Human monoclonal antibodies broadly neutralizing against influenza B virus

Abstract

Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus.

Figure 1. Nucleotide and amino acid sequences of the V_H and V_L of three HuMAbs 5A7 (A), 3A2 (B) and 10C4 (C).

Closest germline sequences in NCBI database by IgBlast softwares were aligned. Complementarity-determining regions (CDRs) are indicated in red, blue and pink (CDRs 1, 2 and 3, respectively). V-D and D-J junctions in VH and V-J junctions in VL are shown in white, light gray and dark gray colors, respectively.

Figure 2. Reactivity of three HuMAbs.

In vitro VN assay was performed with HuMAbs 5A7 (red), 3A2 (blue),10C4 (green) and control IgG (D23-1B3B9; black). HuMAbs (100 µg/ml) were serially four-fold diluted. The percentage of neutralization was estimated as the viral infectivity under HuMAb-treated conditions compared with that without HuMAb. Upper panels are Yamagata lineage viruses and lower panels are Victoria lineage viruses.

Figure 3. Epitope mapping of HuMAbs.

(A) Reactivity of 5A7 with truncated HA. A series of truncated forms of HA (a to f) were prepared in an expression plasmid as depicted in the diagram. Transfected 293T cells were subjected to western blotting with 5A7 or serum from a mouse infected with B/Ibaraki/2/1985 as a control (Ms serum). Black bars in the HA diagram indicate reactivity with 5A7. Arrowed region is the estimated region involved in recognition. (B) Expression plasmids bearing one point mutated B/Florida/4/2006 HA protein were prepared. 293T cells expressing the mutated HA were subjected to IFA with 5A7 (upper panels) or Ms serum as a control (lower panels). Wild type B/Florida/4/2006 (wt) and pCAGGSII vector without an insertion (empty) were used as controls. (C) Epitope map of the HuMAbs in the three-dimensional structure of the HA trimer (left models) and monomer (right models). The amino acid positions identified by epitope mapping are 315 to 324 (dark yellow), 316, 318 and 321 (pink), 194 and 196 (blue), 131 and 227 (green). The epitope region of each HuMAb is circled.

Figure 4. Therapeutic efficacy of 5A7 in mice.

(A) Mice were treated intraperitoneally with HuMAb 5A7 at 1 (red line), 5 (blue), 10 (green), or 15 (orange) mg/kg or with control IgG at 10 mg/kg (black) 4 hours after intranasal injection of a lethal dose (1.47×10³ MLD₅₀/mouse) of mouse-adapted B/Ibaraki/2/1985. Survival and body weight were checked daily. Each group consists of five mice. Body weight is shown as the mean ± SEM of five mice. (B) Mice were treated with 5A7 (+) or control IgG (D23-1B3B9; −) at 10 mg/kg 4 hours post-infection with 1.47×10³ MLD₅₀/mouse of mouse-adapted B/Ibaraki/2/1985 (left panel) or 5.0×10³ FFU/mouse of B/Florida/4/2006 passaged eight times in mouse lung (right panel). The titers in lungs were calculated at day 3 and day 6 post-infection. Each group consists of five mice (except control IgG-treated group with mouse-adapted B/Ibaraki/2/1985 at day 6, which consists of four mice as one accidentally died before the lung could be collected). Black bars are mean values. **: P<0.01 compared to control IgG-treated group. (C) Two independent experiments were similarly performed (Left two panels). Mice were given 10 mg/kg HuMAb 5A7 at 4 (red line), 24 (blue), 48 (green) or 72 (orange) hours post-infection with mouse-adapted B/Ibaraki/2/1985 (1.47×10³ MLD₅₀/mouse). Right panel shows 10 mg/kg control IgG-treated group at 4, 24, 48 or 72 hours post-infection. Survival and body weight were checked daily. Each group consists of five mice per experiment. Body weight is shown as the mean ± SEM of five mice.