A protective vaccine against the toxic activities following Brown spider accidents based on recombinant mutated phospholipases D as antigens.

Accidents involving Brown spiders are reported throughout the world. In the venom, the major toxins involved in the deleterious effects are phospholipases D (PLDs). In this work, recombinant mutated phospholipases D from three endemic species medically relevant in South America (Loxosceles intermedia, L. laeta and L. gaucho) were tested as antigens in a vaccination protocol. In such isoforms, key amino acid residues involved in catalysis, magnesium-ion coordination, and binding to substrates were replaced by Alanine (H12A-H47A, E32A-D34A and W230A). These mutations eliminated the phospholipase activity and reduced the generation of skin necrosis and edema to residual levels. Molecular modeling of mutated isoforms indicated that the three-dimensional structures, topologies, and surface charges did not undergo significant changes. Mutated isoforms were recognized by sera against the crude venoms. Vaccination protocols in rabbits using mutated isoforms generated a serum that recognized the native PLDs of crude venoms and neutralized dermonecrosis and edema induced by L. intermedia venom. Vaccination of mice prevented the lethal effects of L. intermedia crude venom. Furthermore, vaccination of rabbits prevented the cutaneous lesion triggered by the three venoms. These results indicate a great potential for mutated recombinant PLDs to be employed as antigens in developing protective vaccines for Loxoscelism.

Multiple antibodies targeting tumor-specific mutations redirect immune cells to inhibit tumor growth and increase survival in experimental animal models.

BACKGROUND: T cell therapy for cancer involves genetic introduction of a target-binding feature into autologous T cells, ex vivo expansion and single large bolus administration back to the patient. These reprogrammed T cells can be highly effective in killing cells, but tumor heterogeneity results in regrowth of cells that do not sufficiently express the single antigen being targeted. We describe a cell-based therapy that simultaneously targets multiple tumor-specific antigens. METHODS: High-affinity polyclonal rabbit antibodies were generated against nine different surface-related tumor-specific mutations on B16F10 cells. Unsorted splenic effector cells from syngeneic mice were incubated with a cocktail of the nine anti-B16F10 antibodies. These 'armed' effector cells were used to treat mice previously inoculated with B16F10 melanoma cells. RESULTS: The cocktail of nine antibodies resulted in dense homogeneous binding to histological sections of B16F10 cells. Five treatments with the armed effector cells and PD1 inhibition inhibited tumor growth and improved survival. Shortening the interval of the five treatments from every three days to every day increased survival. Arming effector cells with the four antibodies showing best binding to B16F10 cells even further increased survival. CONCLUSIONS: This study demonstrates that ex vivo arming a mixed population of immune effector cells with antibodies targeting multiple tumor-specific mutated proteins in conjunction with PD1 inhibition delayed tumor growth and prolonged survival in mice inoculated with an aggressive melanoma. A remarkably low total antibody dose of less than 5 µg was sufficient to accomplish tumor inhibition. Scaling up to clinical level may be feasible.

Type-III interferons and rheumatoid arthritis: Correlation between interferon lambda 1 (interleukin 29) and antimutated citrullinated vimentin antibody levels.

AIM: To assess serum type III or lambda (λ) interferons (IFN) levels and its clinical and laboratory associations in rheumatoid arthritis (RA). METHODS: A cross-sectional study including 43 patients with RA (86% females; age 45.3 ± 10.3 years) and 43 healthy individuals was performed. Clinical data including disease activity, acute-phase reactants, rheumatoid factor and anticyclic citrullinated peptide (anti-CCP) antibodies were collected. Serum IFNλ1, IFNλ2, IFNλ3, CXCL8 and anti-mutated citrullinated vimentin (anti-MCV) antibody levels were measured. RESULTS: Patients with RA had higher IFNλ1 (113.5 ± 118.6 pg/mL versus 55.9 ± 122.3 pg/mL; p < 0.0001) and IFNλ2 (245.4 ± 327.7 pg/mL versus 5.1 ± 11.0 pg/mL; p = 0.009) levels than controls, but not IFNλ3 levels. Notably, IFNλ1 levels were found to be higher in both patients with active disease (124.9 ± 135.9 pg/mL; p < 0.001) and quiescent disease (99.0 ± 93.7 pg/mL; p < 0.01), while IFNλ2 levels were higher only in patients with active disease (264.0 ± 356.1 pg/mL; p = 0.02). A noteworthy association between serum IFNλ1 levels and anti-MCV antibody titers (Spearman's rho coefficient 0.36, 95% CI 0.36 to 0.61; p = 0.02) was observed. CONCLUSION: Serum IFNλ1 and IFNλ2 levels are abnormally elevated in patients with RA and the former are linearly associated with circulating anti-MCV antibody levels. These results may place type-III IFN as an attractive new therapeutic target in RA.

Evaluation of the allergenicity potential of TcPR-10 protein from Theobroma cacao.

BACKGROUND: The pathogenesis related protein PR10 (TcPR-10), obtained from the Theobroma cacao-Moniliophthora perniciosa interaction library, presents antifungal activity against M. perniciosa and acts in vitro as a ribonuclease. However, despite its biotechnological potential, the TcPR-10 has the P-loop motif similar to those of some allergenic proteins such as Bet v 1 (Betula verrucosa) and Pru av 1 (Prunus avium). The insertion of mutations in this motif can produce proteins with reduced allergenic power. The objective of the present work was to evaluate the allergenic potential of the wild type and mutant recombinant TcPR-10 using bioinformatics tools and immunological assays. METHODOLOGY/PRINCIPAL FINDINGS: Mutant substitutions (T10P, I30V, H45S) were inserted in the TcPR-10 gene by site-directed mutagenesis, cloned into pET28a and expressed in Escherichia coli BL21(DE3) cells. Changes in molecular surface caused by the mutant substitutions was evaluated by comparative protein modeling using the three-dimensional structure of the major cherry allergen, Pru av 1 as a template. The immunological assays were carried out in 8-12 week old female BALB/c mice. The mice were sensitized with the proteins (wild type and mutants) via subcutaneous and challenged intranasal for induction of allergic airway inflammation. CONCLUSIONS/SIGNIFICANCE: We showed that the wild TcPR-10 protein has allergenic potential, whereas the insertion of mutations produced proteins with reduced capacity of IgE production and cellular infiltration in the lungs. On the other hand, in vitro assays show that the TcPR-10 mutants still present antifungal and ribonuclease activity against M. perniciosa RNA. In conclusion, the mutant proteins present less allergenic potential than the wild TcPR-10, without the loss of interesting biotechnological properties.

Endothelial cell signalling induced by trans-sialidase from Trypanosoma cruzi.

The protozoan responsible for Chagas' disease, Trypanosoma cruzi, expresses on its surface an unusual trans-sialidase enzyme thought to play an important role in host-parasite interactions. Trans-sialidase is the product of a multigene family encoding both active and inactive proteins. We have demonstrated that despite lacking enzymatic activity due to a single mutation, Tyr342-His, inactive trans-sialidase displays sialic acid binding activity, with identical specificity to that of its active analogue. In this work we demonstrate that binding of a recombinant inactive trans-sialidase to molecules containing alpha2,3-linked sialic acid on endothelial cell surface triggers NF-kappaB activation, expression of adhesion molecules and upregulation of parasite entry into host cells. Furthermore, inactive recombinant trans-sialidase blocks endothelial cell apoptosis induced by growth factor deprivation. These results suggest that inactive members of the trans-sialidase family play a role in endothelial cell responses to T. cruzi infection.