Amastigotes of Trypanosoma cruzi sustain an infective cycle in mammalian cells.

The two main stages of development of the protozoan parasite Trypanosoma cruzi found in the vertebrate host are the trypomastigote and the amastigote. It has been generally assumed that only trypomastigotes are capable of entering cells and that amastigotes are the intracellular replicative form of the parasite. We show here that after incubation for 4 h with human monocytes in vitro 90% or more of extracellularly derived (24 h) amastigotes of T. cruzi are taken up by the cells. Within 2 h they escape the phagocytic vacuole and enter the cytoplasm, where they divide and after 4-5 d transform into trypomastigotes. Trypomastigotes also invade cultured human monocytes. However, they show a lag of several hours between invasion and the start of DNA duplication, while amastigotes commence replication without an apparent lag. Amastigotes also infect cultured fibroblasts, albeit with lower efficiency. When injected intraperitoneally into mice, amastigotes are as infective as trypomastigotes. Based on these results, and on prior findings that amastigotes are found free in the circulation of mice during the acute stage of the disease (3), it seems likely that the cellular uptake of amastigotes can initiate an alternative subcycle within the life cycle of this parasite in the mammalian host. Also, because trypomastigotes and amastigotes have diverse surface antigens, they may use different strategies to invade host cells.

The exit of Trypanosoma cruzi from the phagosome is inhibited by raising the pH of acidic compartments.

The protozoan parasite Trypanosoma cruzi can infect many distinct mammalian cell types. The parasites enter cells through the formation of phagocytic vacuoles, but later are found free in the cytosol, where they multiply as amastigotes. Using transmission electron microscopy we found that within 2 h after infection 70% of the parasites, including examples of both mammalian forms (trypomastigotes and amastigotes), were inside partially disrupted vacuoles or free in the cytosol. We demonstrated that the pH of vacuoles containing recently interiorized parasites is acidic, through immunocytochemical localization of the acidotropic compound DAMP (18) in their interior. Increasing the vacuolar pH with chloroquine, ammonium chloride, methylamine, or monensin significantly inhibited the escape of the parasites into the cytosol. These results are compatible with the hypothesis that an acid-active hemolysin of T. cruzi (15) might be involved in the escape mechanism.

Developmentally regulated, phospholipase C-mediated release of the major surface glycoprotein of amastigotes of Trypanosoma cruzi.

The surface of amastigotes of Trypanosoma cruzi is covered by Ssp-4, a major stage-specific glycoprotein. Ssp-4 is anchored to the cell membrane by GPI. It can be metabolically labeled with [3H]myristic acid, and is converted into a hydrophilic form by treatment with the glycan-specific phospholipase C of T. brucei, or after lysis of the parasites in non-ionic detergents. The hydrophilic form of Ssp-4 is recognized by antibodies to the cross-reactive determinant of the variant surface glycoprotein of African trypanosomes. Ssp-4 is progressively shed during the intra- or extracellular development of amastigotes preceding their transformation into epi- and trypomastigotes. We show here that T. cruzi contains a phospholipase C and that most shed Ssp-4 is hydrophilic, does not contain myristic acid, and reacts with anti-CRD. These observations provide strong evidence that phospholipase C mediates the release of this glycosyl-phosphatidylinositol-anchored protein under physiological conditions, as the parasite undergoes differentiation.

Synthetic peptide vaccine confers protection against murine malaria.

A synthetic peptide, (DPPPPNPN)2D, representing a subunit of the repeat domain of the Plasmodium berghei circumsporozoite protein, was conjugated to tetanus toxoid using bisdiazobenzidine. Immunization of mice and rats with the conjugate induced high serum titers of antibodies to the parasite, and most of the animals were completely protected from malaria infection when challenged with sporozoites.

Isolation and characterization of the main allergen of Dermatophagoides farinae by monoclonal antibodies that recognize IgE related epitopes.

Mouse monoclonal antibodies (MAbs) with different specificities against Dermatophagoides farinae (D. farinae) extract have been obtained. Fifteen of these antibodies reacted with allergen molecules contained in D. farinae and D. pteronyssinus extracts, immunoprecipitating the main allergen of D. farinae (DF29) and homologous allergen of D. pteronyssinus (DP28). In addition, the monoclonal antibody MADF2 immunoprecipitated DF29 together with two other polypeptides (mol. wt 20,000 and 40,000) from D. farinae extracts. Five monoclonal antibodies (MADF2, MADF5, MADF9, MADF10 and MADF13) were selected to study their epitope specificity and the relationship of the epitope location on the allergen with the IgE binding site. By cross-inhibition studies two different epitopes and two partly overlapping determinants were found. In addition, two of these epitopes, those defined by MADF13 and MADF5, are close to, or overlapping, IgE binding site(s) on the allergen molecule. DF29 allergen from D. farinae extract was purified by affinity chromatography using MADF5 coupled to Sepharose. The purified allergen had capacity to bind mite specific human IgE and demonstrated an allergenic activity of up to 70% of total extract of D. farinae. These results indicate that DF29 molecule is the main allergen from D. farinae extracts.

Isolation of the major IgE-binding protein from Parietaria judaica pollen using monoclonal antibodies.

Allergen molecules from Parietaria judaica pollen, a widely distributed allergy inducer in Southern and Western Europe, have been studied using specific monoclonal antibodies (MAbs). MAbs against IgE-binding components were selected in a 4-step radioimmunoassay. Three different MAbs (AC/1.1, AC/7.1 and AC/15.1) were obtained which recognized epitope(s) located on a polypeptide of 10 Kd (Pj10). This polypeptide displayed the highest IgE-binding ability under either native or SDS-denatured conditions, as determined by immunoadsorption and immunodetection after SDS-PAGE, respectively. The Pj10-containing allergen, purified on an AC/1.1 MAb-Sepharose column, was able to inhibit most of the binding of specific IgE to the pollen extract coupled to paper discs in an inhibition radioallergosorbent test (RAST). The affinity-purified allergen exhibited the same immunoelectrophoretic behaviour as the native allergen.

A four-step sandwich radioimmunoassay for direct selection of monoclonal antibodies to allergen molecules.

A 4-step radioimmunoassay has been devised for direct identification of monoclonal antibodies (MAb) directed to IgE-binding molecules. Polyvinyl chloride wells coated with purified anti-mouse kappa chain MAb (187-1) were successively incubated with: (1) MAb-containing hybridoma supernatants, (2) allergen extract. (3) allergic patients' serum pool, and (4) 125I-labeled anti-human IgE antiserum, to detect MAb-allergen-IgE complexes. MAb to allergens from Parietaria judaica pollen and Dermatophagoides mites have been selected with this screening procedure. The affinity-purified allergen molecules completed the binding of IgE to allergen extracts coated to paper discs in a RAST inhibition assay, confirming the anti-allergen specificity of the selected MAb. This screening method is sensitive enough to allow detection of MAb directed to poorly represented allergens.

Cloning and characterization of a gene coding for a protein (KAP) associated with the kinetoplast of epimastigotes and amastigotes of Trypanosoma cruzi.

We have cloned and characterized a gene of Trypanosoma cruzi which encodes a protein, KAP (kinetoplasts-associated protein), expressed in the kinetoplasts of epimastigotes and amastigotes, the replicative stages of the parasite, but not in kinetoplasts of trypomastigotes. The single-copy gene is transcribed into a 3900-nt polyadenylated mRNA. Its trans-splicing acceptor site is preceded by a run of 15 adenosine residues. An open reading frame of 1052 codons is followed by a 3' untranslated region containing short sequences characteristic of rapidly degradable RNAs. The potential translation product of the KAP gene contains a central region composed of four blocks of repeats of a 9-amino-acid motif. Rabbit antibodies raised against three synthetic peptides containing KAP sequence recognized a 175-kDa protein in epimastigotes and amastigotes which appears by indirect immunofluorescence to be associated with their kinetoplasts. The antibodies do not recognize the kinetoplast of trypomastigotes. The amino terminus of KAP contains features compatible with mitochondrial topogenic sequences.

The structural protein p54 is essential for African swine fever virus viability.

Protein p54, one of the most antigenic structural African swine fever virus (ASFV) proteins, has been localized by immuno-electron microscopy in the replication factories of infected cells, mainly associated with membranes and immature virus particles. Attempts to inactivate the p54 gene from ASFV by targeted insertion of beta-galactosidase selection marker was uniformly unsuccessful, suggesting that this gene is essential for virus viability. To demonstrate that, we inserted in the TK (thymidine kinase) locus of the virus a construction containing a second copy of the p54 gene and beta-glucuronidase selection marker under the control of p54 and p73 promoters, respectively. Virus mutant clones expressing a second copy of p54 and beta-glucuronidase were used to achieve deletion mutants of the original copy of the gene. Virus mutants expressing only the second inserted copy of p54 and the two selection markers mentioned above were successfully obtained. Therefore, we have demonstrated that the p54 gene product plays an essential role in virus growth, characterizing for the first time in ASFV an essential virus gene.

Molecular cloning, expression and immunological analysis of the capsid precursor polypeptide (P1) from swine vesicular disease virus.

Swine vesicular disease virus (SVDV) is the aetiological agent of a highly contagious viral disease of pigs, whose symptoms are indistinguishable from those caused by foot-and-mouth disease virus (FMDV). The gene coding for the capsid protein precursor of SVDV (P1) from a recent spanish isolate (SPA/1/'93) was cloned and expressed in bacteria, and the antigenicity and immunogenicity of the recombinant product were evaluated. The recombinant P1 was recognised by antibodies against SVDV induced in pigs infected experimentally with different SVDV strains. Immunisation of swine with recombinant P1-induced SVDV-specific cellular and humoral immune responses. The implications of these results in SVD diagnostic as well as in vaccine development are discussed.

Identification of a novel allergen molecule from Dermatophagoides by monoclonal antibodies.

Six different monoclonal antibodies (MAb) have been obtained which bind to components contained in extracts from Dermatophagoides. One out of the six MAb recognized molecules displaying IgE binding ability. This MAb (MADP-1) immunoprecipitated allergenic polypeptides of 42 kDa and 30 kDa from 125I-labeled extracts of D. pteronyssinus and D. farinae respectively. Purified allergen preparations from both extracts have been obtained by affinity chromatography using a column of purified MADP-1 coupled to Sepharose. The purified fractions partially compete the binding of specific IgE contained in sera from sensitized patients to the whole extracts, in a radioallergosorbent inhibition test.

Isolation and characterization of a 92-KD surface molecule of Trypanosoma cruzi amastigotes recognized by a monoclonal antibody that induces complement-mediated killing.

The presence of lytic antibodies in the circulation of patients with chronic Chagas' disease might lead to their cure. It has been shown that amastigotes of Trypanosoma cruzi activate complement and accumulate large amounts of the terminal complement components, but without killing the parasites. One plausible explanation for this observation is that the insertion of the membrane attack complex of complement is prevented by inhibitors present in the parasite membrane. To explore this possibility, we raised a panel of monoclonal antibodies (MAbs) against the surface molecules of T. cruzi amastigotes. One of these, MAb M4C12, induced complement-mediated lysis of amastigotes as detected with a 86Rb-release assay. The antigen molecule from the membrane lysate of amastigotes that was recognized by MAb M4C12 was purified, characterized, and designated M4C12Ag. It is a 92-kD molecule structurally related to Ssp4, a previously characterized amastigote surface molecule. However, M4C12Ag is more basic (pI 6.9-7.1) than Ssp4 (pI 5.2-6.0), and is larger (92 kD) than Ssp4 (70 kD and 84 kD). Purified M4C12Ag did not inhibit the terminal components of complement when tested in hemolytic assays. This molecule may not be an inhibitor of complement, yet it is certainly an amastigote-specific surface molecule with immunologic importance because it is the only molecule that could induce formation of a lytic antibody.

Swine vesicular disease virus. Pathology of the disease and molecular characteristics of the virion.

Swine vesicular disease is a highly contagious disease of pigs that is caused by an enterovirus of the family Picornaviridae. The virus is a relatively recent derivative of the human coxsackievirus B5, with which it has high molecular and antigenic homology. The disease is not severe, and affected animals usually show moderate general weakening and slight weight loss that is recovered in few days, as well as vesicular lesions in the mucosa of the mouth and nose and in the interdigital spaces of the feet. However, the similarity of these lesions to those caused by foot-and-mouth disease virus has led to the inclusion of this virus in list A of the Office International des Epizooties. The disease has been eradicated in the European Union except in Italy, where it is considered endemic in the south. Nevertheless, as occasional outbreaks still appear and must be eliminated rapidly, European countries are on the alert and farms are monitored routinely for the presence of the virus. This circumstance has led to a considerable effort to study the pathology of the disease and the molecular biology and antigenicity of the virus, andto the development of optimized methods for the diagnosis of the infection.

The structure and function of MHC molecules. Possible implications for the control of tumor growth by MHC-restricted T cells.

We briefly survey recent basic progress on the structure and function of MHC molecules. We call attention on a number of remaining unanswered questions, and focus on a few points which we think might be of importance in immune surveillance against tumors mediated by MHC restricted T cells. In particular, we discuss the possible peptidic nature of certain tumor antigens. Following these lines, we mention a recent approach for the in vivo activation of specific CTL.

Synthetic peptide vaccines: foot-and-mouth disease virus as a model.

Foot-and-mouth disease virus (FMDV) has been one of the pioneering viral systems in the development of synthetic peptides as vaccines. Protection against FMDV infection is associated with the induction of neutralising antibodies. Therefore, attempts have been made to identify peptides capable of eliciting protective humoral responses. Peptides based on a continuous, immunodominant B cell site on the capsid protein VP1 have been shown to confer limited protection in natural hosts. This probably reflects the difficulties in reproducing the immunogenicity of an entire viral particle by using a much simpler synthetic antigen, due to: (i) the polymorphism of the class II MHC; (ii) the adequate presentation to the immune system of the peptides, and (iii) the difficulties of achieving protection against a highly variable RNA virus, which may favour selection of virus antigenic variants. The improvement of FMD peptide vaccines, and the development of in vitro alternatives to in vivo immunogenic assays require further understanding of the immune mechanisms leading to protection against this important animal virus disease.

Analysis of the B and T cell response in guinea pigs induced with recombinant vaccinia expressing foot-and-mouth disease virus structural proteins.

Recombinant vaccinia viruses expressing foot-and-mouth disease virus (FMDV) P1 and VP1 genes have been used to study the immune response induced by these viral polypeptides in guinea pigs. Anti-FMDV antibodies, but not neutralizing activity, were detected in the sera from immunized animals. The results indicate that both CD4+ and CD8+ FMDV-specific T cells were induced by the vaccinia recombinants. Consistently with the activation of CD4+ T cells, lymphocytes from immunized animals specifically proliferated in vitro in response to whole virus. The induction of virus-specific CD8+ T cells was determined by CTL assay of immune splenocytes restimulated in vitro with FMDV infected cells. Altogether, the results obtained indicate that both B and T cell immune responses to FMDV are elicited upon immunization of guinea pigs with vaccinia recombinants expressing FMDV structural polypeptides.

Infection with foot-and-mouth disease virus results in a rapid reduction of MHC class I surface expression.

The modulation of MHC class I molecule expression on the surface of cells as a consequence of foot-and-mouth disease virus (FMDV) infection has been examined. On cells infected with FMDV, class I expression was reduced to approximately 70% of the initial value 3 h after the infection and to 53% after 6 h. On cells depleted of surface class I complexes by acid treatment, the appearance of newly assembled class I-peptide complexes on the cell surface of non-infected cells increased immediately upon neutralization and original class I levels were recovered in about 20 h. In contrast, the appearance of new peptide-bound class I molecules on the cell surface was inhibited as early as 30 min after FMDV infection. Since the shut-down of FMDV-mediated host protein synthesis occurs approximately 2-3 h post-infection, this result suggests that an earlier event, which prevents the surface expression of newly synthesized complexes, is induced following FMDV infection. Thus, FMDV-infected cells rapidly become unable to present viral peptides in association with MHC class I molecules to T lymphocytes. Such a mechanism would assist virus evasion of the cytotoxic immune response of the host.

The N-terminal region of the VP1 protein of swine vesicular disease virus contains a neutralization site that arises upon cell attachment and is involved in viral entry.

The N-terminal region of VP1 of swine vesicular disease virus (SVDV) is highly antigenic in swine, despite its internal location in the capsid. Here we show that antibodies to this region can block infection and that allowing the virus to attach to cells increases this blockage significantly. The results indicate that upon binding to the cell, SVDV capsid undergoes a conformational change that is temperature independent and that exposes the N terminus of VP1. This process makes this region accessible to antibodies which block virus entry.

Immune recognition of swine vesicular disease virus structural proteins: novel antigenic regions that are not exposed in the capsid.

Swine vesicular disease virus (SVDV) is an enterovirus of the Picornaviridae family that belongs to the coxsackievirus B group. A number of antigenic sites have been identified in SVDV by analysis of neutralizing monoclonal antibody-resistant mutants and shown to be exposed on the surface of the capsid. In this paper we have identified seven new immunodominant antigenic regions in SVDV capsid proteins by a peptide scanning method, using a panel of sera from infected pigs. When these antigenic regions were located in the capsid by using a computer-generated three-dimensional model of the virion, one was readily exposed on the surface of the virus and the remaining sites were located facing the inner side of the capsid shell, at subunit contacts, or in the interior of the subunit structure.