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Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.

Chromikova V, Mader A, Hofbauer S, Göbl C, Madl T, Gach JS, Bauernfried S, Furtmüller PG, Forthal DN, Mach L, Obinger C, Kunert R - Biochim. Biophys. Acta (2015)

Bottom Line: We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization.Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties.Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.

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Ligand-binding activity of 2G12 variants. Interaction of 2G12-IgG and 2G12-IgM with trimeric BG505 SOSIP.664 HIV-1 gp140 as determined by ELISA.
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f0030: Ligand-binding activity of 2G12 variants. Interaction of 2G12-IgG and 2G12-IgM with trimeric BG505 SOSIP.664 HIV-1 gp140 as determined by ELISA.

Mentions: Next we compared the ligand-binding properties of 2G12-IgG, IgM-012 and IgM-012_GL. Binding to trimeric HIV-1 envelope glycoprotein BG505 SOSIP.664 gp140 (which has an architecture very similar to that of native envelope spikes on HIV-1 virions) was assessed by ELISA. As documented by the binding curves depicted in Fig. 6, IgM-012_GL is still able to bind efficiently to HIV-1 gp140. There is a 1.6-fold difference in EC50 values between IgM-012 (EC50 = 0.12 μg/mL) and IgM-012_GL (EC50 = 0.20 μg/mL), suggesting only a minimal loss in affinity. Based on protein concentration, the EC50 of 2G12-IgG for HIV-1 gp140 is considerably lower (0.02 μg/mL) than that of either IgM. However, it is possible that due to steric hindrance only one binding site per IgM molecule is reactive in our ELISA system. In terms of molarity, the ligand-binding capacity of the IgM and IgG molecules would then be comparable, as the molar mass of IgM-012 and IgM-012_GL is > 5 times higher than that of 2G12-IgG.


Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.

Chromikova V, Mader A, Hofbauer S, Göbl C, Madl T, Gach JS, Bauernfried S, Furtmüller PG, Forthal DN, Mach L, Obinger C, Kunert R - Biochim. Biophys. Acta (2015)

Ligand-binding activity of 2G12 variants. Interaction of 2G12-IgG and 2G12-IgM with trimeric BG505 SOSIP.664 HIV-1 gp140 as determined by ELISA.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582045&req=5

f0030: Ligand-binding activity of 2G12 variants. Interaction of 2G12-IgG and 2G12-IgM with trimeric BG505 SOSIP.664 HIV-1 gp140 as determined by ELISA.
Mentions: Next we compared the ligand-binding properties of 2G12-IgG, IgM-012 and IgM-012_GL. Binding to trimeric HIV-1 envelope glycoprotein BG505 SOSIP.664 gp140 (which has an architecture very similar to that of native envelope spikes on HIV-1 virions) was assessed by ELISA. As documented by the binding curves depicted in Fig. 6, IgM-012_GL is still able to bind efficiently to HIV-1 gp140. There is a 1.6-fold difference in EC50 values between IgM-012 (EC50 = 0.12 μg/mL) and IgM-012_GL (EC50 = 0.20 μg/mL), suggesting only a minimal loss in affinity. Based on protein concentration, the EC50 of 2G12-IgG for HIV-1 gp140 is considerably lower (0.02 μg/mL) than that of either IgM. However, it is possible that due to steric hindrance only one binding site per IgM molecule is reactive in our ELISA system. In terms of molarity, the ligand-binding capacity of the IgM and IgG molecules would then be comparable, as the molar mass of IgM-012 and IgM-012_GL is > 5 times higher than that of 2G12-IgG.

Bottom Line: We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization.Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties.Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.

Show MeSH
Related in: MedlinePlus