Evaluation of the PEΔIII-LC3-KDEL3 Chimeric Protein of Entamoeba histolytica-Lectin as a Vaccine Candidate against Amebic Liver Abscess.

Entamoeba histolytica is an intestinal parasite that causes dysentery and amebic liver abscess. E. histolytica has the capability to invade host tissue by union of virulence factor Gal/GalNAc lectin; this molecule induces an adherence-inhibitory antibody response as well as to protect against amebic liver abscess (ALA). The present work showed the effect of the immunization with PEΔIII-LC3-KDEL3 recombinant protein. In vitro, this candidate vaccine inhibited adherence of E. histolytica trophozoites to HepG2 cell monolayer, avoiding the cytolysis, and in a hamster model, we observed a vaccine-induced protection against the damage to tissue liver and the inhibition of uncontrolled inflammation. PEΔIII-LC3-KDEL3 reduced the expression of TNF-α, IL-1ß, and NF-κB in all immunized groups at 4- and 7-day postinfection. The levels of IL-10, FOXP3, and IFN-γ were elevated at 7 days. The immunohistochemistry assay confirmed this result, revealing an elevated quantity of +IFN-γ cells in the liver tissue. ALA formation in hamsters immunized was minimal, and few trophozoites were identified. Hence, immunization with PEΔIII-LC3-KDEL3 herein prevented invasive amebiasis, avoided an acute proinflammatory response, and activated a protective response within a short time. Finally, this recombinant protein induced an increase of serum IgG.

(-)-Epicatechin protects from amebic liver abscess development in hamster.

In this work the effect of (-)-epicatechin on the development of amebic liver abscess in hamsters was evaluated. (-)-epicatechin is a flavonoid present in plants that possesses various biological properties, including its activity against some protozoal parasites; however its antiamebic activity in a living model had not been evaluated. Syrian golden hamsters were intrahepatically inoculated with 1x106E. histolytica trophozoites, three days after inoculation they received nine intraperitoneal doses of (-)-epicatechin (10 mg/100 g) every 48 h. Animals without treatments and treated with metronidazole were included as controls. Macroscopic characteristics of the hepatic abscess, histopathological analysis of the tissue and the levels of inflammatory cytokines were determined. (-)-epicatechin produced a decrease in liver abscess progression being observed only 9.49% of damage compared to 84% shown by untreated animals. During treatment with (-)-epicatechin hepatic tissue showed signs of liver repair and absence of amoebae. Additionally, (-)-epicatechin produced a modulating effect on inflammatory cytokines TNF-α, IL-1ß and IL-10. All these events observed in animals treated with (-)-epicatechin could contribute to the elimination of trophozoites and liver healing.

Linearolactone and Kaempferol Disrupt the Actin Cytoskeleton in Entamoeba histolytica: Inhibition of Amoebic Liver Abscess Development.

Linearolactone (1) and kaempferol (2) have amebicidal activity in in vitro studies. The type of cell death induced by 1 and 2 and their effects on the virulence of E. histolytica were analyzed by transmission and confocal electron microscopy, reactive oxygen species (ROS) production, and apoptosis, detected by flow cytometry with dichlorofluorescein 2',7'-diacetate and annexin-V binding, respectively, and confirmed by TUNEL. The interaction of 1 and 2 with actin was analyzed by docking, and the in vivo amoebicidal activity was established with the Mesocricetus auratus model; amebic liver abscess (ALA) development was evaluated by magnetic resonance (MR) and validated post mortem. In vitro, compounds 1 and 2 caused chromatin condensation, intracellular ROS, and loss of actin structures. Coupling analysis showed that they bind to the allosteric and catalytic sites of actin with binding energies of -11.30 and -8.45 kcal/mol, respectively. Treatments with 1 and 2 induced a decrease in ALA formation without toxic effects on the liver and kidney. Thus, compound 1, but not 2, was able to induce apoptosis-like effects in E. histolytica trophozoites by intracellular production of ROS that affected the actin cytoskeleton structuration. In vivo, compound 1 was more active than compound 2 to reduce the development of ALA.

Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess.

BACKGROUND: Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties. METHODOLOGY/PRINCIPAL FINDINGS: A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene. CONCLUSIONS/SIGNIFICANCE: Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses.

Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment.

In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4.

Immunization with the Entamoeba histolytica surface metalloprotease EhMSP-1 protects hamsters from amebic liver abscess.

Diarrhea and amebic liver abscesses due to invasive Entamoeba histolytica infections are an important cause of morbidity and mortality in the developing world. Entamoeba histolytica adherence and cell migration, two phenotypes linked to virulence, are both aberrant in trophozoites deficient in the metallosurface protease EhMSP-1, which is a homologue of the Leishmania vaccine candidate leishmanolysin (GP63). We examined the potential of EhMSP-1 for use as a vaccine antigen to protect against amebic liver abscesses. First, existing serum samples from South Africans naturally infected with E. histolytica were examined by enzyme-linked immunosorbent assay (ELISA) for the presence of EhMSP-1-specific IgG. Nine of 12 (75%) people with anti-E. histolytica IgG also had EhMSP-1-specific IgG antibodies. We next used a hamster model of amebic liver abscess to determine the effect of immunization with a mixture of four recombinant EhMSP-1 protein fragments. EhMSP-1 immunization stimulated a robust IgG antibody response. Furthermore, EhMSP-1 immunization of hamsters reduced development of severe amebic liver abscesses following intrahepatic injection of E. histolytica by a combined rate of 68% in two independent animal experiments. Purified IgG from immunized compared to control animals bound to the surface of E. histolytica trophozoites and accelerated amebic lysis via activation of the classical complement cascade. We concluded that EhMSP-1 is a promising antigen that warrants further study to determine its full potential as a target for therapy and/or prevention of invasive amebiasis.

Obtaining of three recombinant antigens of Entamoeba histolytica and evaluation of their immunogenic ability without adjuvant in a hamster model of immunoprotection.

A 30-kDa surface collagen binding protein peroxiredoxin of Entamoeba histolytica (EhCBP30) was evaluated either alone or fused to the chaperone (CHP) or ATPase (ATP) domains of heat shock protein 70 of Trypanosoma cruzi (TcHSP70) as a vaccine candidate in a hamster model of experimental amoebic liver abscess (ALA) development. Three constructs were produced containing the EhCBP30 DNA sequence, one expressing EhCBP30 and two expressing EhCBP30 fused to either CHP or ATP domains of TcHSP70. High purity recombinant proteins rEhCBP30, rEhCBP30-CHP and rEhCBP30-ATP with N-terminal His tag were obtained by single step affinity purification. Hamsters were immunized without adjuvant with the antigenic recombinant proteins and then challenged intrahepatically with E. histolytica trophozoites. A 70% decrease in ALA development was detected in hamsters immunized with rEhCBP30 and rEhCBP30-CHP, while animals immunized with rEhCBP30-ATP did not show a statistically significant decrease in ALA formation compared with non-immunized animals. Histological analysis of liver tissue showed that the inflammatory infiltrate was discrete or moderate in hamsters immunized with rEhCBP30 or rEhCBP30-CHP compared with that observed in control hamsters or hamsters immunized with rEhCBP30-ATP. These results suggest that rEhCBP30 and rEhCBP30-CHP are able to induce an effective immune response that may protect hamsters against ALA development.

Mucosal delivery of ACNPV baculovirus driving expression of the Gal-lectin LC3 fragment confers protection against amoebic liver abscess in hamster.

Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine.

Intranasal immunization with Gal-inhibitable lectin plus an adjuvant of CpG oligodeoxynucleotides protects against Entamoeba histolytica challenge.

The development of an effective amebiasis vaccine could improve child health in the developing world, reducing cases of amebic colitis and liver abscess. An ideal vaccine would be comprised of a well-characterized parasite antigen and an adjuvant, which would have high potency while driving the immune response in a Th1 direction. This study describes a mucosal vaccine composed of the Entamoeba histolytica galactose/N-acetyl-D-galactosamine-inhibitable lectin (Gal-lectin) and CpG oligodeoxynucleotides (CpG-ODN). The Gal-lectin is a protein involved in parasite virulence and adherence and is known to activate immune cells, while CpG-ODN are known to be potent inducers of type 1-like immune responses. We demonstrated that intranasal administration of the vaccine resulted in strong Gal-lectin-specific Th1 responses and humoral responses. Vaccination induced the production of Gal-lectin-specific T cells and the production of the proinflammatory cytokine gamma interferon. Vaccinated animals had detectable serum anti-Gal-lectin immunoglobulin G (IgG) and stool anti-Gal-lectin IgA capable of blocking parasite adherence to target cells in vitro. One week after immunization, gerbils were challenged intrahepatically with live trophozoites. Vaccinated gerbils had no detectable abscesses after day 5, whereas control gerbils developed larger abscesses. These results show that mucosal vaccination with Gal-lectin and CpG-ODN can induce both systemic and humoral immune responses.

CpG-oligodeoxynucleotide is a potent adjuvant with an Entamoeba histolytica Gal-inhibitable lectin vaccine against amoebic liver abscess in gerbils.

The protozoan parasite Entamoeba histolytica causes invasive amoebiasis characterized by amoebic dysentery and liver abscesses (ALA). The E. histolytica galactose/N-acetyl-D-galactosamine-inhibitable lectin (Gal-lectin), an immunogenic surface molecule involved in colonization and invasion, is a promising vaccine candidate against amoebiasis. Gal-lectin is known to induce Th1 cytokines in macrophages and spleen cells in vitro, and a Th1 response is thought to be protective against ALA. In this study, we report the use of cytosine guanine oligodeoxynucleotide (CpG-ODN) as adjuvant to augment Th1 responses against Gal-lectin in the gerbil model of ALA. Gerbils were vaccinated intramuscularly with the native Gal-lectin plus CpG-ODN or a paired non-CpG control GpC-ODN, and control gerbils received CpG-ODN alone. One week after the last boost gerbils were challenged intrahepatically with 10(6) amoebae. Gerbils receiving CpG-ODN as adjuvant with Gal-lectin were completely protected against the development of ALA, whereas 50% of gerbils receiving GpC-ODN and Gal-lectin developed ALA and 85% of controls developed ALA. Stronger lymphoproliferation in response to the Gal-lectin and higher prechallenge titers of serum Gal-lectin-specific antibodies, capable of blocking amoebic adherence, were observed when CpG-ODN was used as adjuvant. Gerbils vaccinated with CpG-ODN and Gal-lectin also had significantly higher levels of gamma interferon, interleukin-12 (IL-12), and IL-2 mRNA than controls. These data indicate that CpG-ODN can enhance the Th1 responses, which improve the protective effects of Gal-lectin. This is the first report of the use of CpG as a potent Th1 adjuvant with Gal-lectin to increase protection against ALA formation.

Vaccines for amoebiasis: barriers and opportunities.

Amoebiasis, infection by the protozoan parasite Entamoeba histolytica, remains a global health problem, despite the availability of effective treatment. While improved sanitation could lead to the eradication of this disease, it is unlikely that this will occur worldwide in the foreseeable future; thus alternative measures must be pursued. One approach is to develop a vaccine to prevent this deadly disease. Clinical studies indicate that mucosal immunity may provide some protection against recurrent intestinal infection with E. histolytica, but there is no clear evidence that protective immunity develops after amoebic liver abscess. Over the past decade, progress in vaccine development has been facilitated by new animal models that allow better testing of potential vaccine candidates and the application of recombinant technology to vaccine design. Oral vaccines and DNA-based vaccines have been successfully tested in animals models for immunogenicity and efficacy. There has been significant progress on a number of fronts, but there are unanswered questions regarding the effectiveness of immune responses in preventing disease in man and, as yet, no testing of any of these vaccines in humans has been performed. In addition, there are strong economic barriers to developing an amoebiasis vaccine and questions about how and where an effective vaccine would be utilized.

Oral vaccination with recombinant Yersinia enterocolitica expressing hybrid type III proteins protects gerbils from amebic liver abscess.

Protection against invasive amebiasis was achieved in the gerbil model for amebic liver abscess by oral immunization with live attenuated Yersinia enterocolitica expressing the Entamoeba histolytica galactose-inhibitable lectin that has been fused to the Yersinia outer protein E (YopE). Protection was dependent on the presence of the YopE translocation domain but was independent from the antibody response to the ameba lectin.

Entamoeba histolytica: cDNAs cloned as 30kDa collagen-binding proteins (CBP) belong to an antioxidant molecule family. Protection of hamsters from amoebic liver abscess by immunization with recombinant CBP.

A cDNA expression library of Entamoeba histolytica was screened with antiserum to native amoebic collagen binding proteins (CBPs), and two clones C13 and C7 which partially encode for the 30 kDa CBP were obtained. The sequenced clones were 90% homologous. C7 had a 69 bp deletion at the 5' end that is present in C13 and encodes for a Glu-Cys-Lys rich region and a four amino acids repeat (Glu-Lys-Glu-Cys). Purified fusion proteins from these cDNA clones were able to bind native type I collagen gels in a pH, calcium, ionic strength, and temperature dependent way. The binding of pgtC13 to collagen gel was time and temperature stable, while pgtC7 binding was not, suggesting that the deleted region in C7 is important for the binding. The clones reported here partially encode a 30 kDa CBP that also belong to an antioxidant molecule family. We demonstrated that the fusion protein pgtC13 is immunogenic and partially protective as a subunit vaccine in the hamster model of amoebic liver abscess.

Immunosuppressive treatment inhibits the development of amebic liver abscesses in hamsters.

BACKGROUND: The aim of the present study was to determine if the inflammation and/or immunosuppression induced by Entamoeba histolytica may contribute to amebic invasion. MATERIAL/METHODS: Dexamethasone was administered three days before and three days after inoculation of hamsters with E. histolytica. Seven days alter inoculation the animals were sacrificed and the sizes of their amebic liver abscesses were determined. The number of neutrophils, macrophages, T and B cells in the peritoneum as well as the production of nitric oxide and the susceptibility to Listeria monocytogenes infection was also determined. RESULTS: Dexamethasone treatment significantly reduced the number of T lymphocytes in thymus and spleen. The number of neutrophils, macrophages and T lymphocytes in the peritoneal exudate was also reduced as well as the production of nitric oxide and the microbicidal activity against Listeria monocytogenes. However, in the animals treated with a high dose of dexamethasone the size of the liver abscesses was significantly smaller than in the untreated animals. CONCLUSIONS: The results suggest that macrophage and T cell-mediated immunity is not relevant as a protective mechanism because tissue invasion by E. histolytica was reduced in immunosuppresed animals. On the contrary, the inflammatory process may contribute to the invasion and liver damage.

The EhADH112 recombinant polypeptide inhibits cell destruction and liver abscess formation by Entamoeba histolytica trophozoites.

The Entamoeba histolytica EhCPADH complex, formed by a cysteine proteinase (EhCP112) and an adhesin (EhADH112), is involved in adherence, phagocytosis and cytolysis. This makes this complex an attractive candidate as a vaccine against amoebiasis. Here, we produced the recombinant polypeptide EhADH243, which includes the adherence epitope detected by a monoclonal antibody against the EhCPADH complex. EhADH243 was purified, and the effect of the polypeptide on in vitro and in vivo virulence was studied. Antibodies against EhADH243 reacted with the EhCPADH complex and with the recombinant polypeptide. EhADH243 and antibodies against this polypeptide inhibited adherence, phagocytosis and destruction of cell monolayers by live trophozoites, but had little effect on cell monolayer destruction by trophozoite extracts. EhADH243 recognized a 97 kDa protein in the MDCK membrane fraction that could be a putative receptor for E. histolytica trophozoites. Hamsters immunized with EhADH243 developed humoral response against EhCPADH, and animals were partially protected from amoebic liver abscess.

Immunization with a tetramer derivative of an anti-inflammatory pentapeptide produced by Entamoeba histolytica protects gerbils (Meriones unguiculatus) against experimental amoebic abscess of the liver.

Axenically grown Entamoeba histolytica produces a pentapeptide (Met-Gln-Cys-Asn-Ser) with several anti-inflammatory properties, including the inhibition of human monocyte locomotion (Monocyte Locomotion Inhibitory Factor (MLIF)). A construct displays the same effects as the native material. It remains to be seen if MLIF is used, or even produced in vivo by the tissue-invading parasite. If MLIF were to be relevant in invasive amoebiasis, immunizing against it could diminish this parasite advantage and prevent lesions. KLH-linked MLIF mixed with Freund's adjuvant was too aggressive an immunizing material to answer this question. However, immunization with a tetramer of MLIF (but not a scrambled version of MLIF) around a lysine core (MLIF-MAPS), that displays increased antigenicity, yet lacks excessive innate immunity activation, completely protects gerbils against amoebic abscess of the liver caused by the intraportal injection of virulent E. histolytica. Liver abscesses caused by Listeria monocytogenes were not prevented. Invasive E. histolytica may produce the parent protein of MLIF in vivo, and if appropriately cleaved, it may play a role in invasive amoebiasis. MLIF may join new vaccination strategies against amoebiasis.

Protection of hamsters from amebic liver abscess formation by immunization with the 150- and 170-kDa surface antigens of Entamoeba histolytica.

A monoclonal antibody, EH3015, prevents in vitro adherence of Entamoeba histolytica trophozoites to mammalian cells and inhibits amebic liver abscess formation in hamsters. By immunoaffinity chromatography with the monoclonal antibody, purified E. histolytica antigens with molecular masses of 150 and 170 kDa under non-reduced conditions were obtained. Hamsters were immunized with these antigens (group I) or with fractions further purified by polyacrylamide gel electrophoresis (group II). Pooled immune sera from the two groups inhibited in vitro amebic adherence to Chinese hamster ovary cells by 98% at 1:10 dilutions. The immunized hamsters were challenged by the intrahepatic injection of E. histolytica trophozoites. Complete protection from abscess formation was observed in 38% of hamsters in group I and 67% in group II, whereas all control hamsters inoculated only with adjuvant developed amebic liver abscesses. In the immunized hamsters, the abscesses in the two groups were significantly smaller than in the controls. These results demonstrate that the E. histolytica antigens are possible vaccine candidates for amebiasis.

Protection of gerbils from amebic liver abscess by vaccination with a 25-mer peptide derived from the cysteine-rich region of Entamoeba histolytica galactose-specific adherence lectin.

The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality through intestinal infection and amebic liver abscess. Here we show that immunization of gerbils with a single keyhole limpet hemocyanin-coupled 25-mer peptide derived from the 170-kDa subunit of the E. histolytica galactose-binding adhesin is sufficient to confer substantial protection against experimentally induced amebic liver abscesses. Vaccination provided total protection in 5 of 15 immunized gerbils, and abscesses were significantly smaller (P < 0.01) in the remaining vaccinated animals. The degree of protection correlated with the titer of antibodies to the peptide, and results of passive transfer experiments performed with SCID mice were consistent with a role for antibodies in protection. In addition, parenteral or oral vaccination of gerbils with 13-amino-acid subfragments of the peptide N-terminally fused to the B subunit of cholera toxin also significantly inhibited liver abscess formation (P < 0.05). These data indicate that small peptides derived from the galactose-binding adhesin administered by the parenteral or oral route can provide protection against amebic liver abscess and should be considered as components of a subunit vaccine against invasive amoebiasis.