Miguel Rosado-Vallado

Cross-Protective Efficacy of a Prophylactic Leishmania donovani DNA Vaccine against Visceral and Cutaneous Murine Leishmaniasis

ABSTRACT The fucose-mannose ligand (FML) complex of Leishmania donovani is a promising vaccine candidate against murine and canine visceral leishmaniasis, and its main component is a 36-kDa nucleoside hydrolase (NH36). In this study, we tested the immune response and protection induced by the purified FML, the recombinant NH36 (rNH36), and NH36 DNA vaccines against the agents of visceral (L. chagasi) and cutaneous (L. mexicana) leishmaniasis in BALB/c mice. Mice developed weak humoral response to the vaccines alone, except for those immunized with FML. However, all three vaccine groups presented elevated immunoglobulin G (IgG), IgG1, and IgG2a levels after infection with L. chagasi, whereas no differences were observed between vaccine and control groups after infection with L. mexicana. A strong intradermal reaction to L. donovani and L. mexicana antigens was observed in mice immunized with rNH36 or FML, whereas mice immunized with NH36 DNA only reacted against L. donovani antigens. Experimental infection of immunized mice demonstrated that FML and rNH36 induced significant protection against L. chagasi infection with reductions in parasite loads of 79%. FML also conferred partial protection against L. mexicana infection. The best protection was observed in mice immunized with the VR1012-NH36 DNA vaccine, which induced an 88% reduction in L. chagasi parasite load and a 65% reduction in L. mexicana lesion size. Fluorescence-activated cell sorting analysis indicated the DNA vaccine induced a two- to fivefold increase in gamma interferon-producing CD4+ T cells, indicating a Th1-type immune response. Our results showed that the NH36 DNA vaccine induced a strong immunoprotection against visceral and cutaneous leishmaniasis, suggesting that this DNA vaccine represents a very good candidate for use against several Leishmania species.

Eco-Bio-Social Determinants for House Infestation by Non-domiciliated Triatoma dimidiata in the Yucatan Peninsula, Mexico

Background Chagas disease is a vector-borne disease of major importance in the Americas. Disease prevention is mostly limited to vector control. Integrated interventions targeting ecological, biological and social determinants of vector-borne diseases are increasingly used for improved control. Methodology/principal findings We investigated key factors associated with transient house infestation by T. dimidiata in rural villages in Yucatan, Mexico, using a mixed modeling approach based on initial null-hypothesis testing followed by multimodel inference and averaging on data from 308 houses from three villages. We found that the presence of dogs, chickens and potential refuges, such as rock piles, in the peridomicile as well as the proximity of houses to vegetation at the periphery of the village and to public light sources are major risk factors for infestation. These factors explain most of the intra-village variations in infestation. Conclusions/significance These results underline a process of infestation distinct from that of domiciliated triatomines and may be used for risk stratification of houses for both vector surveillance and control. Combined integrated vector interventions, informed by an Ecohealth perspective, should aim at targeting several of these factors to effectively reduce infestation and provide sustainable vector control.

An innovative ecohealth intervention for Chagas disease vector control in Yucatan, Mexico

Background Non-domiciliated (intrusive) triatomine vectors remain a challenge for the sustainability of Chagas disease vector control as these triatomines are able to transiently (re-)infest houses. One of the best-characterized examples is Triatoma dimidiata from the Yucatan peninsula, Mexico, where adult insects seasonally infest houses between March and July. Methods We focused our study on three rural villages in the state of Yucatan, Mexico, in which we performed a situation analysis as a first step before the implementation of an ecohealth (ecosystem approach to health) vector control intervention. Results The identification of the key determinants affecting the transient invasion of human dwellings by T. dimidiata was performed by exploring associations between bug presence and qualitative and quantitative variables describing the ecological, biological and social context of the communities. We then used a participatory action research approach for implementation and evaluation of a control strategy based on window insect screens to reduce house infestation by T. dimidiata. Conclusions This ecohealth approach may represent a valuable alternative to vertically-organized insecticide spraying. Further evaluation may confirm that it is sustainable and provides effective control (in the sense of limiting infestation of human dwellings and vector/human contacts) of intrusive triatomines in the region.

Expression, purification, immunogenicity, and protective efficacy of a recombinant Tc24 antigen as a vaccine against Trypanosoma cruzi infection in mice.

The Tc24 calcium binding protein from the flagellar pocket of Trypanosoma cruzi is under evaluation as a candidate vaccine antigen against Chagas disease. Previously, a DNA vaccine encoding Tc24 was shown to be an effective vaccine (both as a preventive and therapeutic intervention) in mice and dogs, as evidenced by reductions in T. cruzi parasitemia and cardiac amastigotes, as well as reduced cardiac inflammation and increased host survival. Here we developed a suitable platform for the large scale production of recombinant Tc24 (rTc24) and show that when rTc24 is combined with a monophosphoryl-lipid A (MPLA) adjuvant, the formulated vaccine induces a Th1-biased immune response in mice, comprised of elevated IgG2a antibody levels and interferon-gamma levels from splenocytes, compared to controls. These immune responses also resulted in statistically significant decreased T. cruzi parasitemia and cardiac amastigotes, as well as increased survival following T. cruzi challenge infections, compared to controls. Partial protective efficacy was shown regardless of whether the antigen was expressed in Escherichia coli or in yeast (Pichia pastoris). While mouse vaccinations will require further modifications in order to optimize protective efficacy, such studies provide a basis for further evaluations of vaccines comprised of rTc24, together with alternative adjuvants and additional recombinant antigens.

Medicinal potions used against infectious bowel diseases in Mayan traditional medicine

AIM OF THE STUDY Since one of the main health problems of the indigenous population are infectious bowel diseases, we decided to test Mayan medicinal potions used to treat these conditions against some of the causal agents. MATERIALS AND METHODS Thirty-one herbal formulations used for the treatment of infectious bowel diseases were prepared according to the collected ethnobotanical data. Their activities were tested against some of the causal agents of diarrheic symptoms, such as Entamoeba histolytica, Giardia lamblia, Escherichia coli, Klebsiella pneumoniae, Salmonella typhi and Shigella flexneri. RESULTS Nine formulations were active against bacteria (MIC=0.5 mg/ml), four on Entamoeba histolytica, and seven on Giardia lamblia (IC(50)≤20 μg/ml). CONCLUSIONS This work supports the use of the traditional Mayan formulations against some infectious bowel diseases, and it is the first step towards their study.

Antileishmanial activity of a mixture of Tridax procumbens and Allium sativum in mice

We tested a mixture of Tridax procumbens, known for its direct action against Leishmania mexicana, and Allium sativum, known for its immunomodulatory effect, as an alternative to treat cutaneous leishmaniasis. Acute oral toxicity was tested with the Up-and-Down Procedure (UDP) using a group of healthy mice administered with either T. procumbens or A. sativum extracts and compared with a control group. Liver injury and other parameters of toxicity were determined in mice at day 14. The in vivo assay was performed with mice infected with L. mexicana promastigotes and treated with either a mixture of T. procumbens and A. sativum or each extract separately. The thickness of the mice’s footpads was measured weekly. After the 12-week period of infection, blood samples were obtained by cardiac puncture to determine the total IgG, IgG1 and IgG2a immunoglobulins by a noncommercial indirect ELISA. We showed that the mixture of T. procumbens and A. sativum extracts was better at controlling L. mexicana infection while not being toxic when tested in the acute oral toxicity assay in mice. An increase in the ratio of IgG2a/IgG1 indicated a tendency to raise a Th1-type immune response in mice treated with the mixture. The mixture of T. procumbens and A. sativum extracts is a promising natural treatment for cutaneous leishmaniasis and its healing effects make it a good candidate for a possible new phytomedicine.

Malaria vaccine efficacy: overcoming the helminth hurdle.

Evaluation of: Noland GS, Chowdhury DR, Urban JF Jr, Zavala F, Kumar N. Helminth infection impairs the immunogenicity of a Plasmodium falciparum DNA vaccine, but not irradiated sporozoites, in mice. Vaccine 28(17), 2917–2923 (2010). Several studies have documented that helminth infections can interfere with the development of the immune response of vaccines against different diseases, although some results have been contradictory. The mechanisms involved in the inhibition of the immune response to vaccination by helminth are still unclear, and murine models of helminth–malaria coinfections have proven helpful in investigating some aspects of the interactions involved. The study evaluated here focuses on the effect of helminth infection in mice on the immunogenicity and protective efficacy of two distinct malaria vaccine candidates, a transmission-blocking DNA vaccine based on Pfs25 antigen and a pre-erythrocytic vaccine based on irradiated sporozoites. Interestingly, the authors found that helminth infection dramatically reduced DNA vaccine immunogenicity, while immunization with irradiated sporozoites was able to induce a high level of antibodies and protection, independently of helminth infection. Immune suppression by helminth infection affected all IgG isotypes, suggesting no particular polarization of the immune response, but the generation of memory B cells was not affected. It will be of key interest to understand the mechanisms underlying the efficacy of the sporozoite vaccine, and its ability to overcome helminth immunosuppression, as this may help in the design of more effective vaccines.

From Genome Screening to Creation of Vaccine Against Trypanosoma cruzi by Use of Immunoinformatics

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and activation of CD8(+) T cells is crucial for a protective immune response. Therefore, the identification of antigens with major histocompatibility complex class I epitopes is a crucial step for vaccine development against T. cruzi. Our aim was to identify novel antigens and epitopes by immunoinformatics analysis of the parasite proteome (12 969 proteins) and to validate their immunotherapeutic potential in infected mice. We identified 172 predicted epitopes, using NetMHC and RANKPEP. The corresponding protein sequences were reanalyzed to generate a consensus prediction, and 26 epitopes were selected for in vivo validation. The interferon γ (IFN-γ) recall response of splenocytes from T. cruzi-infected mice confirmed that 10 of 26 epitopes (38%) induced IFN-γ production. The immunotherapeutic potential of a mixture of all 10 peptides was evaluated in infected mice. The therapeutic vaccine was able to control T. cruzi infection, as evidenced by reduced parasitemia, cardiac tissue inflammation, and parasite burden and increased survival. These findings illustrate the benefits of this approach for the rapid development of a vaccine against pathogens with large genomes. The identified peptides and the proteins from which they are derived are excellent candidates for the development of a vaccine against T. cruzi.

Trypanosoma cruzi vaccine candidate antigens Tc24 and TSA-1 recall memory immune response associated with HLA-A and -B supertypes in Chagasic chronic patients from Mexico

Trypanosoma cruzi antigens TSA-1 and Tc24 have shown promise as vaccine candidates in animal studies. We evaluated here the recall immune response these antigens induce in Chagasic patients, as a first step to test their immunogenicity in humans. We evaluated the in vitro cellular immune response after stimulation with recombinant TSA-1 (rTSA-1) or recombinant Tc24 (rTc24) in mononuclear cells of asymptomatic Chagasic chronic patients (n = 20) compared to healthy volunteers (n = 19) from Yucatan, Mexico. Proliferation assays, intracellular cytokine staining, cytometric bead arrays, and memory T cell immunophenotyping were performed by flow cytometry. Peripheral blood mononuclear cells (PBMC) from Chagasic patients showed significant proliferation after stimulation with rTc24 and presented a phenotype of T effector memory cells (CD45RA-CCR7-). These cells also produced IFN-γ and, to a lesser extent IL10, after stimulation with rTSA-1 and rTc24 proteins. Overall, both antigens recalled a broad immune response in some Chagasic patients, confirming that their immune system had been primed against these antigens during natural infection. Analysis of HLA-A and HLA-B allele diversity by PCR-sequencing indicated that HLA-A03 and HLA-B07 were the most frequent supertypes in this Mexican population. Also, there was a significant difference in the frequency of HLA-A01 and HLA-A02 supertypes between Chagasic patients and controls, while the other alleles were evenly distributed. Some aspects of the immune response, such as antigen-induced IFN-γ production by CD4+ and CD8+ T cells and CD8+ proliferation, showed significant association with specific HLA-A supertypes, depending on the antigen considered. In conclusion, our results confirm the ability of both TSA-1 and Tc24 recombinant proteins to recall an immune response induced by the native antigens during natural infection in at least some patients. Our data support the further development of these antigens as therapeutic vaccine against Chagas disease.

Pioneering Neglected Disease Research in Southern Mexico at the ''Dr. Hideyo Noguchi'' Regional Research Center

The Autonomous University of Yucatan is a major public university in Mexico, and the largest institution for superior education in the southern part of the country, with 772 academics attending over 20,000 students. It offers 43 undergraduate and 53 graduate programs in all areas of knowledge. On October 12, 1975, Dr. Alberto Rosado G. Canton, rector of the University at this time, created the Biomedical Research Center to provide the institution with a leading facility to perform scientific medical research. The Center was named in honor of Dr. Hideyo Noguchi, the famous Japanese scientist who worked on yellow fever in the region from 1918–1920 and received a Doctorate Honoris Causa from the School of Medicine of the University in 1920. The Research Center initiated its activities with three departments: Tropical Pathology, headed by Dr. Jorge Zavala-Velazquez; Reproductive Health, headed by Dr. Thelma CetinaCanto; and Physiology, headed by Dr. Heriberto Arcila-Herrera. In 1976, the Center was strengthened by the incorporation of several researchers in social sciences, and renamed ‘‘Dr. Hideyo Noguchi’’ Regional Research Center. In 1987, it became organized in a Social Science and a Biomedical Unit, to accommodate wider research interests encompassing virology, microbiology, parasitology, reproductive health, human genetics, hematology, social medicine, neurosciences, regional development, Mayan identity and culture, and political and social processes. The department of Tropical Pathology initially pioneered some key research in Mexico and in the region in the area of tropical medicine and several neglected diseases. It also served as a seed for the training of several young researchers who contributed to the growth of the Research Center by becoming group leaders themselves in the following years, including Dr. Andrade-Narvaez, Dr. Puerto-Manzano, Dr. Farfan-Ale, and Dr. Barrera-Perez, and more recently Dr. Zavala-Castro, Dr. Garcia-Miss, Dr. Acosta-Vianna, and Dr. Vargas-Gonzalez, among others.