Monoclonal Antibodies with Neutralizing Activity and Fc-Effector Functions against the Machupo Virus Glycoprotein

Abstract

Machupo virus (MACV), the causative agent of Bolivian hemorrhagic fever (BHF), is a New World arenavirus that was first isolated in Bolivia from a human spleen in 1963. Due to the lack of a specific vaccine or therapy, this virus is considered a major risk to public health and is classified as a category A priority pathogen by the U.S. National Institutes of Health. In this study, we used DNA vaccination against the MACV glycoprotein precursor complex (GPC) and murine hybridoma technology to generate 25 mouse monoclonal antibodies (MAbs) against the GPC of MACV. Out of 25 MAbs, five were found to have potent neutralization activity in vitro against a recombinant vesicular stomatitis virus expressing MACV GPC (VSV-MACV) as well as against authentic MACV. Furthermore, the five neutralizing MAbs exhibited strong antibody-dependent cellular cytotoxicity (ADCC) activity in a reporter assay. When tested in vivo using VSV-MACV in a Stat2^-/- mouse model, three MAbs significantly lowered viral loads in the spleen. Our work provides valuable insights into epitopes targeted by neutralizing antibodies that could be potent targets for vaccines and therapeutics and shed light on the importance of effector functions in immunity against MACV.IMPORTANCE MACV infections are a significant public health concern and lead to high case fatality rates. No specific treatment or vaccine for MACV infections exist. However, cases of Junin virus infection, a related virus, can be treated with convalescent-phase serum. This indicates that a MAb-based therapy for MACV could be effective. Here, we describe several MAbs that neutralize MACV and could be used for this purpose.

Keywords: MACV; Machupo virus; arenavirus; monoclonal antibody.

FIG 1

Most antibodies are highly specific and bind only to MACV GPC. Binding of antibodies to several NW GPCs and LASV GPC is shown. A standard ELISA was performed using several dilutions of each respective antibody starting at 30 μg/ml against various NW arenavirus GPCs such as MACV GPC, TCRV GPC, JUNV GPC, GTOV GPC, TAMV GPC, and LASV GPC. An anti-influenza H4 virus antibody was used as a negative control while a pan-arenavirus GPC antibody, KL-AV-2A1, was used as a positive control. The values in the graph correspond to minimal binding concentration, the lowest concentration at which the antibody still demonstrates binding signal. Phylogenetic differences between the GPCs is indicated by a phylogenetic tree (based on amino acid sequence). The scale bar represents a 5% amino acid change.

FIG 2

Antibodies bind to cells infected with VSV-MACV. (A) Binding of antibodies to MACV glycoprotein in an immunofluorescence assay. Vero.E6 cells were infected with VSV-MACV at an MOI of 1.0 overnight and then fixed and immunostained using each antibody at a concentration of 30 μg/ml. An irrelevant antibody, anti-influenza H4 virus (KL-H4-4A11), was used as a negative control while the positive control used was KL-AV-2A1. (B) Western blot analysis. A standard Western blot was performed using recombinant MACV GPC and an irrelevant protein, influenza virus H2 HA. Each blot was stained with 30 μg/ml of each respective antibody, and an anti-mouse IgG conjugated to alkaline phosphatase was used as a secondary antibody at a dilution of 1:3,000. A separate Western blot was run using anti-histidine antibody to ensure proper transfer of protein to the nitrocellulose membrane. The ladder used was a ColorPlus Prestained Protein Ladder, Broad Range (New England BioLabs).

FIG 3

Five antibodies have potent neutralizing activity and ADCC reporter activity against VSV-MACV and can also neutralize pathogenic MACV at low concentrations. (A) PRNA against VSV-MACV. PRNAs were performed with each antibody starting at a concentration of 100 μg/ml with subsequent 1:5 dilutions. Vero.E6 cells were infected with the antibody-virus mixture, and similar concentrations of antibody were also present in the overlay for the duration of the assay. An anti-influenza virus H4 antibody was utilized as a negative control. Percent inhibition with each antibody is plotted. (B) PRNAs against authentic MACV. PRNAs were run with several dilutions of the five MAbs that neutralized VSV-MACV. An antibody-virus mixture was used to infect Vero.E6 cells, and cells were overlaid for 8 days. No antibody was added to the overlay. An irrelevant antibody which does not bind to MACV GPC was used as a negative control for this assay. (C) PRNAs against VSV-LASV. PRNAs were also performed with VSV-LASV to test the few antibodies that had cross-reactivity toward LASV GP, as shown in Fig. 1. Cells were overlaid with antibody present in the overlay for 48 h, and plaques were counted to calculate percent inhibition. (D) ADCC reporter activity using VSV-MACV. An in vitro ADCC reporter assay was performed using a commercial kit (Promega). Vero.E6 cells were infected overnight with VSV-MACV at an MOI of 1.0, and the next day, antibody dilutions and effector cells were added. Six hours later, luciferase substrate was added, and luminescence was measured to assess fold induction of ADCC activity due to FcR engagement.

FIG 4

VSV-MACV is prevalent in the serum and spleen, and prophylactic administration of neutralizing MAbs leads to reduction in viral titer in vivo. (A and B) Assessment of viral load in several organs and the spleen. Stat2^−/− mice (n = 3) were infected with 1,000 PFU of VSV-MACV via the intraperitoneal route, and organs were harvested on day 3 and day 6, as indicated, to assess viral titer via standard plaque assay with organ homogenate. An anti-influenza virus H4 antibody was used as a negative control. (C and D) Prophylactic administration of neutralizing antibodies. Each neutralizing MAb was administered at 10 mg/kg via the intraperitoneal route, and Stat2^−/− mice (n = 4) were then infected with 1,000 PFU 2 h later. Viral titer in the spleen was used as a measure to see if antibody administration can reduce viral titers in vivo. The negative control was a running control as experiments were performed in batches due to limited availability of mice. Spleen was harvested on day 3 and day 6, as indicated. Statistical significance was determined via one-way ANOVA test (***, P ≤ 0.001; ns, not significant).

FIG 5

Antibodies target epitopes on GP1 to inhibit entry into host cell. PRNAs were performed with escape viruses. (A to E) Escape mutant viruses were generated for each neutralizing MAb, and these viruses were used to confirm that the virus had escaped and that the respective MAb could not neutralize the virus any longer. PRNAs were performed using each MAb against VSV-MACV as well as each respective escape virus (EV) as indicated in the figure. (F to I) Structural visualization of the antibody epitopes. Using a previously defined structure of MACV GP1 (PDB accession number 5W1M) and LASV GPC (PDB 5VK2), critical epitopes on GP1, which are targeted by neutralizing MAbs, are shown highlighted via PyMOL. MACV GP1 (gray) was aligned and overlaid onto the LASV GPC trimer (gold) (G). The RBS is shown in green, loop 10 of MACV GP1 is shown in orange (H), and the epitopes are marked with unique colors (I). For ease of visualization, one monomer is shown as a solid surface while the other two monomers are depicted as ribbon structures.

FIG 6

ELISAs using recombinant mutant MACV GPCs. (A to F) To assess if binding was altered after escape virus mutations were introduced in the recombinant MACV GPC, ELISAs were performed using MACV GPC, MACV GPC K170N, and MACV GPC F226L with the antibodies indicated at the top of each panel. MAb KL-AV-2A1 was used as the positive control.