Rodrigo Correa-Oliveira

Tetraspanins on the surface of Schistosoma mansoni are protective antigens against schistosomiasis.

Schistosomes are blood-dwelling flukes that infect 200 million people worldwide and are responsible for hundreds of thousands of deaths annually. Using a signal sequence trap, we cloned from Schistosoma mansoni two cDNAs, Sm-tsp-1 and Sm-tsp-2, encoding the tetraspanin (TSP) integral membrane proteins TSP-1 and TSP-2. We raised antibodies to recombinant TSP fusion proteins and showed that both proteins are exposed on the surface of S. mansoni. Recombinant TSP-2, but not TSP-1, is strongly recognized by IgG1 and IgG3 (but not IgE) from naturally resistant individuals but is not recognized by IgG from chronically infected or unexposed individuals. Vaccination of mice with the recombinant proteins followed by challenge infection with S. mansoni resulted in reductions of 57% and 64% (TSP-2) and 34% and 52% (TSP-1) for mean adult worm burdens and liver egg burdens, respectively, over two independent trials. Fecal egg counts were reduced by 65–69% in both test groups. TSP-2 in particular provided protection in excess of the 40% benchmark set by the World Health Organization for progression of schistosome vaccine antigens into clinical trials. When coupled with its selective recognition by naturally resistant people, TSP-2 seems to be an effective vaccine antigen against S. mansoni.

Antibodies against a secreted protein from hookworm larvae reduce the intensity of hookworm infection in humans and vaccinated laboratory animals

The development of a vaccine would provide an important new tool for the control of human hookworm infection. On the basis of successful vaccination of laboratory animals with living irradiated, third‐stage hookworm larvae (L3), we examined the antibody responses of individuals from hookworm endemic areas of Brazil and China against the most abundant L3 secreted antigens, the ancylostoma secreted proteins, ASP‐1 and ASP‐2. Logistic regression was used to investigate the effects of antibody isotype responses to ASPs on the risk of an individual harboring heavy hookworm infection. A significant protective association was observed between increasing anti‐ASP‐2 IgE levels and the risk of heavy hookworm infection. To confirm that ASP‐2 is a protective antigen, laboratory dogs were immunized with recombinant ASP‐2 formulated with the GlaxoSmithKline Adjuvant, AS03. Sera obtained from the immunized dogs exhibited high geometric mean antibody titers, immunoprecipitated native ASP‐2 from L3 extracts and localized the site of ASP‐2 expression to the glandular esophagus and body channels exiting to the cuticle. The sera also exhibited an increased ability to inhibit migration of L3 through tissue in vitro relative to sera from AS03‐injected controls. Upon L3 challenge, the ASP‐2 vaccinated dogs exhibited significant reductions in fecal egg counts and intestinal hookworm burden. These findings provide strong support for the development of an effective recombinant vaccine against hookworm infection in humans.

Synergistic associations between hookworm and other helminth species in a rural community in Brazil

Objective  To identify possible synergistic associations of hookworm and other helminths.

Progress in the development of a recombinant vaccine for human hookworm disease: The Human Hookworm Vaccine Initiative

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.

Systemic and compartmentalized immune response in canine visceral leishmaniasis

Human visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL) are the most important emerging diseases with high prevalence in Latin American countries and are mainly caused by Leishmania (L.) chagasi (Syn=L. infantum). CVL has a great impact on Brazilian public health because domestic dogs are the most important VL peri-domicile reservoirs in both urban and peri-urban areas. Our findings highlight the complexity of cellular immunological events related to the natural infection from dogs by L. chagasi, additionally correlating major peripheral blood phenotypic markers with clinical status and tissues parasite density. Our main results demonstrated that lower frequency of circulating B cells and monocytes are important markers of severe CVL, whereas increased levels of CD8+ lymphocytes appear to be the major phenotypic feature of asymptomatic disease. Determination of the isotypes patterns during CVL demonstrated that asymptomatic dogs and those with low parasitism are associated with an increase of IgG1, while the symptomatic dogs and those with high parasitism are associated with an increase of IgG, IgG2, IgM, IgA and IgE immunoglobulins. Pioneer findings obtained by our group showed a correlation between clinical status of CVL with degree of tissue parasite density. This data demonstrated that asymptomatic dogs presented low parasitism while symptomatic dogs are associated with high parasite load in various tissues such as skin, bone marrow and spleen. We have also investigated the association between tissue parasitism and CVL clinical forms. Regardless of clinical status, skin and spleen are the major sites of high parasite density during ongoing CVL. Furthermore, we demonstrated that bone marrow and spleen parasite density are the most reliable parasitological markers to decode the clinical status of CVL. In this article, we have reviewed some aspects of the histopathological and immunological events occurring in natural and experimental L. chagasi/L. infantum infection, pointing out the main L. chagasi-parasitized tissue. We have discussed the importance of the association between parasite density, immunological/histopathological aspects and clinical status of the CVL, their current applications, challenges for the future and potential opportunities in CVL research.

Cytokine Regulation of Human Immune Response to Schistosoma mansoni: Analysis of the Role of IL‐4, IL‐5 and IL‐10 on Peripheral Blood Mononuclear Cell Responses

The role of cytokines on the in vitro proliferative response of peripheral blood mononuclear cells (PBMC) from Schistosoma mansoni infected patients to soluble egg (SEA) and adult worm antigens (SWAP) were evaluated. The results obtained demonstrated that the proliferative response of PBMC from chronic intestinal (INT) patients to SEA and SWAP is increased by the blockage of IL‐10 with specific monoclonal antibodies (MAb). The effects of these antibodies were readily reversed by the addition of recombinant IL‐10. In contrast, no effect was observed on the PBMC response of acute and hepatosplenic patients (HS) in the presence of anti‐IL‐10. Anti‐IL‐4 antibodies decreased the PBMC response of the intestinal (INT) and HS individuals to SEA and SWAP, and the PBMC response of acute patients to SEA but not to SWAP. Addition of anti‐IL‐5 MAb did not decrease the PBMC response of acute patients to SEA or SWAP. These results suggested that IL‐10 has an important role in the modulation of the immune response in chronic asymptomatic patients and that this cytokine may be an important factor in controlling morbidity.

Mixed inflammatory/regulatory cytokine profile marked by simultaneous raise of interferon-gamma and interleukin-10 and low frequency of tumour necrosis factor-alpha(+) monocytes are hallmarks of active human visceral Leishmaniasis due to Leishmania chagasi infection.

Considering the complexity of the immunological events triggered during active visceral Leishmaniasis (VL), the relevance of the segregation of the immune response during human VL into type 1 and type 2 still remains unclear. For this purpose, in individuals living in risk areas for VL, we have evaluated especially asymptomatic individuals and patients with active VL, the plasmatic levels of cytokines and reactive nitrogen species under ex vivo conditions. In addition, we have also performed an analysis of intracellular cytokine patterns of circulating leucocytes after short‐term culture, particularly in the absence of antigenic‐specific stimulation, in order to reflect dynamic events of immune response in vivo during Leishmania chagasi infection. Although asymptomatic individuals and non‐infected subjects presented a similar immunological profile, an outstanding inflammatory/regulatory profile, based on higher plasmatic levels of cytokines such as interleukin (IL)‐8, interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α, IL‐6 and IL‐10, was associated with clinical status observed in active VL. In this context, we hypothesize that IL‐10, through its ability to inhibit anti‐leishmanial macrophage activation, associated with the lower frequency of TNF‐α+ monocytes and ordinary levels of nitrite and nitrate are the major mechanisms associated with disease onset.

Hookworm, Ascaris lumbricoides infection and polyparasitism associated with poor cognitive performance in Brazilian schoolchildren.

Objective  To investigate the relationship between hookworm and Ascaris lumbricoides infection and performance on three subsets of the Wechsler Intelligence Scale for Children – third edition (WISC‐III) (Digit Span, Arithmetic and Coding) and Raven Colored Progressive Matrices.

Immunity to schistosomiasis: glycans are potential antigenic targets for immune intervention.

The major humoral immune responses in animals infected with Schistosoma mansoni are directed toward carbohydrate antigens. Among these antigens are complex-type N-glycans expressing LDN [GalNAcbeta1-4GlcNAc-R], LDNF [GalNAcbeta1-4(Fucalpha1-3)GlcNAc-R], and polymeric Lewis x (Lex) [Galbeta1-4(Fucalpha1-3)GlcNAc]n-R epitopes. We have now evaluated the potential of the three glycan antigens as targets for immune-mediated intervention of infections and serodiagnosis. A variety of approaches were employed, including ELISA, Western blot, immunohistology, and in vitro complement lysis assays, to determine the immunogenicity of the glycans in infected humans, their localization on the parasites and their efficacy as targets for parasite lysis. Our results show that S. mansoni-infected patients, with either intestinal or hepatosplenic disease, generate predominantly IgM, but also IgG and IgA, antibodies to LDN, LDNF, and Lex. However, immune responses to Lex are generally lower than responses to LDN and LDNF and less specific to schistosome infections. Western blot analysis with monoclonal antibodies (mAb) to LDN, LDNF, and Lex determinants show that the glycan antigens occur on multiple glycoproteins from cercariae, 3-h, 48-h, and lung stage schistosomula, as well as adults and eggs. Immunohistological studies demonstrate that LDN, LDNF, and Lex are expressed on the parasite surface at all stages of development in the vertebrate host. Importantly, a mAb to LDN in the presence of complement efficiently kills schistosomula in vitro, as demonstrated by flow-cytometric assays that quantify cytolysis by propidium iodide uptake into damaged parasites. These findings raise the possibility that LDN and LDNF may be targets for vaccination and/or serodiagnosis of chronic schistosomiasis in humans.

Age patterns in undernutrition and helminth infection in a rural area of Brazil: associations with ascariasis and hookworm

Objective  To investigate the nutritional status of individuals from a rural area of Brazil, and associations with helminth infections in an age‐stratified sample.