Platform for the generation of oral vaccines based on protozoan surface proteins.

The international spread of infectious diseases is a global problem of health security. Vaccination is one of the most successful and profitable health interventions. Oral immunization has significant advantages over the widely used parental vaccines. Intestinal and free-living protozoa express on their surface a dense layer of proteins that protect them from hostile environmental conditions. The use of variable surface proteins (VSPs), such as those of the intestinal protozoan Giardia lamblia, is a feasible mechanism for the generation of oral vaccines, since they are highly immunogenic as well as resistant to changes in pH and proteases. In a recently published article, we showed that these properties of VSPs can be exploited to protect and enhance the immunogenicity of vaccine antigens, thus enabling their oral administration. We recently generated an oral vaccine against influenza virus composed of virus-like particles (VLPs) containing VSPs of G. lamblia and the HA antigen (viral hemagglutinin) in its envelope. When administered orally to mice, these coated particles elicit HA-specific humoral (systemic and local) and cellular responses, without the need of any additional adjuvant. Treated mice are protected against viral challenge as well as against the development of tumors expressing the HA vaccine antigen.

La propagación internacional de enfermedades infecciosas constituye un problema global de seguridad sanitaria. La vacunación es una de las intervenciones en salud más exitosas y efectivas. La administración por vía oral presenta ventajas significativas sobre la vía parental utilizada comúnmente. Protozoarios intestinales y de vida libre expresan en su superficie una densa capa de proteínas que los protegen de condiciones ambientales hostiles. La utilización de proteínas de superficie variante-específicas o VSPs (del inglés "Variant-specific Surface Proteins") tales como las del protozoario intestinal Giardia lamblia constituye un enfoque eficiente para la generación de vacunas orales, dada su alta inmunogenicidad y su resistencia a cambios de pH y proteasas. En un trabajo reciente mostramos que estas propiedades pueden ser explotadas para proteger antígenos vacunales y potenciar su inmunogenicidad, facilitando así su administración oral. Como modelo inicial, generamos una vacuna oral contra el virus de la influenza compuesta por partículas similares a virus (VLPs, del inglés "virus-like particles") que contienen en su envoltorio VSPs de G. lamblia y el antígeno HA (hemaglutinina del virus de la influenza). La administración oral a ratones de estas partículas recubiertas con VSPs y HA induce una respuesta inmune humoral (sistémica y de mucosa) y celular específica para HA sin la necesidad de adyuvantes externos. La respuesta inmune generada protege frente al desafío con el virus y también frente al desarrollo de tumores que expresan el antígeno vacunal HA.

Efficient oral vaccination by bioengineering virus-like particles with protozoan surface proteins.

Intestinal and free-living protozoa, such as Giardia lamblia, express a dense coat of variant-specific surface proteins (VSPs) on trophozoites that protects the parasite inside the host's intestine. Here we show that VSPs not only are resistant to proteolytic digestion and extreme pH and temperatures but also stimulate host innate immune responses in a TLR-4 dependent manner. We show that these properties can be exploited to both protect and adjuvant vaccine antigens for oral administration. Chimeric Virus-like Particles (VLPs) decorated with VSPs and expressing model surface antigens, such as influenza virus hemagglutinin (HA) and neuraminidase (NA), are protected from degradation and activate antigen presenting cells in vitro. Orally administered VSP-pseudotyped VLPs, but not plain VLPs, generate robust immune responses that protect mice from influenza infection and HA-expressing tumors. This versatile vaccine platform has the attributes to meet the ultimate challenge of generating safe, stable and efficient oral vaccines.

Cytokines, Antibodies, and Histopathological Profiles during Giardia Infection and Variant-Specific Surface Protein-Based Vaccination.

Giardiasis is one of the most common human intestinal diseases worldwide. Several experimental animal models have been used to evaluate Giardia infections, with gerbils (Meriones unguiculatus) being the most valuable model due to their high susceptibility to Giardia infection, abundant shedding of cysts, and pathophysiological alterations and signs of disease similar to those observed in humans. Here, we report cytokine and antibody profiles both during the course of Giardia infection in gerbils and after immunization with a novel oral vaccine comprising a mixture of purified variant-specific surface proteins (VSPs). Transcript levels of representative cytokines of different immune profiles as well as macro- and microtissue alterations were assessed in Peyer's patches, mesenteric lymph nodes, and spleens. During infection, cytokine responses showed a biphasic profile: an early induction of Th1 (gamma interferon [IFN-γ], interleukin-1ß [IL-1ß], IL-6, and tumor necrosis factor [TNF]), Th17 (IL-17), and Th2 (IL-4) cytokines, together with intestinal alterations typical of inflammation, followed by a shift toward a predominant Th2 (IL-5) response, likely associated with a counterregulatory mechanism. Conversely, immunization with an oral vaccine comprising the entire repertoire of VSPs specifically showed high levels of IL-17, IL-6, IL-4, and IL-5, without obvious signs of inflammation. Both immunized and infected animals developed local (intestinal secretory IgA [S-IgA]) and systemic (serum IgG) humoral immune responses against VSPs; however, only infected animals showed evident signs of giardiasis. This is the first comprehensive report of cytokine expression and anti-Giardia antibody production during infection and VSP vaccination in gerbils, a reliable model of the human disease.

In vivo immunomodulatory effect of the lectin from edible mushroom Agaricus bisporus.

Lectins are glycan-binding proteins that are resistant to digestion in the gastrointestinal tract and enter intact to blood circulation. The aim of this study was to evaluate the influence of edible mushroom Agaricus bisporus lectin (ABL) on innate and adaptive immune responses as well as its effect in two different experimental pathologies that involve the immune system. ABL inhibited in vitro nitric oxide (NO) production by mouse peritoneal macrophages in response to the pro-inflammatory stimuli lipopolysaccharides (LPS). However, it did not modify the activity of arginase, showing that while ABL downregulates M1 activation, it does not affect M2 activation. ABL also inhibited mononuclear cell proliferation in response to mitogen Con A, or in a mixed lymphocyte reaction. During the in vivo studies, oral administration of ABL to BALB/c mice induced a marked inhibition of NO production by peritoneal macrophages after LPS stimuli. The influence of ABL on tumor growth was studied in BALB/c mice receiving daily oral doses of ABL and implanted with CT26 tumor cells. ABL treatment induced significantly higher rate of tumor growth when compared with control mice. On the other hand, oral ABL administration in Wistar rats induced a marked diminution of the incidence of the disease and the severity of the clinical signs of experimental autoimmune encephalomyelitis. We can conclude that ABL has an in vivo immunomodulatory effect reducing the innate and adaptive responses. This food lectin shows potential therapeutic application on control of inflammatory autoimmune pathologies.

Vaccination of domestic animals with a novel oral vaccine prevents Giardia infections, alleviates signs of giardiasis and reduces transmission to humans.

Giardia lamblia is a human intestinal parasite and one of the most frequent enteric pathogen of companion animals. Clinical manifestations of giardiasis, such as diarrhoea, anorexia, weight loss and lethargy, have been associated with Giardia infections in both domestic and farm animals. A few anti-parasitic drugs are routinely used to treat giardiasis, but re-infections are common and drug-resistant strains have already been reported. Unfortunately, efficient vaccines against Giardia are not available. Giardia undergoes antigenic variation; through this mechanism, parasites can avoid the host's immune defenses, causing chronic infections and/or re-infections. Antigenic variation is characterised by a continuous switch in the expression of members of a homologous family of genes encoding surface antigens. In a previous report, we indicated that in Giardia, the mechanism responsible for the exchange of variant-specific surface proteins (VSPs) involves the RNA interference (RNAi) pathway. From a repertoire of ~200 VSP genes, only one is expressed on the surface of single trophozoites; however, RNAi machinery disruption generates trophozoites that express the complete VSP repertoire. We also demonstrated that gerbils orally immunised with VSPs isolated from these altered parasites showed high levels of protection. Here we tested this vaccine in cats and dogs, and found that it is highly efficient in preventing new infections and reducing chronic giardiasis in domestic animals both in experimental and natural infections. Remarkably, immunisation of dogs in a highly endemic area strongly decreased the percentage of infected children in the community, suggesting that this vaccine would block the zoonotic transmission of the disease.

Diphenyl diselenide-modulation of macrophage activation: down-regulation of classical and alternative activation markers.

Diphenyl diselenide (PhSe)(2), a simple organoselenium compound, possesses interesting pharmacological properties that are under extensive research. As macrophages respond to microenvironmental stimuli and can display activities engaged in the initiation and the resolution of inflammation, in the present report we describe the ability of (PhSe)(2) to modulate the macrophage activation. Our data indicate that (PhSe)(2) could inhibit the NO production in a dose-dependent fashion in peritoneal macrophages activated by LPS or treated with vehicle alone. We could demonstrate that this effect correlated with a reduction in the expression of the inducible NO synthase in (PhSe)(2)-treated cells. Furthermore, (PhSe)(2) suppressed the production of reactive oxygen species, diminished the activity of the arginase enzyme, and the accumulation of nitrotyrosine modified proteins in LPS-stimulated macrophages. This compound also diminished the antigen presentation capacity of classically activated macrophages, as it reduced MHCII and CD86 expression. In addition, (PhSe)(2) modulated the alternative activation phenotype of macrophages. Dexamethasone-activated macrophages presented higher production of IL-10 and CD206, which were both down-regulated by the addition of (PhSe)(2). These results suggest that (PhSe)(2) possesses antioxidant and anti-inflammatory activities in classically-activated macrophages. We could demonstrate that (PhSe)(2) can be also utilized to modulate the alternative activation phenotype of macrophages.

Protective effect of a synapsin peptide genetically fused to the B subunit of Escherichia coli heat-labile enterotoxin in rat autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease with similarities to multiple sclerosis that requires the activation of auto reactive T cells that infiltrate the central nervous system. In previous studies we have shown that intraperitoneal administration of synaptosomal antigens could suppress EAE. Herein we examined the effect in this animal model of a fusion protein comprising the C domain of synapsin Ia and the B subunit of Escherichia coli heat-labile enterotoxin (LTBSC). Oral administration to rats of low amounts of LTBSC induced immunological systemic tolerance to the encephalitogenic myelin basic protein. Treatment with LTBSC prior to EAE induction diminished disease incidence, DTH reaction to myelin basic protein, and central nervous system inflammation. LTBSC treatment also reduced the specific T-cell proliferative response to myelin basic protein, decreased nitric oxide production, and augmented arginase activity by peritoneal macrophages. All animals challenged for EAE developed antibody response specific for myelin basic protein, but rats treated with LTBSC showed a lower IgG2b/IgG1 ratio, indicating a shift to a Th2-type milieu. The data presented here suggest that well-conserved synapsin peptides conjugated to the B subunit of enterotoxins from the cholera toxin family have a protective role and provide a potential therapeutic tool for intervention in EAE as well as in multiple sclerosis.