Stabilization of HIV-1 Envelope in the CD4-bound Conformation through Specific Cross-linking of a CD4 Mimetic

Abstract

CD4 binding on gp120 leads to the exposure of highly conserved regions recognized by the HIV co-receptor CCR5 and by CD4-induced (CD4i) antibodies. A covalent gp120-CD4 complex was shown to elicit CD4i antibody responses in monkeys, which was correlated with control of the HIV virus infection (DeVico, A., Fouts, T., Lewis, G. K., Gallo, R. C., Godfrey, K., Charurat, M., Harris, I., Galmin, L., and Pal, R. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 17477-17482). Because the inclusion of CD4 in a vaccine formulation should be avoided, due to potential autoimmune reactions, we engineered small sized CD4 mimetics (miniCD4s) that are poorly immunogenic and do not induce anti-CD4 antibodies. We made covalent complexes between such an engineered miniCD4 and gp120 or gp140, through a site-directed coupling reaction. These complexes were recognized by CD4i antibodies as well as by the HIV co-receptor CCR5. In addition, they elicit CD4i antibody responses in rabbits and therefore represent potential vaccine candidates that mimic an important HIV fusion intermediate, without autoimmune hazard.

Range of CD4-Bound Conformations of HIV-1 gp120, as Defined Using Conditional CD4-Induced Antibodies

“…1, the dependence on CD4 for gp120 binding of the selected, representative panel of five CD4i MAbs to gp120 is variable (the MAbs of the panel are described in Table 1). One of these, MAb 17b, has been used extensively as a "gold standard" CD4i MAb (11,24,39,62,(69)(70)(71)(72)(73)(74)(75)(76). The prominent CD4i status of MAb 17b stems from the fact that cell surface trimeric envelope requires CD4 for 17b binding (38), as is also the case for the soluble BG505 SOSIP.664 trimer (Fig.…”

“…MAb 17b has often been taken as a gold standard indicator for the CD4-bound conformation (11,24,39,62,(69)(70)(71)(72)(73)(74)(75)(76). This MAb was originally described by Thali et al, who recognized that native cell surface HIV-1 spike was absolutely dependent on CD4 for 17b…”

Straightforward Selection of Broadly Neutralizing Single-Domain Antibodies Targeting the Conserved CD4 and Coreceptor Binding Sites of HIV-1 gp120

“…The miniCD4 derivatives M48U1 and M64U1-SH were synthesized and purified by reverse-phase chromatography as described earlier (16) and characterized by mass spectrometry.…”

“…The various envelopes were purified as previously described by miniCD4 affinity chromatography (17). They were then used as such or cross-linked to miniCD4 derivative M64U1-SH through disulfide bond formation as described previously (16). Wild-type gp140YU2, gp140YU2 D368R (having a change from Asp to Arg at position 368), gp140YU2 I420R, and the gp140YU2 I423M N425K G431E triple mutant were kindly provided by Richard Wyatt (Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA).…”

“…Interestingly, they possess CD4 functional properties, including the ability to unmask gp120 conserved neutralization epitopes that are cryptic on the unbound glycoprotein (15)(16)(17)(18)(19). In the present work, to increase the chance of selecting neutralizing sdAbs targeting the CD4 and coreceptor binding sites of gp120, we have immunized llamas with gp140 (trimeric version of gp120 bound to the gp41 ectodomain), either free or cross-linked to a CD4 mimic (16). The last version was aimed to enhance CD4i epitope exposure.…”