Identification of autoantigens recognized by the 2F5 and 4E10 broadly neutralizing HIV-1 antibodies

Many human monoclonal antibodies that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian autoantigens. Indeed, the generation of neutralizing antibodies to the 2F5 and 4E10 epitopes of HIV-1 gp41 in man may be proscribed by immune tolerance because mice expressing the VH and VL regions of 2F5 have a block in B cell development that is characteristic of central tolerance. This developmental blockade implies the presence of tolerizing autoantigens that are mimicked by the membrane-proximal external region of HIV-1 gp41. We identify human kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3) as the primary conserved, vertebrate self-antigens recognized by the 2F5 and 4E10 antibodies, respectively. 2F5 binds the H4 domain of KYNU which contains the complete 2F5 linear epitope (ELDKWA). 4E10 recognizes an epitope of SF3B3 that is strongly dependent on hydrophobic interactions. Opossums carry a rare KYNU H4 domain that abolishes 2F5 binding, but they retain the SF3B3 4E10 epitope. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of serum antibody to the 2F5 ELDKWA epitope but little or nothing to the 4E10 determinant. Identification of structural motifs shared by vertebrates and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1.

Antigenic and Immunogenic Study of Membrane-Proximal External Region-Grafted gp120 Antigens by a DNA Prime-Protein Boost Immunization Strategy

ABSTRACT The membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 is a target of broadly neutralizing monoclonal antibodies (MAbs) 2F5, 4E10, and Z13. Here we engrafted the MPER into the V1/2 region of HIV-1 gp120 to investigate the ability of the engineered antigens to elicit virus-neutralizing antibodies (NAbs). To promote the correct folding and presentation of the helical 4E10 epitope, we flanked the epitope with helical domains and manipulated the helix by sequential deletion of residues preceding the epitope. Binding of the recombinant proteins to MAb 4E10 increased four- to fivefold with the deletion of one or two residues, but it returned to the wild-type level when three residues were deleted, suggesting rotation of the 4E10 epitope along the helix. Immunization of mice and rabbits by electroporation-mediated DNA priming and protein boosting with these constructs elicited high levels of gp120-specific antibodies. A consistent NAb response against the neutralization-resistant, homologous JR-FL virus was detected in rabbits but not in mice. Analysis of the neutralizing activity revealed that the NAbs do not target the MPER or the V1, V2, or V3 region. Through this study, we learned the following. (i) The 4E10 epitope can be manipulated using a “rotate-the-helix” strategy that alters the helix register. However, presentation of this epitope in the immunogenic V1/2 region did not render it immunogenic in mice or rabbits. (ii) DNA vaccination with monomeric gp120-based antigens can elicit a consistent NAb response against the homologous neutralization-resistant virus by targeting epitopes outside the V1, V2, and V3 regions.