Ricardo Toshio Fujiwara

Vaccination with recombinant aspartic hemoglobinase reduces parasite load and blood loss after hookworm infection in dogs.

Background Hookworms infect 730 million people in developing countries where they are a leading cause of intestinal blood loss and iron-deficiency anemia. At the site of attachment to the host, adult hookworms ingest blood and lyse the erythrocytes to release hemoglobin. The parasites subsequently digest hemoglobin in their intestines using a cascade of proteolysis that begins with the Ancylostoma caninum aspartic protease 1, APR-1. Methods and Findings We show that vaccination of dogs with recombinant Ac-APR-1 induced antibody and cellular responses and resulted in significantly reduced hookworm burdens (p = 0.056) and fecal egg counts (p = 0.018) in vaccinated dogs compared to control dogs after challenge with infective larvae of A. caninum. Most importantly, vaccinated dogs were protected against blood loss (p = 0.049) and most did not develop anemia, the major pathologic sequela of hookworm disease. IgG from vaccinated animals decreased the catalytic activity of the recombinant enzyme in vitro and the antibody bound in situ to the intestines of worms recovered from vaccinated dogs, implying that the vaccine interferes with the parasites ability to digest blood. Conclusion To the best of our knowledge, this is the first report of a recombinant vaccine from a hematophagous parasite that significantly reduces both parasite load and blood loss, and it supports the development of APR-1 as a human hookworm vaccine.

Randomized, placebo-controlled, double-blind trial of the Na-ASP-2 hookworm vaccine in unexposed adults.

Necator americanus Ancylostoma Secreted Protein-2 (Na-ASP-2) is a leading larval-stage hookworm vaccine candidate. Recombinant Na-ASP-2 was expressed in Pichia pastoris and formulated with Alhydrogel. In a phase 1 trial, 36 healthy adults without history of hookworm infection were enrolled into 1 of 3 dose cohorts (n=12 per cohort) and randomized to receive intramuscular injections of either Na-ASP-2 or saline placebo. Nine participants in the first, second and third cohorts were assigned to receive 10, 50 and 100 microg of Na-ASP-2, respectively, on study days 0, 56 and 112, while 3 participants in each cohort received placebo. The most frequent adverse events were mild-to-moderate injection site reactions; in 8 participants these were delayed and occurred up to 10 days after immunization. No serious adverse events occurred. Anti-Na-ASP-2 IgG endpoint titers as determined by ELISA increased from baseline in all vaccine groups and peaked 14 days after the third injection, with geometric mean titers of 1:7066, 1:7611 and 1:11,593 for the 10, 50 and 100 microg doses, respectively, compared to <1:100 for saline controls (p<0.001). Antibody titers remained significantly elevated in all vaccine groups until the end of the study, approximately 8 months after the third vaccination. In vitro stimulation of PBMCs collected from participants with Na-ASP-2 resulted in robust proliferative responses in those who received vaccine, which increased with successive immunizations and remained high in the 50 and 100 microg dose groups through the end of the study. This first trial of a human hookworm vaccine demonstrates that the Na-ASP-2 vaccine is well-tolerated and induces a prolonged immune response in adults not exposed to hookworm, justifying further testing of this vaccine in an endemic area.

Biochemical Characterization and Vaccine Potential of a Heme-Binding Glutathione Transferase from the Adult Hookworm Ancylostoma caninum

ABSTRACT We report the cloning and expression of Ac-GST-1, a novel glutathione S-transferase from the adult hookworm Ancylostoma caninum, and its possible role in parasite blood feeding and as a vaccine target. The predicted Ac-GST-1 open reading frame contains 207 amino acids (mass, 24 kDa) and exhibited up to 65% amino acid identity with other nematode GSTs. mRNA encoding Ac-GST-1 was detected in adults, eggs, and larval stages, but the protein was detected only in adult hookworm somatic extracts and excretory/secretory products. Using antiserum to the recombinant protein, Ac-GST-1 was immunolocalized to the parasite hypodermis and muscle tissue and weakly to the intestine. Recombinant Ac-GST-1 was enzymatically active, as determined by conjugation of glutathione to a model substrate, and exhibited a novel high-affinity binding site for hematin. The possible role of Ac-GST-1 in parasite heme detoxification during hemoglobin digestion or heme uptake prompted interest in evaluating it as a potential vaccine antigen. Vaccination of dogs with Ac-GST-1 resulted in a 39.4% reduction in the mean worm burden and 32.3% reduction in egg counts compared to control dogs following larval challenge, although the reductions were not statistically significant. However, hamsters vaccinated with Ac-GST-1 exhibited statistically significant worm reduction (53.7%) following challenge with heterologous Necator americanus larvae. These studies suggest that Ac-GST-1 is a possible drug and vaccine target for hookworm infection.

Plasmodium vivax: induction of CD4+CD25+FoxP3+ regulatory T cells during infection are directly associated with level of circulating parasites.

Circulation CD4+CD25+FoxP3+ regulatory T cells (Tregs) have been associated with the delicate balancing between control of overwhelming acute malaria infection and prevention of immune pathology due to disproportionate inflammatory responses to erythrocytic stage of the parasite. While the role of Tregs has been well-documented in murine models and P. falciparum infection, the phenotype and function of Tregs in P. vivax infection is still poorly characterized. In the current study, we demonstrated that patients with acute P. vivax infection presented a significant augmentation of circulating Tregs producing anti-inflammatory (IL-10 and TGF-β) as well as pro-inflammatory (IFN-γ, IL-17) cytokines, which was further positively correlated with parasite burden. Surface expression of GITR molecule and intracellular expression of CTLA-4 were significantly upregulated in Tregs from infected donors, presenting also a positive association between either absolute numbers of CD4+CD25+FoxP3+GITR+ or CD4+CD25+FoxP3+CTLA-4+ and parasite load. Finally, we demonstrate a suppressive effect of Treg cells in specific T cell proliferative responses of P. vivax infected subjects after antigen stimulation with Pv-AMA-1. Our findings indicate that malaria vivax infection lead to an increased number of activated Treg cells that are highly associated with parasite load, which probably exert an important contribution to the modulation of immune responses during P. vivax infection.

Evaluation of an immunochemotherapeutic protocol constituted of N-methyl meglumine antimoniate (Glucantime ® ) and the recombinant Leish-110f ® + MPL-SE ® vaccine to treat canine visceral leishmaniasis

Summary The evaluation of the efficacy of an immunochemotherapy protocol to treat symptomatic dogs naturally infected with Leishmania chagasi was studied. This clinical trial had the purpose to test the combination of N-methyl meglumine antimoniate (Glucantime®) and the second generation recombinant vaccine Leish-110f® plus the adjuvant MPL-SE® to treat the canine leishmaniasis (CanL). Thirty symptomatic naturally infected mongrel dogs were divided into five groups. Animals received standard treatment with Glucantime® or treatment with Glucantime®/Leish-110f® +MPL-SE® as immunochemotherapy protocol. Additional groups received Leish-110f® +MPL-SE® only, MPL-SE® only, or placebo. Evaluation of haematological, biochemical (renal and hepatic function) and plasmatic proteins, immunological (humoral and cellular immune response) and the parasitological test revealed improvement of the clinical parameters and parasitological cure in dogs in both chemotherapy alone and immunochemotherapy cohorts. However, the immunotherapy and immunochemotherapy cohorts had reduced number of deaths, higher survival probability, and specific cellular reactivity to leishmanial antigens, in comparison with chemotherapy cohort only and control groups (adjuvant alone and placebo). These results support the notion of using well-characterized recombinant vaccine as an adjunct to improve the current chemotherapy of CanL.

Genomic analyses, gene expression and antigenic profile of the trans-sialidase superfamily of Trypanosoma cruzi reveal an undetected level of complexity.

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a highly debilitating human pathology that affects millions of people in the Americas. The sequencing of this parasites genome reveals that trans-sialidase/trans-sialidase-like (TcS), a polymorphic protein family known to be involved in several aspects of T. cruzi biology, is the largest T. cruzi gene family, encoding more than 1,400 genes. Despite the fact that four TcS groups are well characterized and only one of the groups contains active trans-sialidases, all members of the family are annotated in the T. cruzi genome database as trans-sialidase. After performing sequence clustering analysis with all TcS complete genes, we identified four additional groups, demonstrating that the TcS family is even more heterogeneous than previously thought. Interestingly, members of distinct TcS groups show distinctive patterns of chromosome localization. Members of the TcSgroupII, which harbor proteins involved in host cell attachment/invasion, are preferentially located in subtelomeric regions, whereas members of the largest and new TcSgroupV have internal chromosomal locations. Real-time RT-PCR confirms the expression of genes derived from new groups and shows that the pattern of expression is not similar within and between groups. We also performed B-cell epitope prediction on the family and constructed a TcS specific peptide array, which was screened with sera from T. cruzi-infected mice. We demonstrated that all seven groups represented in the array are antigenic. A highly reactive peptide occurs in sixty TcS proteins including members of two new groups and may contribute to the known cross-reactivity of T. cruzi epitopes during infection. Taken together, our results contribute to a better understanding of the real complexity of the TcS family and open new avenues for investigating novel roles of this family during T. cruzi infection.

Hookworm products ameliorate dextran sodium sulfate‐induced colitis in BALB/c mice

Background: Several lines of evidence have shown that helminthiasis can significantly reduce disease severity in animal models of intestinal inflammation, airway inflammation/hyperreactivity, diabetes, and multiple sclerosis. Identification and characterization of helminth‐derived immunomodulatory molecules that contribute to anticolitis effects could lead to new therapeutic approaches in inflammatory bowel diseases (IBDs) without the need for helminth infection. We evaluated the therapeutic potential of adult human hookworm, Ancylostoma ceylanicum, crude (Aw) and excreted/secreted (ES) products on dextran sulfate sodium (DSS)‐induced colitis in BALB/c mice. Methods: Colitis was induced by 5% DSS oral administration for 7 days. Clinical disease severity was monitored daily during concomitant intraperitoneal treatment with helminth‐derived products. Additionally, several pathways of immunological modulation induced by A. ceylanicum products (MPO, EPO, Th1, Th2, and Th17 cytokine responses) in the inflamed intestinal microenvironment were assessed. Finally, the histopathological profile of the colon was characterized. Results: Hookworm products are able to modulate the potent proinflammatory response induced by DSS, mainly through the downregulation of Th1 and Th17 cytokines. These proteins also reduce clinical and colonic microscopic inflammation scores as well as EPO and MPO activity. Conclusions: Ancylostoma ceylanicum Aw and ES mediators have an important therapeutic potential in experimental colitis in mice, which may provide a more socially acceptable form of therapy for patients with IBDs as opposed to using living worms. Our results support the urgency of further isolation and recombinant expression of active hookworm products responsible for the beneficial effects on colitis. (Inflamm Bowel Dis 201117:2275–2286)

Necator americanus Infection: A Possible Cause of Altered Dendritic Cell Differentiation and Eosinophil Profile in Chronically Infected Individuals

Background Hookworms survive for several years (5 to 7 years) in the host lumen, inducing a robust but largely ineffective immune response. Among the most striking aspects of the immune response to hookworm (as with many other helminths) is the ablation of parasite-specific T cell proliferative response (hyporesponsiveness). While the role of the adaptive immune response in human helminth infection has been well investigated, the role of the innate immune responses (e.g., dendritic cells and eosinophils) has received less attention and remains to be clearly elucidated. Methodology/Principal Findings We report on the differentiation/maturation of host dendritic cells in vitro and the eosinophil activation/function associated with human hookworm infection. Mature DCs (mDCs) from Necator americanus (Necator)–infected individuals showed an impaired differentiation process compared to the mDCs of non-infected individuals, as evidenced by the differential expression of CD11c and CD14. These same hookworm-infected individuals also presented significantly down-regulated expression of CD86, CD1a, HLA-ABC, and HLA-DR. The lower expression of co-stimulatory and antigen presentation molecules by hookworm-infected–derived mDCs was further evidenced by their reduced ability to induce cell proliferation. We also showed that this alternative DC differentiation is partially induced by excreted-secreted hookworm products. Conversely, eosinophils from the same individuals showed a highly activated status, with an upregulation of major cell surface markers. Antigen-pulsed eosinophils from N. americanus–infected individuals induced significant cell proliferation of autologous PBMCs, when compared to non-infected individuals. Conclusion Chronic N. americanus infection alters the hosts innate immune response, resulting in a possible modulation of the maturation process of DCs, a functional change that may diminish their ability for antigen presentation and thus contribute to the ablation of the parasite-specific T cell proliferative response. Interestingly, a concomitant upregulation of the major cell surface markers of eosinophils was observed in hookworm-infected individuals, indicative of antigen-specific immune responses, especially antigen presentation. We showed that in addition to the postulated role of the eosinophils as effector cells against helminth infection, activated cells may also be recruited to sites of inflammation and contribute to the immune response acting as antigen presenting cells.

Profile of central and effector memory T cells in the progression of chronic human Chagas disease.

Background Chronic Chagas disease presents several different clinical manifestations ranging from asymptomatic to severe cardiac and/or digestive clinical forms. Several studies have demonstrated that immunoregulatory mechanisms are important processes for the control of the intense immune activity observed in the chronic phase. T cells play a critical role in parasite specific and non-specific immune response elicited by the host against Trypanosoma cruzi. Specifically, memory T cells, which are basically classified as central and effector memory cells, might have a distinct migratory activity, role and function during the human Chagas disease. Methodology/Principal Findings Based on the hypothesis that the disease severity in humans is correlated to the quality of immune responses against T. cruzi, we evaluated the memory profile of peripheral CD4+ and CD8+ T lymphocytes as well as its cytokine secretion before and after in vitro antigenic stimulation. We evaluated cellular response from non-infected individuals (NI), patients with indeterminate (IND) or cardiac (CARD) clinical forms of Chagas disease. The expression of CD45RA, CD45RO and CCR7 surface molecules was determined on CD4+ and CD8+ T lymphocytes; the pattern of intracellular cytokines (IFN-γ, IL-10) synthesized by naive and memory cells was determined by flow cytometry. Our results revealed that IND and CARD patients have relatively lower percentages of naive (CD45RAhigh) CD4+ and CD8+ T cells. However, statistical analysis of ex-vivo profiles of CD4+ T cells showed that IND have lower percentage of CD45RAhigh in relation to non-infected individuals, but not in relation to CARD. Elevated percentages of memory (CD45ROhigh) CD4+ T cells were also demonstrated in infected individuals, although statistically significant differences were only observed between IND and NI groups. Furthermore, when we analyzed the profile of secreted cytokines, we observed that CARD patients presented a significantly higher percentage of CD8+CD45RAhigh IFN-γ-producing cells in control cultures and after antigen pulsing with soluble epimastigote antigens. Conclusions Based on a correlation between the frequency of IFN-γ producing CD8+ T cells in the T cell memory compartment and the chronic chagasic myocarditis, we propose that memory T cells can be involved in the induction of the development of the severe clinical forms of the Chagas disease by mechanisms modulated by IFN-γ. Furthermore, we showed that individuals from IND group presented more TCM CD4+ T cells, which may induce a regulatory mechanism to protect the host against the exacerbated inflammatory response elicited by the infection.

Comparative immunology of human and animal models of hookworm infection

Hookworm infection is a major cause of disease burden for animals and humans. Over the past years, the use of animal models in hookworm infections has been driven by the search of new anthelminthic therapies and, especially, vaccine development. These studies also contributed to the advance of knowledge on immunity to hookworms, offering new insights to understand the nature of this parasitic infection. In this article, we will summarize the essential features of the immune response in the two major animal models of hookworm infections (dog and hamster) and then consider its implication for the human immune response.