Engineered Single Human CD4 Domains as Potent HIV-1 Inhibitors and Components of Vaccine Immunogens

Abstract

Soluble forms of the HIV-1 receptor CD4 (sCD4) have been extensively characterized for more than 2 decades as promising inhibitors and components of vaccine immunogens. However, they were mostly based on the first two CD4 domains (D1D2), and numerous attempts to develop functional, high-affinity, stable soluble one-domain sCD4 (D1) have not been successful because of the strong interactions between the two domains. We have hypothesized that combining the power of structure-based design with sequential panning of large D1 mutant libraries against different HIV-1 envelope glycoproteins (Envs) and screening for soluble mutants could not only help solve the fundamental stability problem of isolated D1, but may also allow improvement of D1 affinity while preserving its cross-reactivity. By using this strategy, we identified two stable monomeric D1 mutants, mD1.1 and mD1.2, which were significantly more soluble and bound Env gp120s more strongly (50-fold) than D1D2, neutralized a panel of HIV-1 primary isolates from different clades more potently than D1D2, induced conformational changes in gp120, and sensitized HIV-1 for neutralization by CD4-induced antibodies. mD1.1 and mD1.2 exhibited much lower binding to human blood cell lines than D1D2; moreover, they preserved a ␤-strand secondary structure and stability against thermally induced unfolding, trypsin digestion, and degradation by human serum. Because of their superior properties, mD1.1 and mD1.2 could be potentially useful as candidate therapeutics, components of vaccine immunogens, and research reagents for exploration of HIV-1 entry and immune responses. Our approach could be applied to other cases where soluble isolated protein domains are needed.

Exceptionally Potent and Broadly Cross-Reactive, Bispecific Multivalent HIV-1 Inhibitors Based on Single Human CD4 and Antibody Domains

“…To briefly evaluate the nonspecificity of mD1.22 in vivo, we used a human B cell line that expresses high level of MHC-II (BJAB) and a human T cell line with no detectable MHC-II expression (SUPT1) (12). Because oligomerization of CD4 is required for a stable interaction with MHC-II (37), we generated Fc-fusion proteins of D1D2 (CD4-Ig), mD1.2 (mD1.2Fc), and mD1.22 (mD1.22Fc).…”

“…Several promising monomeric (3)(4)(5), dimeric (6)(7)(8), and tetrameric (9-11) sCD4 derivatives have been tested in animal models and in human clinical trials, but they exhibited modest and transient antiviral activities. Previously, we demonstrated that decreasing the molecular size of D1D2 to a single domain, D1, significantly increased its antiviral activity and reduce its nonspecificity, i.e., interactions with molecules other than the HIV-1 envelope glycoprotein (Env) gp120; a D1 variant (mD1.2) was identified that is also more soluble than D1D2 (12). However, mD1.2 still binds to human B cells and CD4 ϩ T cells without HIV-1 Env expression, although it binds more weakly than D1D2 and its stability is comparable to that of D1D2, which is relatively low (12).…”

“…Other cell lines and plasmids used for expression of various HIV-1 Envs were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (ARRRP). gp140 SC , gp140 MS , gp120 MS , D1D2, CD4-Ig, mD1.2Fc, IgG1 m102.4, IgG1 m909, m36.4, and m36h1Fc were produced in our laboratory as described previously (12,16,17,19,20). gp140 Con-s (21), gp140 CH12.0544.2 , and gp140 89.6 were gifts from Barton F. Haynes (Duke University Medical Center, Durham, NC).…”

“…ELISAs were performed as described previously (12,17). Bound D1 mutants were detected by horseradish peroxidase (HRP)-conjugated anti-FLAG tag antibodies (Sigma-Aldrich, St. Louis, MO) or by using the Penta-His HRP conjugate kit from Qiagen.…”

“…Proteins in PBS were concentrated using an ultrafiltration method and then quantified as described previously (12). Circular dichroism spectroscopy.…”