Immunostimulatory capacity of the Trypanosoma cruzi TcTASV protein family engineered with different adjuvants

CAEIRO, L; MASIP, Y; CORIA, ML; RIZZI, M; RODRIGUEZ, ME; MOLINA, G; PUEBLAS CASTRO, C; SÁNCHEZ, DO; CASSATARO, J; MOLINARI, MP; TEKIEL, V

Cancún

Congreso; XII Congress of the Latin American Association of Immunology & XXIII Congress of the Mexican Society of Immunology.; 2018

Latin American Association of Immunology

Chagas?disease is an endemic illness caused by the protozoan parasiteTrypanosoma cruzi, that affects more than 6 million people in Latin America. T.cruzi has a complex life cycle alternating between a reduviid insect and amammalian host. Proteins differentially expressed on circulating bloodstreamtrypomastigotes and/or on intracellular amastigotes (stages present in themammalian host) are those somehow involved in parasite survival. Most ofthese molecules are either virulence factors of the parasite or involved inimmune system evasion, and, therefore, interesting targets for rationalinterventions, like vaccine design. The Trypomastigote Threonine, Valine andSerine rich (TcTASV) is a medium-size T.cruzi multigene family that is presentin all T. cruzi lineages and has no orthologs in other organisms. All TcTASVshave 3?UTRs as well as amino and carboxi-termini conserved. The variablecentral core of the proteins -along with sequence, protein length, and motifsallowsto distinguish 4 TcTASV subfamilies (A, B, C and W). TcTASV-A andTcTASV-C are the major subfamilies, with 15-20 members each one. Althoughall TcTASV are expressed into trypomastigotes and/or amastigotes, the patternof expression oscillate among the different subfamilies. TcTASV-A is expressedintracellularly both in amastigotes and trypomastigotes, while TcTASV-C issolely expressed in trypomastigotes, at the cell surface and strongly secreted inextracellular vesicles (Garcia et al, 2010; Bernabó et al, 2013; and unpublishedresults). In line with our results, peptides from TcTASV-A and TcTASV-C wererecently found in proteomes of bloodstream trypomastigotes andtrypomastigote?s secretomes of different T. cruzi strains. Also previously, bymeans of genetic library immunization, we identified a fragment of a TcTASV-Cgene among a pool of protective clones (Tekiel et al, 2009). Later, we evaluateTcTASV-C as an individual vaccine in a prime (DNA+GM-CSF) and boost(recombinant protein+AlOH) protocol. After challenged with a highly virulentT. cruzi strain (RA, DTU TcVI) vaccinated mice presented delayed appearanceof bloodstream trypomastigotes but only slight improvement in survival rates.This fact can be related with the immune response triggered by theimmunization scheme: a strong humoral but negligible cellular immuneresponses. Considering that both TcTASV-A and TcTASV-C subfamilies are incontact with the host immune system in vivo (Floridia et al, 2016), altogetherwith their expression dynamics and the previous vaccine results, in the presentstudy we evaluated different immunization protocols employing both TcTASV-Cand TcTASV-A and several delivery systems to trigger both humoral and cellularspecific immune responses. In this context we hypothesize that an adequatevaccination protocol should induce a cellular response against TcTASV-A(intracellular) while a humoral response against TcTASV-C (surface andsecreted) should be desirable. The first vaccination protocol tested consisted inthe administration of both recombinant TcTASV-C and TcTASV-A (rTcTASVC+A) simultaneously adjuvanted with aluminum hydroxide and saponin. MaleC3H/HeJ mice were immunized by s.c. route with 3 doses of 25μg of proteins(12,5 μg each) intervaled by 15 days. Splenocytes from TcTASV immunizedanimals produced IFNg upon restimulation with TcTASV-A, but not afterrestimulation with TcTASV-C. On the other hand, the humoral response elicitedby vaccination was mainly against TcTASV-C and only slightly higher thancontrols against TcTASV-A. The anti-TcTASV-C antibody response showed amixed Th1/Th2 pattern, with a bias to IgG2a>IgG1. A vaccination schemeemploying rTcTASV-A+C delivered with aluminum and boosted with saponingave similar results. We conclude that it is possible to trigger both arms of theimmune response after vaccination with proteins from both subfamiliessimultaneously or sequentially formulated with saponin and aluminum salts.Then, we evaluate another immunization scheme employing a proteaseinhibitor from B. abortus with adjuvant capacity, the unlipidated outermembrane protein 19 (U-Omp19), in our model. U-Omp19 increases the antigenhalf-life in APCs improving cross-presentation and triggers Th1, Th17, CD8+antigen specific responses (Coria et al, 2016). Mice were immunized by the s.c.route with 3 doses of rTcTASV-C+A (25μg each/mice) plus U-Omp19, or withrTcTASV-C+A (25μg each) simultaneously delivered with U-Omp19, saponinand aluminum hydroxide. Vaccinated mice developed anti-TcTASV-A as well asanti TcTASV-C specific antibodies. However the group solely adjuvanted withU-Omp19 presented lower titers than the group immunized with all adjuvants(TcTASV-A: 3200 vs >12800; TcTASV-C: 6400 vs >12800). Moreover, althoughboth groups of vaccinated mice presented lower bloodstream trypomastigotes inthe early infection (p