Prior to the present study, the vast majority of nAbs isolated against influenza A virus have targeted the receptor-binding head and lacked broad cross-neutralizing activity. However, a murine nAb, termed C17939
, was positively selected on the basis of its cross-neutralization properties (of H1 and H2 subtypes), and subsequently shown to neutralize H5, but not Group 2 subtypes39,40
(Supplementary Note 2
). Moreover, C179 was shown to block membrane fusion rather than cell attachment and to protect mice against viral challenge41
, although a detailed mechanism was not reported. We compared the activities of C179 and F10 and found that both showed similar binding towards H5. We also found that F10 efficiently competed with C179 for binding to H5, but not vice versa
(Supplementary Fig. 9
). Furthermore, the point mutant V522
E abrogated binding to both Abs, while T3181
K only affected C179 binding. These results suggest that F10 and C179 have partially overlapping epitopes and that their modes of action are similar.
The manner in which HA was presented to the antibody phage display library in this study seems to have been critical in our success, since similar attempts to isolate broadly nAbs using cell-surface expressed HA showed only partial success against H5, and most Abs recognized linear epitopes42
. As noted above, we repeatedly isolated nAbs that utilize the same VH germline gene (IGHV1-69 or “VH1-69”). Huang et al.43
have pointed out that this is the only VH gene that consistently encodes 2 hydrophobic residues at the tip of its CDR-H2 loop; indeed, it is the only germline gene to encode a Phe at this position, which makes several critical interactions with H5. Moreover, the “type 2” H2 loop, which is long and compact, is only predicted to occur in 4 out of the ~50 human germline genes. These factors may explain at least in part the remarkable ability of nAbs derived from this germline gene to cross-react with viral epitopes: their unusual ability to bind to conserved hydrophobic pockets. Such pockets are likely to have an important function and for this reason they are often cryptic in the unactivated state of the antigen. For example, VH1-69 is the predominant gene utilized by a group of CD4-induced (“CD4i”) nAbs raised against the HIV-1 surface glycoprotein, gp120, where the “pocket” is part of a conserved co-receptor binding site that is only exposed transiently upon binding to its primary receptor, CD443
. Similarly, an antibody raised against the HIV gp41 trimeric “inner-core” fusion protein intermediate utilizes the hydrophobic tip of its VH1-69 CDR-H2 loop to insert into a conserved hydrophobic pocket that blocks further assembly to the fusion-competent 6-helix structure44
. In vivo
, B cells carrying the VH1-69 gene are the primary mediators of innate defense against HCV infection, generating antibodies against its membrane fusion glycoprotein, E245
, although the epitope and mode of action have not been determined. Notably, as we found in the current study, VH1-69 is not the only germline that is suitable for achieving neutralization in a similar manner. Another recent example is a nAb against Ebola virus surface glycoprotein, KZ52, which uses the VH3-21 germline46
. However, their common ability to lock viral envelope proteins into a non-fusogenic conformation offers support for the possibility of a general strategy for broad-spectrum and/or potent viral neutralization.
Recent work using immune-based phage-display libraries generated from B cell populations of patients who survived H5N1 infection resulted in the isolation of three human nAbs that neutralized both H1 and H5 viral strains. The authors postulated that the reason for survival was an effective humoral immune response mediated by such nAb-generating B cells in vivo47
, although no control populations were studied. Analysis of their data (Supplementary Note 3
) indicates that the antibodies are also derived from the VH1-69 germline gene, and share other key characteristics, including the Met-Phe pair in CDR-H2 and a tyrosine at the tip of CDR-H3. Thus it would appear, at least in this case, that our non-immune (H5-naive) donor library approach generated antibodies with characteristics very similar to those found using immunized-donor-based phage-display libraries derived from H5N1 survivors.
Why broad-spectrum nAbs similar to those identified in our study are not similarly generated/expanded during successive rounds of influenza infection and repeated vaccination is not known (Supplementary Note 4
), and warrants further investigation. It is unlikely that the F10 epitope provokes self-tolerance mechanism(s) via auto-antigen mimicry48
(Supplementary Note 5
). Rather, we hypothesize that an immunodominant Ab response to the highly-exposed globular head may overwhelm the Ab response to the F10-epitope, although it remains possible that other immune exclusion mechanism(s) may preclude natural Ab responses against the F10 epitope. It is not surprising that many viruses are highly adept at keeping their most critical (and conserved) determinants of pathogenesis cryptic, in which case subunit-based vaccines, utilizing properly presented fragments of F10 or F10-like epitopes, may offer distinct advantages over whole-virus-based approaches for the induction of broad spectrum nAbs in vivo49,50
In summary, we have used in vitro
methodologies to isolate a family of high affinity broad-spectrum human nAbs against HA that show potent in vitro
and in vivo
efficacy against both highly pathogenic H5N1s and H1N1s. We show that they inhibit the post-attachment fusion process by recognizing a highly conserved epitope within the stem region of HA at a point where key elements of the conformational change are brought into close apposition. Our initial experiments suggest that this region is recalcitrant to the generation of escape mutants. The prospects for their use for passive immunotherapy would therefore seem to be excellent, either alone or in combination with small molecule inhibitors (Supplementary Note 6
). Finally, our structural work pinpoints the reasons why Group 2 HAs do not bind the nAbs described here: despite surface sequence similarities, they form a structurally distinct group, but one that is also highly conserved and therefore may be amenable to a similar panning discovery approach.