Non-human Primates

Rhesus macaques (Macaca mulatta) and African Green monkeys (Chlorocebus pygerythrus) were housed at BIOQUAL's housing facilities in Rockville, MD. Care and husbandry of all non-human primates were provided in compliance with federal laws and guidelines as well as in accordance with recommendations provided in the NIH guide and other accepted standards of laboratory animal care and use. BIOQUAL is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, (AAALAC file #624) and holds an Assurance on file with the National Institute of Health, Office for Protection of Research Risks as required by the US Public Health Service Policy on Humane Care and Use of Laboratory Animals with a PHS Animal Welfare Assurance File Number #A-3086-01. Animals were sedated with ketamine or telazol for all technical procedures. Ketamine was given IM in amounts necessary for short-term procedures such as blood drawing.

Animals were housed in accordance with the recommendations of the Association for Assessment and Accreditation of Laboratory Animal Care International Standards and with the recommendations in the Guide for the Care and Use of Laboratory Animals of the United States—National Institutes of Health. The Institutional Animal Use and Care Committee of BIOQUAL approved these experiments. When immobilization was necessary, the animals were sedated intramuscularly with 10 mg/kg of Ketamine HCl (Parke-Davis, Morris Plains N.J.) before any direct handling or procedures. All efforts were made to minimize suffering. Details of animal welfare and steps taken to ameliorate suffering were in accordance with the recommendations of the Weatherall report, “The use of non-human primates in research”. Animals were housed in an air-conditioned facility with an ambient temperature of 21–25°C, a relative humidity of 40%–60% and a 12 h light/dark cycle. Animals were socially housed when possible or individually housed if no compatible pairing could be found. The animals were housed in suspended, stainless steel, wire-bottomed 6 sq ft cages and provided with a commercial primate diet and fresh fruit and vegetables twice daily with water freely available at all times. Social housing, toys, foraging equipment and mirrors were provided. Animals were monitored at least twice daily for behavior, food intake, activity, and overall health by trained technicians. No macaques were euthanized and all animals were returned to the colony for recycling.

Antibody production in plants

Monoclonal antibody PGT121 was produced in N. benthamiana using a Agrobacterium-mediated transient gene expression system as described previously [7]. Briefly, codon-optimized heavy and light chain DNA fragments were synthesized and separately cloned into pTRAk plant expression vector. In addition, the silencing suppressor protein p19 from tomato bushy stunt virus was also cloned into a separate pTRAk vector. Six-eight week old plants were co-infiltrated with the recombinant Agrobacteria suspension containing the three expression plasmids. After infiltration, plants were incubated at 20°C with 16/8 hour light cycles. At 10–12 days, soluble proteins were extracted and purified by protein A and MEP HyperCel chromatography with a recovery of 1.3 g/kg.

Non-human primate studies

For the pharmacokinetic studies, two African Green monkeys (~4kg) were injected with 5 mg/kg of plant-derived PGT121 either SC in the back or IM in the thigh, bled from the femoral artery at 0 to 14 days and assessed for levels of PGT121 by both neutralizing antibody activity (ID50) using TZM-bl cells as previously described [8] and ELISA. IC50 neutralization titers are purified antibody concentrations, and ID50 are serum dilutions, at which relative luminescence units (RLU) were reduced by 50% compared to RLU in virus control wells after subtraction of background RLU in cell control wells. ELISA assays were performed using 96-well Immuno Module plates (Nunc) coated with anti-human kappa LC (50μL of 1μg/mL) (SIGMA K3502) or with 1μg/mL of either CHO-derived monomeric HIV BaL-gp120 (NIH HIV Reagent Program) or m.CONgp140 env (a kind gift of Dr Bart Haynes, Duke University, NC) as previously described [6].

Two SC protection studies were conducted using 3-6kg Indian rhesus macaques (Macaca mulatta). In the first study, macaques were injected SC with 3.5–7.1mg/kg of PGT121, 24h prior to intravaginal challenge with a high dose (1700 TCID) of SHIV SF162P3 that was expected to infect all control animals after a single challenge. For intravaginal challenge, anesthetized macaques were administered SHIV SF162P3 using a non-leuer-lock syringe inserted ~2 cm into the vaginal vault.

In the second study, the same macaques were injected SC with 5mg/kg of PGT121 at 30–60 mins post-vaginal challenge with SHIV SF162P3 (1700 TCID. The potency of the plant-derived PGT121 against the rhesus (R157) PBMC-derived SF162P3 stock used for challenge was 0.08 ug/ml; similar to the IC50 of CHO-derived PGT121 (0.15 ug/ml). Protection was assessed using a viral RNA assay as described [9]. Fisher’s exact test was performed using the R statistical package [10].

Pharmacokinetics of plant-derived PGT121 following subcutaneous and intramuscular administration

In order to establish the time of peak PGT121 levels (Tmax) following parenteral administration and thus the time of challenge to assess optimal protection, preliminary studies were performed in which two African Green monkeys each received 5mg/kg of PGT121 by either SC or IM delivery and the pharmacokinetics monitored from 4 hr to 14 days. Fig 1 compares the serum neutralizing ID50 clearance profiles for each monkey using a pseudovirus-based TZM-bl assay against SHIV SF162P3. Cmax data were also assessed from the same samples by ELISA. Results indicate that each of the monkeys receiving SC injections (#8338, #8291) exhibited similar pharmacokinetics with Cmax of 60μg/ml and 80μg/ml at Tmax of ~24-30hr. By contrast, clearance profiles and Tmax in monkeys injected IM differed widely. Thus, monkey #8288 exhibited a T max of 4hr and a Cmax of 110ug/kg while the corresponding values for #8390 were 16μg/ml at a Tmax ~18-24hr. Based on minimum predicted protective neutralizing titers, these data suggest that SC injection of PGT121 (5mg/kg) will be protective within a range of <4hr to >14 days.

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Fig 1. Circulatory clearance profiles of 5mg/kg of plant-derived PGT121 in African Green monkeys following SC and IM injections.

Neutralizing ID50s were assessed at different times after injection using a pseudovirus-based TZM-bl assay against the Tier 2 SHIV-SF162P3.

https://doi.org/10.1371/journal.pone.0152760.g001

Efficacy of protection of PGT121 delivered by the subcutaneous route

Based on the ID50 clearance profiles, the efficacy of PGT121 given SC to protect against SHIV SF162P3 challenge at the Cmax was assessed in an initial dose-finding protection study. Thus, doses of 3.5–7.1mg/kg 24hr prior to intra-vaginal challenge with SF162P3 (1700 TCID) resulted in sterilizing protection in all six rhesus macaques (Fig 2A). This protection reached statistical significance compared to the control group by the Fisher’s Exact Test (p = 0.03). This is consistent with the serum PGT121 neutralizing levels at all doses against SHIV-SF162P3, measured just prior to challenge (inset in Fig 2A) with the lowest dose of 3.5mg/kg resulting in an ID50 of 1:295 (12N010) and reaching an ID50 of 1:1,565 at the 7.1mg/kg dose.

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Fig 2. Protection by SC-administered plant-derived PGT121 against intravaginal SHIVSF162P3 challenge in rhesus macaques as measured by log 10 vRNA (copies/ml of plasma).

(A) Protection in 6/6 macaques that received 3.7–7.1 mg/kg doses of PGT121 given 24 hr prior to SHIV SF162P3 challenge. Insert shows a linear correlation between ID50 and dose (3.5–7.1 mg/kg) at 24 hr; ID50 values at doses of 3.86, 4.7 and 5.6 mg/kg were 1:202, 1: 612 and 1: 738 respectively). (B) Protection in 5/6 macaques administered 5 mg/kg SC immediately after SF162P3 challenge. (C) Four control macaques received no passive bnAb. Application of Fisher’s Exact Test showed that protection of animals given PGT121 prior to challenge (panel A) compared to the control group (panel C) was statistically significant (p = 0.03), while protection of animals given PGT121 after challenge was not (p = 0.19).

https://doi.org/10.1371/journal.pone.0152760.g002

In order to mimic aspects of MTCT wherein infants born to HIV+ mothers may be injected immediately after birth, two months later these same six protected animals were administered 5 mg/kg of PGT121 SC immediately (30–60 mins) after intravaginal challenge with 1700 TCID SF162P3; resulting in protection in 5 of the 6 macaques at day 18 (Fig 2B). As observed previously in several published studies [11–13], one of the four untreated SHIVSF162P3-infected controls was not infected even at a high TCID (Fig 2C). Protection of animals given PGT121 after challenge was not significantly different from the control group (p = 0.19, Fisher’s Exact Test), likely due to the one control animal that was not infected during challenge with SHIV-SF162P3.