Adeno-associated viral delivery of Env-specific antibodies prevents SIV rebound after discontinuing antiretroviral therapy

Abstract

An alternative to lifelong antiretroviral therapy (ART) is needed to achieve durable control of HIV-1. Here, we show that adeno-associated virus (AAV) delivery of two rhesus macaque antibodies to the simian immunodeficiency virus (SIV) envelope glycoprotein (Env) with potent neutralization and antibody-dependent cellular cytotoxicity can prevent viral rebound in macaques infected with barcoded SIV_mac239M after discontinuing suppressive ART. After AAV administration, sustained antibody expression with minimal antidrug antibody responses was achieved in all but one animal. After ART withdrawal, SIV replication rebounded within 2 weeks in all control animals but remained <15 copies per milliliter in plasma for more than a year in four of the eight animals that received AAV vectors encoding Env-specific antibodies. Viral sequences from animals that rebounded with delayed kinetics exhibited restricted clonal diversity and antibody escape mutations in Env. Thus, sustained expression of antibodies with potent antiviral activity can afford durable, ART-free containment of pathogenic SIV infection.

Fig. 1.. AAV-delivery of antibodies to the SIV envelope glycoprotein delays viral rebound in SIV_mac239M-infected rhesus macaques.

(A) Fourteen rhesus macaques were infected intravenously with barcoded SIV_mac239M (5,000 IU) (38). From day 9 to week 60 PI, the animals were maintained on a daily subcutaneous ART regimen consisting of dolutegravir (2.5 mg/kg), tenofovir disoproxil fumarate (5.1 mg/kg) and emtricitabine (40 mg/kg). At week 34 PI, eight animals were inoculated with AAV9 vectors encoding the SIV Env-specific antibodies ITS61.01 and ITS103.01 (light red, orange and maroon) and six animals were inoculated with a vector encoding the control antibody 17-HD9 (blue). Viral RNA loads in plasma were measured using a qRT-PCR assay with a detection threshold of 15 copies/ml (dotted line) (56). Plotted values represent the mean of six replicate reactions per sample. The shaded region indicates the period of ART. (B) Differences in the percentage of aviremic animals (< 15 SIV copies/ml) between the treatment group (N=8) and the control group (N=6) were compared (p<0.005, Mantel-Cox test).

Fig. 2.. Serum antibody concentrations and anti-drug antibody responses.

ITS61.01 (A) and ITS103.01 (B) concentrations in serum were measured by ELISA on plates coated with an antibody to a rhodopsin tag appended to the C-terminus of ITS61.01 or with an anti-idiotype antibody to ITS103.01. The median concentration of each antibody in the treatment group (N=8) is shown in black. Anti-drug antibody responses to ITS61.01 (C) and ITS103.01 (D) were measured by probing ITS61.01- or ITS103.01-coated plates with biotinylated IgG purified from serum followed by streptavidin-HRP and development in TMB substrate. (56). Plotted values represent the average of absorbance reads across all dilutions falling within the range of a valid calibration curve, in most cases 3–4 technical replicates. Symbols are color-coded to indicate the animal with lowest concentration of both antibodies and the highest ADA responses (orange), the animals with delayed viral rebound (light red) and the animals that contained viral loads below the limit of detection in plasma (maroon).

Fig. 3.. Restricted barcode diversity of the rebounding virus in animals with Env-specific antibodies.

(A) The virus population in plasma was sequenced on day 9 post-infection (PI) and at the indicated time points after treatment interruption (TI) to determine the number of unique SIV_mac239M barcodes (38). The barcodes detected after viral rebound are shown for animals that received vectors expressing 17-HD9 (blue) or ITS61.01 and ITS103.01 (orange and light red). Differences in the number of rebounding barcodes in the control animals (N=6) and the animals that received vectors encoding Env-specific antibodies (N=5) were compared at the first detectable time point (p=0.019, Mann-Whitney U test) (B) and at peak viral rebound (p=0.0095, Mann-Whitney U test) (C).

Fig. 4.. Substitutions predicted to eliminate glycosylation of Env residues N295 and N479 confer resistance to ITS103.01.

(A) SIV RNA was isolated from plasma after viral rebound and sequenced to look for evidence of antibody escape. Amino acid changes in or near residues N295 and N479 (red), their frequency in the virus population in plasma, and serum concentrations of ITS103.01 and ITS61.01 close to the time of viral rebound are shown. (B) N-linked glycans attached to N295 and N479 (magenta) are located on the periphery of the gp120 CD4-binding site (yellow) (PDB: 6TYB) (41, 42). (C-F) The introduction of Env substitutions into SIV_mac239 predicted to abrogate glycosylation of N295 or N479 confer resistance to neutralization (C) and ADCC (D) by ITS103.01, but not do not alter sensitivity to neutralization (E) or ADCC (F) by ITS61.01. Representative neutralization and ADCC data are shown for two independent experiments.