Long-Term Delivery of an Anti-SIV Monoclonal Antibody With AAV

Abstract

Long-term delivery of anti-HIV monoclonal antibodies using adeno-associated virus (AAV) holds promise for the prevention and treatment of HIV infection. We previously reported that after receiving a single administration of AAV vector coding for anti-SIV antibody 5L7, monkey 84-05 achieved high levels of AAV-delivered 5L7 IgG1 in vivo which conferred sterile protection against six successive, escalating dose, intravenous challenges with highly infectious, highly pathogenic SIVmac239, including a final challenge with 10 animal infectious doses (1). Here we report that monkey 84-05 has successfully maintained 240-350 μg/ml of anti-SIV antibody 5L7 for over 6 years. Approximately 2% of the circulating IgG in this monkey is this one monoclonal antibody. This monkey generated little or no anti-drug antibodies (ADA) to the AAV-delivered antibody for the duration of the study. Due to the nature of the high-dose challenge used and in order to rule out a potential low-level infection not detected by regular viral loads, we have used ultrasensitive techniques to detect cell-associated viral DNA and RNA in PBMCs from this animal. In addition, we have tested serum from 84-05 by ELISA against overlapping peptides spanning the whole envelope sequence for SIVmac239 (PepScan) and against recombinant p27 and gp41 proteins. No reactivity has been detected in the ELISAs indicating the absence of naturally arising anti-SIV antibodies; moreover, the ultrasensitive cell-associated viral tests yielded no positive reaction. We conclude that macaque 84-05 was effectively protected and remained uninfected. Our data show that durable, continuous antibody expression can be achieved after one single administration of AAV and support the potential for lifelong protection against HIV from a single vector administration.

Keywords: AAV vector; HIV/SIV cure; broadly neutralizing antibodies; gene therapy; immunotherapy; long-term expression; prophylaxis; rhesus monkeys.

Figure 1

5L7 antibody levels and the corresponding anti-antibodies (ADAs) in serum following AAV administration. (A) Schematic of the experiment in which rhesus monkey 84-05 received intramuscular administration of AAV1 coding for antibody 5L7 and was subsequently challenged with SIVmac239 (six times: 4 times with a 1 animal infectious doses followed by a 2x dose challenge and a final 10x dose challenge). (B) 5L7 antibody levels (blue; plotted on left axis) and anti-5L7 antibody levels (red; plotted on right axis) in serum from monkey 84-05, quantified by ELISA.

Figure 2

Analysis of antibody responses by PepScan. PepScan (ELISA against overlapping peptides) spanning the whole envelope sequence for SIVmac239, performed with (A) serum from 84-05 (week 328 post-AAV) at a 1:20 dilution; (B) recombinant purified 5L7 antibody at a concentration of 2 μg/ml; and (C) serum from a rhesus monkey experimentally infected with SIVmac239 (week 12 post-SIV infection) at a 1:20 dilution. Note: regions of interest are shadowed in gray and indicated as follows: variable regions 1–2, 3, 4, and 5 are labeled V1–V2, V3, V4, and V5, respectively; the cleavage site and beginning of gp41 are represented by brackets; g123 indicates the location of the N-linked carbohydrate sites found in gp41; TM is the transmembrane domain of gp41; and HIR stands for highly immunogenic region. Data from SIV+ monkey in panel (C) is representative of many such SIV+ monkeys tested previously (47, 48).

Figure 3

Analysis of antibody responses to p27 and gp41. Serum from 84-05 (week 328 post-AAV) at a 1:20 dilution was tested by ELISA against recombinant purified proteins p27 (A) gp41 and (B) sera from two rhesus monkeys experimentally infected with SIVmac239 (week 12 post-SIV infection) at a 1:20 dilution were used as positive controls, and recombinant purified 5L7 as a negative.