A recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo

Abstract

Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is a major cause of lower-respiratory-tract disease. hMPV is associated with more severe disease in infants and persons with underlying medical conditions. Animal studies have shown that the hMPV fusion (F) protein alone is capable of inducing protective immunity. Here, we report the use of phage display technology to generate a fully human monoclonal antibody fragment (Fab) with biological activity against hMPV. Phage antibody libraries prepared from human donor tissues were selected against recombinant hMPV F protein with multiple rounds of panning. Recombinant Fabs then were expressed in bacteria, and supernatants were screened by enzyme-linked immunosorbent assay and immunofluorescent assays. A number of Fabs that bound to hMPV F were isolated, and several of these exhibited neutralizing activity in vitro. Fab DS7 neutralized the parent strain of hMPV with a 60% plaque reduction activity of 1.1 mug/ml and bound to hMPV F with an affinity of 9.8 x10(-10) M, as measured by surface plasmon resonance. To test the in vivo activity of Fab DS7, groups of cotton rats were infected with hMPV and given Fab intranasally 3 days after infection. Nasal turbinates and lungs were harvested on day 4 postinfection and virus titers determined. Animals treated with Fab DS7 exhibited a >1,500-fold reduction in viral titer in the lungs, with a modest 4-fold reduction in the nasal tissues. There was a dose-response relationship between the dose of DS7 and virus titer. Human Fab DS7 may have prophylactic or therapeutic potential against severe hMPV infection.

FIG. 1.

Light-microscopic and immunofluorescent images of hMPV-infected LLC-MK2 cell monolayers stained with human anti-hMPV F Fab and AlexaFluor568-conjugated goat anti-human IgG. (A and B) Fab ACN044; (C and D) Fab DS7. Magnification, ×20.

FIG. 2.

Schematic map of epitopes recognized by hMPV A2 F protein-specific MAbs. Each circle represents an individual epitope on hMPV A2 F protein, with the Fab binding to that epitope shown inside the circle. Fab names inside the intersections of circles are those that have recognition sites composed of a portion of two or three epitopes.

FIG. 3.

SPR analysis of DS7 Fab. (A) Association-dissociation curves of decreasing concentrations of DS7 against immobilized hMPV FΔTM protein. Palivizumab (RSV F-specific MAb) was used as an irrelevant control. (B) Association-dissociation curves of DS7 at 100 nM against immobilized hMPV FΔTM protein and RSV FΔTM protein.

FIG. 4.

Nasal (A) and lung (B) titers of hMPV. Groups are defined in the text. Tissue virus titers were log₁₀ transformed for statistical analysis. Comparisons between groups were made using a Wilcoxon rank sum test. Horizontal bars represent geometric means; the dotted line indicates the limit of detection (5 PFU/g).

FIG. 5.

Dose-response relationship between DS7 and nasal (A) and lung (B) titers of hMPV. Groups are defined in the text. Linear regression was used to examine the relationship between Fab DS7 dose and log₁₀-transformed viral titer, as described in text. The dotted line indicates the limit of detection (5 PFU/g).