Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to Surface Antigen 1 ofToxoplasma gondiiTachyzoites

Abstract

A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant surface antigen 1 (SAG1) of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy-chain gene revealed that the closest germ line V segments were VH3-23. The germ line D segment was D1-7, and the closest germ line J segment was JH4. In the light-chain genes, the closest germ line V segment was V1-17 with the J1 or J4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09 ؋ 10 ؊9 M for Tox203 and 2.01 ؋ 10 ؊8 M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa and infects a variety of warm-blooded domestic and wild animals worldwide. It is an important foodborne parasite transmitted primarily from animals to humans through meat, as well as through oocysts shed by cats into the environment (25). Infection in humans is usually asymptomatic in immunocompetent hosts. However, primary infection during pregnancy can result in severe neonatal malformations and ocular complications in the fetus. In immunocompromised hosts such as patients with human immunodeficiency virus/ AIDS, reactivation of the latent infection results in symptomatic diseases such as toxoplasmic encephalitis (14,22). Transmission of T. gondii by organ transplantation from a seropositive donor to a seronegative recipient is also an important potential cause of disease in heart, heart-lung, kidney, liver, and liver-pancreas transplant patients (25).The surface of T. gondii is the first component to contact the host cells. The T. gondii surface is coated by closely related antigens that belong to the surface antigen 1 (SAG1)-related sequences (SRS) superfamily (13, 16). SAG1 is the most abundant and immunogenic of these antigens and is important for the process of invasion. Treatment of T. gondii with mouse monoclonal or rabbit polyclonal antibodies to SAG1 inhibits parasite attachment to host cells (24). Fab fragments derived from a mouse monoclonal antibody also showed dose-dependent inhibition of parasite attachment (23). Therefore, human monoclonal antibodies to SAG1 may be applicable for prevention of transmission and reactivation ...

Recent Advances in Toxoplasma gondii Immunotherapeutics

“…In an experiment using Fab antibody fragments specific to T. gondii SAG1 antigen, it was shown that the antibodies blocked parasites attachment to host cells in vitro by 52% at an antibody concentration of 200 µg/ml [ 87 ]. Passive immunization of mice with 10 mg of this antibody provided improved survival rates of up to 50% following lethal challenge infection [ 87 ].…”

“…In an experiment using Fab antibody fragments specific to T. gondii SAG1 antigen, it was shown that the antibodies blocked parasites attachment to host cells in vitro by 52% at an antibody concentration of 200 µg/ml [ 87 ]. Passive immunization of mice with 10 mg of this antibody provided improved survival rates of up to 50% following lethal challenge infection [ 87 ]. This survival rate improvement is a significant finding considering that the Fab fragments used in this study is lacking the Fc region of full antibodies which mediates Fc-receptor-dependent phagocytosis of opsonized tachyzoites, cellular cytotoxicity and complement activation.…”

Production, Characterization and Applications for Toxoplasma gondii-Specific Polyclonal Chicken Egg Yolk Immunoglobulins

“…Specific polyclonal antibodies generated in mammals against T. gondii have been extremely useful in the last decades to unravel several aspects related to the parasite biology as host-parasite interactions, parasite invasion and replication mechanisms, candidate epitopes for diagnosis, characterization of T. gondii virulence factors and candidate protein for immunoprophylactic assays [14][15][16][17].…”

Characterization of a Lipopolysaccharide-Targeted Monoclonal Antibody and Its Variable Fragments as Candidates for Prophylaxis against the Obligate Intracellular Bacterial Pathogen Coxiella burnetii

“…The bestdocumented direct effect of the variable region is virus neutralization, which is defined as the abrogation of virus infectivity by the inhibition of virus attachment and early entry into susceptible host cells (30). In contrast, only a few studies have reported that the ability of Abs to block receptors is required for the uptake of intracellular bacterial pathogens, thereby inhibiting bacterial infection (32,33). In the present study, to determine the role of the variable region and Fc segment in 1E4-mediated passive protection, we examined if the variable region of 1E4 (Fab1E4) retains the ability of 1E4 to inhibit C. burnetii infection in both in vitro and in vivo systems.…”