Antiviral effects of different CD4-immunoglobulin constructs against HIV-1 and SIV: immunological characterization, pharmacokinetic data and in vivo experiments

Soluble forms of the human immunodeficiency virus type 1 (HIV-1) primary receptor CD4 (soluble CD4 [sCD4]) have been extensively characterized for a quarter of a century as promising HIV-1 inhibitors, but they have not been clinically successful. By combining a protein cavity-filling strategy and the power of library technology, we identified an engineered cavity-altered single-domain sCD4 (mD1.22) with a unique combination of excellent properties, including broad and potent neutralizing activity, high specificity, stability, solubility, and affinity for the HIV-1 envelope glycoprotein gp120, and small molecular size. To further improve its neutralizing potency and breadth, we generated bispecific multivalent fusion proteins of mD1.22 with another potent HIV-1 inhibitor, an antibody domain (m36.4) that targets the coreceptor-binding site on gp120. The fusion proteins neutralized all HIV-1 isolates tested, with potencies about 10-, 50-, and 200-fold higher than those of the broadly neutralizing antibody VRC01, the U.S. FDA-approved peptide inhibitor T20, and the clinically tested sCD4-Fc fusion protein CD4-Ig, respectively. In addition, they exhibited higher stability and specificity and a lower aggregation propensity than CD4-Ig. Therefore, mD1.22 and related fusion proteins could be useful for HIV-1 prevention and therapy, including eradication of the virus. Soluble forms of human CD4 (sCD4) comprising all four (D1 to D4) or the first two (D1D2) extracellular domains are potent inhibitors of the human immunodeficiency virus type 1 (HIV-1) in vitro (1, 2). Several promising monomeric (3-5), dimeric (6-8), 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).It has been shown previously that some proteins exhibit poor hydrophobic packing, leading to low stability and solubility due to the presence of cavities within or on the surfaces of proteins that are either empty or hydrated (13,14). Identification of such cavities and filling them with bulkier hydrophobic amino acid side chains have proven effective in improving stability and other properties of proteins (15). By combining this cavity-filling strategy with the power of library technology, we identified an mD1.2 mutant, designated mD1.22, that has significantly higher soluble expression, thermal stability, and specificity than mD1.2. Bispecific multivalent fusion proteins of mD1.22 with m36.4, an engineered ...

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confidence: 99%

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

Chen

Yang

Prabakaran

et al. 2014

“…Expression of a soluble form of receptor, p75-Fc. The ectodomain of the human p75NTR receptor (amino acids 1 to 222) was cloned in fusion with the cDNA from the human IgG1 heavy chain Fc domain (2,30). A plasmid (CD4-Fc) was obtained from Brian Seed (Massachusetts General Hospital); the CD4 coding sequence was excised with XhoI and BamHI restriction enzymes and replaced by the human p75NTR coding sequence (kindly provided by Barbara Hempstead, Cornell University, Ithaca, N.Y.).…”

Section: Methodsmentioning

confidence: 99%

“…The most powerful approach for the localization of the receptor binding site is to select mutants that escape neutralization by a soluble form of a viral receptor: they are named "srr (soluble receptor resistant) mutants." Although neutralizing activity of soluble receptors has been reported for many viruses, such as poliovirus (11,26), rhinovirus (23,36), coronavirus (71,72), human immunodeficiency virus (13,24,30,52,56), measles virus (10), and hepatitis A viruses (51), characterization of srr mutants has only been described in the cases of poliovirus (11,26) and coronavirus (37,38,45,46).…”

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confidence: 99%

Mutations Conferring Resistance to Neutralization by a Soluble Form of the Neurotrophin Receptor (p75NTR) Map outside of the Known Antigenic Sites of the Rabies Virus Glycoprotein

Langevin

Tuffereau

2002

The neurotrophin receptor (p75NTR) serves as a receptor for rabies virus (RV). We expressed and purified a soluble chimera consisting of the p75NTR ectodomain fused to the human immunoglobulin G1 (IgG1) Fc fragment (p75-Fc). Although p75-Fc interacts with RV, the infectivity of RV did not decrease significantly when it was incubated in the presence of the soluble receptor alone. However, when it was subsequently incubated with an antihuman IgG directed against the Fc fragment of p75-Fc, the infectivity of RV was significantly lowered (>90%), whereas incubation with antihuman IgG alone had no effect. We then selected eight independent RV mutants that were not neutralized by p75- Rabies virus glycoprotein (RVG) is the only surface-exposed viral coat protein. It is organized in trimers (21) that protrude from the lipid envelope. RVG has three major functions in the RV infection process. First, RVG mediates cell binding through interaction with cellular receptors and is the first determinant of Lyssavirus neurotropism (3). Second, RVG mediates fusion between viral and cell membranes through a pH-dependent process (22,39). Third, RVG, the main viral antigen, induces both humoral (neutralizing antibodies) (66) and cellular immune responses.RVG is a 505-amino-acid type I membrane glycoprotein (1, 70) with three potential N-glycosylation sites: the first one, located at amino acid 37, is very inefficiently glycosylated (50); the second one, at position 204 or 247, is glycosylated in some strains, but not in others; and the third one, located at amino acid 319, is always glycosylated (67). RVG folding, intracellular transport, and oligomerization have been analyzed for Pasteur virus (PV) and Evelyn Rokitnicki-Abelseth (ERA) virus, and it has been shown that RVG acquired its trimeric organization in the endoplasmic reticulum (18, 65). The putative fusion peptide has also been localized between amino acids 102 and 179 (17).RVG has been extensively mapped with specific anti-RVG monoclonal antibodies (MAbs) by different laboratories. Our group has selected about 50 RVG mutants from the challenge virus strain (CVS) that are resistant to neutralization by anti-RVG MAbs. Several independent antigenic sites have been defined, depending on their interaction with neutralizing anti-RVG MAbs and on the effect of mutations (6, 29). Site II is the major antigenic site (70% of the MAbs recognize this region). It contains two stretches of amino acid residues located at positions 34 to 42 and 184 to 210 (43) joined by a disulfide bridge between cysteines 35 and 207 (15,17,62). Site III is a conformational site (amino acids 330 to 340) and plays a major role in RV propagation in the nervous system and pathogenicity (12,16,49,59). Two minor sites (site a at amino acids 342 and 343 [6] and a linear site at amino acids 251 to 264 [14,28,60]) and several other epitopes have also been described (28). RVG can adopt at least two conformations (20): N (for neutral) and I (for fusion inactive). In the I state, RVG is no longer recognized by site I...

“…A previous study (4) showed that monomeric sCD4 did not inhibit immune functions in monkeys, suggesting it was unlikely to be immunosuppressive in humans. However, the experiment needs to be repeated in humans with higher-order oligomers, because oligomerization of CD4 is required for interaction with MHCII or, presumably, other targets, and the highly potent sCD4 derivatives described above are exclusively multimeric (1,14,22,26). Although sCD4-Fc fusion proteins have been safely administered to a number of humans in several clinical trials, it was uncertain whether long-term treatment would be safe.…”

Section: Vol 85 2011 Engineered Single Human Cd4 Domains 9401mentioning

confidence: 99%

“…Third, the D1 mutants could be more useful than D1D2 to generate candidate HIV-1 therapeutics, not only because of higher potency, but also due to smaller size. The antiviral activities of monomeric (18,32,33), dimeric (22,26), and tetrameric (1,14) human sCD4 as Fc fusion proteins have been evaluated in animal models and human clinical trials. They were well tolerated by patients with no significant clinical or immunologic toxicities and exhibited significant inhibitory activities, such as reductions in plasma viremia and virus titer.…”

Section: Vol 85 2011 Engineered Single Human Cd4 Domains 9401mentioning

confidence: 99%

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

Chen

Yang

Gong

et al. 2011

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.