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AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges.

Gardner MR, Kattenhorn LM, Kondur HR, von Schaewen M, Dorfman T, Chiang JJ, Haworth KG, Decker JM, Alpert MD, Bailey CC, Neale ES, Fellinger CH, Joshi VR, Fuchs SP, Martinez-Navio JM, Quinlan BD, Yao AY, Mouquet H, Gorman J, Zhang B, Poignard P, Nussenzweig MC, Burton DR, Kwong PD, Piatak M, Lifson JD, Gao G, Desrosiers RC, Evans DT, Hahn BH, Ploss A, Cannon PM, Seaman MS, Farzan M - Nature (2015)

Bottom Line: Rhesus macaques inoculated with an AAV vector stably expressed 17-77 μg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8.Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs.Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, The Scripps Research Institute, Jupiter, Florida 33458, USA.

ABSTRACT
Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs). However, even the best bNAbs neutralize 10-50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 μg ml(-1)), suggesting that high concentrations of these antibodies would be necessary to achieve general protection. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 μg ml(-1)). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17-77 μg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

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Functional characterization of eCD4-Iga, CD4-Ig is comprised of CD4 domains 1 and 2 (blue) fused to the human IgG1 Fc domain (grey). In eCD4-Ig, the sulfopeptide CCR5mim1 is fused to the carboxy-terminus of CD4-Ig. The sequence of the CCR5 amino terminus is provided for comparison. Common residues, including four CCR5 sulfotyrosines, are shown in red. CCR5mim1 alanine 4 (blue) is substituted with tyrosine in CCR5mim2, described below. b, HIV-1 pseudotyped with the Envs of the indicated HIV-1 or SIV isolates was incubated with GHOST-CCR5 cells and varying concentrations of CD4-Ig (red) or eCD4-Ig (blue). Infection was measured as GFP-expression by flow cytometry. Errors of replicates are less than 20% of indicated values but not indicated for clarity. c, 293T cells transfected to express 89.6 or ADA Envs were incubated with the indicated concentrations of CD4-Ig (red), eCD4-Ig (blue), or IgG (grey) and analyzed by flow cytometry. d, HIV-1 expressing luciferase and pseudotyped with the Envs of the indicated isolates was incubated with Cf2Th-CCR5 cells in the presence of varying concentrations of CD4-Ig (red) or eCD4-Ig (blue). Experiment was controlled with HIV-1 pseudotyped with the VSV-G protein (grey). Infection normalized to the maximum value observed for each pseudovirus. e, HIV-1 pseudotyped with the 89.6 Env was incubated with TZM-bl cells and varying concentration of CD4-Ig (red), eCD4-Ig (blue), or a CD4-Ig/eCD4-Ig heterodimer (grey). Similar experiments using additional Envs are presented in Extended Data Figs. 2c and d. f, Infection curves of humanized NSG mice with 2–4 mg/ml of serum eCD4-Ig at time of HIV-1NL4-3 challenges (blue line, n = 5), or mock treated (red line, n = 6) are shown. Three uninfected eCD4-Ig treated mice and the sole uninfected mock treated mouse were rechallenged 5 weeks post-first challenge. Significant protection (p=0.002; Mantel-Cox test) was observed in the eCD4-Ig treated group. Viral load measurements are shown in Extended Data Fig. 2h. Experiments shown in panels b-e were performed at least twice with each indicated isolate with similar results. Errors bars of duplicates denote one s.e.m.
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Figure 1: Functional characterization of eCD4-Iga, CD4-Ig is comprised of CD4 domains 1 and 2 (blue) fused to the human IgG1 Fc domain (grey). In eCD4-Ig, the sulfopeptide CCR5mim1 is fused to the carboxy-terminus of CD4-Ig. The sequence of the CCR5 amino terminus is provided for comparison. Common residues, including four CCR5 sulfotyrosines, are shown in red. CCR5mim1 alanine 4 (blue) is substituted with tyrosine in CCR5mim2, described below. b, HIV-1 pseudotyped with the Envs of the indicated HIV-1 or SIV isolates was incubated with GHOST-CCR5 cells and varying concentrations of CD4-Ig (red) or eCD4-Ig (blue). Infection was measured as GFP-expression by flow cytometry. Errors of replicates are less than 20% of indicated values but not indicated for clarity. c, 293T cells transfected to express 89.6 or ADA Envs were incubated with the indicated concentrations of CD4-Ig (red), eCD4-Ig (blue), or IgG (grey) and analyzed by flow cytometry. d, HIV-1 expressing luciferase and pseudotyped with the Envs of the indicated isolates was incubated with Cf2Th-CCR5 cells in the presence of varying concentrations of CD4-Ig (red) or eCD4-Ig (blue). Experiment was controlled with HIV-1 pseudotyped with the VSV-G protein (grey). Infection normalized to the maximum value observed for each pseudovirus. e, HIV-1 pseudotyped with the 89.6 Env was incubated with TZM-bl cells and varying concentration of CD4-Ig (red), eCD4-Ig (blue), or a CD4-Ig/eCD4-Ig heterodimer (grey). Similar experiments using additional Envs are presented in Extended Data Figs. 2c and d. f, Infection curves of humanized NSG mice with 2–4 mg/ml of serum eCD4-Ig at time of HIV-1NL4-3 challenges (blue line, n = 5), or mock treated (red line, n = 6) are shown. Three uninfected eCD4-Ig treated mice and the sole uninfected mock treated mouse were rechallenged 5 weeks post-first challenge. Significant protection (p=0.002; Mantel-Cox test) was observed in the eCD4-Ig treated group. Viral load measurements are shown in Extended Data Fig. 2h. Experiments shown in panels b-e were performed at least twice with each indicated isolate with similar results. Errors bars of duplicates denote one s.e.m.

Mentions: The breadth of an antibody depends on the conservation of its epitope. The two most conserved epitopes of HIV-1 Env are its CD4- and coreceptor-binding sites9–11. The immunoadhesin form of CD4, CD4-Ig, has been extensively studied as a therapeutic. It neutralizes most isolates, irreversibly inactivates Env, and is demonstrated safe for use in humans12–15. However, its affinity for Env is lower than those of bNAbs16, and its potency is further compromised by its parallel ability to promote infection17. Mimetics of the primary HIV-1 coreceptor CCR5, in particular peptides based on its tyrosine-sulfated amino-terminus, have also been characterized18,19. These sulfopeptides bind Env specifically but with low affinity in the absence of CD4, in part because they include hydrophobic residues and O-linked glycosylation that impede their association with Env18,20. CCR5mim1, a 15-amino acid sulfopeptide derived from the HIV-1 neutralizing antibody E5121, lacks these interfering elements (Fig. 1a) and binds Env with higher affinity than CCR5-based peptides20,22. Reflecting the conservation of the sulfotyrosine-binding pockets of Env9,10, CCR5mim1 binds both CCR5- and CXCR4-dependent Envs from all HIV-1 clades20,22.


AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges.

Gardner MR, Kattenhorn LM, Kondur HR, von Schaewen M, Dorfman T, Chiang JJ, Haworth KG, Decker JM, Alpert MD, Bailey CC, Neale ES, Fellinger CH, Joshi VR, Fuchs SP, Martinez-Navio JM, Quinlan BD, Yao AY, Mouquet H, Gorman J, Zhang B, Poignard P, Nussenzweig MC, Burton DR, Kwong PD, Piatak M, Lifson JD, Gao G, Desrosiers RC, Evans DT, Hahn BH, Ploss A, Cannon PM, Seaman MS, Farzan M - Nature (2015)

Functional characterization of eCD4-Iga, CD4-Ig is comprised of CD4 domains 1 and 2 (blue) fused to the human IgG1 Fc domain (grey). In eCD4-Ig, the sulfopeptide CCR5mim1 is fused to the carboxy-terminus of CD4-Ig. The sequence of the CCR5 amino terminus is provided for comparison. Common residues, including four CCR5 sulfotyrosines, are shown in red. CCR5mim1 alanine 4 (blue) is substituted with tyrosine in CCR5mim2, described below. b, HIV-1 pseudotyped with the Envs of the indicated HIV-1 or SIV isolates was incubated with GHOST-CCR5 cells and varying concentrations of CD4-Ig (red) or eCD4-Ig (blue). Infection was measured as GFP-expression by flow cytometry. Errors of replicates are less than 20% of indicated values but not indicated for clarity. c, 293T cells transfected to express 89.6 or ADA Envs were incubated with the indicated concentrations of CD4-Ig (red), eCD4-Ig (blue), or IgG (grey) and analyzed by flow cytometry. d, HIV-1 expressing luciferase and pseudotyped with the Envs of the indicated isolates was incubated with Cf2Th-CCR5 cells in the presence of varying concentrations of CD4-Ig (red) or eCD4-Ig (blue). Experiment was controlled with HIV-1 pseudotyped with the VSV-G protein (grey). Infection normalized to the maximum value observed for each pseudovirus. e, HIV-1 pseudotyped with the 89.6 Env was incubated with TZM-bl cells and varying concentration of CD4-Ig (red), eCD4-Ig (blue), or a CD4-Ig/eCD4-Ig heterodimer (grey). Similar experiments using additional Envs are presented in Extended Data Figs. 2c and d. f, Infection curves of humanized NSG mice with 2–4 mg/ml of serum eCD4-Ig at time of HIV-1NL4-3 challenges (blue line, n = 5), or mock treated (red line, n = 6) are shown. Three uninfected eCD4-Ig treated mice and the sole uninfected mock treated mouse were rechallenged 5 weeks post-first challenge. Significant protection (p=0.002; Mantel-Cox test) was observed in the eCD4-Ig treated group. Viral load measurements are shown in Extended Data Fig. 2h. Experiments shown in panels b-e were performed at least twice with each indicated isolate with similar results. Errors bars of duplicates denote one s.e.m.
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Figure 1: Functional characterization of eCD4-Iga, CD4-Ig is comprised of CD4 domains 1 and 2 (blue) fused to the human IgG1 Fc domain (grey). In eCD4-Ig, the sulfopeptide CCR5mim1 is fused to the carboxy-terminus of CD4-Ig. The sequence of the CCR5 amino terminus is provided for comparison. Common residues, including four CCR5 sulfotyrosines, are shown in red. CCR5mim1 alanine 4 (blue) is substituted with tyrosine in CCR5mim2, described below. b, HIV-1 pseudotyped with the Envs of the indicated HIV-1 or SIV isolates was incubated with GHOST-CCR5 cells and varying concentrations of CD4-Ig (red) or eCD4-Ig (blue). Infection was measured as GFP-expression by flow cytometry. Errors of replicates are less than 20% of indicated values but not indicated for clarity. c, 293T cells transfected to express 89.6 or ADA Envs were incubated with the indicated concentrations of CD4-Ig (red), eCD4-Ig (blue), or IgG (grey) and analyzed by flow cytometry. d, HIV-1 expressing luciferase and pseudotyped with the Envs of the indicated isolates was incubated with Cf2Th-CCR5 cells in the presence of varying concentrations of CD4-Ig (red) or eCD4-Ig (blue). Experiment was controlled with HIV-1 pseudotyped with the VSV-G protein (grey). Infection normalized to the maximum value observed for each pseudovirus. e, HIV-1 pseudotyped with the 89.6 Env was incubated with TZM-bl cells and varying concentration of CD4-Ig (red), eCD4-Ig (blue), or a CD4-Ig/eCD4-Ig heterodimer (grey). Similar experiments using additional Envs are presented in Extended Data Figs. 2c and d. f, Infection curves of humanized NSG mice with 2–4 mg/ml of serum eCD4-Ig at time of HIV-1NL4-3 challenges (blue line, n = 5), or mock treated (red line, n = 6) are shown. Three uninfected eCD4-Ig treated mice and the sole uninfected mock treated mouse were rechallenged 5 weeks post-first challenge. Significant protection (p=0.002; Mantel-Cox test) was observed in the eCD4-Ig treated group. Viral load measurements are shown in Extended Data Fig. 2h. Experiments shown in panels b-e were performed at least twice with each indicated isolate with similar results. Errors bars of duplicates denote one s.e.m.
Mentions: The breadth of an antibody depends on the conservation of its epitope. The two most conserved epitopes of HIV-1 Env are its CD4- and coreceptor-binding sites9–11. The immunoadhesin form of CD4, CD4-Ig, has been extensively studied as a therapeutic. It neutralizes most isolates, irreversibly inactivates Env, and is demonstrated safe for use in humans12–15. However, its affinity for Env is lower than those of bNAbs16, and its potency is further compromised by its parallel ability to promote infection17. Mimetics of the primary HIV-1 coreceptor CCR5, in particular peptides based on its tyrosine-sulfated amino-terminus, have also been characterized18,19. These sulfopeptides bind Env specifically but with low affinity in the absence of CD4, in part because they include hydrophobic residues and O-linked glycosylation that impede their association with Env18,20. CCR5mim1, a 15-amino acid sulfopeptide derived from the HIV-1 neutralizing antibody E5121, lacks these interfering elements (Fig. 1a) and binds Env with higher affinity than CCR5-based peptides20,22. Reflecting the conservation of the sulfotyrosine-binding pockets of Env9,10, CCR5mim1 binds both CCR5- and CXCR4-dependent Envs from all HIV-1 clades20,22.

Bottom Line: Rhesus macaques inoculated with an AAV vector stably expressed 17-77 μg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8.Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs.Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, The Scripps Research Institute, Jupiter, Florida 33458, USA.

ABSTRACT
Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs). However, even the best bNAbs neutralize 10-50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 μg ml(-1)), suggesting that high concentrations of these antibodies would be necessary to achieve general protection. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 μg ml(-1)). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17-77 μg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

Show MeSH
Related in: MedlinePlus