RESUMO
As part of on-going efforts to control hookworm infection, the "human hookworm vaccine initiative" has recognised blood feeding as a feasible therapeutic target for inducing immunity against hookworm infection. To this end, molecular approaches have been used to identify candidate targets, such as Necator americanus (Na) haemoglobinase aspartic protease-1 (APR-1), with immunogenicity profiled in canine and hamster models. We sought to accelerate the immune analysis of these identified therapeutic targets by developing an appropriate mouse model. Here we demonstrate that Nippostrongylus brasiliensis (Nb), a phylogenetically distant strongylid nematode of rodents, begins blood feeding early in its development and that immunisation with Na-APR-1 can block its growth and completion of its life cycle. Furthermore, we identify a new haem detoxification pathway in Nb required for blood feeding that can be blocked by drugs of the quinolone family, reducing both infection burden and the associated anaemia in rodents. Collectively, our findings show that haem metabolism has potential as a checkpoint for interrupting hookworm development in early stages of the hookworm life cycle and that the Nippostrongylus brasiliensis rodent model is relevant for identifying novel therapeutic targets against human hookworm.
Assuntos
Anticorpos Anti-Helmínticos/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Eritrócitos/efeitos dos fármacos , Infecções por Uncinaria/prevenção & controle , Necator americanus/enzimologia , Nippostrongylus/crescimento & desenvolvimento , Infecções por Strongylida/prevenção & controle , Ancylostomatoidea/efeitos dos fármacos , Ancylostomatoidea/crescimento & desenvolvimento , Animais , Antígenos de Helmintos/imunologia , Ácido Aspártico Endopeptidases/imunologia , Eritrócitos/parasitologia , Feminino , Infecções por Uncinaria/parasitologia , Estágios do Ciclo de Vida , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nippostrongylus/efeitos dos fármacos , Infecções por Strongylida/parasitologiaRESUMO
A molecular modeling study giving structural, functional, and mutagenesis insights into the anti-BACE1 Fab fragment that recognizes the BACE1 exosite is reported. Our results allow extending experimental data resulting from X-ray diffraction experiments in order to examine unknown aspects for the Fab-BACE1 recognition and its binding mode. Thus, the study performed here allows extending the inherently static nature of crystallographic structures in order to gain a deeper understanding of the structural and dynamical basis at the atomic level. The characteristics and strength of the interatomic interactions involved in the immune complex formation are exhaustively analyzed. The results might explain how the anti-BACE1 Fab fragment and other BACE1 exosite binders are capable to produce an allosteric modulation of the BACE1 activity. Our site-directed mutagenesis study indicated that the functional anti-BACE1 paratope, residues Tyr32 (H1), Trp50 (H2), Arg98 (H3), Phe101 (H3), Trp104 (H3) and Tyr94 (L3), strongly dominates the binding energetics with the BACE1 exosite. The mutational studies described in this work might accelerate the development of new BACE1 exosite binders with interesting pharmacological activity.
Assuntos
Secretases da Proteína Precursora do Amiloide/imunologia , Ácido Aspártico Endopeptidases/imunologia , Fragmentos Fab das Imunoglobulinas/química , Epitopos , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Modelos Moleculares , Mutagênese Sítio-DirigidaRESUMO
Rhipicephalus (Boophilus) microplus is one of the most widely distributed tick in the world. The control of the parasite is based mainly on the use of chemical acaricides, which are produced from a limited set of molecules. These drugs induce selection of acaricide-resistant ticks, and are an important source of environmental pollution. An approach based on anti-tick vaccines may circumvent these obstacles. Characterization of the physiological function of tick molecules may be useful to develop new vaccines. Previously, we reported the ability of some tick proteins as inducers of protective immune response. Vaccination studies using tick proteins like native (nBYC), recombinant (rBYC) egg-yolk aspartic endopeptidase and cysteine endopeptidase (VTDCE) from R. microplus and glutathione S-transferase (Hl-GST) from Haemaphysalis longicornis demonstrated the immunogenicity and antigenicity of these proteins in bovines. Eventually, immunization with these proteins triggered a partial immune response against R. microplus infestation in cattle, manifested mainly as a reduction in egg fertility (7.7% and 13.9% for nBYC, 5.9% for rBYC; 4.7% for VTDCE, 7.9% for Hl-GST), and in the number of fully engorged ticks (18.2% for rBYC, 14.6% for VTDCE, 53% for Hl-GST). The data so far obtained suggest that these proteins have potential to be used as antigens in an anti-tick vaccine. Other proteins involved in tick embryogenesis also have this potential, like THAP and BmCl1, which are enzymes with key roles in vitellin and hemoglobin hydrolysis. Moreover, the identification of analogous proteins present in other tick species may bring information about the way to develop a vaccine against multiple tick species which can help to solve the problem faced by numerous countries where animals are parasitized by more than one tick species. The aim of the present review is to comprehensibly summarize the data obtained in the last few years by our collaborative research, discussing the efforts we have made to find antigens efficient enough for a cattle tick-controlling vaccine. This review discusses tick physiology studies aimed at the selection of possible targets, characterization of the selected proteins with emphasis on their biochemical and immunological aspects and results of vaccine trials on bovines.
Assuntos
Ácido Aspártico Endopeptidases/imunologia , Doenças dos Bovinos/parasitologia , Cisteína Endopeptidases/imunologia , Proteínas do Ovo/imunologia , Rhipicephalus/imunologia , Infestações por Carrapato/veterinária , Vacinas/imunologia , Animais , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Imunização/veterinária , Rhipicephalus/enzimologia , Infestações por Carrapato/imunologia , Infestações por Carrapato/parasitologia , Infestações por Carrapato/prevenção & controleRESUMO
VTDCE (Vitelin-Degrading Cysteine Endopeptidase) is a peptidase with an active role in Rhipicephalus (Boophilus) microplus embryogenesis. VTDCE is found in the tick's eggs and was shown to be the most active protein in vitellin (VT) hydrolysis of the three peptidases already characterized in R. microplus eggs (Boophilus Yolk pro-cathepsin (BYC), Tick Heme Binding Aspartic Proteinase (THAP) and VTDCE). VTDCE activity was assessed in vitro using the natural substrate and a synthetic substrate (N-Cbz-Phe-Arg-MCA). The activity was inhibited by anti-VTDCE antibodies. In the present study, it was shown that VTDCE acts differently from BYC and THAP in VT hydrolysis and that the vaccination of bovines with VTDCE induces a partial protective immune response against R. microplus infestation. Immunized bovines challenged with R. microplus larvae presented an overall protection of 21%, and a reduction in the weight of fertile eggs of 17.6% was observed. The data obtained indicate that VTDCE seems to be important for tick physiology, and that it induces partial protective immune response when inoculated in bovines. This suggests that VTDCE can be useful to improve the protective capacity observed for other antigens.
Assuntos
Doenças dos Bovinos/parasitologia , Cisteína Endopeptidases/imunologia , Rhipicephalus/imunologia , Infestações por Carrapato/veterinária , Vacinas/imunologia , Animais , Anticorpos/sangue , Ácido Aspártico Endopeptidases/imunologia , Western Blotting/veterinária , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Cisteína Endopeptidases/metabolismo , Precursores Enzimáticos/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Infestações por Carrapato/imunologia , Infestações por Carrapato/prevenção & controle , Vacinas/farmacologia , Vitelinas/metabolismoRESUMO
Secreted acid proteinases (SAP) constitute an important group of virulence factors in Candida albicans. In the present work, an acid proteinase from C. albicans was sequentially purified from the supernatant of a yeast culture by precipitation with ammonium sulfate, ion exchange chromatography, and molecular exclusion chromatography, yielding a specific enzymatic activity of 204.1 IU/mg on bovine serum albumin (BSA). The molecular mass of the purified proteinase was estimated at 43 kd after exclusion chromatography and at 41 kd by nondenaturating sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified proteinase was able to degrade BSA at pH 2.5, but was not active on collagen, and it was significantly inhibited by pepstatin A. The immunization of BALB/c mice with the purified proteinase and later fusion of their spleen cells with myeloma cells resulted in 19 monoclonal antibody secreting hybridomas (MAbs) capable of detecting SAP in enzyme-linked immunosorbent assay (ELISA) assays. All MAbs obtained are isotype IgG1 kappa (kappa) immunoglobulins and develop a 41 kd protein band by Western blot (WB) in samples of SAP obtained from C. albicans (12-A) and C. dubliniensis (strain 778) crude extracts. The anti-SAP MAbs were used in capture ELISA and two combinations of these antibodies proved suitable for SAP detection, that is, MAP1 (1B1B3) or MAP2 (2D2C10) as coat antibodies, and biotinylated MAP3 (2A6E8) as detect antibody. Capture ELISA using these sets of MAbs detected over 32 ng/mL protein in purified SAP samples as well as in crude C. albicans and C. dubliniensis extracts. The results herein obtained allow for the prediction of how this set of antibodies can be useful for SAP detection in biologic specimens.
Assuntos
Anticorpos Monoclonais/biossíntese , Ácido Aspártico Endopeptidases/análise , Candida albicans/enzimologia , Candida/enzimologia , Hibridomas/metabolismo , Imunoglobulina G/biossíntese , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Ácido Aspártico Endopeptidases/imunologia , Ácido Aspártico Endopeptidases/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Boophilus Yolk pro-Cathepsin (BYC) is an aspartic proteinase found in Boophilus microplus eggs that is involved in the embryogenesis and has been tested as antigen to compose an anti-tick vaccine. The vaccine potential of a recombinant BYC expressed in Escherichia coli (rBYC) was investigated. rBYC was purified and used to immunize Hereford cattle. The sera of bovines immunized with rBYC recognized the native BYC with a titer ranging from 125 to 4000. Furthermore, immunized bovines challenged with 20,000 larvae presented an overall protection of 25.24%. The partial protection obtained against B. microplus infestation with the recombinant protein immunization was similar to the already described for native BYC immunization.
Assuntos
Ácido Aspártico Endopeptidases/imunologia , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/parasitologia , Precursores Enzimáticos/imunologia , Ixodidae/imunologia , Infestações por Carrapato/prevenção & controle , Infestações por Carrapato/veterinária , Vacinação/veterinária , Animais , Formação de Anticorpos , Ácido Aspártico Endopeptidases/genética , Western Blotting/veterinária , Bovinos , Doenças dos Bovinos/imunologia , Precursores Enzimáticos/genética , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Imunoglobulina G/sangue , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Infestações por Carrapato/imunologia , Infestações por Carrapato/parasitologiaRESUMO
The capacity of the Boophilus Yolk pro-Cathepsin (BYC) to induce a protective immune response in cattle against Boophilus microplus infestation was tested by vaccination experiments and by inoculation of monoclonal antibody (MAb) against BYC into fully engorged tick females. In immunization experiments the measurement of various biological parameters demonstrated a partial protection against B. microplus. A continuous decrease in the levels of specific antibodies was observed over 11 months when six bovines were maintained in field conditions. The inoculation of the MAb into tick females produced a dose-dependent decrease in oviposition and survival of the ectoparasite compared to the control.