Preparation and evaluation of chitosan nanoparticles containing CtxB antigen against Vibrio cholera.

Vibrio cholera is a Gram-negative pathogen that causes diarrheal disease. The B subunit of Chlora toxin (CtxB) is one of the most important antigens of Vibrio cholera in which mediates the attachment of the bacteria to target cells. The aim of this study was to prepare chitosan nanoparticles containing CtxB and evaluate the effect of the antigen entrapment on the immunogenicity of this antigen. For this, the pET28a vector was induced using IPTG. Recombinant CtxB protein was expressed and purified using Ni-NTA column and finally was confirmed by western blotting. Following the confirmation of the protein entrapment onto the chitosan nanoparticles, the formulation was prescribed to BALB/c mice in three groups, including oral, oral-injection and injection groups. Serum and fecal IgA and IgG were evaluated by ELISA test. Finally, challenge of immunized mice was performed using Ctx toxin and rabbit ileal loop test. Using SDS-PAGE and western blotting, the 17.5 kDa recombinant CtxB was confirmed. Size electrical charge and of nanoparticles were determined and approved by Zetasizer. Nanoparticles prescription showed 1/102400 IgG endpoint titers for injection groups and 1/1600, 1/6400 for oral, oral-injection groups respectively and Serum and fecal IgA endpoint titers showed above 1/160 in all groups. Furthermore, immunized mice were able to neutralize Ctx toxin by ileal loop test. The CtxB is a suitable immunogen of V. cholera to be incorporated in both protective and preventive vaccines. Chitosan nanoparticles improve the immune responses and it may be used as a carrier for vaccine delivery.

The Occurrence of Antibodies Against Gluten in Children with Autism Spectrum Disorders Does Not Correlate with Serological Markers of Impaired Intestinal Permeability.

There is evidence that children with autism spectrum disorders (ASDs) display an increased immune reactivity against gluten, which is supposed to be the effect of intestinal barrier abnormalities. The aim of study was to evaluate the relation of antibody induced by gluten to zonulin and intestinal fatty acid binding proteins (I-FABP), that is, serological markers of an impaired gut barrier. The study included 77 patients with ASDs. Zonulin, I-FABP, celiac-specific antibodies, anti-gliadin antibodies (AGA), and antibodies against neural transglutaminase 6 (TG6) of immunoglobulin (Ig) A and IgG classes were detected in sera. Celiac-specific antibodies were negative in all ASD children, four children (5.2%) had positive anti-TG6 antibodies, and increased AGA-IgG production was found in 21 patients (27.3%). Mean levels of zonulin and I-FABP in ASD patients were similar to those found in healthy controls and revealed a negative correlation with age, whereas regression analysis revealed a significant positive relationship between antibody production and the age. Serum concentrations of zonulin and I-FABP showed no statistically significant association with antibody positivity. An increased production of antibodies related to gliadin and neural TG6 in ASD children is not related to serological markers of an impaired intestinal barrier.

A New Way to Beat Intestinal Pathogens.

In the gastrointestinal tract, the tug of war for iron may provide a new way to vaccinate. Recent work shows that immunizing mice with siderophores (small molecules that microbes produce to capture iron) foils pathogen colonization and may instead allow a commensal to expand.

Cocoa Diet Prevents Antibody Synthesis and Modifies Lymph Node Composition and Functionality in a Rat Oral Sensitization Model.

Cocoa powder, a rich source of polyphenols, has shown immunomodulatory properties in both the intestinal and systemic immune compartments of rats. The aim of the current study was to establish the effect of a cocoa diet in a rat oral sensitization model and also to gain insight into the mesenteric lymph nodes (MLN) activities induced by this diet. To achieve this, three-week-old Lewis rats were fed either a standard diet or a diet with 10% cocoa and were orally sensitized with ovalbumin (OVA) and with cholera toxin as a mucosal adjuvant. Specific antibodies were quantified, and lymphocyte composition, gene expression, and cytokine release were established in MLN. The development of anti-OVA antibodies was almost totally prevented in cocoa-fed rats. In addition, this diet increased the proportion of TCRγδ+ and CD103+CD8+ cells and decreased the proportion of CD62L+CD4+ and CD62L+CD8+ cells in MLN, whereas it upregulated the gene expression of OX40L, CD11c, and IL-1ß and downregulated the gene expression of IL-17α. In conclusion, the cocoa diet induced tolerance in an oral sensitization model accompanied by changes in MLN that could contribute to this effect, suggesting its potential implication in the prevention of food allergies.

Mucosally administered Lactobacillus surface-displayed influenza antigens (sM2 and HA2) with cholera toxin subunit A1 (CTA1) Induce broadly protective immune responses against divergent influenza subtypes.

The development of a universal influenza vaccine that provides broad cross protection against existing and unforeseen influenza viruses is a critical challenge. In this study, we constructed and expressed conserved sM2 and HA2 influenza antigens with cholera toxin subunit A1 (CTA1) on the surface of Lactobacillus casei (pgsA-CTA1sM2HA2/L. casei). Oral and nasal administrations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and their isotypes (IgG1 & IgG2a) as well as mucosal IgA. The mucosal administration of pgsA-CTA1sM2HA2/L. casei may also significantly increase the levels of sM2- or HA2-specific cell-mediated immunity because increased release of both IFN-γ and IL-4 was observed. The recombinant pgsA-CTA1sM2HA2/L. casei provided better protection of BALB/c mice against 10 times the 50% mouse lethal doses (MLD50) of homologous A/EM/Korea/W149/06(H5N1) or A/Aquatic bird/Korea/W81/2005 (H5N2) and heterologous A/Puerto Rico/8/34(H1N1), or A/Chicken/Korea/116/2004(H9N2) or A/Philippines/2/08(H3N2) viruses, compared with L. casei harboring sM2HA2 and also the protection was maintained up to seven months after administration. These results indicate that recombinant L. casei expressing the highly conserved sM2, HA2 of influenza and CTA1 as a mucosal adjuvant could be a potential mucosal vaccine candidate or tool to protect against divergent influenza viruses for human and animal.

Cutaneous or respiratory exposures to peanut allergens in mice and their impacts on subsequent oral exposure.

BACKGROUND: Recent data suggested that non-gastrointestinal exposure can lead to sensitisation to food allergens. We thus assessed the immune impact of respiratory or cutaneous exposure to peanut proteins on non-altered epithelium and investigated the effect of such pre-exposure on subsequent oral administration of peanut. METHODS: BALB/cJ mice were exposed to purified Ara h 1 or to a non-defatted roasted peanut extract (PE) by simple deposit of allergens solutions on non-altered skin or in the nostrils. Exposures were performed 6 times at weekly intervals. Pre-exposed mice then received intra-gastric administrations of PE alone or in the presence of the Th2 mucosal adjuvant cholera toxin (CT). The specific humoral and cellular immune response was assessed throughout the protocol. RESULTS: Both cutaneous and respiratory exposures led to the production of specific IgG1. Local and systemic IL-5 and IL-13 production were also evidenced, demonstrating activation of specific Th2 cells. This effect was dose-dependent and most efficient via the respiratory route. Moreover, these pre-exposures led to the production of specific IgE antibodies after gavage with PE, whatever the presence of CT. CONCLUSIONS: Cutaneous or respiratory exposures to peanut induce Th2 priming in mice. Moreover, pre-exposures promote further sensitisation via the oral route without the use of CT; this proposes a new adjuvant-free experimental model of sensitisation to food that may reflect a realistic exposure pattern in infants. These results also suggest that non-gastrointestinal peanut exposure should be minimised in high-risk infants, even those with non-altered skin, to potentially reduce allergic sensitisation to this major food allergen.

Regulation of intestinal IgA responses by dietary palmitic acid and its metabolism.

Enhancement of intestinal IgA responses is a primary strategy in the development of oral vaccine. Dietary fatty acids are known to regulate host immune responses. In this study, we show that dietary palmitic acid (PA) and its metabolites enhance intestinal IgA responses. Intestinal IgA production was increased in mice maintained on a PA-enriched diet. These mice also showed increased intestinal IgA responses against orally immunized Ag, without any effect on serum Ab responses. We found that PA directly stimulates plasma cells to produce Ab. In addition, mice receiving a PA-enriched diet had increased numbers of IgA-producing plasma cells in the large intestine; this effect was abolished when serine palmitoyltransferase was inhibited. These findings suggest that dietary PA regulates intestinal IgA responses and has the potential to be a diet-derived mucosal adjuvant.

As a genetic adjuvant, CTA improves the immunogenicity of DNA vaccines in an ADP-ribosyltransferase activity- and IL-6-dependent manner.

Cholera toxin (CT) and its subunits (A and B) have been intensively investigated as adjuvants for protein-based vaccines. Their underlying mechanisms vary with respect to the inoculation route used. By fusing the CTA gene to either the HIV-1-derived Tat-Rev-Vif-Integrase-Nef fusion gene or the OVA gene, our study showed that the fusion of CTA in these DNA vaccines had no cytotoxic effect in vitro and significantly improved both the quantity and quality of the elicited CD8(+) T cell responses. Further experiments identified that the fusion of CTA in these DNA vaccines augmented the secretion of IL-6 in a manner that was dependent on its ADP-ribosyltransferase activity, and protein kinase A (PKA) was found to be the major mediator of its downstream signaling. By site-directed mutagenesis of the ADP-ribosyltransferase catalytic center and in vivo RNAi, we demonstrated that the ADP-ribosyltransferase activity and the upregulation of IL-6 were required for the CTA gene-mediated adjuvant effect. These findings demonstrate that when fused to an immunogen gene, the CTA gene could serve as a potent genetic adjuvant, providing new insights into the mechanisms of CTA as an adjuvant.

Cross-allergic reactions to legumes in lupin and fenugreek-sensitized mice.

Several legumes may induce allergy, and there is extensive serological cross-reactivity among legumes. This cross-reactivity has traditionally been regarded to have limited clinical relevance. However, the introduction of novel legumes to Western countries may have changed this pattern, and in some studies cross-allergy to lupin has been reported in more than 60% of peanut-allergic patients. We wanted to explore cross-reactions among legumes using two newly established mouse models of food allergy. Mice were immunized perorally with fenugreek or lupin with cholera toxin as adjuvant. The mice were challenged with high doses of fenugreek, lupin, peanut or soy, and signs of anaphylactic reactions were observed. Cross-allergic mechanisms were investigated using serum mouse mast cell protease-1 (MMCP-1), antibody responses, immunoblotting and ex vivo production of cytokines by spleen cells. Signs of cross-allergy were observed for all the tested legumes in both models. The cross-allergic symptoms were milder and affected fewer mice than the primary allergic responses. The cross-allergy was reflected to a certain extent in the antibody and T-cell responses, but not in serum MMCP-1 levels. Cross-allergy to peanut, soy, fenugreek and lupin was observed in lupin-sensitized and fenugreek-sensitized mice. Differences in serological responses between primary allergy and cross-allergy might be due to mediation through different immune mechanisms or reflect different epitope affinity to IgE. These differences need to be further investigated.

Oral-nasopharyngeal dendritic cells mediate T cell-independent IgA class switching on B-1 B cells.

Native cholera toxin (nCT) as a nasal adjuvant was shown to elicit increased levels of T-independent S-IgA antibody (Ab) responses through IL-5- IL-5 receptor interactions between CD4+ T cells and IgA+ B-1 B cells in murine submandibular glands (SMGs) and nasal passages (NPs). Here, we further investigate whether oral-nasopharyngeal dendritic cells (DCs) play a central role in the induction of B-1 B cell IgA class switch recombination (CSR) for the enhancement of T cell-independent (TI) mucosal S-IgA Ab responses. High expression levels of activation-induced cytidine deaminase, Iα-Cµ circulation transcripts and Iµ-Cα transcripts were seen on B-1 B cells purified from SMGs and NPs of both TCRß⁻/⁻ mice and wild-type mice given nasal trinitrophenyl (TNP)-LPS plus nCT, than in the same tissues of mice given nCT or TNP-LPS alone. Further, DCs from SMGs, NPs and NALT of mice given nasal TNP-LPS plus nCT expressed significantly higher levels of a proliferation-inducing ligand (APRIL) than those in mice given TNP-LPS or nCT alone, whereas the B-1 B cells in SMGs and NPs showed elevated levels of transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI) expression. Interestingly, high frequencies of IgA+ B-1 B cells were induced when peritoneal IgA⁻ IgM+ B cells were stimulated with mucosal DCs from mice given nasal TNP-LPS plus nCT. Taken together, these findings show that nasal nCT plays a key role in the enhancement of mucosal DC-mediated TI IgA CSR by B-1 B cells through their interactions with APRIL and TACI.

Cholera toxin: a paradigm of a multifunctional protein.

Cholera toxin (CT) was discovered exactly half a century ago by S.N. De. We have come a long way since this epoch-making discovery. Retrospectively, science had to wait a long time since Koch's prediction of the existence of a toxin, and its actual discovery by De. CT is not just another enterotoxin that causes the signs and symptoms of the dreaded disease, cholera. It is unique in many respects, starting from its structure to its functions. CT is a multifunctional protein that is capable of influencing the immune system in many ways. It not only has remarkable adjuvant properties, but also acts as an anti-inflammatory agent, by modulating specific signal transduction pathways. Its immunomodulatory properties can be harnessed for treatment of various autoimmune disorders, and have shown great promise in the area of immunotherapeutics. CT can truly be considered as a paradigm of a multifunctional protein.

Expression of truncated CTB::VP60 in tobacco exhibited no immunogenicity in rabbits.

Despite several optimizations, the production of CTB::VP60 antigen fusion proteins in tobacco is still very low. This might be due to the size of the fusion partner VP60 (579 aa). Hence, two different N-terminal truncations of VP60 were fused to CTB, either with or without an ER retention signal. CTB::VP60 expression levels, in vitro and in vivo antigenicity and immunogenicity were analyzed in plants carrying one of four different transgenes. Only one of the truncated CTB::VP60 fusions (365 aa) directed to the endoplasmic reticulum led to similar but not enhanced expression levels as compared to the complete protein in tobacco and possessed similar in vitro antigenicity. In contrast to the complete protein, no anti-VP60-specific antibodies were induced in rabbits after the intramuscular application of plant extracts containing the truncated protein.

Cholera toxin B subunit-domain III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants.

Envelope glycoprotein E of the dengue virus, which plays a crucial role in its entry into host cells, has an immunogenic domain III (EIII, amino acids 297-394), which is capable of inducing neutralizing antibodies. However, mice immunized with EIII protein without adjuvant elicited low immune responses. To improve low immune responses, a DNA fragment, consisting of cholera toxin B subunit and EIII gene (CTB-EIII), was constructed and introduced into tobacco plant cells (Nicotiana tabacum L. cv. MD609) by Agrobacterium tumefaciens-mediated transformation methods. The integration and transcription of CTB-EIII fusion gene were confirmed in transgenic plants by genomic DNA PCR amplification and Northern blot analysis, respectively. The results of immunoblot analysis with anti-CTB and anti-dengue virus antibodies showed the expression of the CTB-EIII fusion protein in transgenic plant extracts. Based on the G(M1)-ELISA results, the CTB-EIII protein expressed in plants showed the biological activity for intestinal epithelial cell membrane glycolipid receptor, G(M1)-ganglioside, and its expression level was up to about 0.019% of total soluble protein in transgenic plant leaf tissues. The feasibility of using a plant-produced CTB-EIII fusion protein to generate immunogenicity against domain III will be tested in future animal experiments.

Enteric reovirus infection stimulates peanut-specific IgG2a responses in a mouse food allergy model.

IgE-mediated food allergies are an important cause of life-threatening hypersensitivity reactions. Orally administered peanut antigens mixed with the mucosal adjuvant cholera toxin (CT) induce a strong peanut extract (PE)-specific serum IgE response that is correlated with T-helper type 1 (Th1) and type 2 (Th2)-like T-cell responses. This study was conducted to determine if respiratory enteric orphan virus (reovirus), a non-pathogenic virus that induces robust Th1-mediated mucosal and systemic responses could modulate induction of PE-specific allergic responses when co-administered with PE. Young mice were orally exposed to PE mixed with CT, reovirus, or both CT and reovirus. As expected, CT promoted PE-specific serum IgE, IgG1, and IgG2a and intestinal IgA production as well as splenic Th1- and Th2-associated cytokine recall responses. Reovirus did not alter PE-specific serum IgE and IgG1 levels, but substantially increased the PE-specific IgG2a response when co-administered with PE with or without CT. Additionally, reovirus significantly decreased the percentage of the Peyer's patch CD8+ T-cells and Foxp3+CD4+ T-regulatory cells when co-administered with PE. These results demonstrate that an acute mucosal reovirus infection and subsequent Th1 immune response is capable of modulating the Th1/Th2 controlled humoral response to PE. The reovirus-mediated increase in the PE-specific IgG2a antibody response may have therapeutic implications as increased levels of non-allergenic PE-specific IgG2a could block PE antigens from binding to IgE-sensitized mast cells.

Intranasal immunization with phosphorylcholine induces antigen specific mucosal and systemic immune responses in mice.

Phosphorylcholine (PC) is a structural component of a wide variety of pathogens including Streptococcus pneumoniae and Haemophilus influenzae, and anti-PC immune responses are known to protect mice against invasive bacterial diseases. The present study tested the capability of PC as an intranasal plurispecific vaccine against upper airway infections. BALB/c mice immunized with intranasal PC-keyhole limpet hemocyanin (KLH) plus cholera toxin (CT) as a mucosal adjuvant showed increased PC-specific IgM in serum, IgA in nasal wash and saliva, and numbers of PC-specific nasal and splenic antibody producing cells. Enhanced production of IL-4 and IFN-gamma by CD4+ T cells indicated the participation of Th2- and Th1-type cells. Salivary IgA antibodies produced by intranasal immunization with PC-KLH plus CT reacted to most strains of S. pneumoniae and H. influenzae. Further we demonstrated that the clearance of S. pneumoniae and H. influenzae from the nasal tract was significantly enhanced by nasal immunization with PC-KLH and CT. Thus, intranasal vaccination to induce PC-specific immune responses might help to prevent upper airway infections caused by S. pneumoniae and H. influenzae.

Oral immunogenicity of the plant proteinase bromelain.

Bromelain is a natural mixture of proteolytic enzymes derived from pineapple stem that has been shown to have anti-inflammatory activity when administered orally. Although most proteins given orally without adjuvant (e.g., food) result in tolerance, we previously reported that long-term oral exposure to bromelain stimulated the development of high serum anti-bromelain antibody titers. The purpose of these studies was to further investigate the mechanisms responsible for the immunogenicity of oral bromelain. Results showed that repeated exposure was required for development of anti-bromelain antibodies, with strong antibody responses in all mice that received at least 12 doses of bromelain either orally or intragastrically over 3-6 weeks. Proteolytic activity was required for strong oral immunogenicity in the absence of conventional adjuvant, with strong serum antibody responses generated against proteolytically active bromelain and trypsin, but not against ovalbumin, lysozyme, or inactivated bromelain. Significantly higher anti-bromelain antibody titers were seen in IL-10-deficient versus wild-type mice, suggesting that simultaneous treatments that decrease IL-10 activity may further enhance systemic antibody responses following oral exposure. The antibodies generated did not affect the proteolytic activity of bromelain. The data demonstrate that proteolytically active antigens such as bromelain can stimulate both systemic and mucosal immune responses following repeated oral exposure. Further studies of the mechanisms involved in generation of immune responses following oral exposure to proteolytically active antigens can lead to a better understanding of mechanisms of oral tolerance and to the development of novel adjuvants for oral vaccines.

Splenic marginal zone dendritic cells mediate the cholera toxin adjuvant effect: dependence on the ADP-ribosyltransferase activity of the holotoxin.

The in vivo mechanisms of action of most vaccine adjuvants are poorly understood. In this study, we present data in mice that reveal a series of critical interactions between the cholera toxin (CT) adjuvant and the dendritic cells (DC) of the splenic marginal zone (MZ) that lead to effective priming of an immune response. For the first time, we have followed adjuvant targeting of MZ DC in vivo. We used CT-conjugated OVA and found that the Ag selectively accumulated in MZ DC following i.v. injections. The uptake of Ag into DC was GM1 ganglioside receptor dependent and mediated by the B subunit of CT (CTB). The targeted MZ DC were quite unique in their phenotype: CD11c(+), CD8alpha(-), CD11b(-), B220(-), and expressing intermediate or low levels of MHC class II and DEC205. Whereas CTB only delivered the Ag to MZ DC, the ADP-ribosyltransferase activity of CT was required for the maturation and migration of DC to the T cell zone, where these cells distinctly up-regulated CD86, but not CD80. This interaction appeared to instruct Ag-specific CD4(+) T cells to move into the B cell follicle and strongly support germinal center formations. These events may explain why CT-conjugated Ag is substantially more immunogenic than Ag admixed with soluble CT and why CTB-conjugated Ag can tolerize immune responses when given orally or at other mucosal sites.

Induction of neutralizing antibodies by a tobacco chloroplast-derived vaccine based on a B cell epitope from canine parvovirus.

The 2L21 epitope of the VP2 protein from the canine parvovirus (CPV), fused to the cholera toxin B subunit (CTB-2L21), was expressed in transgenic tobacco chloroplasts. Mice and rabbits that received protein-enriched leaf extracts by parenteral route produced high titers of anti-2L21 antibodies able to recognize the VP2 protein. Rabbit sera were able to neutralize CPV in an in vitro infection assay with an efficacy similar to the anti-2L21 neutralizing monoclonal antibody 3C9. Anti-2L21 IgG and seric IgA antibodies were elicited when mice were gavaged with a suspension of pulverized tissues from CTB-2L21 transformed plants. Combined immunization (a single parenteral injection followed by oral boosters) shows that oral boosters help to maintain the anti-2L21 IgG response induced after a single injection, whereas parenteral administration of the antigen primes the subsequent oral boosters by promoting the induction of anti-2L21 seric IgA antibodies. Despite the induced humoral response, antibodies elicited by oral delivery did not show neutralizing capacity in the in vitro assay. The high yield of the fusion protein permits the preparation of a high number of vaccine doses from a single plant and makes feasible the oral vaccination using a small amount of crude plant material. However, a big effort has still to be done to enhance the protective efficacy of subunit vaccines by the oral route.

Peanut- and cow's milk-specific IgE, Th2 cells and local anaphylactic reaction are induced in Balb/c mice orally sensitized with cholera toxin.

BACKGROUND: The development of animal models developing specific immunoglobulin (Ig)E presenting the same specificity as human IgE and similar clinical symptoms as those observed in allergic patients are of great interest for the understanding of mechanisms involved in the induction and regulation of food allergy. METHODS: Balb/c female mice were sensitized with whole peanut protein extract (WPPE) by means of intraperitoneal (i.p.) injections with alum or gavages with cholera toxin (CT). The WPPE specific IgE, IgG1 and IgG2a were monitored. Th2 cells activation was analysed assaying interleukin (IL)-4 and IL-5 vs IFNgamma on reactivated splenocytes. Local anaphylactic reaction was evaluated by assaying histamine in faecal samples. The oral sensitization protocol was further extended to cow's milk proteins (CMP). RESULTS: Balb/c mice developed high peanut-specific IgE and IgG1 responses either after i.p. or oral sensitizations. In both cases, antibodies were specific to polymer of glycinin fragments, containing polypeptides from Ara h3/4, and to a lesser extent to Ara h1 and Ara h2. Interleukin-4 and IL-5 production were evidenced. Balb/c mice could also be sensitized to CMP, as demonstrated by CMP-specific IL-4 and IL-5 secretions and induction of IgE specific for whole caseins, beta-lactoglobulin, serum bovine albumin and lactoferrin. Of interest was the occurrence of a local anaphylactic reaction in the peanut and CM models. CONCLUSIONS: In contrast with previous authors, Balb/c mice were sensitized and evidenced an allergic reaction after oral administrations of peanut or CMP plus CT, providing an interesting model for further studies on immunopathogenic mechanisms.