Protection of gerbils from amebic liver abscess by immunization with a recombinant protein derived from the 170-kilodalton surface adhesin of Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality worldwide through intestinal infection and amebic liver abscess. Here we show that vaccination of gerbils, a standard model for amebic liver abscess, with recombinant proteins derived from the 170-kDa galactose-binding adhesin of E. histolytica and the serine-rich E. histolytica protein or a combination of the two recombinant antigens provides excellent protection against subsequent hepatic challenge with virulent E. histolytica trophozoites.     

Protection of gerbils from amebic liver abscess by immunization with the galactose-specific adherence lectin of Entamoeba histolytica.

No protective antigens from Entamoeba histolytica have been previously defined. We tested the ability of the galactose-specific adherence lectin of E. histolytica to elicit a protective immune response in conjunction with Freund's incomplete and complete adjuvants. The gerbil (Meriones unguiculatus) model of an experimental amebic liver abscess was used. Gerbils were immunized intraperitoneally or subcutaneously with 10 micrograms of the affinity-purified lectin in complete Freund's adjuvant and then at 2 and 4 weeks with 10 micrograms of the lectin in incomplete Freund's adjuvant. All of the immunized animals developed antilectin antibody titers of greater than 1/1,024 as measured by a radioimmunoassay. The gerbil antilectin antibodies were shown by Western immunoblotting to be directed to the heavy subunit but not the light subunit of the lectin. Immune gerbil sera inhibited amebic adherence by 100% at a 1/10 dilution. Immune and control gerbils were challenged at 6 weeks by the intrahepatic injection of 5 x 10(5) E. histolytica trophozoites. Four independent trials demonstrated complete protection from amebic liver abscess formation in 67% of lectin-immunized gerbils. Unexpectedly, liver abscess weights were significantly higher in the gerbils that failed to become immune than in the control animals. Our results demonstrate that the galactose lectin is a protective antigen and provide an immune-animal model to study the mechanisms of protection and potential disease exacerbation conferred by the antilectin immune response.     

Protection of gerbils from amebic liver abscess by immunization with recombinant Entamoeba histolytica 29-kilodalton antigen.

The goal of our study was to obtain a highly conserved Entamoeba histolytica recombinant antigen for study as a subunit amebiasis vaccine. We screened a Uni-Zap cDNA library of E. histolytica (strain HM1:IMSS) with human immune sera and isolated a dominant 804-bp cDNA clone. A 33-kDa fusion protein expressed from the cDNA clone was determined by monoclonal antibody binding, DNA hybridization, and nucleotide sequence to be the complete E. histolytica 29-kDa antigen. Serum antibodies to the recombinant protein were detected by enzyme-linked immunosorbent assay in 80% of subjects from Egypt and South Africa with amebic liver abscess. Similar results were found with the native 29-kDa protein. Native and recombinant 29-kDa antigens induced proliferation of lymphocytes harvested from patients with amebic liver abscess (P < 0.01 compared with controls). Intraperitoneal immunization of gerbils with the recombinant fusion protein (10 micrograms) with Titermax adjuvant elicited an antigen-specific serum immunoglobulin G antibody response and was partially protective (54%) against intrahepatic challenge with 5 x 10(5) virulent axenic trophozoites (strain HM1:IMSS). In summary, the recombinant form of the E. histolytica 29-kDa antigen demonstrated serologic specificity for amebic liver abscess, exhibited conserved T-cell epitopes, and was effective as a subunit vaccine in an experimental animal model of amebic liver abscess.     

Protection of gerbils from amebic liver abscess by immunization with a recombinant Entamoeba histolytica antigen.

Amebiasis, infection by the intestinal protozoan parasite Entamoeba histolytica, is a leading parasitic cause of death. As a step in the development of a recombinant antigen vaccine to prevent E. histolytica infection, we looked at the ability of a recombinant version of the serine-rich E. histolytica protein (SREHP) to elicit a protective immune response against invasive amebic disease. Gerbils, a standard model for amebic liver abscess, were immunized with either a recombinant SREHP/maltose-binding protein (MBP) fusion, recombinant MBP alone, or phosphate-buffered saline (PBS), all combined with complete Freund's adjuvant. In the first trial (group 1), gerbils received a primary and two booster immunizations intraperitoneally; in the second trial (group 2), gerbils were immunized by a single intradermal injection. SREHP/MBP-immunized gerbils in both groups produced antibody to native SHEHP and developed delayed-type hypersensitivity responses to recombinant SREHP. All gerbils were challenged by an intrahepatic injection with 5 x 10(4) virulent E. histolytica HM1-IMSS trophozoites. Complete protection from amebic liver abscess was seen in 64% of the SHEHP/MBP-immunized gerbils in group 1 and in 100% of the SREHP/MBP-immunized gerbils in group 2. There was no protection observed in MBP- or PBS-immunized gerbils in either group. Our results indicate that the SREHP molecule has potential as a vaccine to prevent amebic infection and demonstrate that successful vaccination of animals with recombinant E. histolytica antigen vaccines is possible.     

Oral immunization with an attenuated vaccine strain of Salmonella typhimurium expressing the serine-rich Entamoeba histolytica protein induces an antiamebic immune response and protects gerbils from amebic liver abscess.

Attenuated salmonellae represent attractive candidates for the delivery of foreign antigens by oral vaccination. In this report, we describe the high-level expression of a recombinant fusion protein containing the serine-rich Entamoeba histolytica protein (SREHP), a protective antigen derived from virulent amebae, and a bacterially derived maltose-binding protein (MBP) in an attenuated strain of Salmonella typhimurium. Mice and gerbils immunized with S. typhimurium expressing SREHP-MBP produced mucosal immunoglobulin A antiamebic antibodies and serum immunoglobulin G antiamebic antibodies. Gerbils vaccinated with S typhimurium SREHP-MBP were protected against amebic liver abscess, the most common extraintestinal complication of amebiasis. Our findings indicate that the induction of mucosal and immune responses to the amebic SREHP antigen is dependent on the level of SREHP-MBP expression in S. typhimurium and establish that oral vaccination with SREHP can produce protective immunity to invasive amebiasis.     

Role of the Entamoeba histolytica cysteine proteinase in amebic liver abscess formation in severe combined immunodeficient mice.

Evidence from in vitro studies suggest that the Entamoeba histolytica cysteine proteinase plays a role in the tissue lysis and cytopathic effects seen in invasive amebiasis. We used affinity-purified antibodies against a recombinant E. histolytica cysteine proteinase to demonstrate that the proteinase is present extracellularly in amebic liver abscesses in mice with severe combined immunodeficiency (SCID mice). Treatment of E. histolytica trophozoites with specific cysteine proteinase inhibitor E-64 blocked or greatly reduced liver abscess formation at 48 h in SCID mice. Our study suggests an important role for a functional cysteine proteinase in amebic liver abscess formation.     

Suppression of T-lymphocyte responses to Entamoeba histolytica antigen by immune sera.

Lymphocytes from patients cured of amebic liver abscesses proliferate and produce gamma interferon upon incubation with soluble Entamoeba histolytica antigen: however, amebic liver abscesses exhibit a relentless progression without treatment. To determine whether suppressive factors are present in sera, we studied T-lymphocyte responses to total soluble E. histolytica antigen by using cells from five patients treated for amebic liver abscesses in the presence of 15 different immune sera and 10 control sera. In the presence of immune sera, E. histolytica antigen-induced lymphocyte proliferation decreased by 63% and production of gamma interferon was reduced by 93.2% (P less than 0.01). Immune sera had no effect on the mitogenic responses of patient lymphocytes to phytohemagglutinin or on the proliferative responses of control lymphocytes to phytohemagglutinin or tetanus toxoid. The suppressive activity of immune sera diminished as the time between therapy for amebic liver abscesses and serum collection increased (P less than 0.05). Suppressive activity did not correlate with the titers of serum anti-amebic antibody and was not affected when serum was absorbed with viable amebic trophozoites. In conclusion, soluble factors present in the sera of amebic liver abscess patients suppressed in vitro lymphocyte responses to E. histolytica antigen and may have contributed to the lack of development of effective in vivo cell-mediated immune responses following the onset of amebic liver abscesses.     

Progress towards development of a vaccine for amebiasis.

The application of molecular biologic techniques over the past decade has seen a tremendous growth in our knowledge of the biology of Entamoeba histolytica, the causative agent of amebic dysentery and amebic liver abscess. This approach has also led to the identification and structural characterization of three amebic antigens, the serine-rich Entamoeba histolytica protein (SREHP), the 170-kDa subunit of the Gal/GalNAc binding lectin, and the 29-kDa cysteine-rich protein, which all show promise as recombinant antigen-based vaccines to prevent amebiasis. In recent studies, an immunogenic dodecapeptide derived from the SREHP molecule has been genetically fused to the B subunit of cholera toxin, to create a recombinant protein capable of inducing both antiamebic and anti-cholera toxin antibodies when administered by the oral route. Continued progress in this area will bring us closer to the goal of a cost-effective oral combination "enteric pathogen" vaccine, capable of inducing protective mucosal immune responses to several clinically important enteric diseases, including amebiasis.     

Neutrophils play a critical role in early resistance to amebic liver abscesses in severe combined immunodeficient mice.

Animal models of liver abscess formation with Entamoeba histolytica suggest that the neutrophil is the first cell of the host immune system to interact with the invading ameba. In vitro studies have suggested that lysis of neutrophils by virulent amebae may exacerbate the damage seen in amebic liver abscesses. To investigate the role of neutrophils in vivo, we used the severe combined immunodeficient (SCID) mouse model of amebic liver abscess formation and compared liver damage in neutrophil-depleted and control mice. We found that neutrophil-depleted animals have significantly larger amebic liver abscesses at early stages of infection and that abscesses in neutrophil-depleted SCID mice lack the prominent inflammatory cell ring seen in amebic liver abscesses in control SCID mice. These data suggest that neutrophils play a protective role in the early host response to amebic infection of the liver.     

Diagnosis of amebic liver abscess and intestinal infection with the TechLab Entamoeba histolytica II antigen detection and antibody tests

A noninvasive diagnostic test for amebic liver abscess is needed, because amebic and bacterial abscesses appear identical on ultrasound or computer tomography and because it is rarely possible to identify Entamoeba histolytica in stool specimens from patients with amebic liver abscess. Here we report a method of detection in serum of circulating E. histolytica Gal/GalNAc lectin to diagnose amebic liver abscess, which was used in patients from Dhaka, Bangladesh. The TechLab E. histolytica II test (which differentiates the true pathogen E. histolytica from Entamoeba dispar) detected Gal/GalNAc lectin in the sera of 22 of 23 (96%) amebic liver abscess patients tested prior to treatment with the antiamebic drug metronidazole and 0 of 70 (0%) controls. After 1 week of treatment with metronidazole, 9 of 11 (82%) patients became serum lectin antigen negative. The sensitivity of the E. histolytica II antigen detection test for intestinal infection was also evaluated. Antigen detection identified E. histolytica infection in 50 samples from 1, 164 asymptomatic preschool children aged 2 to 5 years, including 16 of 16 (100%) culture-positive specimens. PCR analysis of stool specimens was used to confirm that most antigen-positive but culture-negative specimens were true-positive: PCR identified parasite DNA in 27 of 34 (79%) of the antigen-positive, culture-negative stool specimens. Antigen detection was a more sensitive test for infection than antilectin antibodies, which were detected in only 76 of 98 (78%) amebic liver abscess patients and in 26 of 50 (52%) patients with intestinal infection. We conclude that the TechLab E. histolytica II kit is a sensitive means to diagnose hepatic and intestinal amebiasis prior to the institution of metronidazole treatment.     

Opportunities and obstacles in developing a vaccine for Entamoeba histolytica

The development of a vaccine against Entamoeba histolytica, the causative agent of amebic colitis and liver abscess, would reduce childhood mortality in countries such as Bangledesh where community-based studies have demonstrated a high prevalence of E. histolytica. Immunological studies from this population have shown that protection from amebiasis is associated with mucosal anti-E. histolytica Gal/GalNAc lectin antibodies, suggesting that a vaccine is an achievable goal. However, garnering resources for vaccine development is a challenge when the vaccine is targeted to poor people living in developing countries.     

Sequential histopathology of cavitary liver abscess. Formation induced by axenically grown Entamoeba histolytica

Multiple hamster liver passage of Entamoeba histolytica trophozoites with intervening recovery into axenic culture caused increased virulence as measured by increase in the size of the lesion produced. Lesions produced by amebae that had not been liver-passaged did not persist; however, multiply liver-passaged substrains produced large, fluid-filled abscesses one month to six weeks after inoculation. Six days after inoculation, lesions consisted of multiple granulomas, lymphocytes, and E histolytica trophozoites. Large, fluid-filled abscesses produced by liver-passaged substrains lacked the granulomatous appearance of the earlier lesions. The abscesses had a fibrous wall, with E histolytica trophozoites at the inner aspect. To our knowledge, the evolution of early granulomatous lesions into a cavitary abscess with features closely resembling those of human amebic abscess has not been reported previously in the experimental disease in the hamster.     

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.     

Diagnosis of invasive amebiasis by enzyme-linked immunosorbent assay of saliva to detect amebic lectin antigen and anti-lectin immunoglobulin G antibodies

Saliva from subjects with amebic liver abscess (ALA), acute amebic colitis, asymptomatic infection with Entamoeba histolytica or Entamoeba dispar, and uninfected controls was tested by enzyme-linked immunosorbent assay (ELISA) for the presence of E. histolytica galactose-inhibitable lectin antigen and salivary immunoglobulin (IgG) antibodies to a recombinant cysteine-rich lectin-derived protein (LC3). Salivary lectin antigen was found in 65.8% of subjects with acute colitis, compared to 22.2% of those convalescent from ALA, 10.0% with asymptomatic E. histolytica infection, 9.8% with E. dispar infection, and 2.6% of controls (subjects from the United States and study patients with nonamebic diarrhea) (P < 0.001 for each compared to values for subjects with colitis). Salivary anti-LC3 IgG antibodies were found in 92% of ALA patients regardless of duration of illness and in 83.3% of colitis patients who were symptomatic for at least 7 days (P < 0.001 compared to other study groups). Serum anti-LC3 IgG antibodies were detected in 56.3% of subjects with acute colitis, 100% of subjects with ALA or prolonged colitis, 45% of subjects with asymptomatic E. histolytica infection, 32.3% of subjects with E. dispar infection, and 23.4% of diarrhea controls. In comparison to ELISA for serum anti-LC3 IgG antibodies, the salivary lectin antigen assay is a more sensitive and specific test for acute amebic colitis. Detection of salivary anti-LC3 IgG antibodies by ELISA is an effective means for the diagnosis of ALA and prolonged cases of amebic colitis.     

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.     

Thiol proteinase expression and pathogenicity of Entamoeba histolytica.

Expression of the 56-kilodalton (kDa) neutral thiol proteinase has been shown to correlate with the potential of clinical isolates of Entamoeba histolytica to produce invasive disease. A 56-kDa band was identified by gelatin substrate gel electrophoresis in 10 of 10 isolates from patients with colitis or amebic liver abscesses, but in only 1 of 10 isolates from asymptomatic patients. Pathogenic isolates appear capable of releasing significantly larger quantities of the proteinase, as measured by cleavage of a synthetic peptide substrate, ZRR-AMC (benzyloxy-carbonyl-arginine-arginine-4-amino-7-methylcoumarin). We have also shown that the proteinase is released during the course of clinical invasive amebic disease, as demonstrated by the presence of circulating antibodies detectable by enzyme-linked immunosorbent assay. These studies support the importance of the 56-kDa thiol proteinase in the pathogenesis of invasive amebiasis.     

Expression of amoebapores is required for full expression of Entamoeba histolytica virulence in amebic liver abscess but is not necessary for the induction of inflammation or tissue damage in amebic colitis

Entamoeba histolytica trophozoites produce amoebapores, a family of small amphipathic peptides capable of insertion into bacterial or eukaryotic membranes and causing cellular lysis. Recently, E. histolytica trophozoites that are totally deficient in the production of amoebapore-A were created through a gene silencing mechanism (R. Bracha, Y. Nuchamowitz, and D. Mirelman, Eukaryot. Cell 2:295-305, 2003). Here we tested the virulence of amoebapore A(-) trophozoites in models of the two major forms of amebic disease: amebic liver abscess and amebic colitis. We demonstrate that amoebapore expression is required for full virulence in the SCID mouse model of amebic liver abscess, but E. histolytica trophozoites that do not express amoebapore-A can still cause inflammation and tissue damage in infected human colonic xenografts. These data are consistent with the concept that tissue damage may proceed by different mechanisms in amebic liver abscess compared to amebic colitis.     

Innate immunity to amebic liver abscess is dependent on gamma interferon and nitric oxide in a murine model of disease

Evidence from in vitro studies suggests that gamma interferon (IFN-gamma) and nitric oxide (NO) are important in host defense against the protozoan parasite Entamoeba histolytica. We used SCID mice with targeted disruption of the IFN-gamma receptor gene and mice with targeted disruption of the gene encoding inducible NO synthase to show that IFN-gamma plays a role in the innate immunity to amebic liver abscess seen in SCID mice while NO is required for control of amebic liver abscess in immunocompetent mice.     

Identification of the Entamoeba histolytica galactose-inhibitable lectin epitopes recognized by human immunoglobulin A antibodies following cure of amebic liver abscess

Immunity to Entamoeba species intestinal infection is associated with the presence of intestinal IgA antibodies against the parasite's galactose-inhibitable adherence lectin. We determined the epitope specificity of serum and intestinal antilectin IgA antibodies by enzyme-linked immunosorbent assay using overlapping fragments of a recombinant portion of the lectin heavy subunit, designated LC3. These findings were correlated with the effects of epitope-specific murine antilectin immunoglobulin A (IgA) monoclonal antibodies (MAbs) on amebic in vitro galactose-specific adherence. LC3 is a highly antigenic and immunogenic cysteine-rich protein (amino acids [aa] 758 to 1150) that includes the lectin's carbohydrate binding domain. The study subjects, from Durban, South Africa, were recently cured of amebic liver abscess (ALA) with or without concurrent Entamoeba histolytica intestinal infection or were infection free 1 year after cure. We also studied seropositive subjects that were infected with E. histolytica, disease free, and asymptomatic. Serum anti-LC3 IgA antibodies from all study groups exclusively recognized the third (aa 868 to 944) and the seventh (aa 1114 to 1134) LC3 epitopes regardless of clinical status; epitope 6 (aa 1070 to 1114) was also recognized by serum anti-LC3 IgG antibodies. However, IgG antibody recognition of epitope 6 but not 3 or 7 was lost 1 year following cure of ALA. We produced 14 murine anti-LC3 IgA MAbs which collectively recognized five of the seven LC3 epitopes. The majority of the murine MAbs recognized the first epitope (aa 758 to 826), which was not recognized by human IgA antibodies. Interestingly, adherence of E. histolytica trophozoites to CHO cells was inhibited by MAbs against epitopes 1, 3, 4 (aa 944 to 987), and 6 (P < 0.01). The LC3 epitopes recognized by human IgA antibodies (3 and 7) were further characterized by use of overlapping synthetic peptides. We identified four peptides (aa 891 to 903, 918 to 936, 1114 to 1134, and 1128 to 1150) that in linear or cyclized form were recognized by pooled intestinal IgA antibodies and serum IgG antibodies from subjects with ALA and asymptomatic, seropositive infected subjects. This study identifies the lectin epitopes to be studied in an amebiasis subunit vaccine designed to elicit mucosal immunity mimicking that of humans cured of ALA.     

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.