Neutralizing IgG at the portal of infection mediates protection against vaginal simian/human immunodeficiency virus challenge

Abstract

Neutralizing antibodies may have critical importance in immunity against human immunodeficiency virus type 1 (HIV-1) infection. However, the amount of protective antibody needed at mucosal surfaces has not been fully established. Here, we evaluated systemic and mucosal pharmacokinetics (PK) and pharmacodynamics (PD) of 2F5 IgG and 2F5 Fab fragments with respect to protection against vaginal challenge with simian-human immunodeficiency virus-BaL in macaques. Antibody assessment demonstrated that 2F5 IgG was more potent than polymeric forms (IgM and IgA) across a range of cellular and tissue models. Vaginal challenge studies demonstrated a dose-dependent protection for 2F5 IgG and no protection with 2F5 Fab despite higher vaginal Fab levels at the time of challenge. Animals receiving 50 or 25 mg/kg of body weight 2F5 IgG were completely protected, while 3/5 animals receiving 5 mg/kg were protected. In the control animals, infection was established by a minimum of 1 to 4 transmitted/founder (T/F) variants, similar to natural human infection by this mucosal route; in the two infected animals that had received 5 mg 2F5 IgG, infection was established by a single T/F variant. Serum levels of 2F5 IgG were more predictive of sterilizing protection than measured vaginal levels. Fc-mediated antiviral activity did not appear to influence infection of primary target cells in cervical explants. However, PK studies highlighted the importance of the Fc portion in tissue biodistribution. Data presented in this study may be important in modeling serum levels of neutralizing antibodies that need to be achieved by either vaccination or passive infusion to prevent mucosal acquisition of HIV-1 infection in humans.

Fig 1

Inhibition of HIV-1_BaL by 2F5 IgG, IgA, IgM, and Fab in human primary cells. The inhibitory activities of 2F5 IgG, IgA, IgM, and Fab were tested in PHA-stimulated PBMCs, macrophages, and MDDCs. 2F5 IgG, IgA, IgM, or Fab dilutions were incubated with HIV-1_BaL for 1 h at 37°C prior to addition of 5 × 10⁴ PBMCs (A), macrophages (B), or 4 × 10⁴ MDDCs (C). After 2 h of incubation at 37°C, the cells were washed and incubated with medium in the presence of the antibodies for 7 days. (D) To determine inhibition of HIV-1 trans-infection by DCs to CD4⁺ T cells, 2 × 10⁴ MDDCs were incubated with HIV-1_BaL for 1 h at 37°C. After the incubation, the cells were washed and 2F5 IgG, IgA, IgM, or Fab dilutions together with 4 × 10⁴ PM-1 T cells were added and incubated for 7 days. HIV-1 replication was assessed by measurement of p24 release into culture supernatant. Reduction in p24 levels was expressed as percent inhibition ± standard errors of the means (SEM) in relation to the corresponding isotype control. Data are represented as the means from three independent experiments, where each dilution was tested in triplicate.

Fig 2

Ability of 2F5 to inhibit HIV-1 infection of cervical tissue and transmission by cells migrating out from cervical tissue explants. (A) 2F5 IgG and 2F5 Fab were preincubated with HIV-1_BaL for 1 h at 37°C prior to being added to cervical tissue explants for 2 h. After washing, the explants were cultured in the presence of the antibodies for a further 14 days. (B) Twenty-four h after the initiation of explant culture, migratory cells that emigrate out of the tissue were recovered and cocultured with 4 × 10⁴ PM-1 T cells in the presence of the antibody. HIV-1 infection was determined by p24 ELISA. Reduction in p24 levels was expressed as percent inhibition ± SEM in relation to the virus control. Data are represented as the means from three independent donors, where each dilution was tested in triplicate.

Fig 3

Pharmacokinetics of infused monoclonal antibody 2F5 IgG or 2F5 Fab in plasma. Female Indian rhesus macaques were i.v. infused with 25 mg/kg of 2F5 IgG (A) or 2F5 Fab (B). Plasma samples were taken prior to infusion and 4, 6, 12, 24, 48, 72, and 144 h after infusion. The concentration of antibody in plasma (in μg/ml) was determined by direct ELISA and compared to a corresponding standard curve. Each sample was done in triplicate. The symbols in the key identify different macaques.

Fig 4

Pharmacokinetics of 2F5 IgG and 2F5 Fab in vaginal and rectal secretions and saliva following intravenous administration. Female Indian rhesus macaques were i.v. infused with 25 mg/kg of 2F5 IgG or 2F5 Fab. Mucosal secretions were collected by Weck-cel sponges prior to infusion and 4, 6, 12, 24, 48, 72, and 144 h after infusion. The concentrations (in μg/ml) of 2F5 IgG in vaginal secretions (A), rectal secretions (B), and saliva (C) and of 2F5 Fab in vaginal secretions (D), rectal secretions (E), and saliva (F) were determined by direct ELISA (each sample in triplicate) and compared to a corresponding standard curve. The symbols in the key identify different macaques.

Fig 5

Plasma viral loads after vaginal SHIV-BaL challenge. Female rhesus macaques were i.v. infused with 50 mg/kg (A), 25 mg/kg (B), 5 mg/kg (C), and 0 mg/kg (D) of 2F5 IgG. Six h after antibody infusion, macaques were vaginally challenged with SHIV-BaL. Serum samples were taken at week 0 (6 h after infusion and just prior to challenge) and 1, 2, 3, 4, 6, 9, 12, 16, and 20 weeks postchallenge. Plasma was analyzed for viral RNA levels.

Fig 6

Pharmacokinetics of infused 2F5 IgG after SHIV-BaL challenge. Each group consists of five female Indian rhesus macaques. Animals were i.v. infused with 50 (A), 25 (B), or 5 (C) mg/kg 2F5 IgG or received no antibodies as a negative-control group (D). Six h after infusion, all macaques were challenged intravaginally with SHIV-BaL (day 0 indicates the time point 6 h after infusion). Plasma samples were taken prior to infusion, 6 h after infusion (time point of intravaginal SHIV-BaL challenge), and 7, 14, and 21 days after infusion. The concentration of antibody in plasma (in μg/ml) was determined by direct ELISA and compared to a corresponding standard curve. Each sample was analyzed in triplicate. The symbols in the key identify different macaques.

Fig 7

Comparison of 2F5 IgG levels in plasma and mucosal secretions at time of challenge. Each group consists of five female Indian rhesus macaques. Animals were i.v. infused with 50 (A), 25 (B), or 5 (C) mg/kg 2F5 IgG or received no antibodies as a negative-control group (E). Plasma, vaginal (vag) and rectal (rec) secretion, and saliva (sal) samples were taken 6 h after infusion and just prior to intravaginal challenge with SHIV-BaL. The concentration of antibody in plasma (in μg/ml) was determined by direct ELISA and compared to a corresponding standard curve. Each sample was done in triplicate. The symbols in the key identify different macaques.

Fig 8

Correlation of 2F5 Fab concentrations and protection following vaginal challenge with SHIV-BaL. Four female rhesus macaques were i.v. infused with 25 mg/kg of 2F5 Fab. Six h after antibody infusion, macaques were vaginally challenged with SHIV-BaL. Serum samples were taken prior to infusion, 6 h after infusion (time point of intravaginal SHIV-BaL challenge), and 7, 14, and 21 days after infusion and analyzed for viral RNA levels (A) and for 2F5 Fab concentration (B). (C) Comparison of 2F5 Fab levels in plasma, vaginal and rectal secretion, and saliva samples at the time of challenge. Antibody levels (in μg/ml) were determined by direct ELISA and compared to a corresponding standard curve. Each sample was tested in triplicate. The symbols in the key identify different macaques.

Fig 9

Multiplicity of SHIV-BaL infection in rhesus macaques. Numbers of T/F viruses were estimated from analysis of the first plasma viral RNA-positive plasma sample from each infected rhesus macaque by single-genome sequencing. In untreated animals, numbers of T/F viruses ranged from 0 to 4 with a median of 2. In 2F5 IgG-treated animals, there was a significant dose-related reduction in numbers of T/F viruses establishing productive clinical infection (P = 0.01393 by Fisher's exact test).

Fig 10

Neighbor-joining phylogenetic tree of SHIV-BaL sequences from the inoculum and from each of 6 infected animals. The tree reveals discrete low-diversity sequence lineages corresponding to the progeny of T/F viral genomes (colored rectangles) interspersed among diverse sequences from the virus inoculum represented by black lines. Sequences with G-A hypermutation were excluded from the tree.