Vaccination with parasite-specific TcTASV proteins combined with recombinant baculovirus as a delivery platform protects against acute and chronic Trypanosoma cruzi infection.

Chagas' is a neglected disease caused by the eukaryotic kinetoplastid parasite, Trypanosoma cruzi. Currently, approximately 8 million people are infected worldwide, most of whom are in the chronic phase of the disease, which involves cardiac, digestive, or neurologic manifestations. There is an urgent need for a vaccine because treatments are only effective in the initial phase of infection, which is generally underdiagnosed. The selection and combination of antigens, adjuvants, and delivery platforms for vaccine formulations should be designed to trigger mixed humoral and cellular immune responses, considering that T. cruzi has a complex life cycle with both intracellular and bloodstream circulating parasite stages in vertebrate hosts. Here, we report the effectiveness of vaccination with a T. cruzi-specific protein family (TcTASV), employing both recombinant proteins with aluminum hydroxide and a recombinant baculovirus displaying a TcTASV antigen at the capsid. Vaccination stimulated immunological responses by producing lytic antibodies and antigen-specific CD4+ and CD8+ IFNÉ£ secreting lymphocytes. More than 90% of vaccinated animals survived after lethal challenges with T. cruzi, whereas all control mice died before 30 days post-infection. Vaccination also induced a strong decrease in chronic tissue parasitism and generated immunological memory that allowed vaccinated and infected animals to control both the reactivation of the infection after immunosuppression and a second challenge with T. cruzi. Interestingly, inoculation with wild-type baculovirus partially protected the mice against T. cruzi. In brief, we demonstrated for the first time that the combination of the baculovirus platform and the TcTASV family provides effective protection against Trypanosoma cruzi, which is a promising vaccine for Chagas disease.

Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCCs) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The former group exhibits reduced proliferation, genome instability, and heightened sensitivity to genotoxic agents like PARP1/2 inhibitors. Conversely, H3K36M HNSCC models with constant H3K27me3 levels lack these characteristics unless H3K27me3 is elevated by DNA hypomethylating agents or inhibiting H3K27me3 demethylases KDM6A/B. Mechanistically, H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, aberrant H3K27me3 levels induced by H3K36M expression are not a bona fide epigenetic mark because they require continuous expression of H3K36M to be inherited. Moreover, increased sensitivity to PARP1/2 inhibitors in H3K36M HNSCC models depends solely on elevated H3K27me3 levels and diminishing BRCA1- and FANCD2-dependent DNA repair. Finally, a PARP1/2 inhibitor alone reduces tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a model with consistent H3K27me3, a combination of PARP1/2 inhibitors and agents that up-regulate H3K27me3 proves to be successful. These findings underscore the crucial balance between H3K36 and H3K27 methylation in maintaining genome instability, offering new therapeutic options for patients with H3K36me-deficient tumors.

Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCC) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The first group shows decreased proliferation, genome instability, and increased sensitivity to genotoxic agents, such as PARP1/2 inhibitors. In contrast, the H3K36M HNSCC models with steady H3K27me3 levels do not exhibit these characteristics unless H3K27me3 levels are elevated, either by DNA hypomethylating agents or by inhibiting the H3K27me3 demethylases KDM6A/B. Mechanistically, we found that H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, we found that aberrant H3K27me3 levels induced by H3K36M expression is not a bona fide epigenetic mark in HNSCC since it requires continuous expression of H3K36M to be inherited. Moreover, increased sensitivity of H3K36M HNSCC models to PARP1/2 inhibitors solely depends on the increased H3K27me3 levels. Indeed, aberrantly high H3K27me3 levels decrease BRCA1 and FANCD2-dependent DNA repair, resulting in higher sensitivity to DNA breaks and replication stress. Finally, in support of our in vitro findings, a PARP1/2 inhibitor alone reduce tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a H3K36M HNSCC xenograft model with consistent H3K27me3 levels, a combination of PARP1/2 inhibitors and agents that upregulate H3K27me3 proves to be successful. In conclusion, our findings underscore a delicate balance between H3K36 and H3K27 methylation, essential for maintaining genome stability. This equilibrium presents promising therapeutic opportunities for patients with H3K36me-deficient tumors.

The Trypanosoma cruzi TcTASV-C protein subfamily administrated with U-Omp19 promotes a protective response against a lethal challenge in mice.

The development of a Chagas disease vaccine has yet the need for the identification of novel combinations of antigens and adjuvants. Here, the performance of TcTASV-C proteins that are virulence factors of trypomastigotes and belong to a novel surface protein family specific for T. cruzi, have been evaluated as antigens for a prophylactic vaccine. Several immunization schemes in which TcTASV-C was combined with aluminum hydroxide, saponin and/or U-Omp19 were assayed. Aluminum hydroxide and saponin were assayed together to trigger different pathways of the immune response simultaneously. U-Omp19 is a promising novel adjuvant able to promote a Th1 immune response with IFNg production, thus an interesting molecule to be tested as adjuvant for the control of T. cruzi infection. Therefore, U-Omp19 was added to the aluminum hydroxide-saponin formulation as well as assayed individually with TcTASV-C. The immunization with TcTASV-C and U-Omp19 had the best performance as a prophylactic vaccine. Mice presented the lowest parasitemias and improved survival by 40% after being challenged with a highly virulent T. cruzi strain, which promoted 100% mortality in all other immunized groups. Immunization with TcTASV-C and U-Omp19 triggered cellular responses with IFN-γ and IL-17 production and with lytic antibodies that could explain the protection achieved by this vaccination scheme. To our knowledge, this is the first time that U-Omp19 is tested with a defined T. cruzi antigen in a vaccine formulation.

Transmigration of Trypanosoma cruzi trypomastigotes through 3D cultures resembling a physiological environment.

To disseminate and colonise tissues in the mammalian host, Trypanosoma cruzi trypomastogotes should cross several biological barriers. How this process occurs or its impact in the outcome of the disease is largely speculative. We examined the in vitro transmigration of trypomastigotes through three-dimensional cultures (spheroids) to understand the tissular dissemination of different T. cruzi strains. Virulent strains were highly invasive: trypomastigotes deeply transmigrate up to 50 µm inside spheroids and were evenly distributed at the spheroid surface. Parasites inside spheroids were systematically observed in the space between cells suggesting a paracellular route of transmigration. On the contrary, poorly virulent strains presented a weak migratory capacity and remained in the external layers of spheroids with a patch-like distribution pattern. The invasiveness-understood as the ability to transmigrate deep into spheroids-was not a transferable feature between strains, neither by soluble or secreted factors nor by co-cultivation of trypomastigotes from invasive and non-invasive strains. Besides, we demonstrated that T. cruzi isolates from children that were born congenitally infected presented a highly migrant phenotype while an isolate from an infected mother (that never transmitted the infection to any of her children) presented significantly less migration. In brief, we demonstrated that in a 3D microenvironment each strain presents a characteristic migration pattern that can be associated to their in vivo behaviour. Altogether, data presented here repositionate spheroids as a valuable tool to study host-pathogen interactions.

The protein family TcTASV-C is a novel Trypanosoma cruzi virulence factor secreted in extracellular vesicles by trypomastigotes and highly expressed in bloodstream forms.

TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes. In contrast, TcTASV-C is highly expressed in bloodstream trypomastigotes; its upregulation in bloodstream parasites was observed in different T. cruzi strains and was specific for TcTASV-C, suggesting that some host-molecules trigger TcTASV-C expression. TcTASV-C is also strongly secreted by bloodstream parasites. A DNA prime-protein boost immunization scheme with TcTASV-C was only partially effective to control the infection in mice challenged with a highly virulent T. cruzi strain. Vaccination triggered a strong humoral response that delayed the appearance of bloodstream trypomastigotes at the early phase of the infection. Linear epitopes recognized by vaccinated mice were mapped within the TcTASV-C family motif, suggesting that blockade of secreted TcTASV-C impacts on the settlement of infection. Furthermore, although experimental and naturally T. cruzi-infected hosts did not react with antigens from extracellular vesicles, vaccinated and challenged mice recognized not only TcTASV-C but also other vesicle-antigens. We hypothesize that TcTASV-C is involved in the establishment of the initial T. cruzi infection in the mammalian host. Altogether, these results point towards TcTASV-C as a novel secreted virulence factor of T. cruzi trypomastigotes.

TcTASV-C, a protein family in Trypanosoma cruzi that is predominantly trypomastigote-stage specific and secreted to the medium.

Among the several multigene families codified by the genome of T. cruzi, the TcTASV family was the latest discovered. The TcTASV (Trypomastigote, Alanine, Serine, Valine) family is composed of ∼40 members, with conserved carboxi- and amino-termini but with a variable central core. According to the length and sequence of the central region the family is split into 3 subfamilies. The TcTASV family is conserved in the genomes of - at least - lineages TcI and TcVI and has no orthologues in other trypanosomatids. In the present work we focus on the study of the TcTASV-C subfamily, composed by 16 genes in the CL Brener strain. We determined that TcTASV-C is preferentially expressed in trypomastigotes, but it is not a major component of the parasite. Both immunoflourescence and flow cytometry experiments indicated that TcTASV-C has a clonal expression, i.e. it is not expressed by all the parasites of a certain population at the same time. We also determined that TcTASV-C is phosphorylated and glycosylated. TASV-C is attached to the parasite surface by a GPI anchor and is shed spontaneously into the medium. About 30% of sera from infected hosts reacted with TcTASV-C, confirming its exposition to the immune system. Its superficial localization and secretory nature suggest a possible role in host-parasite interactions.