The hybrid protein BTH2 suppresses allergic airway inflammation in a murine model of HDM-specific immunotherapy.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the only disease-modifying treatment approach to change disease-causing allergens. Hypoallergenic derivatives show promise as potential therapeutics, amongst which BTH2 was designed to induce tolerance against Blomia tropicalis allergy. Our aim was to investigate the hypoallergenicity and immunoregulatory activity of BTH2 in vitro and its therapeutic potential in a mouse model of AIT. METHODS: Recombinant Blo t 5 and Blo t 21 allergens and their hybrid derivatives (BTH1 and BTH2) were expressed and purified. IgE binding capacity was tested by ELISA using sera from Brazilian, Colombian, and Ecuadorian subjects. Secretion of cytokines in supernatants from human cell cultures was measured following stimulation with the four recombinants and controls. The capacity of BTH2 to ameliorate allergic airway inflammation induced by B. tropicalis extract was evaluated in a murine model of AIT. RESULTS: rBlo t 5 and rBlo t 21 were identified as major allergens in Latin American patients, and BTH2 had the lowest IgE binding. In vitro stimulation of human cells induced greater levels of IL-10 and IFN-γ and reduced the secretion of Th2 cytokines. BTH2 ameliorated allergic airway inflammation in B. tropicalis-challenged A/J mice, as evidenced by the histopathological and humoral biomarkers: decreased Th2 cytokines and cellular infiltration (especially eosinophils), lower activity of eosinophil peroxidase, an increase in IgG blocking antibodies and strong reduction of mucus production by goblet cells. CONCLUSIONS: Our study shows that BTH2 represents a promising candidate for the treatment of B. tropicalis allergy with hypoallergenic, immune regulatory and therapeutic properties. Further pre-clinical studies are required in murine models of chronic asthma to further address the efficacy and safety of BTH2 as a vaccine against B. tropicalis-induced allergy.

Immunogenicity and protection induced by recombinant Toxocara canis proteins in a murine model of toxocariasis.

Toxocariasis, a natural helminth infection of dogs and cats caused by Toxocara canis and T. cati, respectively, that are transmitted to mammals, including humans. Infection control is based currently on periodic antihelmintic treatment and there is a need for the development of vaccines to prevent this infection. MATERIALS AND METHODS: Eight potential vaccine candidate T. canis recombinant proteins were identified by in silico (rTcGPRs, rTcCad, rTcVcan, rTcCyst) and larval proteomics (rTES26, rTES32, rMUC-3 and rCTL-4) analyses. Immunogenicity and protection against infectious challenge for seven of these antigens were determined in a murine model of toxocariasis. C57BL/6 female mice were immunized with each of or combinations of recombinant antigens prior to challenge with 500 T. canis embryonated eggs. Levels of specific antibodies (IgG, IgG1, IgG2a and IgE) in sera and cytokines (IL-5, INF-É£ and IL-10) produced by antigens-stimulated splenocytes, were measured. Presence of specific antibodies to the molecules was measured in sera of T. canis-seropositive dogs and humans. RESULTS: All seven molecules were immunogenic in immunized mice; all stimulated significantly elevated levels of specific IgG, IgG1 or IgG2a and six were associated with elevated levels of specific IgE; all induced elevated production of IFN- É£ and IL-10 by splenocytes, but only the in silico-identified membrane-associated recombinants (rTcCad, rTcVcan, and rTcCyst) induced significantly increased IL-5 production. Vaccination with two of the latter (rTcCad and rTcVcan) reduced larval loads in the T. canis challenged mice by 54.3% and 53.9% (P < 0.0001), respectively, compared to unimmunized controls. All seven recombinants were recognized by T. canis-seropositive dog and human sera. CONCLUSION: The identification of vaccine targets by in silico analysis was an effective strategy to identify immunogenic T. canis proteins capable of reducing larval burdens following challenge with the parasite. Two recombinant proteins, rTcCad and rTcVcan, were identified as promising vaccine candidates for canine toxocariasis.

PCR-RFLP screening of polymorphisms associated with benzimidazole resistance in Necator americanus and Ascaris lumbricoides from different geographical regions in Brazil.

Ascaris lumbricoides and Necator americanus are soil-transmitted parasites with global geographic distribution, and they represent some of the most common and neglected infections in the world. Periodic treatment with mass drug administration (MDA) in endemic areas is the recommended action put forth by the World Health Organization. However, MDA can cause the selection of subpopulations that possess the genetic ability to overcome the mechanism of drug action. In fact, beta-tubulin gene mutations (codons 167, 198 and 200) are correlated with benzimidazole resistance in nematodes of veterinary importance. It is possible that these SNPs also have strong correlation with treatment resistance in the human geohelminths A. lumbricoides, Trichuris trichiura and hookworms. Here, we aimed to investigate the presence of some of these canonical molecular markers associated with parasite resistance to benzimidazole in N. americanus and A. lumbricoides collected from six Brazilian states. Nested-PCR and PCR-RFLP were used to detect mutations at codons 167 and 198 in 601 individual eggs of A. lumbricoides collected from 62 human stool samples; however, no mutations were found. Codons 198 and 200 were tested in 552 N. americanus eggs collected from 48 patients using the same methodology, which presented a relative frequency of 1.4% and 1.1%, respectively. The presence of these SNPs in N. americanus eggs is an important finding, indicating that with high benzimidazole drug pressure there is potential for benzimidazole resistance to be selected in this hookworm. However, at these low frequencies it does not indicate that there is at present any benzimidazole resistance problem. This is the first systematic study performed in South America, and the study yielded a landscape of the genetic variants in the beta-tubulin gene and anthelmintic resistance to soil-transmitted parasites detected by a simple, rapid and affordable genotyping assay of individual eggs.

Immunological characterization of a chimeric form of Schistosoma mansoni aquaporin in the murine model.

Aquaporin (SmAQP) is the most abundant transmembrane protein in the tegument of Schistosoma mansoni. This protein is expressed in all developmental stages and seems to be essential in parasite survival since it plays a crucial role in osmoregulation, nutrient transport and drug uptake. In this study, we utilized the murine model to evaluate whether this protein was able to induce protection against challenge infection with S. mansoni cercariae. A chimeric (c) SmAQP was formulated with Freund's adjuvant for vaccination trial and evaluation of the host's immune response was performed. Our results demonstrated that immunization with cSmAQP induced the production of high levels of specific anti-cSmAQP IgG antibodies and a Th1/Th17 type of immune response characterized by IFN-γ, TNF-α and IL-17 cytokines. However, vaccination of mice with cSmAQP failed to reduce S. mansoni worm burden and liver pathology. Finally, we were unable to detect humoral immune response anti-cSmAQP in the sera of S. mansoni-infected human patients. Our results lead us to believe that SmAQP, as formulated in this study, may not be a good target in the search for an anti-schistosomiasis vaccine.

Evaluation of the use of C-terminal part of the Schistosoma mansoni 200kDa tegumental protein in schistosomiasis diagnosis and vaccine formulation.

Schistosoma mansoni tegument is involved in essential functions for parasite survival and represents a target for screening candidates for vaccine and diagnosis. Our group using reverse vaccinology selected six candidates, previously demonstrated by proteomics studies to be expressed in the parasite tegument, among them was Sm200. In this work we have cloned and expressed a recombinant form of Sm200 C-terminal (1069-1520) region. The efficacy of rSm200 (1069-1520) in the diagnosis of schistosomiasis and in the formulation of a vaccine against S. mansoni was assessed respectively in an ELISA based diagnostic assay and immunization protocols in mice. Significant differences between non-infected and acutely infected or chronically infected animals were observed and no cross-recognition was observed with sera from Ascaris suum or Ancylostoma ceylanicum infected mice. rSm200-ELISA test could also discriminate infected individuals from healthy donors not living in endemic area for schistosomiasis but failed to discriminate between individuals from a low endemic area for schistosomiasis known to have positive or negative stools after examination. Recombinant Sm200 also failed to induce protection against schistosomiasis, demonstrating that the C-terminal part of Sm200 is unable to induce protective immune response in mice. Therefore rSm200 (1069-1520)-ELISA represents an important tool to be used in the diagnosis of schistosomiasis.