Immunological and Metabolomic Impacts of Administration of Cry1Ab Protein and MON 810 Maize in Mouse

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab–induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab–specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight “cultivar” effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen

Data for: Therapy with multi-epitope virus-like particles of B19 parvovirus reduce tumor growth and lung metastasis in an aggressive breast cancer mouse model

the results of the experiments corresponding to mice with triple negative breast cancer immunized with virus-like particles of parvovirus B19 containing multiple neoepitopes are shown.Female, BALB/c mice, 6–8 weeks of age were used for this study. The mice were kept in micro-isolators, with sterile filter covers, had ad libitum access to water and food, 4T1 mouse mammary carcinoma cells (ATCC, Manassas, Virginia, USA) were maintained for a limited time in vitro by passages in RPMI-1640 medium (Gibco®, Grand Island, New York, USA), containing penicillin (100 U/mL), streptomycin (100 μg/mL) and fungicide (0.75 μg/mL) (Sigma Aldrich, St. Luis, Misuri, USA) and supplemented with 10% FBS (Gibco®)The BALB/c mice were randomly separated into experimental groups (n = 5–8) as follows: vehicle (PBS), WT-VLPs (50μg), WT-VLPs + adjuvant (protoxin Cry1Ac 50 μg), rMe-VLPs (50 μg,) and rMe-VLPs + adjuvant (protoxin Cry1Ac 50 μg). The tumors were induced by subcutaneous injection, into the sixth breast of the mice, of 3 x 103 4T1 cells that were freshly obtained from cell culture, with a viability greater than 90% (evaluated by trypan blue exclusion). The treatments were administered intraperitoneally and peritumorally during the experiment every 7 days, from day 7 post tumor induction. Tumor growth was monitored with a digital caliper every 3 days and tumor volume was calculated according to the formula V = L x S2 / 2, where L is the longest side and S the shortest. On day 36 post-tumor induction, the animals were sacrificed using humane methods. The spleens, inguinal nodes, lungs, and the tumors were obtained for further analysis at indicated days.It is important to mention that in this work, we used a lower number of 4T1 cells (3 x 103) to induce orthotopic tumors in BALB/c mice. This cell concentration was chosen to avoid the induction of necrotic processes in the tumors. In fact, when a higher concentration of cells was used, we observed necrosis. We wanted to avoid the induction of necrosis because it provokes painful tissue lesions in the animals, making it necessary to sacrifice the mice prematurely, and thus complicating the evaluation of the antitumor treatments.Lung metastasisFor the analysis of the macro-metastases in the lungs, the same scheme described above was used, resulting in an orthotopic model of metastasis. During sacrifice, the lungs were filled with 1 mL of a 10% India Ink solution (Winsor & Newton, London, UK), in PBS, through the trachea with the help of a cannula. The trachea was blocked by surgical thread and the lungs were extracted and washed 3 times with 10 mL of Fekete´s solution (85 mL 70% ethanol, 10 mL 10% paraformaldehyde, and 5 mL acetic acid) and, the lungs were fixed in the same solution overnight. The macroscopic foci were counted and photographed with a stereoscopic microscope, OLYMPUS SZ CTV (Olympus, Tokyo, Japan)

Data for: Therapy with multi-epitope virus-like particles of B19 parvovirus reduce tumor growth and lung metastasis in an aggressive breast cancer mouse model

the results of the experiments corresponding to mice with triple negative breast cancer immunized with virus-like particles of parvovirus B19 containing multiple neoepitopes are shown.Female, BALB/c mice, 6–8 weeks of age were used for this study. The mice were kept in micro-isolators, with sterile filter covers, had ad libitum access to water and food, 4T1 mouse mammary carcinoma cells (ATCC, Manassas, Virginia, USA) were maintained for a limited time in vitro by passages in RPMI-1640 medium (Gibco®, Grand Island, New York, USA), containing penicillin (100 U/mL), streptomycin (100 μg/mL) and fungicide (0.75 μg/mL) (Sigma Aldrich, St. Luis, Misuri, USA) and supplemented with 10% FBS (Gibco®)The BALB/c mice were randomly separated into experimental groups (n = 5–8) as follows: vehicle (PBS), WT-VLPs (50μg), WT-VLPs + adjuvant (protoxin Cry1Ac 50 μg), rMe-VLPs (50 μg,) and rMe-VLPs + adjuvant (protoxin Cry1Ac 50 μg). The tumors were induced by subcutaneous injection, into the sixth breast of the mice, of 3 x 103 4T1 cells that were freshly obtained from cell culture, with a viability greater than 90% (evaluated by trypan blue exclusion). The treatments were administered intraperitoneally and peritumorally during the experiment every 7 days, from day 7 post tumor induction. Tumor growth was monitored with a digital caliper every 3 days and tumor volume was calculated according to the formula V = L x S2 / 2, where L is the longest side and S the shortest. On day 36 post-tumor induction, the animals were sacrificed using humane methods. The spleens, inguinal nodes, lungs, and the tumors were obtained for further analysis at indicated days.It is important to mention that in this work, we used a lower number of 4T1 cells (3 x 103) to induce orthotopic tumors in BALB/c mice. This cell concentration was chosen to avoid the induction of necrotic processes in the tumors. In fact, when a higher concentration of cells was used, we observed necrosis. We wanted to avoid the induction of necrosis because it provokes painful tissue lesions in the animals, making it necessary to sacrifice the mice prematurely, and thus complicating the evaluation of the antitumor treatments.Lung metastasisFor the analysis of the macro-metastases in the lungs, the same scheme described above was used, resulting in an orthotopic model of metastasis. During sacrifice, the lungs were filled with 1 mL of a 10% India Ink solution (Winsor & Newton, London, UK), in PBS, through the trachea with the help of a cannula. The trachea was blocked by surgical thread and the lungs were extracted and washed 3 times with 10 mL of Fekete´s solution (85 mL 70% ethanol, 10 mL 10% paraformaldehyde, and 5 mL acetic acid) and, the lungs were fixed in the same solution overnight. The macroscopic foci were counted and photographed with a stereoscopic microscope, OLYMPUS SZ CTV (Olympus, Tokyo, Japan).THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV

Bacillus thuringiensis Cry1Ac protoxin is a potent systemic and mucosal adjuvant

Recently we demonstrated that recombinant Cry1Ac protoxin from Bacillus thuringiensis is a potent systemic and mucosal immunogen. In this study we compared the adjuvant effects of Cry1Ac and cholera toxin (CT) for the hepatitis B surface antigen (HBsAg) and bovine serum albumin (BSA). The antibody responses of intestinal secretions and serum were determined by ELISA in Balb/c mice immunized through the intragastric (IG) or intraperitoneal (IP) routes. When HBsAg was administered via IG, the anti-HBsAg intestinal response was not enhanced by either Cry1Ac or CT, whereas via IP Cry1Ac increased the anti-HBsAg intestinal immunoglobulin (Ig)G response and CT increased the intestinal IgA and IgM responses. Serum anti-BSA antibodies increased when BSA was co-administered with CT or Cry1Ac by both routes. Cholera toxin and Cry1Ac co-administered via IP increased the IgG anti-BSA response in fluid of the large intestine and CT also increased the IgA and IgM responses slightly. When co-administered via IP, CT and Cry1Ac did not affect the IgG anti-BSA response of the small intestine significantly. We conclude that Cry1Ac is a mucosal and systemic adjuvant as potent as CT which enhances mostly serum and intestinal IgG antibody responses, especially at the large intestine, and its effects depend on the route and antigen used. These features make Cry1Ac of potential use as carrier and/or adjuvant in mucosal and parenteral vaccines

Characterization of the mucosal and systemic immune response induced by Cry1Ac protein from Bacillus thuringiensis HD 73 in mice

The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins