A broadly neutralizing HuMAb, 5A7, was established using PBMC from a vaccinated healthy volunteer. This antibody neutralizes influenza B virus strains isolated between 1985 and 2006 that belong to the Yamagata or Victoria lineage. Moreover, therapeutic efficacy was shown in mice even when the HuMAb was administered 48 or 72 hours after viral challenge. As previously reported for almost all MAbs broadly neutralizing influenza virus, 5A7 recognized the stalk region of the HA protein 
. Importantly, the epitope region recognized by 5A7 is highly conserved in influenza B virus and the divergent strains occur only sparsely (Table S2
). Although influenza B virus has a much slower mutational rate than that observed for influenza A subtypes like H1 and H3, cocirculation of two phylogenetically and antigenically distinct lineages of influenza B virus leads to annual epidemics in humans 
. Dreyfus et al.
first reported broadly neutralizing HuMAbs against influenza B virus in September 2012 
. The epitope regions they identified were distinct from that of our HuMAb, 5A7. Characterization of the epitope region recognized by such a HuMAb could therefore provide insight for the development of a universal vaccine.
The high degree of conservation of amino acid residues in the epitope region implies that influenza B virus would not easily induce mutation in this region. Indeed, amino acid residues in the epitope region did not mutate even when the virus was passaged ten times under 5A7-treatment conditions. It could be considered that the poor inhibitory activity of 5A7 (weak HI, fusion inhibition and VN50
) is the cause of failure to establish escape mutants. However, a previous report showed that escape mutants could be prepared even from an MAb with weak fusion inhibition and complete neutralizing activities (25 and 50 µg/ml, respectively), and without HI activity, in order to map its epitope 
. Since 5A7 shows similarly weak activity, we would also expect to have been able to prepare 5A7 escape mutants and determine the epitope region. Failure to establish escape mutants in the presence of 5A7 could be an advantage for its development as a therapeutic tool, and also for designing the next generation of globally effective vaccines.
Generally, MAbs recognizing the globular head show strong HI activity, whereas those against the stalk region usually show none 
. Thus, it is considered that MAbs against the globular head inhibit the receptor binding step, while MAbs against the stalk region inhibit the fusion step, in viral replication 
. In fact, HuMAbs, 3A2 and 10C4, that recognize the 190-helix in the globular head near the receptor binding site, showed strong HI activity, which suggests that they can inhibit viral binding to the receptor on the host cells. Their ability to inhibit the fusion process (Figure S1
) implies that they could secondarily disturb the low pH-dependent structural change in HA by binding to the globular head. Surprisingly, although 5A7 reacted to the stalk region it also showed specific and weak HI activity. This suggests that MAb recognizing the stalk region distal from the viral membrane could affect the ability of the virus to bind to the receptor. As reported for MAbs reacting to the stalk region, 5A7 also showed specific fusion inhibition activity 
. These results suggest that HuMAb 5A7 could inhibit viral entry by preventing receptor binding, and the subsequent fusion process.
Previous reports show that the concentration of MAbs necessary for viral neutralization was much higher for those recognizing the stalk region of the envelope protein than for those reacting with the globular head in influenza viruses 
, as well as other viruses 
. In agreement with these reports, the concentration required for viral neutralization in this study was higher for 5A7 than 3A2 and 10C4 (). Such results can be explained by either a difference in binding affinity or in physical accessibility of HuMAbs to the epitope region. 5A7 showed similar KD
with 10C4 in binding kinetics analysis ( and Figure S2
), indicating that 5A7 has more difficulty physically accessing the epitope region of the HA protein. Modifying the HuMAb structure to enable easier access to the epitope region and improving its binding affinity, as described 
, could lead to the development of better therapeutic HuMAbs for influenza.
HuMAb 5A7 had specific therapeutic efficacy in mice even when administered after viral challenge (). Two independent passive transfer experiments were performed. Surprisingly, in both experiments, mice treated with 5A7 HuMAb 72 hours post-infection had a better survival rate than those treated 48 hours post-infection. It is reported that in the first 24 hours after infection, the levels of lymphocyte apoptosis increase transiently in both nasal-associated lymphoid tissue and spleen, and cellular immune suppression occurs 
. Temporal cellular immune suppression or infection-mediated endogenous signals could therefore interfere with the efficacy of exogenous HuMAb. These results imply that the timing of HuMAb treatment could be critical for efficacy, and therefore, injection at several time points may be necessary.
Mouse-adapted B/Ibaraki/2/1985 was used to examine the kinetics of survival rate and body weight change in passive transfer experiments because other viral strains are not lethal to mice, even if passaged several times in vivo. HuMAb 5A7 protected mice against mouse-adapted B/Ibaraki/2/1985 viral challenge although 5A7 was obtained from a volunteer vaccinated with B/Florida/4/2006 and had shown the lowest sensitivity to this viral strain in vitro (). These results suggested that 5A7 would have therapeutic efficacy against a wide spectrum of influenza B viruses and, in fact, lung viral titers of both mouse-adapted B/Ibaraki/2/1985 and B/Florida/4/2006 were reduced significantly under 5A7-treatment conditions (). Further study using both mice and ferrets with several viral strains is needed to confirm the wide-ranging therapeutic potential of 5A7 in vivo.