Rhoptry Antigen Research Articles

Tandem repeats in the genome of Toxoplasma gondii display compositional bias that impacts in protein structure

Repetitive elements in DNA sequences are a hallmark of Apicomplexan protozoa. A genome-wide screening for Tandem Repeats was conducted in Toxoplasma gondii and related Coccidian parasites with a novel strategy to assess compositional bias. A conserved pattern of GC skew and purine-pyrimidine bias was observed. Compositional bias was also present at the protein level. Glutamic acid was the most abundant amino acid in the purine (GA) rich cluster, while Serine prevailed in pyrimidine (CT) rich cluster. Purine rich repeats, and consequently glutamic acid abundance, correlated with high scores for intrinsically disordered protein regions/domains. Finally, variability was established for repetitive regions within a well-known rhoptry antigen (ROP1) and an uncharacterized hypothetical protein with similar features. The approach we present could be useful to identify potential antigens bearing repetitive elements.

Cell biological analysis reveals an essential role for Pfcerli2 in erythrocyte invasion by malaria parasites

Merozoite invasion of host red blood cells (RBCs) is essential for survival of the human malaria parasite Plasmodium falciparum. Proteins involved with RBC binding and invasion are secreted from dual-club shaped organelles at the apical tip of the merozoite called the rhoptries. Here we characterise P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 2 (PfCERLI2), as a rhoptry bulb protein that is essential for merozoite invasion. Phylogenetic analyses show that cerli2 arose through an ancestral gene duplication of cerli1. We show that PfCERLI2 is essential for blood-stage growth and localises to the cytosolic face of the rhoptry bulb. Inducible knockdown of PfCERLI2 led to a proportion of merozoites failing to invade and was associated with elongation of the rhoptry organelle during merozoite development and inhibition of rhoptry antigen processing. These findings identify PfCERLI2 as a protein that has key roles in rhoptry biology during merozoite invasion.

PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes

The disease-causing blood-stage of the Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we identify an essential role for the conserved protein P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. We show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, biochemical techniques and semi-quantitative super-resolution microscopy show that PfCERLI1 knockdown prevents secretion of key rhoptry antigens that coordinate merozoite invasion. PfCERLI1 is a rhoptry associated protein identified to have a direct role in function of this essential merozoite invasion organelle, which has broader implications for understanding apicomplexan invasion biology.

Structural predication and antigenic analysis of ROP16 protein utilizing immunoinformatics methods in order to identification of a vaccine against Toxoplasma gondii: An in silico approach

Toxoplasmosis, caused by Toxoplasma gondii, is a common parasitic disease, affecting almost one-third of the world's population. Currently, there are no effective treatments for inhibiting the formation of chronic tissue cysts in infected hosts. Thus, the production of appropriate vaccines against this pathogen is an important goal to avoid toxoplasmosis. considering the role of rhoptry antigens like ROP16 in virulence and satisfactory immunogenicity, they can be used as promising vaccine candidates against T. gondii. In the present study, an in silico approach was used to analyze various aspects of the ROP16 protein, including physicochemical characteristics, the potential epitopes of B and T-cells, the secondary and tertiary structure, the subcellular localization, the transmembrane domain, and other important features of this protein using several bioinformatics tools to design a proper vaccine against T. gondii. The results showed that ROP16 protein includes 93 potential post-translational modification sites. The secondary structure of the ROP16 protein comprises 34.23% alpha-helix, 54.46% random coil, and 11.32% extended strand. Moreover, several potential B- and T-cell epitopes were identified for ROP16. Based on the results of Ramachandran plot, 84.64% of the amino acid residues were located in the favored, 10.34% in allowed, and 5.02% in outlier regions. Furthermore, the results of the antigenicity and allergenicity assessment noted that this protein was immunogenic and non-allergenic. Our findings suggested that structural and functional predictions applied to ROP16 protein using in silico tools can reduce the failure risk of the laboratory studies. This research provided an important basis for further studies and also developed an effective vaccine against acute and chronic toxoplasmosis by various strategies. Further studies are needed on the development of vaccines in vivo using ROP16 alone or in combination with other antigens in the future.

Toxoplasma gondii Tetravalent Chimeric Proteins as Novel Antigens for Detection of Specific Immunoglobulin G in Sera of Small Ruminants.

Simple SummaryToxoplasma gondii infection leads to large economic losses in the sheep and goat industry worldwide and is considered to be one of the main causes of infectious ovine and caprine abortion. Moreover, in countries where sheep and goat meat are frequently consumed, T. gondii infection in small ruminants may also pose a public health risk. Due to its medical and veterinary importance, it is essential to study the seroprevalence of T. gondii infection among farm animals and humans. This requires the development of new, low-cost diagnostic methods such as enzyme immunoassays based on recombination antigens. Thus, the study aimed to evaluate the reactivity of four different tetravalent chimeric proteins containing immunodominant regions from the AMA1 (apical membrane antigen 1), SAG2 (surface antigen 2), GRA1 (dense granule antigen 1), GRA2 (dense granule antigen 2), and ROP1 (rhoptry antigen 1) T. gondii antigens with specific IgG from the sera of small ruminants. The results demonstrate that an IgG ELISA (enzyme-linked immunosorbent assay) based on one of these chimeric proteins (AMA1-SAG2-GRA1-ROP1) may be a useful test for the determination of T. gondii infection in small ruminants.The detection of Toxoplasma gondii infection in small ruminants has important significance for public health and veterinary medicine. This study, for the first time, describes the reactivity of four tetravalent chimeric proteins (AMA1N-SAG2-GRA1-ROP1, AMA1C-SAG2-GRA1-ROP1, AMA1-SAG2-GRA1-ROP1, and SAG2-GRA1-ROP1-GRA2) containing immunodominant regions from the AMA1 (apical membrane antigen 1), SAG2 (surface antigen 2), GRA1 (dense granule antigen 1), GRA2 (dense granule antigen 2), and ROP1 (rhoptry antigen 1) with specific IgG antibodies from the sera of small ruminants with the use of an indirect enzyme-linked immunosorbent assay (ELISA). The reactivity of individual chimeric antigens was analyzed in relation to the results obtained in IgG ELISA based on a Toxoplasma lysate antigen (TLA). All chimeric proteins were characterized by high specificity (between 96.39% to 100%), whereas the sensitivity of the IgG ELISAs was variable (between 78.49% and 96.77%). The highest sensitivity was observed in the IgG ELISA test based on the AMA1-SAG2-GRA1-ROP1. These data demonstrate that this chimeric protein can be a promising serodiagnostic tool for T. gondii infection in small ruminants.

The persistence of naturally acquired antibodies and memory B cells specific to rhoptry proteins of Plasmodium vivax in patients from areas of low malaria transmission

BackgroundRhoptries are the large, paired, secretory organelles located at the apical tip of the malaria merozoite that are considered important for parasite invasion processes. Plasmodium vivax rhoptry proteins have been shown to induce humoral immunity during natural infections. Therefore, these proteins may be potential novel vaccine candidates. However, there is a lack of data on the duration of antibody and memory B cell (MBC) responses. Here, the longitudinal analysis of antibody and MBC responses to the P. vivax rhoptry proteins PvRALP1-Ecto and PvRhopH2 were monitored and analysed in individuals to determine their persistence.MethodsThirty-nine samples from P. vivax-infected subjects (age 18–60 years) were recruited to explore the frequency and persistence of antibody and MBC responses against rhoptry proteins (PvRALP1-Ecto and PvRhopH2) using both cross-sectional and longitudinal cohort study designs. Antibody levels were determined by ELISA during clinical malaria, and at 3, 9 and 12 months post-infection. The frequency of MBC sub-sets and presence of rhoptry-specific MBCs in subjects 18 months after treatment were detected by flow cytometry and ELISPOT assay.ResultsThe seroprevalence of antibodies against PvRALP1-Ecto and PvRhopH2 proteins was found to be high during acute infection, with IgG1, IgG2 and IgG3 sub-classes predominant. However, these anti-rhoptry responses were short-lived and significantly decreased at 9 months post-infection. To relate the durability of these antibody responses to MBC persistence at post-infection, 18-month post-infection peripheral blood mononuclear cells (PBMCs) samples were taken to detect rhoptry-specific MBCs and frequency of MBC sub-sets, and correlate with antibody responses. These late post-infection samples revealed that rhoptry-specific MBCs were present in about 70% of total subjects. However, the persistence of specific MBCs was not correlated with antibody responses as the majority of malaria subjects who were positive for PvRALP1-Ecto- or PvRhopH2-specific MBCs were seronegative for the rhoptry antigens. The frequencies of classical MBCs were increased after infection, whereas those of activated and atypical MBCs were decreased, indicating that MBC responses could switch from activated or atypical MBCs to classical MBCs after parasite clearance, and were maintained in blood circulating at post-infection.ConclusionThe study showed that rhoptry antigens induced the development and persistence of MBC responses in P. vivax-infected subjects who lived in a region of low malaria transmission, which were not related to the longevity of antibody responses.

DNA-Loaded Cationic Liposomes Efficiently Function as a Vaccine against Malarial Proteins

The delivery of antigens as DNA vaccines is an efficient alternative to induce immune responses against antigens, which are difficult to produce in recombinant form. However, the delivery of naked DNA is ineffective or relies on sophisticated ballistic devices. Here, we show a combination of liposome application and naked DNA vaccine that successfully overcomes these problems. Upon entrapment of plasmids encoding different antigens in cationic particles, transfection efficiencies similar to commercial kits were achieved in in vitro cell cultures. The liposome-based approach provided strong humoral responses against three malarial antigens, namely the Circumsporozoite protein and the C terminus of merozoite surface protein 1 from Plasmodium vivax (titers 104 or 103–104, respectively) and P. falciparum Rhoptry antigen 5 from Plasmodium falciparum (titers 103–104). When employed in P. falciparum growth-inhibition assays, antibodies demonstrated consistent reinvasion-blocking activities that were dose dependent. Liposome-formulated DNA vaccines may prove useful when targets cannot be produced as recombinant proteins and when conformation-dependent and highly specific antibodies are mandatory.

Evaluation of the Protective Effect of Deoxyribonucleic Acid Vaccines Encoding Granule Antigen 2 and 5 Against Acute Toxoplasmosis in BALB/c Mice.

AbstractToxoplasma gondii infects a broad range of warm-blooded hosts, including humans. Important clinical manifestations include encephalitis in immunocompromised patients as well as miscarriage and fetal damage during early pregnancy. Toxoplasma gondii dense granule antigen 2 and 5 (GRA2 and GRA5) are essential for parasitophorous vacuole development of the parasite. To evaluate the potential of GRA2 and GRA5 as recombinant DNA vaccine candidates, these antigens were cloned into eukaryotic expression vector (pcDNA 3.1C) and evaluated in vaccination experiments. Recombinant DNA vaccines constructed with genes encoding GRAs were validated in Chinese hamster ovary cells before evaluation using lethal challenge of the virulent T. gondii RH strain in BALB/c mice. The DNA vaccines of pcGRA2 and pcGRA5 elicited cellular-mediated immune response with significantly higher levels of interferon-gamma, interleukin-2 (IL-2), IL-4, and IL-10 (P < 0.05) compared with controls. A mixed T-helper cell 1 (Th1)/Th2 response was associated with slightly prolonged survival. These findings provide evidence that DNA vaccination with GRA2 and GRA5 is associated with Th1-like cell-mediated immune responses. It will be worthwhile to construct recombinant multiantigen combining full-length GRA2 or/and GRA5 with various antigenic proteins such as the surface antigens and rhoptry antigens to improve vaccination efficacy.

Influenza M1 Virus-Like Particles Consisting of Toxoplasma gondii Rhoptry Protein 4.

Toxoplasma gondii infections occur throughout the world, and efforts are needed to develop various vaccine candidates expressing recombinant protein antigens. In this study, influenza matrix protein (M1) virus-like particles (VLPs) consisting of T. gondii rhoptry antigen 4 (ROP4 protein) were generated using baculovirus (rBV) expression system. Recombinant ROP4 protein with influenza M1 were cloned and expressed in rBV. SF9 insect cells were coinfected with recombinant rBVs expressing T. gondii ROP4 and influenza M1. As the results, influenza M1 VLPs showed spherical shapes, and T. gondii ROP4 protein exhibited as spikes on VLP surface under transmission electron microscopy (TEM). The M1 VLPs resemble virions in morphology and size. We found that M1 VLPs reacted with antibody from T. gondii-infected mice by western blot and ELISA. This study demonstrated that T. gondii ROP4 protein can be expressed on the surface of influenza M1 VLPs and the M1 VLPs containing T. gondii ROP4 reacted with T. gondii-infected sera, indicating the possibility that M1 VLPs could be used as a coating antigen for diagnostic and/or vaccine candidate against T. gondii infection.

A long-lasting protective immunity against chronic toxoplasmosis in mice induced by recombinant rhoptry proteins encapsulated in poly (lactide-co-glycolide) microparticles.

Toxoplasma gondii infection in humans and animals is a worldwide zoonosis. Prevention and control of toxoplasmosis based on vaccination is one of the promising strategies. In the present study, recombinant T. gondii rhoptry proteins 38 and 18 (TgROP38 and TgROP18) were encapsulated into poly (lactide-co-glycolide) (PLG) (1:1), respectively, to obtain the stable water-in-oil-in-water double emulsion. Female Kunming mice were then immunized with the protein vaccines twice at a 2-week interval. Eight weeks after the second immunization, 10 mice from each group were challenged with T. gondii PRU strain (genotype II). The entrapment rates of PLG-rROP38 and PLG-rROP18 ranged from 65.5 to 77.7% and 58.1 to 72.3%, respectively. Immunization of mice with rROP38 and rROP18 proteins encapsulated into PLG microparticles elicited strongly humoral and cell-mediated responses against T. gondii, associated with relatively high levels of total IgG, IgG2a isotype, and IFN-γ, as well as the mixed Th1/Th2 immunity responses. Immunization with various protein vaccines induced significant reduction of the brain cysts after chronic infection with the T. gondii PRU strain, and the most effective protection was achieved in the PLG-rROP38-rROP18-immunized mice, with a cyst reduction of 81.3%. The findings of the present study indicated that recombinant rhoptry antigens encapsulated in PLG could maintain the protein immunogenicity in an extended period and elicit effective protection against chronic T. gondii infection, which has implications for the development of long-lasting vaccines against chronic toxoplasmosis in animals.

A vaccine formulation combining rhoptry proteins NcROP40 and NcROP2 improves pup survival in a pregnant mouse model of neosporosis

Currently there are no effective vaccines for the control of bovine neosporosis. During the last years several subunit vaccines based on immunodominant antigens and other proteins involved in adhesion, invasion and intracellular proliferation of Neospora caninum have been evaluated as targets for vaccine development in experimental mouse infection models. Among them, the rhoptry antigen NcROP2 and the immunodominant NcGRA7 protein have been assessed with varying results. Recent studies have shown that another rhoptry component, NcROP40, and NcNTPase, a putative dense granule antigen, exhibit higher expression levels in tachyzoites of virulent N. caninum isolates, suggesting that these could be potential vaccine candidates to limit the effects of infection. In the present work, the safety and efficacy of these recombinant antigens formulated in Quil-A adjuvant as monovalent vaccines or pair-wise combinations (rNcROP40+rNcROP2 and rNcGRA7+rNcNTPase) were evaluated in a pregnant mouse model of neosporosis. All the vaccine formulations elicited a specific immune response against their respective native proteins after immunization. Mice vaccinated with rNcROP40 and rNcROP2 alone or in combination produced the highest levels of IFN-γ and exhibited low parasite burdens and low IgG antibody levels after the challenge. In addition, most of the vaccine formulations were able to increase the median survival time in the offspring. However, pup survival only ensued in the groups vaccinated with rNcROP40+rNcROP2 (16.2%) and rNcROP2 (6.3%). Interestingly, vertical transmission was not observed in those survivor pups immunized with rNcROP40+rNcROP2, as shown by PCR analyses. These results show a partial protection against N. caninum infection after vaccination with rNcROP40+rNcROP2, suggesting a synergistic effect of the two recombinant rhoptry antigens.

Comparing the effect of IL-12 genetic adjuvant and alum non-genetic adjuvant on the efficiency of the cocktail DNA vaccine containing plasmids encoding SAG-1 and ROP-2 of Toxoplasma gondii

Various methods are available for enhancing the potency of DNA vaccines, including employment of different forms of adjuvant. The current study was carried out to evaluate and compare the effects of genetic and non-genetic adjuvants on the immune response stimulated by DNA vaccine. Thus, two adjuvants, IL-12 (genetic adjuvant) and aluminum hydroxide (alum, non-genetic adjuvant), were used with cocktail DNA vaccine containing plasmids encoding complete rhoptry antigen 2 (ROP-2) and surface major antigen 1 (SAG-1) of Toxoplasma gondii. The efficacy of pcROP2+pcSAG1 in stimulation of the immune response against toxoplasmosis with and without adjuvant was evaluated in female BALB/c mice by measuring the level of total IgG antibody and cytokines. The results obtained indicated that after challenging the mice with the fatal RH strain of T. gondii, the survival rates of mice immunized with pcROP2+pcSAG1 (DNA cocktail), pcSAG1+pcROP2+alum, and pcSAG1+pcROP2+IL-12 were significantly greater than that of the control groups (p<0.05). Moreover, measurement of total IgG antibody indicated the significant difference between the control and experimental groups (p<0.05). Finally, the results obtained by measurement of cytokines (IFN-γ and IL-4) showed high levels of IFN-γ and low levels of IL-4 in groups vaccinated with pcROP2+pcSAG1 (DNA cocktail), pcSAG1+pcROP2+alum, and pcSAG1+pcROP2+IL-12 as the experiment groups, in comparison with the controls groups (PBS, pc-DNA3, alum+PBS, and pCAGGS-IL-12+pcDNA3). The results of the study showed that use of adjuvants (IL-12 and alum) coincident with DNA cocktail leads to significant change in the survival rates of the experiment groups in comparison with control groups. Also, there is no significant difference between adjuvants to induce immune responses.

Determination of Toxoplasma gondii Recombinant ROP2 and ROP4 Antigens Diagnostic Value on Mouse Experimental Model

The aim of this study was to test the potential diagnostic usefulness of recombinant Toxoplasma gondii rhoptry antigens, ROP2 and ROP4, with respect to toxoplasmosis detection and infection phase distinction in laboratory mouce by determining specific serum IgM and IgG antibodies with the use of indirect ELISA technique. The mice antibody response to ROP antigens was significantly higher in the IgM than in the IgG class with the peak on the turn of acute and latent infection, whereas the response to recombinant SAG1 antigen, used as control, revealed preferential synthesis of IgG antibodies with the highest absorbance values measured during latent toxoplasmosis.

Characterization and antigenicity of the promising vaccine candidate Plasmodium vivax 34 kDa rhoptry antigen (Pv34)

This study describes the identification of the Plasmodium vivax rhoptry antigen Pv34 whose sequence was obtained based on homology comparison with the Plasmodium falciparum Pf34. The pv34 gene product was characterized by molecular biology and immunological techniques. Additionally, association of Pv34 to detergent-resistant microdomains (DRMs), expression in late blood-stage parasites and recognition of recombinant Pv34 (rPv34) by sera from P. vivax-infected Aotus monkeys and patients was assessed. Lymphoproliferation and cytokine secretion was also evaluated in individuals living in malaria endemic areas. Altogether, the data support carrying out further studies to assess the immunogenicity and protection-inducing ability of rPv34 as component of a multi-antigenic, multi-stage vaccine against vivax malaria.

Vaccination of mice with recombinant NcROP2 antigen reduces mortality and cerebral infection in mice infected with Neospora caninum tachyzoites

Rhoptry antigens are involved in a variety of cellular functions related to host cell invasion, formation of the parasitophorous vacuole and parasite–host cell interplay. The cDNA sequence of one of these antigens, NcROP2 was identified from Neospora caninum expressed sequence tags (ESTs), amplified by reverse transcription-PCR, expressed in Escherichia coli as a (His)6-tagged recombinant protein (recNcROP2) and purified over Ni2+-affinity chromatography. Both recNcROP2 and antibodies directed against recNcROP2 had a negative impact on N. caninum tachyzoite host cell invasion in vitro, indicating that this protein participates in the host cell entry process. Subsequently, the protective efficacy of NcROP2 as a potential vaccine candidate was evaluated in a C57BL/6 mouse cerebral disease model. Mice were vaccinated three times at 2-week intervals with recNcROP2 emulsified either in Freund’s incomplete adjuvants (FIA) or saponin, and control groups were treated with adjuvants alone (adjuvants control) or PBS (infection control). Subsequently, mice were challenged with 2×106N. caninum tachyzoites. Nine mice, all belonging to the infection control or adjuvants control groups, exhibited clinical signs of cerebral neosporosis and succumbed to infection, whilst no clinical signs were noted for recNcROP2-vaccinated mice. For all other animals, the experiment was terminated 35 days p.i. Cerebral parasite burdens were assessed by quantitative PCR in all mice, and were revealed to be significantly reduced in the recNcROP2-vaccinated mice. ELISA of sera revealed IgG1 to be elevated in recNcROP2–saponin vaccinated mice, whilst IgG2a was higher in recNcROP2–FIA vaccinated animals. This shows that, depending on the adjuvants used, vaccination with NcROP2 induces a protective Th-1- or Th-2-biased immune response against experimental N. caninum infection.

Toxoplasma Rhoptries: Unique Secretory Organelles and Source of Promising Vaccine Proteins for Immunoprevention of Toxoplasmosis

Toxoplasma gondii is an obligate intracellular protozoan parasite classified in the phylum Apicomplexa, which includes numerous notable human and animal pathogens (Plasmodium species, Cryptosporidium species, Neospora caninum, etc.). The invasive stages of apicomplexans are characterized by the presence of an apical complex composed of specialized cytoskeletal and secretory organelles, including rhoptries. Rhoptries, unique apical secretory organelles shared exclusively by all apicomplexan parasites, are known to be involved in an active parasite's penetration into the host cell associated with the biogenesis of specific intracellular compartment, parasitophorous vacuole in which the parasite multiplies intensively, avoiding intracellular killing. Due to the key biological role of rhoptries, rhoptry proteins have recently become vaccine candidates for the prevention of several parasitoses, toxoplasmosis among them. The article presents current data on T. gondii rhoptries biology and new approaches to the development of effective vaccines against toxoplasmosis using rhoptry antigens.

Genetic characteristics of the Korean isolate KI-1 of Toxoplasma gondii

Toxoplasma gondii tachyzoites were isolated from an ocular patient in the Republic of Korea and maintained in the laboratory (designated KI-1). In the present study, its genotype was determined by analyzing dense granule antigen 6 (GRA6) gene and surface antigen 2 (SAG2) gene as typing markers. Digestion of the amplification products of GRA6 and of the 5o and 3o ends of SAG2, respectively, with Mse I, Sau3A I, and Hha I, revealed that KI-1 is included in the genotype I, which includes the worldwide virulent RH strain. In addition, when the whole sequences of the coding regions of SAG1, rhoptry antigen 1 (ROP1), and GRA8 genes of KI-1 were compared with those of RH, minor nucleotide polymorphisms and amino acid substitutions were identified. These results show that KI-1 is a new geographical strain of T. gondii that can be included in the genotype I.

Kinetics study of the localization and quantitation of target antigens of immunoglobulin a antibodies in acquired and congenital toxoplasmosis.

The cellular distribution (localization and quantitation) of the target parasite's antigens in the tachyzoite along the IgA kinetics was determined in the course of acquired toxoplasmosis and congenital toxoplasmosis. In the case of acquired toxoplasmosis, throughout the IgA kinetics a correlation was noted between the membrane and submembrane immunolabeling and the results of the immunocapture and enzyme-linked immunosorbent assay IgA (ELISA-A) tests. The rhoptries' immunolabeling remained higher. The immunolabeling evolution and the results of the immunology tests were not closely related to the treatment (Rovamycin). From the congenital toxoplasmosis cases it was observed that membrane immunolabeling correlated with the results of the serology tests and with the treatment (Fansidar). The rhoptry antigens were recognized throughout the IgA kinetics; even when the serology tests became negative, immunolabeling persisted. Rhoptries appeared as secretory organelles of antigens recognized during acute, chronic, and congenital stages of Toxoplasma infection.

Toxoplasma gondii: Characterization of a Monoclonal Antibody Recognizing Antigens of 36 and 38 kDa with Acid Phosphatase Activity Located in Dense Granules and Rhoptries

Acid phosphatase activity in Toxoplasma gondii was studied by electron microscopy and found in the rhoptries and dense granules. On Western blots, six bands were found to have acid phosphatase activity; a double band of 36-38 kDa showed the strongest activity. In order to raise monoclonal antibodies to antigens of potential importance in parasite invasion of the host cell, mice were immunized with live T. gondii of the cyst-forming ME119 isolate and later boosted with tachyzoites of the RH strain. One of the monoclonal antibodies, S16, recognized the 36- to 38-kDa antigen. Immunogold labeling and electron microscopy showed that the antigens were located in dense granules and rhoptries. Using immuno-precipitation with S16, SDS-PAGE, IEF, and Western blotting, we show that the 36- to 38-kDa rhoptry antigen recognized by S16 has acid phosphatase activity.

Subcellular localization and quantitative analysis of Toxoplasma gondii target antigens of specific immunoglobulins G, M, A, and E

The target antigens of specific immunoglobulins G, M, A, and E from patients with acquired acute toxoplasmosis were determined using immunocytochemistry. The relative repartition of these antigens in four cellular compartments of Toxoplasma (membrane complex, apical area, rhoptries, and dense granules) was quantitatively evaluated. Rhoptry antigens mainly react positively with IgA. Membrane, submembrane area (membrane complex), and rhoptry antigens are immunodominant for IgA and IgM. Apical area antigens are recognized by IgM two times more than IgG and IgA. IgE recognized only rhoptry antigens. The localization of pathogenetically antigenic components and their identification by the immune system appeared to be of importance for selection of immunodominant or recombinant antigens. Such localization would improve laboratory diagnosis of serious congenital toxoplasmosis or in immunocompromised patients with toxoplasmic complications after cyst reactivation.