Protective efficacy of the oral vaccine Tc24:Co1 produced in Schizochytrium sp. against Trypanosoma cruzi infection in a mouse model.

Trypanosoma cruzi parasite - causal Chagas disease agent - affects about 7 million people; no vaccine is available, and current medications have not been entirely effective. Multidisciplinary efforts are necessary for developing clinical vaccine prototypes. Thus, this research study aims to assess the expressed and whole-cell administration protection of the oral vaccine prototype Tc24:Co1 using Schizochytrium sp. microalga. High recombinant protein expression yields (675 µg/L) of algal culture were obtained. Additionally, Schizochytrium sp.-Tc24:Co1 resulted stable at 4 °C for up to six months and at 25 °C for three months. After receiving four oral doses of the vaccine, the mice showed a significant humoral immune response and a parasitemia reduction associated with a lack of heart inflammatory damage compared with the unvaccinated controls. The Schizochytrium sp.-Tc24:Co1 vaccine demonstrates to be promising as a prototype for further development showing protective effects against a T. cruzi challenge in a mouse model.

A short-term method to evaluate anti-leishmania drugs by inhibition of stage differentiation in Leishmania mexicana using flow cytometry.

Leishmaniasis is a vector-borne neglected tropical disease caused by the Leishmania spp. Parasite. The disease is transmitted to humans and animals by the bite of infected female sandflies during the ingestion of bloodmeal. Because current drug treatments induce toxicity and parasite resistance, there is an urgent need to evaluate new drugs. Most therapeutics target the differentiation of promastigotes to amastigotes, which is necessary to maintain Leishmania infection. However, in vitro assays are laborious, time-consuming, and depend on the experience of the technician. In this study, we aimed to establish a short-term method to assess the differentiation status of Leishmania mexicana (L. mexicana) using flow cytometry. Here, we showed that flow cytometry provides a rapid means to quantify parasite differentiation in cell culture as reliably as light microscopy. Interestingly, we found using flow cytometry that miltefosine reduced promastigote-to-amastigote differentiation of L. mexicana. We conclude that flow cytometry provides a means to rapidly assay the efficacy of small molecules or natural compounds as potential anti-leishmanials.

Vaccine-linked chemotherapy with a low dose of benznidazole plus a bivalent recombinant protein vaccine prevents the development of cardiac fibrosis caused by Trypanosoma cruzi in chronically-infected BALB/c mice.

BACKGROUND: Chagas disease (CD) is caused by Trypanosoma cruzi and affects 6-7 million people worldwide. Approximately 30% of chronic patients develop chronic chagasic cardiomyopathy (CCC) after decades. Benznidazole (BNZ), one of the first-line chemotherapy used for CD, induces toxicity and fails to halt the progression of CCC in chronic patients. The recombinant parasite-derived antigens, including Tc24, Tc24-C4, TSA-1, and TSA-1-C4 with Toll-like receptor 4 (TLR-4) agonist-adjuvants reduce cardiac parasite burdens, heart inflammation, and fibrosis, leading us to envision their use as immunotherapy together with BNZ. Given genetic immunization (DNA vaccines) encoding Tc24 and TSA-1 induce protective immunity in mice and dogs, we propose that immunization with the corresponding recombinant proteins offers an alternative and feasible strategy to develop these antigens as a bivalent human vaccine. We hypothesized that a low dose of BNZ in combination with a therapeutic vaccine (TSA-1-C4 and Tc24-C4 antigens formulated with a synthetic TLR-4 agonist-adjuvant, E6020-SE) given during early chronic infection, could prevent cardiac disease progression and provide antigen-specific T cell immunity. METHODOLOGY/ PRINCIPAL FINDINGS: We evaluated the therapeutic vaccine candidate plus BNZ (25 mg/kg/day/7 days) given on days 72 and 79 post-infection (p.i) (early chronic phase). Fibrosis, inflammation, and parasite burden were quantified in heart tissue at day 200 p.i. (late chronic phase). Further, spleen cells were collected to evaluate antigen-specific CD4+ and CD8+ T cell immune response, using flow cytometry. We found that vaccine-linked BNZ treated mice had lower cardiac fibrosis compared to the infected untreated control group. Moreover, cells from mice that received the immunotherapy had higher stimulation index of antigen-specific CD8+Perforin+ T cells as well as antigen-specific central memory T cells compared to the infected untreated control. CONCLUSIONS: Our results suggest that the bivalent immunotherapy together with BNZ treatment given during early chronic infection protects BALB/c mice against cardiac fibrosis progression and activates a strong CD8+ T cell response by in vitro restimulation, evidencing the induction of a long-lasting T. cruzi-immunity.

A canine model of experimental infection with Leishmania (L.) mexicana.

BACKGROUND: Cutaneous leishmaniasis is a tropical disease affecting over one million patients annually and Leishmania (L.) mexicana is one of the major etiological agents in the Americas. Here we established the first experimental infection of L. (L.) mexicana in canids. METHODS: Beagle dogs were infected intradermally with culture-derived L. (L.) mexicana. We followed skin ulcer development, histopathological signs, parasite burden and the immune status of the infected dogs. RESULTS: All infected dogs developed uniform oval-craterform ulcers similar to those observed in humans, associated with mixed T helper 1/T helper 2 immune responses. Parasites were detected in the healed lesions 15 weeks post-infection. Higher anti-Leishmania IgG levels correlated with larger lesions and high IgG1/IgG2 ratio was associated with some level of splenomegaly. CONCLUSIONS: The canine model described in this work will be of use for further understanding of L. (L.) mexicana immunopathogenensis, and for drug and vaccine development.

Cloning and sequence analysis of Peromyscus yucatanicus (Rodentia) Th1 (IL-12p35, IFN-γ and TNF) and Th2 (IL-4, IL-10 and TGF-ß) cytokines.

The Yucatan deer mouse, Peromyscus yucatanicus (order Rodentia), is the principal reservoir of Leishmania (Leishmania) mexicana in the Yucatan peninsula of Mexico. Experimental infection results in clinical and histopathological features similar to those observed in humans with cutaneous leishmaniasis (CL) as well as peritoneal macrophage production of nitric oxide. These results support the possible use of P. yucatanicus as a novel experimental model to study CL caused by L. (L.) mexicana. However, immunological studies in these rodents have been limited by the lack of specific reagents. To address this issue, we cloned and analyzed cytokine sequences of P. yucatanicus as part of an effort to develop this species as a CL model. We cloned P. yucatanicus interleukin 4 (IL-4), IL-10, IL-12p35, gamma interferon, transforming growth factor beta and tumor necrosis factor partial cDNAs. Most of the P. yucatanicus sequences were highly conserved with orthologs of other mammalian species and the identity of all sequences were confirmed by the presence of conserved amino acids with possible biological functions in each putative polypeptide. The availability of these sequences is a first step which will allow us to carry out studies characterizing the immune response during pathogenic and nonpathogenic L. (L.) mexicana infections in P. yucatanicus.

Nitric oxide production by Peromyscus yucatanicus (Rodentia) infected with Leishmania (Leishmania) mexicana.

Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10 2 and 2.5 x 10 6 promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.

Nitric oxide production by Peromyscus yucatanicus (Rodentia) infected with Leishmania (Leishmania) mexicana

Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10 2 and 2.5 x 10 6 promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.