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 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. Following AAV administration, sustained antibody expression with minimal anti-drug antibody responses was achieved in all but one animal. After ART withdrawal, SIV replication rebounded within two weeks in all of the control animals but remained below the threshold of detection in plasma (<15 copies/mL) for more than a year in four of the eight animals that received AAV vectors encoding Env-specific antibodies. Viral sequences from animals with delayed rebound exhibited restricted barcode diversity and antibody escape. Thus, sustained expression of antibodies with potent antiviral activity can afford durable, ART-free containment of pathogenic SIV infection.

Extended Data Fig. 1.. ADCC and neutralization activity of SIV Env-specific antibodies.

The SIV Env-specific antibodies ITS61.01 and ITS103.01 were tested for a, ADCC and b, neutralization activity against SIV_mac239. a, ADCC responses were measured by infecting CEM.NKR-_CCR5-sLTR-Luc cells with SIV_mac239 and incubating with an NK cell line (KHYG-1 cells) expressing rhesus macaque CD16 in the presence of the indicated concentrations of antibody. ADCC responses were calculated as the remaining luciferase activity (% RLU) after an 8-hour incubation at a 10:1 effector-to-target ratio. The values indicate the mean and standard deviation (error bars) for triplicate wells at each antibody concentration and the dotted line indicates half-maximal killing of SIV-infected cells. b, SIV neutralization was measured by incubating virus dilutions with the indicated antibody concentrations for one hour before addition to TZM-bl cells. Three days post-infection, neutralization was calculated as the reduction of luciferase activity (% RLU) in cells inoculated with virus plus antibody relative to cells inoculated with virus without antibody. The values indicate the mean and standard deviation (error bars) for triplicate wells and the dotted line indicates 50% neutralization.

Extended Data Fig. 2.. Adeno-associated virus vector for antibody expression.

ITS61.01, ITS103.01 and 17-HD9 sequences were cloned into an AAV vector downstream of a CMV enhancer, chicken β-actin promoter and an SV40 intron and upstream of a WPRE and SV40 polyadenylation site. The heavy and light chain reading frames were separated by a P2A ribosomal skip sequence and a furin cleavage site for proteolytic removal of the P2A peptide. Three tandem repeats of the miRNA binding site miRNA-142T were included in the 3’UTR to minimize off-target expression in professional antigen presenting cells^,. M428L and N434S (LS) substitutions were introduced into the heavy chain of each antibody to extend their in vivo half-lives through increased affinity for the neonatal Fc receptor^,. A rhodopsin (C9) tag was also appended to the C-terminus of ITS61.01 and 17-HD9 for quantification by ELISA.

Extended Data Fig. 3.. Cell-associated SIV loads in PBMCs and lymph nodes.

a, Viral DNA in PBMCs b, viral RNA in PBMCs c, viral DNA in lymph nodes and d, viral RNA in lymph nodes were measured before (week 30 PI) and after (week 58 PI) the administration of AAV9 vectors to assess the impact of Env-specific antibodies on the size of the viral reservoir. Cell-associated SIV DNA and RNA were measured by quantitative hybrid real-time/digital RT-PCR and PCR assays as previously described.

Extended Data Fig. 4.. Neutralization and ADCC responses after treatment interruption.

a, Neutralization and b, ADCC responses to SIV_mac239 were measured in serum collected 4 weeks after TI (week 64 PI). The binding of c, purified ITS103.01 in PBS-BSA and d, ITS103.01 in serum from an AAV9-ITS103.01-inoculated animal to ELISA plates coated with a mouse anti-ITS103.01 idiotype antibody was determined with and without heat treatment at 55°C for 20 minutes. e, Neutralization and f, ADCC were measured after heat treatment of serum for 20 minutes at 55°C. Neutralization was assessed by the ability to block infection of TZM-bl cells with replication-competent SIV_mac239. ADCC was measured as the ability to direct NK cell killing of SIV_mac239-infected CEM._NKR-CCR5-sLTR-Luc cells. Neutralization and ADCC were calculated as the dose-dependent reduction in luciferase activity for virus or virus-infected cells incubated with antibody relative to virus or infected cells without antibody after subtracting the background luciferase signal in uninfected cells (% RLU). Values reflect the mean and standard deviation (error bars) for triplicate wells at each dilution. The dotted lines indicate half-maximal responses.

Extended Data Fig. 5.. Overview of SIV Env substitutions.

SIV RNA was isolated from plasma at the indicated timepoints after treatment interruption. The SIV env gene was amplified by RT-PCR at limiting dilution favoring amplification from a single viral genome and at least fifteen independent cDNA products were sequenced at each time point. The frequency and position of predicted amino acid changes in Env are shown.

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). 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 (red), 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). The shaded region indicates the period of ART (gray). b, The percentage of animals with viral loads below the threshold of detection (< 15 copies/ml) are shown after treatment interruption. Differences in the percentage of aviremic animals between the treatment and control groups were compared (p<0.005, Mantel-Cox test).

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

a, ITS61.01 and b, ITS103.01 concentrations in serum were measured by ELISA on plates coated with an antibody to the rhodopsin tag appended to the C-terminus of ITS61.01 or with an anti-idiotype antibody to ITS103.01. Anti-drug antibody responses to c, ITS61.01 and d, ITS103.01 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.

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

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. The frequency of each of the barcodes detected in the animals that received vectors expressing 17-HD9 (blue) or ITS61.01 and ITS103.01 (red) is shown.

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 Env (red) surrounding residues N295 and N479, their frequency, and serum concentrations of ITS103.01 and ITS61.01 are shown for each animal at the indicated time points after treatment interruption. b, N-linked glycans attached to N295 and N479 (magenta) are located on the periphery of the CD4-binding site (yellow)^,. c-f, The introduction of substitutions into SIV_mac239 predicted to abrogate glycosylation of N295 or N479 confer resistance to c, neutralization and d, ADCC by ITS103.01, but not does not alter sensitivity to e, neutralization or f, ADCC by ITS61.01.