Analysis of Clonal Type-Specific Antibody Reactions in Toxoplasma gondii Seropositive Humans from Germany by Peptide-Microarray

BACKGROUND: Different clonal types of Toxoplasma gondii are thought to be associated with distinct clinical manifestations of infections. Serotyping is a novel technique which may allow to determine the clonal type of T. gondii humans are infected with and to extend typing studies to larger populations which include infected but non-diseased individuals. METHODOLOGY: A peptide-microarray test for T. gondii serotyping was established with 54 previously published synthetic peptides, which mimic clonal type-specific epitopes. The test was applied to human sera (n = 174) collected from individuals with an acute T. gondii infection (n = 21), a latent T. gondii infection (n = 53) and from T. gondii-seropositive forest workers (n = 100). FINDINGS: The majority (n = 124; 71%) of all T. gondii seropositive human sera showed reactions against synthetic peptides with sequences specific for clonal type II (type II peptides). Type I and type III peptides were recognized by 42% (n = 73) or 16% (n = 28) of the human sera, respectively, while type II-III, type I-III or type I-II peptides were recognized by 49% (n = 85), 36% (n = 62) or 14% (n = 25) of the sera, respectively. Highest reaction intensities were observed with synthetic peptides mimicking type II-specific epitopes. A proportion of the sera (n = 22; 13%) showed no reaction with type-specific peptides. Individuals with acute toxoplasmosis reacted with a statistically significantly higher number of peptides as compared to individuals with latent T. gondii infection or seropositive forest workers. CONCLUSIONS: Type II-specific reactions were overrepresented and higher in intensity in the study population, which was in accord with genotyping studies on T. gondii oocysts previously conducted in the same area. There were also individuals with type I- or type III-specific reactions. Well-characterized reference sera and further specific peptide markers are needed to establish and to perform future serotyping approaches with higher resolution

Epidemiological analysis of toxoplasmosis and identification of immunogenic parasitic antigens

Die Toxoplasmose ist eine Infektionskrankheit, die durch den Parasiten Toxoplasma gondii verursacht wird. Während eine Infektion bei den meisten immunkompetenten Menschen asymptomatisch abläuft, kann bei einer Primärinfektion in der Schwangerschaft der Parasit auf den Föten übergehen und zu einem Abort oder zu schweren Erkrankungen dessen führen.Eine pränatale Therapie soll eine Übertragung verhindern oder klinische Auswirkungen beim Kind mindern. Um die Effektivität der in Deutschland durchgeführten Therapie beurteilen zu können, wurde im Rahmen dieser Arbeit eine retrospektive Untersuchung von 685 schwangeren Frauen durchgeführt, deren serologische Befunde auf eine Primärinfektion hindeuteten. In Folge dieser Studie konnte gezeigt werden, dass eine innerhalb von vier Wochen nach Infektion eingeleitete antiparasitäre Therapie das Risiko für die Ausprägung von klinisch-manifesten Erkrankungen des Kindes mindert.Um valide epidemiologische Untersuchungen durchführen zu können ist eine zuverlässige Diagnostik unabdingbar. Hierfür ist von Bedeutung, dass T. gondii ein intrazellulärer Parasit ist, welcher in drei verschiedenen Stadien vorkommt: als Oozysten, Tachy- und Bradyzoiten. Bei einer akuten Infektion sind vorwiegend sich schnell vermehrende Tachyzoiten vorhanden, während bei einer chronischen Infektion Bradyzoiten-enthaltene Zysten dominieren. Es wird davon ausgegangen, dass jedes Parasitenstadium spezifische Antigene exprimiert.Im Zuge dieser Arbeit sollten daher diagnostische Marker identifiziert werden, die eine Differenzierung zwischen einer akuten und einer chronischen Infektion erlauben. Über 2D-Gelelektrophoresen und Massenspektrometrie konnten dabei u. a. die Proteine GRA1, GRA7, ROP9, MIC5 und SUB1 als Marker für eine akute Infektion ermittelt werden. Diese, sowie sieben weitere Proteine, wurden rekombinant in E. coli hergestellt und in einem Lineblot-Verfahren an Humanseren auf ihre diagnostischen Eigenschaften untersucht.Durch den Nachweis spezifischer IgG-Antikörper gegen rGRA1, rGRA2 und rGRA6 konnte eine Toxoplasma-Infektion in bis zu 100 % erkannt werden. Eine Diskriminierung des Infektionsstadiums war allerdings nicht möglich. Eine akute Infektion ließ sich jedoch durch den Nachweis von rSUB1- und rGRA6-spezifischen IgG- und IgA Antikörper mit einer Wahrscheinlichkeit von bis zu 92 % von einer chronischen Infektion unterscheiden.Des Weiteren wurden mit Hilfe des Lineblot-Verfahrens untersucht, ob die rekombinanten Antigene auch Potenzial hatten eine der häufigsten klinisch-manifesten Erkrankungen - die okuläre Toxoplasmose (Retinochorioiditis) - zu identifizieren. Dabei konnten durch den Nachweis GRA2-spezifischer IgA- und BAG1-spezifischer IgG-Antikörper signifikante Unterschiede bei Patienten mit einer Retinochorioiditis im Vergleich zu T. gondii infizierten Patienten ohne okuläre Läsionen festgestellt werden.Um festzustellen, ob der Lineblot-Assay unter der Verwendung rekombinanter Antigene auch in der Veterinärmedizin eingesetzt werden kann, wurden Verlaufseren von Hühnern, Puten und Schweinen untersucht, die experimentell mit Oozysten oder Tachyzoiten infiziert worden waren. Dabei zeigten die Tiere, in Abhängigkeit von dem getesteten Antigen, eine teilweise Infektionsdosis- und Infektionsart-abhängige Antikörper-Kinetik.In der Zusammenschau können die in dieser Arbeit erzielten Ergebnisse zur Verbesserung der Diagnostik und Therapie der Toxoplasmose insbesondere in der Schwangerschaft beitragen

Experimental Porcine Toxoplasma gondii Infection as a Representative Model for Human Toxoplasmosis

Porcine infections are currently not the state-of-the-art model to study human diseases. Nevertheless, the course of human and porcine toxoplasmosis is much more comparable than that of human and murine toxoplasmosis. For example, severity of infection, transplacental transmission, and interferon-gamma-induced antiparasitic effector mechanisms are similar in pigs and humans. In addition, the severe immunosuppression during acute infection described in mice does not occur in the experimentally infected ones. Thus, we hypothesise that porcine Toxoplasma gondii infection data are more representative for human toxoplasmosis. We therefore suggest that the animal model chosen must be critically evaluated for its assignability to human diseases

Strongest reaction intensities were recorded for clonal type II specific peptides.

<p>To evaluate the intensities (MSIVs) by which single peptides as well as peptide cohorts (I, II, III, I–II, I–III, or II–III) were recognized by <i>T. gondii</i> seropositive patient and volunteer groups, ANOVA and the Least Significant Difference (LSD)-Post-Hoc-Test were performed. Whiskers in barplots represent 95% confidence intervals of the means of MSIVs. The differences between the means of MSIVs for single peptides or peptide cohorts within tested groups were regarded as statistically significant, when the differences were equal or higher than the LSD values. Different letters above the whiskers indicate significant differences between the mean intensities in the Post-Hoc-LSD test. Means of MSIVs for each peptide cohort are presented in (A) for the acutely infected patient group (LSD>0.36, p-value<0.05); in (C) for the latently infected patient group (LSD>0.16, p-value<0.05); and in (E) for the seropositive volunteer group (LSD>0.10, p-value<0.05). Means of MSIVs for each single peptide are presented in (B) within the acutely infected patient group (LSD>0.67, p-value<0.05); in (D) within the latently infected patient group (LSD>0.36, p-value<0.05); and in (F) within the seropositive volunteer group (LSD>0.24, p-value<0.05).</p

Proportion of human sera showing peptide reactions compatible with <i>T. gondii</i> infections by clonal types I, II, or III.

<p>Reactions are sorted according to their compatibility with infections of <i>T. gondii</i> of the clonal type I, II, or III.</p>*<p>Data resolved for seropositive patients with acute toxoplasmosis (AP), patients with latent toxoplasmosis (LP) and seropositive volunteers (V).</p

Labyrinthopeptins exert broad-spectrum antiviral activity through lipid-binding-mediated virolysis.

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re-)emerging infections, for which direct acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including Dengue virus, Zika virus, West Nile virus, Hepatitis C virus, Chikungunya virus, Karposi's Sarcoma-associated Herpes virus, Cytomegalovirus, and Herpes Simplex virus, in the low μM to nM range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to IC10-IC90 values of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (PC/PE/Chol/SM (17:10:33:40)) are particularly sensitive to labyrinthopeptins compared to PC/PE (90:10) LUVs, even though the overall PE-amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (t1/2= 10.0 h), which designates them as promising antiviral compounds acting by an unusual viral lipid targeting mechanism.Importance For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses - well-known as well as (re-)emerging species - has gained attention, especially for the treatment of viral co-infections. While most known broad spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including Chikungunya virus, Dengue virus, Zika virus, Karposi's Sarcoma-associated Herpes virus, or Cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity to host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections

Labyrinthopeptins exert broad-spectrum antiviral activity through lipid-binding-mediated virolysis

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re-)emerging infections, for which direct acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including Dengue virus, Zika virus, West Nile virus, Hepatitis C virus, Chikungunya virus, Karposi's Sarcoma-associated Herpes virus, Cytomegalovirus, and Herpes Simplex virus, in the low μM to nM range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to IC10-IC90 values of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (PC/PE/Chol/SM (17:10:33:40)) are particularly sensitive to labyrinthopeptins compared to PC/PE (90:10) LUVs, even though the overall PE-amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (t1/2= 10.0 h), which designates them as promising antiviral compounds acting by an unusual viral lipid targeting mechanism.Importance For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses - well-known as well as (re-)emerging species - has gained attention, especially for the treatment of viral co-infections. While most known broad spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including Chikungunya virus, Dengue virus, Zika virus, Karposi's Sarcoma-associated Herpes virus, or Cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity to host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.Andreas Meyerhans and Javier P. Martinez were supported by a grant from the Spanish Ministry of Economy, Industry and Competitiveness and FEDER grant no. SAF2016-75505-R (AEI/MINEIC/FEDER, UE). Mark Brönstrup, Andreas Meyerhans and Javier P. Martinez would like to acknowledge a networking contribution from the COST Action CM1407 “Challenging organic syntheses inspired by nature – from natural products chemistry to drug discovery”. Martin Messerle and Thomas F. Schulz were supported by funding from DZIF (project 07.802 TTU IICH). Christine Goffinet, Thomas Pietschmann and Mark Brönstrup were supported by a grant provided by “Innovationsfonds der Helmholtz-Zentren”. Christine Goffinet was supported by a DFG grant within German African Cooperation Projects in Infectiology (GO2153/3-1) and by funding of the Helmholtz Center for Infection Research (HZI) and of Berlin Institute of Health (BIH). Sergej Franz was supported by the Infection Biology international PhD program of Hannover Biomedical Research Schoo

Clonal type-specific anti-peptide reactivity of <i>T. gondii</i> positive humans.

<p>Reactions are sorted according to the specificities of peptides.</p