Isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo

Abstract

Human metapneumovirus (hMPV) is a recently described member of the Paramyxoviridae family/Pneumovirinae subfamily and shares many common features with respiratory syncytial virus (RSV), another member of the same subfamily. hMPV causes respiratory tract illnesses that, similar to human RSV, occur predominantly during the winter months and have symptoms that range from mild to severe cough, bronchiolitis, and pneumonia. Like RSV, the hMPV virus can be subdivided into two genetic subgroups, A and B. With RSV, a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Because of the high level of sequence conservation of the F protein across all the hMPV subgroups, this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies. Here we describe the generation of a panel of neutralizing monoclonal antibodies that bind to the hMPV F protein. A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. The data suggest that a monoclonal antibody could be used prophylactically to prevent lower respiratory tract disease caused by hMPV.

FIG. 1.

Depiction of the epitopes recognized by the hMPV F protein-specific monoclonal antibodies. Each circle represents an individual epitope on hMPV F, with the MAb binding to that epitope shown inside the circle. MAb numbers inside the intersection of circles are those monoclonal antibodies that have recognition sites comprised of a portion of two epitopes.

FIG. 2.

In vivo protection against NL100 challenge. Golden Syrian hamsters were injected 24 h prior to intranasal challenge with NL100 with different does of MAb 234 (solid circles), MAb 338 (solid triangles), or with BSA (solid squares). Animals were bled prior to challenge to determine the levels of serum antibodies present at time of challenge. At 4 days postinfection, lungs (panel A) and nasal turbinates (panel B) were harvested, and virus titers were determined as described in Materials and Methods. The limit of detection (LOD) for the viral titers was 1.2 log/g tissue. For MAb quantification, animal serum samples were diluted 1:100 and 1:500 (panel C). The limit of the quantitation (LOQ) for this assay as performed was 0.1 μg MAb/ml serum. P values for MAb 234 were <0.0001, 0.0004, 0.0013, and 0.0042 for doses of 3 mg/kg, 1 mg/kg, 0.3 mg/kg, and 0.1 mg/kg, respectively. P values for MAb 338 were <0.0001, <0.0001, 0.0014, and 0.0145 for doses of 3.0 mg/kg, 1.0 mg/kg, 0.3 mg/kg, and 0.1 mg/kg, respectively. The nasal turbinate P value, indicated by an asterisk, was 0.008. IgG, immunoglobulin G.

FIG. 3.

In vivo protection against NL199 challenge. Golden Syrian hamsters were injected 24 h prior to intranasal challenge with NL199 with different doses of MAb 234 (solid circles), MAb 338 (solid triangles), or with BSA (solid squares). Animals were bled prior to challenge to determine the levels of serum antibodies present at time of challenge. At 4 days postinfection, lungs (panel A) and nasal turbinates (panel B) were harvested, and virus titers were determined as described in Materials and Methods. Limit of detection (LOD) for the viral titers was 1.2 log/g tissue. For MAb quantification, animal serum samples were diluted 1:100 and 1:500 (panel C). The limit of quantitation (LOQ) for this assay as performed was 0.1 μg MAb/ml serum. P values for MAb 234 were <0.0001, <0.0001, 0.0016, and 0.325 for doses of 3 mg/kg, 1 mg/kg, 0.3 mg/kg, and 0.1 mg/kg, respectively. P values for MAb 338 were <0.0001, <0.0001, <0.0001, and 0.0064 for doses of 3.0 mg/kg, 1.0 mg/kg, 0.3 mg/kg, and 0.1 mg/kg, respectively. Nasal turbinate P values of statistical significance were 0.0006 () and <0.0001 (#). IgG, immunoglobulin G.