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

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.

Extended Data Figure 1. Sequences of CD4-Ig and eCD4-Ig variants

The amino-acid sequences of CD4-Ig, eCD4-Ig, fusion1, fusion2, eCD4-Ig^mim2, eCD4-Ig^Q40A, eCD4-Ig^Q40A,mim2 and rh-eCD4-Ig (rh-eCD4-IgG2^I39N,mim2) are shown. Leader peptides are underlined, CD4 domains 1 and 2 are indicated in red, Fc domains are indicated in cyan, CCR5-mimetics peptides are indicated in green, and linker sequences are shown in black.

Extended Data Figure 2. Extended Data for Figure 1

a, b, Experiments similar to those of Fig. 1b except that CD4-Ig (red), fusion1 (grey), fusion2 (green), and fusion3 (eCD4-Ig; blue) are compared using HIV-1 pseudotyped with the envelope glycoproteins of the 89.6 (a) or ADA (b) isolates. c, d, Experiments similar to those in Fig. 1e except that CD4-Ig (red), eCD4-Ig (blue), or heterodimers thereof (grey) are compared. e, CD4-Ig, eCD4-Ig, and the CD4-Ig/eCD4-Ig heterodimer assayed in Fig. 1e and (c) and (d) were analyzed by SDS-PAGE and stained with Coomassie blue under reducing (left) and non-reducing (right) conditions. f, g, Infectious 89.6 (f) or SG3 (g) HIV-1 was incubated with human PBMC in the presence of the indicated concentrations of CD4-Ig (red) or eCD4-Ig (blue), or without either inhibitor (grey). Culture supernatants were collected on the indicated day and viral p24 levels were measured by ELISA. h, Viral loads in RNA copies/mL are shown for each humanized mouse of Fig. 1f. Mice treated with eCD4-Ig are indicated with blue lines and mice treated with PBS are indicated with red lines. The 800 copies/mL limit of detection of this assay is indicated by a dashed line. Experiments shown in panels a–g were performed at least twice with similar results. Error bars denote s.e.m. of triplicates.

Extended Data Figure 3. A model of eCD4-Ig bound to the HIV-1 Env trimer

a, The structure (2QAD) of gp120 (YU2 isolate) bound to the tyrosine-sulfated CD4i antibody 412d and CD4 domains 1 and 2, was fitted into a cryoelectron micrograph of the HIV-1 envelope glycoprotein trimer (Env; Bal isolate) bound to CD4. gp120 and CD4 domains 1 and 2 are shown in blue and red, respectively. 412d sulfotyrosines are represented as green (carbon), red (oxygen), and yellow (sulfur) spheres. The remainder of 412d was excluded for clarity. b, The same structure shown in (a) rotated 90 degrees about the horizontal axis. Note that the sulfotyrosine-binding pockets are proximal to the trimer axis, whereas the carboxy-terminus of CD4 domain 2 is distal from the trimer axis, preventing both CD4 domains of CD4-Ig from simultaneously binding the same Env trimer. c, A model of how eCD4-Ig may associate with Env is presented. The Fc domain of human IgG1 (1FCC, cyan) was positioned to be proximal to the gp120 sulfopeptide-binding pocket occupied by sulfotyrosine 100 (Tys 100) of the 412d heavy chain while avoiding steric interaction with Env. Tys 100 occupies a pocket in gp120 thought to bind CCR5 sulfotyrosine 10⁵⁰. This pocket is also critical for binding of CCR5mim1 and CCR5mim2^,. In this model, the Fc domain is oriented to allow each eCD4-Ig sulfopeptide to engage a different gp120 protomer. A single CD4 domain also binds one of the sulfopeptide-bound protomers. Distances between the carboxy-terminus of CD4 and the amino-terminus of one Fc domain monomer (38.1 angstroms), between the carboxy-terminus of the Fc domain and Tys 100 pocket of the CD4-bound gp120 protomer (30.6 angstroms), and between the carboxy-terminus of the Fc domain and Tys 100 pocket of an adjacent gp120 protomer (33.3 angstroms), are indicated. d, Residues not visible in the crystal structures used to construct this model are shown between brackets. In the model shown in (c), these residues span the distances indicated. Note that these distances are well under the extension of a typical beta strand. CD4-, IgG1- and CCR5mim1-derived residues are shown in red, cyan, and green, respectively, with linker regions shown in black. Residues visible in the crystal structures, including the CCR5mim1 sulfotyrosine presumed to fill the Tys 100 pocket, are highlighted in grey. Modeling was performed using UCSF Chimera version 1.8.

Extended Data Figure 4. IC₅₀ values of eCD4-Ig variants against neutralization-resistant isolates.

a, The IC₅₀ values (μg/mL) of CD4-Ig, eCD4-Ig, eCD4-Ig^mim2 (mim2), eCD4-Ig^Q40A (Q40A), and eCD4-Ig^Q40A,mim2 (Q40A,mim2) against 24 HIV-1 and SIV isolates selected for their neutralization resistance are shown. The clade and tier of each isolate is listed. HIV-1 pseudotyped with the indicted envelope glycoprotein was incubated in triplicate with TZM-bl cells and varying concentrations of CD4-Ig or eCD4-Ig variant. Luciferase activity was determined two days post-infection. ‘Fold’ indicates the ratio of the IC₅₀ value of CD4-Ig to the geometric mean of the IC₅₀ values of the assayed eCD4-Ig variants. The geometric mean of eCD4-Ig variants and the CD4bs antibodies 3BCN117, NIH45-46, and VRC01 calculated from values reported in Huang et al. and Sheid et al.^, are shown in the two rightmost columns. b, Neutralization studies similar to those in (a) except that the IC₅₀ values of CD4-Ig, eCD4-Ig^mim2 (mim2), eCD4-Ig^Q40A,mim2 (Q40A,mim2) and NIH45-46 were determined for a panel of 40 viral isolates selected for their resistance to the CD4bs bNAbs 3BNC117 and NIH45-46. IC₅₀ values of the CD4bs antibodies VRC01 and 3BNC117 listed in the two rightmost columns were reported in Huang et al. and Scheid et al.^,

Extended Data Figure 5. IC₈₀ values of eCD4-Ig variants against neutralization-resistant isolates.

a, b, The IC₈₀ values (μg/mL) of the experiments described in Extended Data Fig. 5a (a) and 5b (b) are shown.

Extended Data Figure 6. Extended Data for Figure 2

a, IC₉₀ values for the same experiments shown in Fig. 2a, presented in the same format. b, Numeric IC₅₀ and IC₉₀ values of the experiment shown in (a) and Fig. 2a are shown, using the same color coding of Extended Data Figs. 4 and 5. S.e.m of triplicates are indicated below their IC₅₀ and IC₉₀ values. c, Experiments similar to those in Fig. 2b except that HIV-1 pseudotyped with the Env of the HIV-2 isolate ST were incubated with the indicated concentrations of CD4-Ig, eCD4-Ig variants, or the CD4bs antibodies IgG-b12, VRC01, or NIH45-46. Error bars denote s.e.m. of triplicates.

Extended Data Figure 7. Extended Data for Figure 3 (IC₈₀ values)

The IC₈₀ values from studies of Figs. 1b, 2a, 2b, and Extended Data Figs. 4–6 are plotted. The number of isolates resistant to 50 μg/ml of the indicated inhibitors are indicated on top. Geometric means are calculated for neutralized isolates and indicated with horizontal lines.

Extended Data Figure 8. Extended Data for Figure 4

a, An experiment similar to that in Fig. 2b, except that rhesus and human CD4-Ig and eCD4-Ig are compared for their ability to neutralize HIV-1 pseudotyped with the SF162 envelope glycoprotein. All variants have wild-type rhesus or human CD4 domains. Note that variants bearing rhesus CD4 are markedly less potent at neutralizing HIV-1. b, Experiment similar to Fig. 2b and to (a) except that human eCD4-Ig^mim2 and its rhesus analog bearing or not the I39N mutation are compared using SHIV-AD8. Note that the I39N mutation largely restores the neutralization activity of rhesus eCD4-Ig^mim2. c, A representation of the AAV vectors used in the non-human primate studies of Fig. 4. Rh-eCD4-Ig (rh-eCD4-IgG2^I39N,mim2; blue) and rhesus tyrosine protein sulfotransferase 2 (TPST2; green) were introduced into a single-stranded AAV vector downstream of a CMV promoter. A woodchuck response element (WPRE), used to promote expression, and the SV40 polyadenylation signal (SV40pA) were also included. AAV inverted terminal repeats (ITR) are indicated in grey arrows. d, An experiment similar to that in Fig. 4d except that sera from week 6 were analyzed. e, f, g, Experiments similar to those in Figs.4f–h except that the reactivity of rhesus sera was examined for a construct bearing wild-type rhesus CD4 domains 1 and 2 fused to the human IgG1 Fc domain (e), one bearing rhesus CD4 domains 1 and 2 with the I39N mutation, again fused to the human IgG1 Fc domain (f), or the antibody NIH45-46 fused to the rhesus IgG2 constant regions, used here to present the rhesus IgG2 Fc domain (g). Experiments shown in a,b, and d–g represent at least two with similar results. Error bars denote s.e.m. of triplicates.

Figure 1. Functional characterization of eCD4-Ig

a, 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-1_NL4-3 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.

Figure 2. Comparison of eCD4-Ig variants and HIV-1 neutralizing antibodies

a, HIV-1 pseudotyped with the Envs of the indicated isolates were incubated with TZM-bl cells and varying concentrations of the indicated entry inhibitors, and the resulting IC₅₀ values are plotted. IC₉₀ values and standard errors are presented in Extended Data Figs. 6a and 6b. b, Experiments similar to those in (a) except that HIV-1 pseudotyped with the SIVmac239 Env was incubated with varying concentrations of CD4-Ig, eCD4-Ig variants, or CD4bs antibodies. Extended Data Fig. 6c shows a similar study using the HIV-2 ST Env. Errors bars of triplicates denote one s.e.m. of triplicates. c, ADCC activity was assessed using CEM.NKR-CCR5 target cells incubated with infectious HIV-1 NL4-3, SHIV_KB9 or SIVmac239 for four days. Cells were then incubated with KHYG-1 NK effector cells for 8 hours in the presence of the indicated inhibitors. ADCC activity was measured as loss of luciferase activity from the target cells. All experiments represented in this figure were performed at least twice with each isolate and inhibitor with similar results.

Figure 3. Summary of HIV-1, HIV-2 and SIV neutralization studies

The IC₅₀ values from studies of Figs. 1b, 2a, 2b, and Extended Data Figs. 4–6 are plotted. The number of isolates resistant to 50 μg/ml of the indicated inhibitors are indicated at top of figure. Geometric means are calculated for neutralized isolates and indicated with horizontal lines. Note that these data include 38 HIV-1 isolates selected for resistance to NIH45-46 or 3BNC117, so that isolates resistant to these antibodies are over-represented. Nonetheless, the geometric mean values of neutralized viruses are consistent with previous reports (Extended Data Table 1). Data for VRC01 and 3BNC117 were reported in Huang et al. and Scheid et al.^,. IC₈₀ values are presented in Extended Data Fig. 7.

Figure 4. AAV-rh-eCD4-Ig protects rhesus macaques from SHIV-AD8

a, Infection analysis comparing four male Indian-origin rhesus macaques inoculated intramuscularly with 2×10¹³ AAV particles delivering rh-eCD4-Ig (blue) and four age- and gender-matched controls (red). At 8, 11, 16, 20, 26, and 34 weeks post-inoculation, macaques were challenged with the indicated p27 titers of SHIV-AD8. Significant protection (p=0.006; Mantel-Cox test) was observed in the AAV-rh-eCD4-Ig treated group. b, Viral loads of inoculated (blue) and control macaques (red) are shown, with the time and titer of challenge indicated above the graph. c, Concentrations of rh-eCD4-Ig in the sera of inoculated macaques were measured by ELISA through week 40 post-inoculation. d, The neutralizing potency of macaque sera obtained 4 weeks post-AAV-inoculation was compared to pre-inoculation sera (pre-sera), and pre-sera mixed with laboratory produced rh-eCD4-Ig, as in Fig. 2b. e, Anti-transgene antibody responses in AAV-rh-eCD4-Ig inoculated macaques were compared to those in macaques inoculated with AAV expressing the indicated bNAbs bearing constant regions of rhesus IgG2. Sera from four weeks post-inoculation were analyzed. Plates were coated with equivalent amounts of rh-eCD4-Ig or rhesus forms of bNAbs and incubated with sera and anti-rhesus lambda chain (left panel) or -kappa chain (right panel) antibody conjugated to horseradish peroxidase. Note that 3BNC117 and NIH45-46 bear a kappa light chain, whereas PGT121 and 10-1074 bear a lambda light chain, so that only host antibody responses were detected. Values indicate absorbance at 450 nM. P-values (Student’s 2-tailed t test) are indicated above the figures. f, The sensitivity of the assay in (e) was increased to measure longitudinally the anti-rh-eCD4-Ig activity in the sera of inoculated macaques. Both anti-kappa and anti-lamda secondary antibodies were used. Values are scaled for comparison to values in (e). g, h, The same assay as in (f) except that responses to rh-CD4-Ig, lacking CCR5mim2 (g) or to CCR5mim2 fused to a human IgG1 Fc domain (h) were measured. Experiments of panels c-h were performed at least twice with similar results. Errors bars denote one s.e.m. of duplicates.