A hTfR1 Receptor-Specific VHH Antibody Neutralizes Pseudoviruses Expressing Glycoproteins from Junín and Machupo Viruses

Abstract

The Junín virus (JUNV) is one of the New World arenaviruses that cause severe hemorrhagic fever. Human transferrin receptor 1 (hTfR1) has been identified as the main receptor for JUNV for virus entry into host cells. To date, no treatment has been approved for JUNV. Herein, we investigated 12 anti-hTfR1 VHH (variable domain of the heavy chain of heavy-chain antibody) antibodies and confirmed their interaction with hTfR1. Most of them could bind to the hTfR1 apical domain, which is the glycoprotein 1 (GP1) binding domain of JUNV. Among them, 18N18 exhibited neutralizing activity against both the human immunodeficiency virus (HIV)-vectored lentiviral Junín pseudoviruses and the recombinant vesicular stomatitis virus (VSV)-vectored Junín pseudoviruses. We also verified that 18N18 blocked the interaction between hTfR1 and JUNV GP1. In addition, 18N18 could neutralize another New World arenavirus, the Machupo virus. Using AlphaFold 3-based simulation of 18N18-hTfR1 docking, we determined that 18N18's binding epitope was located at the JUNV GP1 binding epitope. 18N18 represents a candidate for JUNV treatment and provides a potential approach that could be applied to New World arenaviruses.

Keywords: Junín viruses; Machupo viruses; VHH antibody; transferrin receptor 1.

Figure 1

Construction and panning of the anti-human transferrin receptor 1 (hTfR1) nanobody library. (A) Schematic of phage-displayed VHH (variable domain of the heavy chain of heavy-chain antibody) antibody screening and expression. (B) Detection of anti-hTfR1 immunoglobulin G (IgG) titer in alpaca serum using an enzyme-linked immunosorbent assay (ELISA). The secondary antibody was horseradish peroxidase (HRP)–Goat anti-Llama IgG H&L antibody for specific IgG detection. (C) Monoclonal antibody identification from the second-round screening: The binding of phage-displayed monoclonal VHHs and hTfR1 was measured using ELISA with an HRP-labeled M13 monoclonal antibody. Bovine serum albumin (BSA) and an irrelevant protein with a His-tag served as two negative controls. The X-axis represents the number of monoclonal phage-displayed VHH antibodies. For a single VHH, its interactions with antigen hTfR1 and two controls are shown in different colors, in which a red dot represents its interaction with hTfR1 and a blue dot or a gray dot represents its interaction with BSA or control-His, respectively. OD—optical density.

Figure 2

Basic characteristics of the candidate antibodies. (A) The binding ability of the candidates to hTfR1, as measured using ELISA. The initial concentrations of all candidates were 1 μg/mL, followed by a 3-fold serial dilution. Data are representative of at least two independent experiments. Means with the standard deviation (SD) are shown. (B) Affinity detection of the candidates binding to hTfR1 using surface plasmon resonance (SPR). (C) Thermostability of the candidate antibodies. Tm1 represents the temperature of the first thermal melting. (D) Protein size of the candidate antibodies: Pk1 Mode diameter represents the mode particle size of the first peak, which is also the main peak in the current test. The thermostability (C) and protein size (D) were measured using Uncle.

Figure 3

Binding of VHH antibodies to the hTfR1 apical domain. (A) The hTfR1 apical domain region is labeled in pink, hTfR1^D184-E383. (B) Western blot detection of hTfR1^D184-E383 expression. The detection antibody was the HRP-conjugated His-tag monoclonal antibody. M: Marker; Lane 1: purified hTfR1^D184-E383. (C) Analysis of the glycosylation sites of hTfR1^D184-E383. The N glycosylation sites are marked in green, and the O glycosylation sites are marked in blue. The numbers above the sequence represent the amino acid positions. (D) The binding ability of candidate antibodies to hTfR1^D184-E383 was measured using ELISA. The expressed hTfR1^D184-E383 was first coated on 96-well microplates, and then the binding activities of different antibodies with hTfR1^D184-E383 were tested at three different concentrations of candidates (0.1, 1, and 10 μg/mL). An anti-TfR1 antibody, ch128.1, was added as the positive control.

Figure 4

Screening of neutralizing antibodies against the Junín pseudovirus and the detection of the cross-neutralization against the Machupo pseudovirus. (A) Schematic diagram of HIV-vectored Junín pseudovirus construction. (B) Neutralization of JUNV by all candidates was measured at concentrations of 0.1, 1, and 10 μg/mL. (C) Neutralizing curves of 18N18, 18N20-1, and 18N20-2 against HIV-vectored Junín pseudoviruses. Ch128.1 was used as a positive control. The initial concentrations of all candidates were 60 nM, followed by a three-fold serial dilution. Data are representative of at least two independent experiments. Means with SDs are shown. (D) Neutralization of candidates against recombinant VSV (vesicular stomatitis virus) expressing JUNV GP (rVSVΔG-eGFP/JUNV GPC). The candidates start at 5 μM, followed by a four-fold serial dilution. (E) Detection of the blocking of hTfR1-JUNV GP1 interaction using Bio-Layer Interferometry (BLI). hTfR1 was first loaded onto nitrilotriacetic acid (NTA) sensors, and then allowed to interact with 18N18, ch128.1, or buffer. The interaction of GP1-Fc with the bio-sensor was finally measured. (F) Cross-neutralization ability against Machupo pseudoviruses of 18N18 at concentrations of 0.1, 1, and 10 μg/mL were measured using the same methods of neutralization against JUNV. Data are presented as means with SDs. IC₅₀—half-maximal inhibitory concentration; GP1—glycoprotein 1.

Figure 5

Epitope analysis of 18N18. (A) Alignment of two JUNV GP1 crystal structures, 5EN2 and 5NUZ. (B) Alignment of JUNV GP1 and MACV GP1 crystal structures, 5EN2 and 3KAS. (C) Simulation of the JUNV GP1-hTfR1 complex. (D) Prediction of the interaction between 18N18 and hTfR1 using AlphaFold 3. (E) Alignment of the JUNV GP1-hTfR1 complex and the 18N18-hTfR1 complex. (F) Epitope analysis of 18N18 and JUNV GP1 against hTfR1. For (C–F), the structure or epitope of 18N18 is shown in blue, and that for JUNV GP1 is shown in pink. The orange epitopes in F represent the overlap between 18N18 and JUNV GP1 binding epitopes.