Effect of Passive Administration of Monoclonal Antibodies Recognizing Simian Immunodeficiency Virus (SIV) V2 in CH59-Like Coil/Helical or β-Sheet Conformations on Time of SIVmac251 Acquisition

Abstract

The monoclonal antibodies (MAbs) NCI05 and NCI09, isolated from a vaccinated macaque that was protected from multiple simian immunodeficiency virus (SIV) challenges, both target an overlapping, conformationally dynamic epitope in SIV envelope variable region 2 (V2). Here, we show that NCI05 recognizes a CH59-like coil/helical epitope, whereas NCI09 recognizes a β-hairpin linear epitope. In vitro, NCI05 and, to a lesser extent, NCI09 mediate the killing of SIV-infected cells in a CD4-dependent manner. Compared to NCI05, NCI09 mediates higher titers of antibody-dependent cellular cytotoxicity (ADCC) to gp120-coated cells, as well as higher levels of trogocytosis, a monocyte function that contributes to immune evasion. We also found that passive administration of NCI05 or NCI09 to macaques did not affect the risk of SIV_mac251 acquisition compared to controls, demonstrating that these anti-V2 antibodies alone are not protective. However, NCI05 but not NCI09 mucosal levels strongly correlated with delayed SIV_mac251 acquisition, and functional and structural data suggest that NCI05 targets a transient state of the viral spike apex that is partially opened, compared to its prefusion-closed conformation. IMPORTANCE Studies suggest that the protection against SIV/simian-human immunodeficiency virus (SHIV) acquisition afforded by the SIV/HIV V1 deletion-containing envelope immunogens, delivered by the DNA/ALVAC vaccine platform, requires multiple innate and adaptive host responses. Anti-inflammatory macrophages and tolerogenic dendritic cells (DC-10), together with CD14⁺ efferocytes, are consistently found to correlate with a vaccine-induced decrease in the risk of SIV/SHIV acquisition. Similarly, V2-specific antibody responses mediating ADCC, Th1 and Th2 cells expressing no or low levels of CCR5, and envelope-specific NKp44⁺ cells producing interleukin 17 (IL-17) also are reproducible correlates of decreased risk of virus acquisition. We focused on the function and the antiviral potential of two monoclonal antibodies (NCI05 and NCI09) isolated from vaccinated animals that differ in antiviral function in vitro and recognize V2 in a linear (NCI09) or coil/helical (NCI05) conformation. We demonstrate that NCI05, but not NCI09, delays SIV_mac251 acquisition, highlighting the complexity of antibody responses to V2.

Keywords: CH59; NCI05; NCI09; V2; monoclonal antibodies; simian immunodeficiency virus; trogocytosis.

FIG 1

Anti-V2 MAb epitopes, binding, neutralization, and kinetics. (A) Binding of NCI05 to deglycosylated SIV_mac251 gp120, denatured SIV_mac251 gp120, and native SIV_mac251 gp120 measured by ELISA. (B) Surface plasmon resonance measuring NCI05 and NCI09 binding to deglycosylated SIV_mac251-M766 gp120 ligand. (C) Binding of NCI05 to SIV_smE543 gp120, SIV_mac251 cyclic V2 (cV2), and SIV_mac251 linear V2 peptide measured by ELISA. (D) SIV_E543 V2 amino acid sequence with colored lettering showing amino acids which augment (red) or decrease (orange) NCI05 binding. (E and F) ELISA showing binding of ITS12, NCI05, and NCI09 to (E) linear peptide 115 and (F) coil/helical peptide 150.

FIG 2

NCI05 binds to the coil/helical conformation of V2. (A) Crystal structure of NCI05 in complex with V2 peptide V2c at 2.35 Å resolution. (Left) the refined structure of antibody and V2 peptide complex with NCI05 heavy chain (orange), light chain (gray), and V2 peptide (green). CDRs are colored as follows: H1, magenta; H2, olive; H3, orange; L1, light blue; L2, navy blue; and L3, purple. (Center) Two 90° rotations of the surface representation show that the V2c peptide binds to the groove formed by CDRs H2, H3, and L3. (Right) The electrostatic potential surface is shown in the same views, with the V2c peptide in sticks representation. (B) Detailed interactions between NCI05 and the V2c peptide. The peptide and NCI05 paratope residues are shown in sticks representation, with other regions of NCI05 shown as cartoon. Hydrogen bonds and salt bridges between the interacting atoms are indicated with gray dashed lines. (C) Sequence and paratope of NCI05. Heavy- and light-chain paratope residues are highlighted in green. NCI05 residues were numbered according to Kabat nomenclature.

FIG 3

Gp120/gp140 binding, neutralization, inhibition of gp120-α₄β₇ binding, inhibition of gp120 T-cell costimulation, ADCC, ADCP, and trogocytosis by MAbs NCI05 and NCI09. (A) Binding of SIV_mac251-M766 gp120 to α₄β₇-expressing RPMI8866 cells in the presence of vedolizumab (positive control) and NCI MAbs produced in either 293 or CHO cells. (B) Inhibition of gp120-induced T-cell activation by ITS12 (negative control), ITS03 (positive control), NCI05, and NCI09. (C) Titration curves of normalized ADCC by NCI05 and NCI09 with ΔV1 gp120-coated CEM.NKR-CCR5 target cells. (D) Trogocytosis by CD14⁺ monocytes incubated with ΔV1 and wild-type gp120-coated CEM.NKR-CCR5 target cells labeled with PKH26 in the presence of NCI05 and NCI09. (E) ADCP by THP-1 cells measured in the presence of ΔV1 and wild-type gp120-coated CEM.NKR-CCR5 target cells and titrations of NCI05 and NCI09 antibodies. (F) Binding of PGT145 to V2 of various SIV-infected cells in the absence of soluble CD4. (G) ADCC mediated by KHYG-1 NK cells recognizing PG145 bound to V2 of various SIV-infected CEM.NKR-CCR5-sLTR-Luc cells in the absence of soluble CD4. (H and I) ADCC mediated by (H) NCI05 and (I) NCI09 against various SIV-infected CEM.NKR-CCR5-sLTR-Luc cells in the presence of sCD4.

FIG 4

Mucosal and plasma NCI05 and NCI09 levels in passively immunized animals. (A) Study design of passive immunization (NCI05 in red, NCI09 in blue) and 1:250 intrarectal SIV_mac251 challenges (black arrows). (B and C) Concentrations of (B) NCI05 and (C) NCI09 measured in the plasma by ΔV1 gp120 ELISA during the SIV_mac251 challenge phase. Colored arrows below the x axis indicate the times of passive immunization 4 days prior to each challenge. (D and E) ELISA detection of V2 MAb binding in the plasma of animals 1 week after the first passive immunization with NCI05 and NCI09 to (D) peptide 150 (biotin-GGG-LKSDKKIEYNETWYSRD) and (E) peptide 115 (biotin-GGG-GLKRDKTKEYN), respectively. (F and G) Mucosal concentrations of (F) NCI05 and (G) NCI09 measured by ΔV1 gp120 ELISA during the SIV_mac251 challenge phase. (H) Geometric means of V2 MAbs measured in the rectal mucosa of the NCI05 and NCI09 groups. (I and J) Spearman rank correlation between the geometric mean of total mucosal IgG and mucosal (I) NCI05 and (J) NCI09. (K) Percent ADCC killing in plasma from passively immunized macaques mediated by macaque NKG2A⁺ NK effectors from the NCI05 and NCI09 groups. (L) Percent ADCC killing in plasma from passively immunized macaques mediated by human PBMC effectors. (M) Efferocytosis by CD14⁺ efferocytes in PBMCs collected prior to passive immunization from the NCI05 and NCI09 groups.

FIG 5

Virus acquisition curves, viral load, and NCI05-specific and NCI09-specific ADCC correlations with time of virus acquisition. (A and B) Kaplan-Meier curves showing SIV acquisition rates in animals passively immunized with (A) NCI05 and (B) NCI09 and controls (black lines). (C to E) SIV viremia in individual animals following infection in (C) NCI05, (D) NCI09, and (E) control groups. (F) Geometric mean of SIV viremia in control, NCI05, and NCI09 groups. (G) SIV DNA measured in the rectal mucosa at 2 weeks postinfection in control, NCI05, and NCI09 groups. (H) Spearman rank correlation between the geometric mean of NCI05 measured in the rectal mucosa by ΔV1 gp120 ELISA and the time of SIV acquisition. (I) Normalized percentage of ADCC mediated by human PBMC effectors incubated with NCI05 plasma obtained 1 week after infection (J) Spearman rank correlation between the percent efferocytosis by CD14⁺ cells in macaque pre-PBMCs after 12 h and the time of SIV acquisition. (K) Spearman rank correlation between the geometric mean of NCI09 measured in plasma prior to infection by peptide 115 ELISA and the time of SIV acquisition. (L) Normalized percentage of ADCC mediated by macaque NKG2A⁺ NK effectors obtained prior to passive immunization and incubated with NCI09 plasma obtained 1 week after infection.