Opsonic monoclonal antibodies enhance phagocytic killing activity and clearance of Mycobacterium tuberculosis from blood in a quantitative qPCR mouse model

Abstract

BackgroundPatients with impaired immunity often have rapid progression of tuberculosis (TB) which can lead to highly lethal Mycobacterium tuberculosis (MTB) sepsis. Opsonic monoclonal antibodies (MABs) directed against MTB that enhance phagocytic killing activity and clearance of MTB from blood may be useful to enhance TB immunity.MethodsBALB/c mice were immunized with ethanol-killed MTB (EK-MTB) and MABs were produced and screened by ELISA for binding to killed and live Mycobacterium smegmatis (SMEG) and MTB. MAB opsonophagocytic killing activity (OPKA) was examined using SMEG with HL60 and U-937 cells and MTB with U-937 cells. Clearance of MTB from blood was evaluated in Institute of Cancer Research (ICR) mice given opsonic anti-MTB MABs or saline (control) 24 h prior to intravenous infusion with 108 CFUs gamma-irradiated MTB (HN878). MTB levels in murine blood collected 0.25, 4 and 24 h post-challenge were assessed by qPCR. MAB binding to peptidoglycan (PGN) was examined by ELISA using PGN cell wall mixture and ultra-pure PGN.ResultsTwo MABs (GG9 and JG7) bound to killed and live SMEG and MTB (susceptible and resistant), and promoted OPKA with live MTB. MAB JG7 significantly enhanced OPKA of MTB. Both MABs significantly enhanced clearance of killed MTB from murine blood at 4 and 24 h as measured by qPCR. These opsonic MABs bound to PGN, a major cell wall constituent.ConclusionsAnti-MTB MABs that promote bactericidal phagocytic activity of MTB and enhance clearance of killed MTB from the blood, may offer an immunotherapeutic approach for treatment of MTB bacteremia or sepsis, and augment treatment of multi-drug resistant (MDR) or extensively drug resistant (XDR) TB.

In vitro evaluation of the binding activity of novel mouse IgG1 opsonic monoclonal antibodies to Mycobacterium tuberculosis and other selected mycobacterial species

“…These MABs were produced by fusing single spleen cells from immunized BALB/c mice, with SP2/0 myeloma cells to form hybridoma cells which were then tested, selected, and subsequently cloned. The MABs produced were purified by chromatography, quantified by IgG capture ELISA, and characterized as opsonic monoclonal antibodies [24] . These MABs have been deposited at ATCC (designated as GG9-01 and JG7-01) and are available for research use.…”

“…Working solutions of MABs GG9 and JG7 were prepared in PBS-T according to calculated dilutions from different stock concentrations to final concentrations of 5 µg/mL, 10 µg/mL, and 25 µg/mL. These concentrations were chosen based on previous titration and optimization assays [24] ; concentrations > 25 µg/mL (up to 100 µg/mL were tested in our initial pilot study (Shey B, 2016; unpublished data), but did not yield discriminatory binding data.…”

“…In a previous study, Sei and colleagues used enzyme-linked immunosorbent assays (ELISAs) to assess the binding activity of two novel mouse IgG1 anti-TB opsonic MABs, GG9 II G2 (GG9) and JG7 III D3 F9 (JG7), to inactivated and live clinical M. tuberculosis strains [24] .They further demonstrated that these MABs at concentrations less than 25 µg/mL, significantly enhanced the phagocytic killing of M. tuberculosis in vitro and also significantly enhanced its clearance from blood in mice in vivo [24] . In addition, the results also indicated that the binding target of these MABs is peptidoglycan (PGN), a component of the mycobacterial cell wall.…”