CpG-containing ODN has a limited role in the protection against Toxoplasma gondii.

Bacterial DNA containing immunostimulatory motifs (CpG) induces the development of a T(H1) immune response. Since protection against Toxoplasma gondii is correlated with this type of response, the aim of this work was to determine if a synthetic oligodeoxynucleotide (ODN) containing CpG sequences could be useful as adjuvant for the induction of a long-lasting protective immune response against T. gondii. BALB/c mice immunized with a total soluble antigen of T. gondii (TSA2) mixed with ODN-containing CpG sequences developed a typical TH1 response, as determined by antibody isotypes and interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production by spleen cells. However, they did not resist a challenge with the virulent RH strain of the parasite. Absence of protection paralleled with lower levels of IFN-gamma, when compared with mice vaccinated with the live tachyzoites of the attenuated ts.4 strain of the parasite, which resisted this challenge. Intraperitoneal injection of ODN alone to mice induced a high degree of resistance to a lethal challenge inoculated by the same route. Nevertheless, this nonspecific protection was transient. Thus, the use of ODN containing CpG motifs as adjuvant is of limited value for the induction of a protective immune response against T. gondii.

MVA ROP2 vaccinia virus recombinant as a vaccine candidate for toxoplasmosis.

Toxoplasma gondii is the aetiological agent of toxoplasmosis and is the most frequent and best known of the parasitic diseases. In the United States, a serological survey from the Third National Health and Nutrition Examination Survey found that an estimated 23% of adolescents and adults have laboratory evidence of infection with T. gondii. Although toxoplasmosis is asymptomatic or shows self-limited symptoms in adults, in pregnant women infections can cause severe health problems to the fetus if the parasites are transmitted. Also, in immunodeficient patients, chronic infection with T. gondii can reactivate and produce encephalitis, which is frequently lethal. In addition, in veterinary medicine, T. gondii infection is of economic importance due to abortion and neonatal loss in sheep and goats. Recently, the development of vaccines against toxoplasmosis has progressed considerably. The live attenuated S48 strain of Toxoplasma has been broadly used for veterinary purposes. DNA vaccines containing the full-length of SAG1/P30, ROP2 or ROP1 genes have proved to be a promising candidate to induce protection against toxoplasmosis. Viral vectors have proved to be the best candidates for vaccination in different diseases. A recombinant Herpes virus carrying the ROP2 gene is able to induce protective immunity in cats. In the present work we describe the potential of the MVA ROP2 recombinant vaccinia virus as a vaccine against toxoplasmosis. MVA ROP2 induces antibodies against the ROP2 protein in similar amount and types as the thermo-sensible strain ts-4 of T. gondii, which is able to fully protect mice against challenge with the virulent RH strain of T. gondii. Also, the life-span of mice is increased in MVA ROP2 vaccinated animals. We conclude that MVA ROP2 vaccine can possibly generate an immune response, which could be useful in protection against toxoplasmosis.

Genetic immunization with plasmid DNA coding for the ROP2 protein of Toxoplasma gondii.

The ROP2 protein of Toxoplasma gondii has previously been proposed as a vaccine candidate against toxoplasmosis. In this work we characterize the immune response induced by injection of plasmid DNA coding for this protein in three strains of mice (BALB/c, C57BL/6, and CBA/J) displaying different levels of susceptibility to toxoplasmosis and compare it with that obtained by vaccination with the live attenuated ts-4 strain of T. gondii. The ROP2 gene was cloned in the eukaryotic expression vector pcDNA3 and the resulting plasmid, named pcDNA3/ROP2, was used to immunize mice. After three immunizations with the plasmid, mice developed antibodies that could be detected by ELISA using a recombinant truncated form of ROP2; and these antibodies also recognized the natural protein by Western blot. Plasmid immunization generated antibodies against the ROP2 of both of the IgG1 and IgG2a isotypes in CBA/J and BALB/c mice and both of the IgG1 and IgG2c isotypes in C57BL/6 mice. However, animals vaccinated with the ts-4 strain generated only IgG2a (in CBA/J and BALB/c mice) or IgG2c (in C57BL/6 mice) against ROP2. Kinetic studies of the generation of isotypes indicated that both isotypes were generated at the same time. Mice immunized with the plasmid DNA did not resist a challenge with the virulent RH strain of T. gondii, while mice vaccinated with the ts-4 strain resisted the same challenge. However, in pcDNA3/ROP2-immunized BALB/c mice, death was significantly delayed with respect to the pcDNA3-immunized control group. These results suggest that plasmid immunization using the ROP2 gene generates a mixed T(H1)/T(H2) response against ROP2, which is different from that obtained by vaccination with live tachyzoites of the ts-4 strain (T(H1) response) and is not protective against the highly virulent RH strain of the parasite.

Cloning, characterization and functional expression of a cyclophilin of Entamoeba histolytica.

Full-length Entamoeba histolytica cyclophilin gene (EhCyp) was isolated, characterized and recombinantly expressed in bacterial cells. The deduced amino acid sequence of EhCyp shows 60-70% identity with cyclophilins from other organisms and has conserved the cyclophilin signature motifs and residues involved in cyclosporin A binding. Upstream of the 501 bp open reading frame of EhCyp, sequences resembling the putative consensus E. histolytica CE1, CE2 and CE3 regulatory elements were found. Northern blot assays revealed a single transcript of 0.63 kb. The transcription start was determined by primer extension at position -13 relative to the initial ATG codon. Cyclosporin A binding and peptidyl-proplyl cis-trans isomerase activities characteristic of cyclophilin were detected in soluble extracts of E. histolytica trophozoites and in the recombinant protein. In both cases, the isomerase activity was inhibited by nanomolar concentrations of cyclosporin A. Treatment of cultured trophozoites with cyclosporin A decreased their proliferation with a 50% inhibition value of 1 microg/ml and was lethal in doses over 50 microg/ml.

Isolation and structure-functional characterization of phage display library-derived mimotopes of noxiustoxin, a neurotoxin of the scorpion Centruroides noxius Hoffmann.

Noxiustoxin (NTX) is a short-chain toxin from the venom of the scorpion Centruroides noxius Hoffmann, whose molecular structure and physiological effects have been characterized in detail, whereas the antigenic properties of this and other K(+) channel-blocking toxins are poorly studied. A monoclonal antibody against NTX, BNTX18, able to inhibit the binding of NTX to rat brain synaptosomes, was used in the present study for selecting immunoreactive peptides, mimotopes, from a 12mer and a 7mer phage library. The peptides were characterized immunologically and used for mapping the epitope on NTX. In total, 75 phage clones carrying 43 different peptides were analyzed of which 42 clones carrying 17 different peptides, twelve 12mer and five 7mer peptides, presented a single consensus motif: Leu(Ile, Val)-Tyr(Phe, Trp, Leu)-Gly-Met(Ala). All but three of the peptides containing this motif were reactive with selected mAb BNTX18 in a dot-blot assay of which eight were clearly positive in ELISA and exhibited in competition-inhibition assay the antibody binding specificity of the NTX epitope recognized by BNTX18. The two most reactive mimotopes injected into mice showed the ability to induce antibodies reacting with NTX, thus, to mimic the epitope of NTX antigenically. Sequence comparison and the analysis of the three-dimensional structure of NTX led to the proposal that residues Glu19-Leu20-Tyr21-Gly22 and the hydrophobic part of the side chain of Lys18 form the C-terminal part of the epitope. Due to the frequent presence of residues Pro, Leu, Thr, Arg, and Gln in the N-terminal part of the mimotopes, corresponding homologous residues in the N-terminal proximity of the partial epitope may be part of an additional more hydrophilic epitope element.

Epitopes recognized by human T lymphocytes in the ROP2 protein antigen of Toxoplasma gondii.

The ROP2 protein of Toxoplasma gondii possesses immunological and biological properties which have led to its proposal as a vaccine candidate. To identify epitopes recognized by human T cells in the ROP2 antigen, we submitted the sequence of this protein to three reported T-cell epitope prediction algorithms. Three sequences that were predicted by all three methods were selected (sequences 197 to 216, 393 to 410, and 501 to 524), and the corresponding peptides were synthesized. The peptides were first tested in a proliferation assay with a DPw4-restricted, ROP2-specific human T-cell clone, and the peptide corresponding to residues 197 to 216 was shown to stimulate the T-cell clone. The three peptides were further tested in proliferation assays with peripheral blood mononuclear cells from a panel of T. gondii-seropositive and -seronegative individuals. We found that cells from a high proportion of the seropositive donors (64%) recognized at least one of the three peptides. The most frequently recognized ones were peptides 197 to 216 (45%) and 501 to 524 (36%). None of the seronegative donors responded to any peptide. These results show that the ROP2 antigen of T. gondii contains T-cell epitopes recognized by a high percentage of the immune population and further strengthen its potential as a vaccine candidate.

Monoclonal antibodies against noxiustoxin.

Noxiustoxin, a 39-amino acid residue peptide isolated from the venom of the Mexican scorpion Centruroides noxius, has previously been shown to affect voltage-dependent K+ channels. Here we describe the isolation and characterization of monoclonal antibodies (MAbs) against this toxin and their use in structure-function relationship studies. Six hybridoma clones (BNTX4, -12, -14, -16, -18, and -21) producing MAbs against noxiustoxin were isolated. The epitopes defined by the MAbs are overlapping or in close proximity because no MAb pair could bind simultaneously to the toxin. All the MAbs inhibited to various degrees the binding of the toxin to its receptor sites on rat brain synaptosomal membranes. The venom from other Centruroides species was shown to contain components cross-reacting with the MAbs, suggesting the existence of other NTX-like toxins.

Subcellular localization of the 54-kDa antigen of Toxoplasma gondii.

A 54-kDa protein antigen of Toxoplasma gondii recently was cloned and expressed in Escherichia coli and shown to display immunoprotective properties. To determine the subcellular localization of this antigen, a fusion protein containing the 330 carboxy-terminal residues of the sequence coded by cDNA clone Tg34 was expressed in E. coli, purified, and used to raise antibodies in mice. Western blot analysis confirmed that the resulting antibody reacted with the 54-kDa antigen of T. gondii. Immunofluorescence and immunoelectron-microscopy showed that the antibody reacted with an antigen localized in the rhoptries, 1 of the organelles of the apical complex of the zoites involved in host cell invasion. Western blot studies using a recombinant fusion protein containing the full amino acid sequence encoded by the cDNA clone Tg34 and rhoptry protein-specific monoclonal antibodies (mAbs) previously described showed a reactivity of the recombinant antigen with 2 mAbs (T4 2F8 and T5 2D1) specific for the ROP2 protein, and with a mAb (T3 4A7) directed against an epitope shared by ROP2 and ROP4, but not with a mAb (T2 2H3) specific for the ROP4 protein. We thus conclude that the 54-kDa T. gondii antigen encoded by cDNA clone Tg34 is the previously described rhoptry protein ROP2.

Serodiagnosis of toxoplasmosis by using a recombinant form of the 54-kilodalton rhoptry antigen expressed in Escherichia coli.

A 330-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii 54-kDa rhoptry protein (ROP2) was expressed in Escherichia coli as a fusion polypeptide containing a 48-amino-acid sequence derived from phage lambda protein Cro and E. coli protein LacI followed by six consecutive histidyl residues. Metal chelate affinity chromatography provided an easy way to isolate the recombinant product in a highly purified form (> 95%). When this material was used to develop an immunoglobulin G (IgG) enzyme-linked immunosorbent assay for diagnosis of toxoplasmosis, the test reached a sensitivity of 89%. The sensitivity of the assay was similar whether the sera contained T. gondii-specific IgM or were devoid of such IgM. It was also found that ROP2 is a conserved antigen since antibodies against the recombinant antigen could be detected in mice experimentally infected with 11 independent T. gondii isolates originating from infected human tissues tested. Thus, the 54-kDa rhoptry antigen could advantageously complement other previously described T. gondii antigens for the development of more-sensitive and more-informative recombinant antigen-based tests for toxoplasmosis diagnosis.

Amino acid sequence and immunological characterization with monoclonal antibodies of two toxins from the venom of the scorpion Centruroides noxius Hoffmann.

Two toxins, which we propose to call toxins 2 and 3, were purified to homogeneity from the venom of the scorpion Centruroides noxius Hoffmann. The full primary structures of both peptides (66 amino acid residues each) was determined. Sequence comparison indicates that the two new toxins display 79% identity and present a high similarity to previously characterized Centruroides toxins, the most similar toxins being Centruroides suffusus toxin 2 and Centruroides limpidus tecomanus toxin 1. Six monoclonal antibodies (mAb) directed against purified fraction II-9.2 (which contains toxins 2 and 3) were isolated in order to carry out the immunochemical characterization of these toxins. mAb BCF2, BCF3, BCF7 and BCF9 reacted only with toxin 2, whereas BCF1 and BCF8 reacted with both toxins 2 and 3 with the same affinity. Simultaneous binding of mAb pairs to the toxin and cross-reactivity of the venoms of different scorpions with the mAb were examined. The results of these experiments showed that the mAb define four different epitopes (A-D). Epitope A (BCF8) is topographically unrelated to epitopes B (BCF2 and BCF7), C (BCF3) and D (BCF9) but the latter three appear to be more closely related or in close proximity to each other. Epitope A was found in all Centruroides venoms tested as well as on four different purified toxins of C. noxius, and thus seems to correspond to a highly conserved structure. Based on the cross-reactivity of their venoms with the mAb, Centruroides species could be classified in the following order: Centruroides elegans, Centruroides suffusus suffusus = Centruroides infamatus infamatus, Centruroides limpidus tecomanus, Centruroides limpidus limpidus, and Centruroides limpidus acatlanensis, according to increasing immunochemical relatedness of their toxins to those of Centruroides noxius. All six mAb inhibited the binding of toxin 2 to rat brain synaptosomal membranes, but only mAb BCF2, which belongs to the IgG2a subclass, displayed a clear neutralizing activity in vivo.

Human T cell clone identifies a potentially protective 54-kDa protein antigen of Toxoplasma gondii cloned and expressed in Escherichia coli.

Protective immunity against Toxoplasma gondii is recognized to be cell mediated and IFN-gamma is considered to be the major mediator of resistance. Thus, protective Ag of the parasite must induce IFN-gamma-producing T cells. In order to identify such Ag, we have constructed a T. gondii cDNA library in the cloning/expression vector lambda gt11, screened this library with a pool of sera of immune donors, and further screened the set of selected recombinant Ag using, as probe, a T. gondii-reactive T-cell clone (TCC) derived from an infected/immune individual and producing a high level of IFN-gamma. One recombinant Ag was shown to induce TCC proliferation and was characterized. The corresponding mature T. gondii Ag has an apparent molecular mass of 54 kDa and the sequence of the cDNA clone suggests that it is membrane associated. The epitope defined by the TCC on this Ag was found to be present in three Toxoplasma strains independently of their phenotype (virulent or cyst forming). Recognition of this Ag by the TCC was shown to be restricted by HLA-DPw4, the most frequent allele in the Caucasian population (approximately 40%). The use of this Ag as a vaccine component is proposed.

Human T-cell clones against Toxoplasma gondii: production of interferon-gamma, interleukin-2, and strain cross-reactivity.

The soluble fraction from a sonicate of Toxoplasma gondii tachyzoites (termed F3) was shown to induce dose-dependent blastic transformation of peripheral-blood mononuclear cells (PBMC) from seropositive individuals only and was used to isolate a panel of T-cell clones from the PBMC of an immune donor. Proliferation assays using F3 showed that 15 (14 CD4+ and 1 CD8+) of the 18 isolated clones were specific for T. gondii. In response to antigen stimulation, 5 of the 15 clones produced detectable levels of interleukin-2 (IL-2, 0.2-15 u/ml) and 9 clones produced significant levels of interferon-gamma (IFN-gamma, 17.5-1400 IU/ml). Seven of the 7 T-cell clones tested reacted with two different Toxoplasma strains (RH and Wiktor). When used as antigen-presenting cells, an autologous B-lymphoblastoid cell line could efficiently present the antigen to only three of the six T-cell clones tested. This study identifies and characterizes cellular probes that could be useful for future vaccine design.

Monoclonal antibodies identify new Toxoplasma gondii soluble antigens.

In order to characterize Toxoplasma gondii antigens, we have produced a panel of monoclonal antibodies specific for the parasite. A total of 22 hybridomas were derived from the spleen cells of mice immunized either with a 100,000 g supernatant of a sonicate from the RH strain (called F3), or chronically infected with the Wiktor or the 76K strain. Except for one hybridoma producing an IgM, all the hybridomas derived from mice immunized with F3 produced IgG1 antibodies while those obtained from chronically infected mice produced antibodies belonging to the IgG2b, IgG2a and IgM subclasses. Western-blot analysis showed that the panel of monoclonal antibodies defines at least 7 distinct antigens or antigen families. An antigen of apparent Mw 25 kD present exclusively in the 100,000 g supernatant of the T. gondii sonicate was recognized by the majority of monoclonal antibodies derived from mice immunized with the F3 fraction. Two other antigens of apparent Mw 27 kD and 29 kD present in the soluble and insoluble fractions of the sonicate were also identified. Monoclonal antibodies against the previously described 21 kD and 31 kD surface antigens and belonging to the IgG2a but also to the IgG1 subclasses were able to mediate lysis of the parasite in the presence of human non immune serum. The 22 monoclonal antibodies did not identify antigenic differences between the two independently isolated RH and Wiktor strains.

Characterization of high affinity monoclonal antibodies against the luteinizing hormone-releasing hormone.

Four hybridoma clones (BKL1, BKL2, BKL5 and BKL6), secreting monoclonal antibodies against the decapeptide luteinizing hormone releasing hormone (LHRH), were obtained by the fusion of Sp 2/0-Ag14 myeloma cells with spleen cells of a Balb/k mouse immunized with a conjugate of thyroglobulin-LHRH. All four monoclonal antibodies belong to the IgG1 subclass. The antibodies cross-reacted in RIA from 9.3 to 21% with 4-10 LHRH and less than 1% with 7-10 LHRH, 1-3 LHRH and LHRH-OH; 4-6 LHRH showed no cross-reaction. A RIA based on the BKL2 antibody and able to detect 4 pg LHRH per tube was developed. An extract of rat hypothalamus was submitted to Sephadex G50 separation and a single peak of LHRH immunoreactivity corresponding to synthetic LHRH, was detected with the antibody BKL2. When this material was further analyzed by HPLC, we found that the major peak of immunoreactivity co-migrated with synthetic LHRH. The data shows that the antibodies should be useful tools for biochemical and physiological studies on LHRH.

Production and characterization of a rat monoclonal antibody against leu5 enkephalin.

A rat X mouse hybridoma line producing a monoclonal antibody against leu5 enkephalin has been obtained. The monoclonal antibody belongs to the IgG2b class and has an affinity constant of 8.0 X 10(8) M-1 at 4 degrees C. This antibody exhibits approximately 40% cross-reactivity with 1-6 dynorphin but very weak cross-reactivities with met5 enkephalin (1.4%), 1-13 dynorphin (1.3%) and beta-endorphin (0.0045%). The detailed study, by competitive assay, of the interaction between this antibody and various enkephalin derivatives shows that the carboxy-terminal part of the molecule and, particularly, the leu side chain constitutes the immunodominant group. Nevertheless, the tyrosyl residue also contribute considerably to the binding, probably via a peculiar conformation effect, although we can not exclude the tyrosyl residue acting as a secondary contact residue. This monoclonal antibody has been used in a radioimmunoassay to determine leu5 enkephalin like immunoreactive material present in rat brain and hypothalamus.

Monoclonal antibodies against plasma protease inhibitors: production and characterization of 15 monoclonal antibodies against human antithrombin III. Relation between antigenic determinants and functional sites of antithrombin III.

Fifteen hybridomas secreting monoclonal antibodies against human antithrombin III, originating from two mouse strains, have been produced by the cell fusion technique. Eight monoclonal antibodies belong to the class IgG1, five to the class IgG2a, and two to the class IgG2b. All light chains belong to the kappa group. No cross-reaction of the monoclonal antibodies have been observed with a crude preparation of albumin nor with alpha 1-antitrypsin and alpha 2-antiplasmin. Five of these monoclonal antibodies exhibit a relatively high avidity for antithrombin III. Inhibition experiments showed that the 15 monoclonal antibodies define seven more or less independent antigenic regions on the antithrombin III molecule. Examination of the effects of these antibodies on the inhibitory capacity of antithrombin III toward thrombin activity, either in the presence or in the absence of heparin, showed that several monoclonal antibodies inhibit the antithrombin III activity and allowed to relate some of the antigenic determinants to functional sites on the antithrombin III molecule.

Expression of human alpha 1-antitrypsin cDNA in the yeast Saccharomyces cerevisiae.

Nucleotide sequences coding either for the precursor or the mature form of human alpha 1-antitrypsin have been placed under the control of the yeast ARG3 expression signals. Recombinant plasmids pRIT10782 and pRIT10787 express the precursor or the mature alpha 1-antitrypsin species, respectively, in two different yeast strains, with yields ranging between 0.3 and 1% of total soluble proteins. The alpha 1-antitrypsin synthesized in yeasts was specifically recognized by polyclonal and monoclonal antibodies raised against human alpha 1-antitrypsin. In addition, it was shown to be biologically active in its mature form only, with optimal activity in a peptidase-deficient yeast strain.

Monoclonal antibodies against plasma protease inhibitors: II. Production and characterization of 25 monoclonal antibodies against human alpha 1-antitrypsin. Correlation between antigenic structure and functional sites.

Twenty-five hybridomas secreting monoclonal antibodies against human alpha 1-antitrypsin have been produced by the cell-fusion technique (Köhler and Milstein, 1976). All antibodies are specific for alpha 1-antitrypsin and carry gamma 1 heavy chains and kappa light chains. Inhibition experiments showed that these monoclonal antibodies define three independent antigenic regions on the alpha 1-antitrypsin molecule; one of these domains appears to be involved in the interaction between alpha 1-antitrypsin and trypsin. In addition, one monoclonal antibody, AATY39, was used to develop an enzyme-linked immunosorbent assay capable of detecting low levels of alpha 1-antitrypsin in the range of 1 to 2 ng/ml.