In this study, we show that prevaccination serum samples have baseline heterosubtypic HA Ab binding activity to both Group 1 and 2 HA subtypes, including H5 and H7, to which these subjects are most likely unexposed because of their US geographic location. The IgM and IgG Abs to H5 increased significantly after the H5N1 vaccination, whereas this did not happen for IgG Abs to H3. F10-like IgG Abs are also detected in pre-immune serum samples and increased significantly in H5N1 vaccinees. A low level of serum anti-HA Abs that bind and neutralize H5N1 viruses has been reported to be age and influenza exposure dependent [9
]. Other investigators have reported enhanced levels of HA-directed anti-H5N1 neutralizing Abs in healthy donors after boosting with unrelated human influenza H1N1/H3N2 seasonal vaccines [5
]. These serum Abs may be directed to the Group 1 stem pocket on HA. Indeed, Corti et al [5
] showed that a majority of 2007 and 2008 seasonal vaccinees had preexisting neutralizing Ab titers against pseudotyped H5N1 viruses that markedly increased after seasonal vaccination and that the HA stem-pocket directed Abs were present at relatively low levels, compared with Abs that bind to the globular head of HA.
We also observed an increase of H7 (Group 2) reactivity after H5N1 (Group 1) vaccination (). The precise location of the cross-binding epitope(s) is currently not known. Observations that support the possible presence of heterosubtypic Abs of the broader type that bind Group 1 and 2 viruses have also been seen in children after primary influenza infection [13
] and mucosal vaccination of experimental animals [14
]. In one study, a cross-neutralizing murine mAb with hemagglutination inhibition activity against Group 1 (H1, H2, H5, H9, and H13) and Group 2 (H3) was recovered from an intranasally vaccinated mouse. In contrast to the F10-like Abs, this mAb was directed to the globular head, and escape mutants were readily obtained [16
]. Other animal vaccine studies have shown that antibodies to the fusion peptide and an HA2 linear peptide can have broad reactivity to Group 1 and 2 HAs [17
] and neutralizing activity [20
], respectively. Whether different quantitative amounts of Group 1–specific BnAbs and/or Group 1 and 2–directed BnAbs are elicited by vaccination or natural infection remains an important but unanswered question of our study.
To quantify the baseline levels of anti-influenza heterosubtypic Abs in human serum, we used IVIG as representative of pre-immune IgG Ab composition in the general population, which includes individuals who are likely exposed to seasonal influenza A virus infection and/or vaccinations (H1N1 and H3N2). Other investigators have shown that a low level of heterotypic anti-HA Abs that bind and neutralize H5N1 viruses is present in IVIG from diverse geographic locations [9
]. These investigations further showed that these heterosubtypic anti-H5N1 Abs cross-react with H3N2 and H1N1; however, efforts to purify and characterize these Abs (beyond neutralization titers) have not been reported. Our data show that there are 2 populations of heterosubtypic Abs with different HA binding ability in IVIG: one can bind to HAs from both Group 1 and Group 2 viruses; the other is specifically directed against Group 1 stem pocket. Approximately 0.01% of IVIG (most certainly derived from H5- and H7-naive donors), which is purified by acid elution from H5-beads, has heterosubtypic binding activity to both Group 1 and Group 2 HAs. This fraction of IVIG Abs also demonstrated neutralizing activity against the H1N1 and H5N1 pseudotyped viruses. The F10-like stem pocket-directed Abs were also detected in IVIG and were recovered at ~10% of the levels of H5 binding Abs. As expected, these F10-like Abs displayed similar binding and neutralization profiles among the tested HAs and viruses as the Group 1 specific mAbs that are directed to the stem pocket of HA [1
]. For both Ab fractions, a broader range of other subtypes were not tested due to limited amount of the Abs that we could purify from IVIG (~1 μg and 0.1 μg/100 mg IVIG, respectively), larger-scale purification of H5 protein and other materials will be required to characterize these heterosubtypic BnAbs in more detail.
Although we quantitatively show that BnAbs that bind to Group 1 and 2 HAs are present at very low levels, as well as that stem pocket-directed F10-like BnAbs exist at even lower levels, the question of whether serum or IVIG has protective levels of either type of heterosubtypic BnAbs is not answered in our study. However, our quantitative data support the notion that the levels of these BnAbs are borderline or below titers that would traditionally be considered protective. For example, there is up to 1 μg of F10-like Abs/100 mg IVIG; assuming 10 mg/mL IgG in normal human serum, then concentrations up to 0.1 μg/ml of F10-like Ab could be present. Likewise, the fraction of acid eluted BnAbs with activities against Groups 1 and 2 could also be in this range. Furthermore, we did observe variability in these levels in our study patients (), and it remains possible that host factors, including VH
polymorphism, may impact the baseline and inducible BnAb levels. In addition, the origins of these 2 populations of heterosubtypic anti-HA Abs are unknown. The possibility that they may be a component of “natural” polyreactive Abs cannot be excluded [22
]. However, it is most likely that both our H5N1 vaccine study subjects and the IVIG donors had prior exposure to other Group 1 (H1N1) and Group 2 (H3N2) influenza A viruses, either through seasonal vaccinations and/or natural infection, and this may have given rise to heterosubtypic H5 and H7 binding Abs, respectively.
In summary, our findings show that the human immune system is capable of making BnAbs—not only to the conserved pocket on the HA stem of Group 1 viruses, but also to another unknown epitope(s) that are shared by Group 1 and 2 influenza A viruses. These observations provide the basis for further investigations aimed at obtaining a better understanding of these BnAbs, their origins, and the host genetic factors that restrict or enable their induction. [23
] These additional studies should bring us closer to developing a universal influenza vaccine that provides durable protection beyond seasonal vaccines and mitigates that ability of the viruses to undergo neutralization escape. Indeed, a recently reported vaccine regimen—which induced protective level of the Group 1 stem-pocket directed BnAbs in animals—provides experimental evidence that the same may be possible in man [24