Immunization with Tc52 or its amino terminal domain adjuvanted with c-di-AMP induces Th17+Th1 specific immune responses and confers protection against Trypanosoma cruzi.

The development of new adjuvants enables fine modulation of the elicited immune responses. Ideally, the use of one or more adjuvants should result in the induction of a protective immune response against the specific pathogen. We have evaluated the immune response and protection against Trypanosoma cruzi infection in mice vaccinated with recombinant Tc52 or its N- and C-terminal domains (NTc52 and CTc52) adjuvanted either with the STING (Stimulator of Interferon Genes) agonist cyclic di-AMP (c-di-AMP), a pegylated derivative of α-galactosylceramide (αGC-PEG), or oligodeoxynucleotides containing unmethylated CpG motifs (ODN-CpG). All groups immunized with the recombinant proteins plus adjuvant: Tc52+c-di-AMP, NTc52+c-di-AMP, CTc52+c-di-AMP, NTc52+c-di-AMP+αGC-PEG, NTc52+CpG, developed significantly higher anti-Tc52 IgG titers than controls. Groups immunized with c-di-AMP and Tc52, NTc52 or CTc52 showed the highest Tc52-specific IgA titers in nasal lavages. All groups immunized with the recombinant proteins plus adjuvant developed a strong specific cellular immune response in splenocytes and lymph node cells with significant differences for groups immunized with c-di-AMP and Tc52, NTc52 or CTc52. These groups also showed high levels of Tc52-specific IL-17 and IFN-γ producing cells, while NTc52+CpG group only showed significant difference with control in IFN-γ producing cells. Groups immunized with c-di-AMP and Tc52, NTc52 or CTc52 developed predominantly a Th17 and Th1immune response, whereas for NTc52+CpG it was a dominant Th1 response. It was previously described that αGC-PEG inhibits Th17 differentiation by activating NKT cells. Thus, in this work we have also included a group immunized with both adjuvants (NTc52+c-di-AMP+αGC-PEG) with the aim to modulate the Th17 response induced by c-di-AMP. This group showed a significant reduction in the number of Tc52-specific IL-17 producing splenocytes, as compared to the group NTc52+c-di-AMP, which has in turn correlated with a reduction in protection against infection. These results suggest that the Th17 immune response developed after immunizing with NTc52+c-di-AMP could have a protective role against T. cruzi infection. Groups NTc52+c-di-AMP, Tc52+c-di-AMP and NTc52PB, were the ones that showed better protection against infection with lower parasitemia and weight loss, and higher survival.

Oral multicomponent DNA vaccine delivered by attenuated Salmonella elicited immunoprotection against American trypanosomiasis.

We have reported that attenuated Salmonella (S) carrying plasmids encoding the cysteine protease cruzipain (Cz) protects against Trypanosoma cruzi infection. Here, we determined whether immunoprotection could be improved by the oral coadministration of 3 Salmonella carrying the plasmids that encode the antigens Cz, Tc52, and Tc24. SCz+STc52+STc24-immunized mice presented an increased antibody response against each antigen compared with those in the single antigen-immunized groups, as well as higher trypomastigotes antibody-mediated lyses and cell invasion inhibition compared with controls. SCz+STc52+STc24-immunized and -challenged mice rendered lower parasitemia. Weight loss after infection was detected in all mice except those in the SCz+STc52+STc24 group. Moreover, cardiomyopathy-associated enzyme activity was significantly lower in SCz+STc24+STc52-immunized mice compared with controls. Few or no abnormalities were found in muscle tissues of SCz+STc24+STc52-immunized mice, whereas controls presented with inflammatory foci, necrosis, and amastigote nests. We conclude that a multicomponent approach that targets several invasion and metabolic mechanisms improves protection compared with single-component vaccines.

Transgenic mice overexpressing GH exhibit hepatic upregulation of GH-signaling mediators involved in cell proliferation.

Chronically elevated levels of GH in GH-transgenic mice result in accelerated growth and increased adult body weight. We have previously described that the GH-induced JAK2/STAT5-signaling pathway is desensitized in the liver of transgenic mice overexpressing GH. However, these animals present increased circulating IGF-I levels, increased hepatic GHR expression, and liver organomegaly due to hypertrophy and hyperplasia, which frequently progress to hepatomas as the animals age, indicating that action of GH on the liver is not prevented. In the present study, we have evaluated other GH-signaling pathways that could be activated in the liver of GH-transgenic mice. Upon GH administration, normal mice showed an important increment in STAT3 phosphorylation level, but transgenic mice did not respond to acute GH stimulation. However, STAT3 was constitutively phosphorylated in transgenic mice, whereas its protein content was not increased. GH-transgenic mice showed overexpression of c-Src, accompanied by an elevation of its activity. Other signaling mediators including focal adhesion kinase, epidermal growth factor receptor, Erk, Akt, and mammalian target of rapamycin displayed elevated protein and basal phosphorylation levels in these animals. Thus, GH-overexpressing transgenic mice exhibit hepatic upregulation of signaling mediators related to cell proliferation, survival, and migration. The upregulation of these proteins may represent GH-signaling pathways that are constitutively activated in the presence of dramatically elevated GH levels throughout life. These molecular alterations could be implicated in the pathological alterations observed in the liver of GH-transgenic mice.