Neutralizing Monoclonal Antibodies against the Gn and the Gc of the Andes Virus Glycoprotein Spike Complex Protect from Virus Challenge in a Preclinical Hamster Model

Abstract

Hantaviruses are the etiological agent of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The latter is associated with case fatality rates ranging from 30% to 50%. HCPS cases are rare, with approximately 300 recorded annually in the Americas. Recently, an HCPS outbreak of unprecedented size has been occurring in and around Epuyén, in the southwestern Argentinian state of Chubut. Since November of 2018, at least 29 cases have been laboratory confirmed, and human-to-human transmission is suspected. Despite posing a significant threat to public health, no treatment or vaccine is available for hantaviral disease. Here, we describe an effort to identify, characterize, and develop neutralizing and protective antibodies against the glycoprotein complex (Gn and Gc) of Andes virus (ANDV), the causative agent of the Epuyén outbreak. Using murine hybridoma technology, we generated 19 distinct monoclonal antibodies (MAbs) against ANDV GnGc. When tested for neutralization against a recombinant vesicular stomatitis virus expressing the Andes glycoprotein (GP) (VSV-ANDV), 12 MAbs showed potent neutralization and 8 showed activity in an antibody-dependent cellular cytotoxicity reporter assay. Escape mutant analysis revealed that neutralizing MAbs targeted both the Gn and the Gc. Four MAbs that bound different epitopes were selected for preclinical studies and were found to be 100% protective against lethality in a Syrian hamster model of ANDV infection. These data suggest the existence of a wide array of neutralizing antibody epitopes on hantavirus GnGc with unique properties and mechanisms of action.IMPORTANCE Infections with New World hantaviruses are associated with high case fatality rates, and no specific vaccine or treatment options exist. Furthermore, the biology of the hantaviral GnGc complex, its antigenicity, and its fusion machinery are poorly understood. Protective monoclonal antibodies against GnGc have the potential to be developed into therapeutics against hantaviral disease and are also great tools to elucidate the biology of the glycoprotein complex.

Keywords: Andes virus; MAb; Sin Nombre virus; hantavirus.

FIG 1

Phylogenetic tree and conservation analysis of the Hantaviridae family. (A) Phylogenetic tree of M segments from hantavirus clades I to IV based on amino acid sequences shown in Table S1 in the supplemental material. ANDV, Andes virus; DOBV, Dobrava-Belgrade virus; HTNV, Hantaan virus; MJNV, Imjin virus; PHV, Prospect Hill virus; PUUV, Puumala virus; SEOV, Seoul virus; SNV, Sin Nombre virus; TPMV, Thottapalayam virus; TULV, Tula virus. TPMV and MJNV (thottimviruses) are included as part of an outgroup. The scale bar represents 5 variants per 100 amino acids. (B) Conservation map of the M segment of the Orthohantavirus genus based on amino acid sequences from Table S1. Transmembrane (TM) domains are based upon analysis via TMHMM. Protease cleavage sites for the signal peptidase are indicated (including the conserved WAASA motif). Black color indicates less conserved and white color indicates more conserved residues.

FIG 2

ELISA reactivity against purified VSV-ANDV and rGn. (A) ELISAs of MAbs from the homologous “AN” hybridoma fusions against VSV-ANDV. Plates were coated with 5 μg/ml of purified VSV-ANDV, while MAbs were used in 1:3 serial dilutions beginning with 30 μg/ml. Data shown are the products from two replicates. The positive control was an anti-VSV-N MAb and common between panels A and C. The negative control was KL-2G12, an IgG2a MAb against Zaire ebolavirus GP. All trend lines are logarithmic regressions except where such regressions were not converged; in such cases, connecting lines were used. (B) ELISAs against recombinant ANDV Gn. The recombinant ANDV Gn was produced in insect cells via baculovirus expression. Plates were coated with 2 μg/ml of ANDV rGn, while MAbs were used in 1:3 serial dilutions beginning with 30 μg/ml. Data shown are the products from two separate experiments, two replicates each. The positive control was an anti-hexahistidine tag antibody and common between panels B and D. Negative control was KL-2G12, an IgG2a MAb against Zaire ebolavirus GP. (C) ELISAs conducted against VSV-ANDV as for panel A but with MAbs sourced from the heterologous “HAP” hybridoma fusions as described for Fig. S1. (D) ELISAs conducted against ANDV rGn as for panel B but with MAbs sourced from HAP fusions as described for Fig. S1. (E) Schematic of full GnGc with features indicated. (F) Schematic of recombinant Gn used in panels B and D. Transmembrane domains, the signal peptide, and the Gn hydrophobic region were annotated based on TMHMM analysis. Both schematics are to scale.

FIG 3

Neutralization and effector functions of the isolated MAbs. (A) FRNAs of “AN” fusion MAbs against VSV-ANDV. (B) FRNAs of “HAP” fusion MAbs against VSV-ANDV. For both A and B, each MAb was run in duplicates in 3-fold serial dilutions starting at 30 μg/ml. Any MAb exhibiting >15% neutralization at the last concentration was repeated with lower dilutions. The negative-control MAb was a murine IgG2a specific for Zaire ebolavirus (KL-2G12). All trend lines are logarithmic regressions except where such regressions were not converged. (C) FRNAs conducted as for panels A and B but against authentic ANDV. Dashed lines in panels A, B, and C demonstrate 50% inhibition. (D) Comparison of IC₅₀ values of each MAb against VSV-ANDV and authentic ANDV. Error bars indicate 95% confidence intervals. (E and F) ADCC reporter assays of each MAb against VSV-ANDV-infected Vero.E6 cells (MOI, 1.0). Data shown are the result from one experiment with a shared positive control in panels E and F (serum from homologous fusion). This positive control was used in 3-fold serial dilutions with a starting dilution of 1:300.

FIG 4

Visualizing VSV-ANDV escape mutations on a computationally fit model of ANDV Gn. A model of ANDV Gn was created using TULV Gn as the template (PDB 5FXU). This model was then computationally fit into a TULV cryo-EM tomograph to depict the relationship between the Gn and the plasma membrane. (A) Structural alignment of ANDV model (red) and TULV Gn structure (blue). (B) Top view of the glycoprotein complex with escape mutations visualized on the model using colors as indicated. The tetramer is the assumed arrangement for ANDV, confirmed for TULV. (C) Side view including the plasma membrane shown in blue. (D) To-scale schematic of the ANDV GnGc showing the location of escape mutations and the alignment sequence used for structural modeling. RMSD, root mean square deviation.

FIG 5

Visualizing VSV-ANDV escape mutations on a computational model of ANDV Gc. Models of ANDV Gc pre- and postfusion were created using HTNV Gc as the template (prefusion, PDB 5LJY; postfusion, PDB 5LK3). (A) Structural fit of the ANDV prefusion Gc model with 5LJY. (B to D) visualization of escape mutations on the prefusion ANDV Gc model. (E) Structural fit of the ANDV postfusion Gc model (monomer) with 5LK3. (F) Visualization of escape mutations on the postfusion Gc model as a monomer. (G to I) Visualization of escape mutations on the postfusion Gc model as a trimer. Colors shown below panel D are representative for all structures included here. (J) Schematic of ANDV GnGc with escape variants. RMSD, root mean square deviation.

FIG 6

In vivo studies in the Syrian hamster model of HCPS. (A) Syrian hamsters were inoculated intranasally (i.n.) with 200 FFU of ANDV^CHI-9717869 or PBS (mock) and then injected i.p. with 25 mg/kg of MAb or PBS on day 3 and day 8 postinfection. Experimental overview, including necropsy endpoints. (B) Survival of the indicated treatment groups shown to day 21 (though all experimental groups survived until the predetermined survival endpoint of day 42). ***, P < 0.0001 via Mantel-Cox log-rank test. (C) Respiratory rates of each hamster depicted as an average/group. (D) Viral genome copies per milligram of homogenized lung tissue on day 10 postinfection. *, P = 0.0161; **, P = 0.0048 via Kruskal-Wallis test incorporating Dunn’s test for multiple comparisons. (E) Same as for panel D but on day 42 postinfection.