In this study we have built combinatorial libraries and selected a panel of human monoclonal antibodies against H5 HA from a particular compartment of memory B cells that are characterized by their continued expression of IgM on the surface. Our results suggest that IgM+
memory B cells express mAbs recognizing T-dependent antigens at high affinity, and consistent with a hallmark of memory B cells, all selected H5N1 mAbs contained mutated VH genes. Memory B cells are also generally defined by their rapid proliferation and differentiation into antibody secreting plasma cells on re-exposure to antigen. Yet from this study it is not clear that the IgM+
memory B cells expressing the cross-neutralizing HA specificities participate in a true recall response upon vaccination. An increase in serum neutralizing activity was measured in all vaccinated donors for H1N1 but not H5N1, even in donor #1020 from which the heterosubtypic neutralizing mAbs were isolated. Thus if the B cell clones expressing CR6261 and the other cross-reactive mAbs were present in the donor prior to vaccination (as would be expected) then a true recall response associated with differentiation into plasma cells has not likely occurred. Although we can not exclude the possibility that at later time points serological differences could have been measured the results are consistent with the observation that measurable heterosubtypic immunity is rare in the general population 
We have not made a comparative analysis of other B cell compartments to investigate whether related clones are present in isotype switched memory cells or plasma cells of donor #1020 or other donors. However, preliminary data for a mAb isolated from the bone marrow of an H5N1 infected patient was recently reported that possesses cross-neutralizing activity between H5N1 and H1N1 
. Remarkably this mAb uses the IGHV1-69 germline gene and conserves the hydrophobic residues at the tip of the HCDR2 loop that we show in this study to be critical in binding the conserved hydrophobic pocket of HA. Unfortunately without further data it can only be inferred that this mAb is binding to the same region as CR6261 and other mAbs described in this study. The ease at which the IgM+
memory B cell repertoire can be accessed in normal, infected and vaccinated donors compared to bone marrow and the diversity in this repertoire compared to antibody secreting cells 
supports this approach to generate therapeutic mAbs against other pathogenic targets and should be further explored.
The epitopes of CR6261, CR6323 and the other cross-neutralizing mAbs were localised to the stem domain of HA using a combination of homology modelling, mutagenesis and mapping with escape variants. This region is highly conserved and, based on comparisons at a sequence and structural level, appears to segregate into two groups one containing H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16 subtypes and the other H3, H4, H7, H10, H14 and H15 subtypes. The neutralizing mAbs described in this study recognize antigenic determinants in the first group that confer different levels of neutralization potency against these subtypes but do not recognise representative virus strains from subtypes in the second group tested so far. Escape mutants of a murine anti-H2N2 mAb (C179) with cross-neutralizing activity to H1 subtypes but not H3 subtypes have also placed its epitope in this region 
. C179 has relatively weak neutralizing and protective activity against H1 and H5 viruses (e.g. protection from a lab adapted LPAI H5N2 strain required 70 mg/kg of C179) 
. Preliminary evidence suggests C179's mechanism of action is related to fusion inhibition rather than blocking of attachment 
. Although we have not examined the neutralisation mechanism of the human mAbs identified in this study they, like C179, do not have hemagglutination inhibition (HAI) activity. Importantly, we show that mAbs like CR6261 targeting this region can have potent prophylactic and therapeutic activity and that humans are capable of generating antibodies directed to this region. Vaccines with broad heterosubtypic protective activity are being actively pursued, particularly against matrix protein 2 (M2) 
, however many uncertainties remain about their effectiveness 
. Vaccination studies have been performed with the HA2 subunit but have not demonstrated robust protective responses in experimental models 
. The information provided here on the conserved antigenic site in the HA stem domain could provide a new avenue for targeted vaccine strategies.
Development of clinical interventions against a potential influenza pandemic are challenged by uncertainty over future pandemic viral subtypes 
, antigenic variability within subtypes and the potential for antiviral resistance to emerge 
, as recently observed in H1N1 strains 
. The mAb CR6261 identified in this study potentially addresses these issues. First, it neutralises most of the virus subtypes that currently show pandemic potential (i.e. H2, H5, H6 and H9 viruses). Second, it was demonstrated to neutralise all five H5N1 virus clades and subclades tested, which represent viruses isolated from 1997 to 2006. The generation of CR6261 neutralisation escape variants required extensive viral passaging suggesting that alteration of its epitope is not easily accomplished without negatively influencing virus infectivity. The prophylactic and therapeutic utility of CR6261 against different H5N1 strains and influenza A subtypes argue for its development as an important alternative or adjunct to currently stockpiled pandemic interventions.