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Objective Broadly neutralizing antibodies (bNt Abs) against HIV-1 are rarely produced during natural infection, and efforts to induce such Abs by vaccination have been unsuccessful. Thus, elucidating the nature and cellular origins of bNt Abs is a high priority for vaccine research. As the bNt monoclonal Abs (MAbs) 2F5, 4E10 and 2G12 have been reported to bind select autoantigens, we investigated whether these MAbs display a broader range of autoreactivity and how their autoreactivity compares with that of pathogenic autoAbs. Methods An autoantigen microarray comprising 106 connective tissue disease-related autoantigens and control antigens was developed and used, in combination with ELISAs, to compare the reactivity profiles of MAbs 4E10, 2F5 and 2G12 to those of four pathogenic autoAbs derived from patients with antiphospholipid-syndrome (APS), and to serum from a patient with systemic lupus erythematosus (SLE). Results The APS MAbs and SLE serum reacted strongly with multiple autoantigens on the microarray, whereas anti-HIV-1 MAb reactivity was limited mainly to HIV-1-related antigens. The APS autoAbs reacted strongly with CL, yet only 4E10 bound CL at high concentrations; both 2F5 and 4E10 bound their HIV-1 epitopes with a 2–3-log higher apparent affinity than CL. Moreover, the polyreactivity of 4E10, but not CL15, could be blocked with dried milk. Conclusion The reactivity profiles of bNt anti-HIV-1 MAbs are fundamentally distinct from those of pathogenic autoAbs that arise from dysregulated tolerance mechanisms. This suggests that the limited polyreactivity observed for the bNt MAbs, and for HIV-1-Nt Abs in general, may arise through alternative mechanisms, such as extensive somatic mutation due to persistent antigen selection during chronic infection.

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The limited success of vaccines targeting the MPER, a target of three broadly neutralizing (bNt) monoclonal antibodies (MAbs), we hypothesize, reflects the difficulty of mimicking the neutralization-competent structure (NCS) of the MPER. We determined the contribution of the amino-acid sequence and the transmembrane domain (TM), to the antigenicity of the MPER in the context of the plasma membrane. DNA constructs encoding various gp41 ectodomain fragments, and the TM of either the platelet-derived growth factor receptor (PGDFR), or that of gp41, were produced and transiently expressed in COS-7 cells. Constructs expressing the MPER tethered to the gp41 TM followed by a 27-residue cytoplasmic tail fragment, MPER-TM1, produced optimal binding of MAbs 2F5, 4E10 and Z13e1. A series of 24 single amino-acid substitutions in the MPER-TM1 revealed critical binding residues for the three MAbs; similar substitutions were previously shown to ablate Ab-mediated viral neutralization. Neutralization-incompetent 2F5 Fab and 4E10 IgG mutant Abs failed to bind MPER-TM1, yet retained the ability to bind to peptide epitopes, indicating the plasma-membrane expressed MPER-TM1 closely approaches the NCS of the MPER. Substitution of the TM of gp41 with that from the PGDFR reduced binding by MAb 4E10, but not MAbs 2F5 or Z13e1. Our studies reveal that the gp41 TM appears to play a pivotal role both in orienting the 4E10 epitope, and affecting exposure of the MPER epitopes for all three bNtMAbs.