Broadly neutralizing monoclonal antibodies 2F5 and 4E10 directed against the human immunodeficiency virus type 1 gp41 membrane-proximal external region protect against mucosal challenge by simian-human immunodeficiency virus SHIVBa-L

Abstract

The membrane-proximal external region (MPER) of HIV-1, located at the C terminus of the gp41 ectodomain, is conserved and crucial for viral fusion. Three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, 4E10, and Z13e1, are directed against linear epitopes mapped to the MPER, making this conserved region an important potential vaccine target. However, no MPER antibodies have been definitively shown to provide protection against HIV challenge. Here, we show that both MAbs 2F5 and 4E10 can provide complete protection against mucosal simian-human immunodeficiency virus (SHIV) challenge in macaques. MAb 2F5 or 4E10 was administered intravenously at 50 mg/kg to groups of six male Indian rhesus macaques 1 day prior to and again 1 day following intrarectal challenge with SHIV(Ba-L). In both groups, five out of six animals showed complete protection and sterilizing immunity, while for one animal in each group a low level of viral replication following challenge could not be ruled out. The study confirms the protective potential of 2F5 and 4E10 and supports emphasis on HIV immunogen design based on the MPER region of gp41.

FIG. 1.

Protection by 2F5 and 4E10 against mucosal SHIV_Ba-L challenge. A total of 16 male Indian rhesus macaques were divided into two antibody treatment groups of six for administration of 2F5 and 4E10. The control group was comprised of two isotype control-treated animals and two control animals (96068 and 2025) that were challenged prior to the beginning of the protection study to confirm viral infectivity but not treated with antibody. All animals were intrarectally challenged with 2,000 TCID₅₀ SHIV_Ba-L. Animals in the antibody treatment groups received an intravenous dose of 50 mg/kg of either 2F5, 4E10, or isotype control antibody 1 day prior to viral challenge (day −1) and again 1 day following viral challenge (day +1). All control animals (4/4) experienced peak viremia on the order of 10⁷ between days 14 and 21. A single animal (2F5-treated 93060), experienced very low-level transient viremia, but at the end of 6 weeks there was no detectable virus in any of the animals. The presence of virus was measured in an assay with a minimum detection limit of 150 SIV RNA copies per ml (2.1 log₁₀ vRNA copies/ml) with a 95% confidence level. A second assay with a minimum detection limit of 10 SIV RNA copies per ml was used to retest available samples of sufficient volume (Table 2).

FIG. 2.

HIV-1_Ba-L gp120 ELISA of macaque sera at day 21, day 42, and month 6 following challenge. Serum samples from all animals were assayed for detection of an anti-Env response in ELISA. (A) No antibody responses were detected in any of the 2F5-treated animals, but controls that were treated with control antibody and that became infected showed an anti-gp120 response. Although a very low-level transient viremia was measured in animal 93060 at day 35 postchallenge, no humoral response was detected. (B) Similarly, antibody responses were detected in controls but not in 4E10-treated animals.

FIG. 3.

Detection of SIVmac239-specific cellular immune responses by intracellular staining. We detected no SIVmac239 Gag-specific CD8 or CD4 responses in 9 of the 10 animals that were tested for cellular immune responses at the time of viral rechallenge. PBMCs were stimulated with overlapping 15-mers of Gag aa 1 to 291 and stained for intracellular IFN-γ and IL-2. (A) CD8⁺ cells within the lymphocyte gate: (a) animal 01050; (b) animal 01090; (c) animal 01094; (d) animal 87083; (e) 95049; (f) animal 97001; (g) animal 97099; (h) animal 99081; (i) animal AV44; (j) animal 93060. (B) CD4⁺ cells within the lymphocyte gate: (a) animal 01050; (b) animal 01090; (c) animal 01094; (d) animal 87083; (e) 95049; (f) animal 97001; (g) animal 97099; (h) animal 99081; (i) animal AV44; (j) animal 93060.

FIG. 4.

Serum concentrations of 2F5 and 4E10 during challenge experiments. For each antibody treatment group, means and standard deviations of serum antibody concentrations averaged over three ELISA formats and then over all the animals in the group are shown. Elimination half-lives of the antibodies were determined by a single-exponential decay formula starting from day 7 (solid line) and are 4.6 and 4.1 days for 2F5 and 4E10, respectively. The modeled dashed curves account for the decay following the first antibody dose and the peak resulting from the second antibody dose.

FIG. 5.

Antibody-dependent cell-mediated viral inhibition (ADCVI) of 2F5 and 4E10 in comparison to b12. Target cells (CEM.NKR-CCR5) were infected with SHIV_Ba-L, incubated for 48 h, and then washed to remove cell-free virus. Serially diluted antibody was added at 50, 2, and 0.4 μg/ml to effector cells (rhesus PBMCs) and incubated for 30 min. Effectors and diluted antibody were combined with targets at an effector-to-target ratio of 10:1 and incubated for 7 days at 37°C in 5% CO₂. Supernatant was collected and assayed for p27 by ELISA to measure viral inhibition.

FIG. 6.

SHIV_Ba-L rechallenges of all protected macaques treated with either 2F5 or 4E10. (A) To assess the vulnerability of the protected animals caused by SHIV infection, rechallenges were conducted with 2,000 TCID₅₀ of SHIV_Ba-L at 6 months after the initial challenge. All animals in each antibody treatment group except 2F5-treated animal 93060 were infected. (B) Subsequently (6 months later), a second rechallenge was conducted with 93060 and the animal became infected. (Note that 2F5-treated animal 95118 was not rechallenged; this animal was euthanized for unrelated medical reasons prior to rechallenge.)