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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)1,2. 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 protection3,4,5,6. 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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Figure 1: Functional characterization of eCD4-Ig.
Figure 2: Comparison of eCD4-Ig variants and HIV-1 neutralizing antibodies.
Figure 3: Summary of HIV-1, HIV-2 and SIV neutralization studies.
Figure 4: AAV-rh-eCD4-Ig protects rhesus macaques from SHIV-AD8.

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Acknowledgements

This project was supported by National Institutes of Health (NIH) grants R01 AI091476 and R01 AI080324 (M.F.), P01 AI100263 (G.G., R.C.D., M.F.), RR000168 (M.R.G., L.M.K., D.T.E., R.C.D., M.F.), R01 AI058715 (B.H.H.), by the Intramural Research program of the Vaccine Research Center, NIAID, NIH (J.G., B.Z., P.D.K.), and by federal funds from the National Cancer Institute, NIH under contract no. HHSN261200800001E. The authors would like to thank H. Choe and M. Martin for critical advice.

Author information

Authors and Affiliations

Authors

Contributions

M.R.G. and L.M.K. contributed equally to this work. M.R.G., L.M.K., H.R.K., M.V.S., T.D., J.J.C., M.D.A., M.P., J.D.L., R.C.D., D.T.E., B.H.H., P.M.C., M.S.S., A.P. and M.F. designed experiments. M.R.G., L.M.K., H.R.K., M.V.S., T.D., J.J.C., K.G.H., J.M.D., M.D.A., C.C.B., C.H.F., V.R.J., B.D.Q. and A.Y.Y. performed experiments. L.M.K. conducted all non-human primate studies. J.G. and P.D.K. assisted with modelling. J.M.M.-N., H.M., B.Z., P.P., M.S.S., M.C.N. and D.R.B. contributed advice and critical reagents. M.F. conceived the study and, with important assistance from M.R.G. and L.M.K., wrote the manuscript.

Corresponding author

Correspondence to Michael Farzan.

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The authors declare no competing financial interests.

Extended data figures and tables

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

The amino-acid sequences of CD4-Ig, eCD4-Ig, fusion1, fusion2, eCD4-Igmim2, eCD4-IgQ40A, eCD4-IgQ40A,mim2 and rh-eCD4-Ig (rh-eCD4-IgG2I39N,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 Additional characterization of eCD4-Ig.

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 c, d and Fig. 1e were analysed 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−1 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−1 limit of detection of this assay is indicated by a dashed line. Experiments in ag 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 (ref. 10), was fitted into a cryoelectron micrograph of the HIV-1 envelope glycoprotein trimer (Env; Bal isolate) bound to CD4 (ref. 48). 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 (sulphur) spheres. The remainder of 412d was excluded for clarity. b, The same structure shown in a rotated 90° about the horizontal axis. Note that the sulfotyrosine-binding pockets are proximal to the trimer axis, whereas the C 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)49 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 10 (ref. 50). This pocket is also critical for binding of CCR5mim1 and CCR5mim 2 (refs 20, 22). In this model, the Fc domain is oriented to allow each eCD4-Ig sulfopeptide to engage a different gp120 protomer24. A single CD4 domain also binds one of the sulfopeptide-bound protomers. Distances between the C terminus of CD4 and the N terminus of one Fc domain monomer (38.1 Å), between the C terminus of the Fc domain and Tys 100 pocket of the CD4-bound gp120 protomer (30.6 Å), and between the C terminus of the Fc domain and Tys 100 pocket of an adjacent gp120 protomer (33.3 Å), 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. Modelling 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−1) of CD4-Ig, eCD4-Ig, eCD4-Igmim2 (mim2), eCD4-IgQ40A (Q40A) and eCD4-IgQ40A,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 indicated envelope glycoprotein was incubated in triplicate with TZM-bl cells and varying concentrations of CD4-Ig or eCD4-Ig variant. Luciferase activity was determined 2 days after 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 previously4,6 are shown in the two right-most columns. b, Neutralization studies similar to those in a except that the IC50 values of CD4-Ig, eCD4-Igmim2 (mim2), eCD4-IgQ40A,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 right-most columns were previously reported4,6.

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

a, b, The IC80 values (μg ml−1) of the experiments described in Extended Data Fig. 5a (a) and Extended Data Fig. 5b (b) are shown.

Extended Data Figure 6 Further comparison of eCD4-Ig and HIV-1 neutralizing antibodies.

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 colour coding of Extended Data Figs 4 and 5. The 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 was 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 Summary of IC80 values for HIV-1, HIV-2 and SIV neutralization studies.

The IC80 values from studies of Figs 1b, 2a, b, and Extended Data Figs 4, 5, 6 are plotted. The number of isolates resistant to 50 μg ml−1 of the indicated inhibitors are indicated at the top. Geometric means are calculated for neutralized isolates and indicated with horizontal lines.

Extended Data Figure 8 Additional characterization of rh-eCD4-Ig.

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 a and Fig. 2b except that human eCD4-Igmim2 and its rhesus analogue bearing or not bearing the Ile39Asn mutation are compared using SHIV-AD8. Note that the Ile39Asn mutation largely restores the neutralization activity of rhesus eCD4-Igmim2. c, A representation of the AAV vectors used in the non-human primate studies of Fig. 4. Rh-eCD4-Ig (rh-eCD4-IgG2I39N,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 analysed. eg, Experiments similar to those in Fig. 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 Ile39Asn 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 in a, b and dg represent at least two with similar results. Error bars denote s.e.m. of triplicates.

Extended Data Table 1 Potencies and breadth of well-characterized broadly neutralizing antibodies

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Gardner, M., Kattenhorn, L., Kondur, H. et al. AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges. Nature 519, 87–91 (2015). https://doi.org/10.1038/nature14264

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