Date Published: June 6, 2019
Publisher: Public Library of Science
Author(s): Bartłomiej Tomasz Ferra, Lucyna Holec-Gąsior, Justyna Gatkowska, Bożena Dziadek, Katarzyna Dzitko, Weronika Grąźlewska, Dariusz Lautenbach, Paulo Lee Ho.
This study presents an evaluation of four tetravalent recombinant chimeric proteins containing fragments of the Toxoplasma gondii antigens, SAG2, GRA1, ROP1 and AMA1, as potential replacements of a the soluble, whole-cell tachyzoite lysate (TLA) used in serological assays. Recombinant chimeric proteins (SAG2-GRA1-ROP1-AMA1N, AMA1N-SAG2-GRA1-ROP1, AMA1C-SAG2-GRA1-ROP1, and AMA1-SAG2-GRA1-ROP1) obtained by genetic engineering were tested for their reactivity with specific IgM and IgG antibodies from sera of experimentally infected mice and humans with T. gondii infection using an enzyme-linked immunosorbent assay (ELISA). In total 192 serum samples from patients with acquired T. gondii infection and 137 sera from seronegative individuals were examined. The reactivity of chimeric antigens with antibodies generated during T. gondii invasion was measured and compared to the results obtained in assays based on whole-cell Toxoplasma antigen. Chimeric proteins proved effective in differentiation between T. gondii-infected and uninfected individuals (100% sensitivity and specificity in the IgG ELISAs) which shows their potential usefulness as a replacements for TLA in standardized commercial tests for the serodiagnosis of toxoplasmosis. In addition, the chimeric proteins were tested for use in avidity determination. Obtained results were comparable to those of the corresponding commercial assays, suggesting the utility of these proteins for avidity assessment. Furthermore, this study demonstrated that the AMA1-SAG2-GRA1-ROP1 chimeric protein has the potential to distinguish specific antibodies from serum samples of individuals with the early and chronic phase of T. gondii infection.
Toxoplasmosis is a widespread disease caused by the ubiquitous obligate intracellular parasite Toxoplasma gondii, which infects a wide range of hosts, including humans . The World Health Organization (WHO) estimates that one-third of the human population is infected with T. gondii. In healthy individuals, the primary infection is generally asymptomatic or causes relatively mild flu-like symptoms. From the medical point of view, the reliable recognition of T. gondii invasion is very important in pregnant women, where the significant risk of tachyzoite transmission via the placenta to the fetus, can lead to miscarriages or cause neonatal malformations, neurological damage, and blindness in newborns [2,3]. Detection of the parasite invasion is also significant for patients with immunodeficiency, for whom even the chronic phase of toxoplasmosis can be a serious threat. For immunocompromised patients, infection with T. gondii may cause serious clinical complication, such as fever, headache, encephalitis, pneumonia, myocarditis, conjunctivitis, and nervous system damage . In light of many recent studies, it should be emphasized that the diagnosis of long-acquired T. gondii infection is also important and may explain the biological basis of some behavioral disorders and neurological diseases. It is assumed that the chronic parasite infection can be associated inter alia with the occurrence of seizures, slow response time, memory loss, increased risk of bipolar disorder, schizophrenia and depression, and even suicide rates [5–11].
Currently serological tools such as ELISA and immunoblots are commonly used in diagnosing T. gondii infection. Progress in research methodology has allowed well-purified recombinant proteins to be obtained via molecular biology that are attractive alternatives for the detection of serum antibodies compared to commercial tests based mainly on TLA lysate. In our study, for the first time, four new tetravalent recombinant chimeric antigens of T. gondii (SAG2-GRA1-ROP1-AMA1N, AMA1N-SAG2-GRA1ROP1, AMA1C-SAG2-GRA1-ROP1, and AMA1-SAG2-GRA1-ROP1), were expressed and purified, and their performance was evaluated in the detection of specific IgG and IgM antibodies in human and mouse sera.