine was delivered by nasal route, encapsulation into NPs resulted beneficial in increasing protection from 40% up to 100% when low dose was administered. These results are extraordinary promising and put in relevance the positive effect of nanoencapsulation of the OMV subcellular vaccine.

Immunogenicity of Peanut Proteins Containing Poly(Anhydride) Nanoparticles

ABSTRACT In the last decade, peanut allergy has increased substantially. Significant differences in the prevalence among different countries are attributed to the type of thermal processing. In spite of the high prevalence and the severe reaction induced by peanuts, there is no immunotherapy available. The aim of this work was to evaluate the potential application of poly(anhydride) nanoparticles (NPs) as immunoadjuvants for peanut oral immunotherapy. NPs loaded with raw or roasted peanut proteins were prepared by a solvent displacement method and dried by either lyophilization or spray-drying. After physicochemical characterization, their adjuvant capacity was evaluated after oral immunization of C57BL/6 mice. All nanoparticle formulations induced a balanced TH1 and TH2 antibody response, accompanied by low specific IgE induction. In addition, oral immunization with spray-dried NPs loaded with peanut proteins was associated with a significant decrease in splenic TH2 cytokines (interleukin 4 [IL-4], IL-5, and IL-6) and enhancement of both TH1 (gamma interferon [IFN-γ]) and regulatory (IL-10) cytokines. In conclusion, oral immunization with poly(anhydride) NPs, particularly spray-dried formulations, led to a pro-TH1 immune response.

Mimicking microbial strategies for the design of mucus-permeating nanoparticles for oral immunization

Abstract Dealing with mucosal delivery systems means dealing with mucus. The name mucosa comes from mucus, a dense fluid enriched in glycoproteins, such as mucin, which main function is to protect the delicate mucosal epithelium. Mucus provides a barrier against physiological chemical and physical aggressors (i.e., host secreted digestive products such as bile acids and enzymes, food particles) but also against the potentially noxious microbiota and their products. Intestinal mucosa covers 400m2 in the human host, and, as a consequence, is the major portal of entry of the majority of known pathogens. But, in turn, some microorganisms have evolved many different approaches to circumvent this barrier, a direct consequence of natural co-evolution. The understanding of these mechanisms (known as virulence factors) used to interact and/or disrupt mucosal barriers should instruct us to a rational design of nanoparticulate delivery systems intended for oral vaccination and immunotherapy. This review deals with this mimetic approach to obtain nanocarriers capable to reach the epithelial cells after oral delivery and, in parallel, induce strong and long-lasting immune and protective responses.

Towards a non-living vaccine against Shigella flexneri: from the inactivation procedure to protection studies

Shigellosis is one of the leading causes of diarrhea worldwide with more than 165 million cases annually. Hence, a vaccine against this disease is a priority, but no licensed vaccine is still available. Considering target population as well as intrinsic risks of live attenuated vaccines, non-living strategies appear as the most promising candidates. Remarkably, the preservation of antigenic properties is a major concern since inactivation methods of bacteria affect these qualities. We previously reported the use of a subcellular antigen complex for vaccination against shigellosis, based on outer membrane vesicles (OMVs) released from Shigella flexneri. Now, we describe in more detail the employment of binary ethylenimine (BEI) for inactivation of Shigella and its subsequent effect on the antigenic conservation of the vaccinal product. Results demonstrate the effectiveness of BEI treatment to completely inactivate Shigella cells without disturbing the antigenicity and immunogenicity of the OMVs. Thus, OMVs harvested after BEI inactivation were able to protect mice against an experimental infection with S. flexneri.

Long-term follow-up in patients with congenital myasthenic syndrome due to RAPSN mutations

Rapsyn (RAPSN) mutations are a common cause of postsynaptic congenital myasthenic syndromes. We present a comprehensive description of the clinical and molecular findings of ten patients with CMS due to mutations in RAPSN, mostly with a long-term follow-up. Two patients were homozygous and eight were heterozygous for the common p.Asn88Lys mutation. In three of the heterozygous patients we have identified three novel mutations (c.869T > C; p.Leu290Pro, c.1185delG; p.Thr396Profs*12, and c.358delC; p.Gln120Serfs*8). In our cohort, the RAPSN mutations lead to a relatively homogeneous phenotype, characterized by fluctuating ptosis, occasional bulbar symptoms, neck muscle weakness, and mild proximal muscle weakness with exacerbations precipitated by minor infections. Interestingly, episodic exacerbations continue to occur during adulthood. These were characterized by proximal limb girdle weakness and ptosis, and not so much by respiratory insufficiency after age 6. All patients presented during neonatal period and responded to cholinergic agonists. In most of the affected patients, additional use of 3,4-diaminopyridine resulted in significant clinical benefit. The disease course is stable except for intermittent worsening.