Antigenic sites of HA that are targeted by antibodies to neutralize influenza viruses have been defined previously by Brownlee and Fodor,31
using H1 residue numbering, as the strain-specific epitopes Sa (141–142, 170–174, 176–181) and Sb (201–212), as well as more conserved epitopes Ca1 (183–187, 219–222, 252–254), Ca2 (154–160, 238–239), and Cb (87–92). Comparison of the amino acid sequences of these epitopes in A/South Carolina/1/1918 (H1N1) (1918) with the 1976 swH1N1 vaccine strain, A/New Jersey/11/76 (H1N1) (1976), the 2009 pH1N1, A/California/07/2009 (H1N1), and the 2009 TIV H1N1 component, A/Brisbane/59/2007 (H1N1), shows 86·5%, 80·8%, and 53·8% homology, respectively. Human seasonal H1N1 viruses have been undergoing rapid antigenic drift from 1918–1957 and again since the late 1970s.32
The seasonal H1N1 viruses have also gained two highly conserved glycosylation sites, whereas the pandemic viruses do not have any glycosylation sites that can mask antigenic regions and further reduce binding of pre-existing cross-reactive antibodies.23
This suggests that HA-specific immunity, primarily in antigenic regions of HA, that is induced by the 1976 and 2009 pH1N1 vaccines would provide greater protection against 1918 challenge than seasonal TIV.
Antigenic regions of NA, specifically N1, have not been defined, but the total sequence homology to 1918 is similar among the H1N1 viruses examined: 87·4% for 1976 swH1N1, 87·2% for 2009 pH1N1, and 86·6% for the seasonal A/Brisbane/59/2007. Future research should be conducted to better define regions of influenza NA antigenicity. The 2009 pH1N1 received the NA from a Eurasian swine H1N1 virus and the seasonal H1N1 virus has undergone substantial antigenic drift, so one could hypothesize that the antigenic regions of the NA of the 2009 pandemic and seasonal H1N1 viruses would be less homologous to the antigenic regions of the 1918 NA than the NA of 1976 swH1N1 that was derived from 1918.
Mice immunized with the 1976 swH1N1 or commercial human 2009 pH1N1 vaccines survived a lethal challenge with 1918, lost no weight, and had 2–3 logs less virus in the lungs early in infection when compared to mock-vaccinated mice. This was comparable to recently reported protection from a lethal infection with 1918 observed in mice immunized with an inactivated 2009 pH1N1 reassortant virus.24
Similarly, laboratory and epidemiological data support cross-protection between 2009 pH1N1 and 1918.17–19,21–23
Infection induces T-cell-mediated responses (e.g. CD8+
T-cell responses), in addition to humoral responses, which may contribute to observations of greater protection against challenge with a non-homologous, but closely related, virus than protection following vaccination with inactivated influenza viruses that primarily induces humoral immunity.33,34
In contrast to 1976 and 2009 pH1N1 vaccination, immunization with the 2009 seasonal TIV offered only partial protection from a lethal 1918 infection. This is consistent with other human and animal studies that demonstrate little protection against a challenge with 2009 pH1N1 or 1918 after infection or immunization with seasonal H1N1 viruses or vaccines.16,20–24,35
The protection from death for some mice vaccinated with the 2009 TIV may be a result of antibodies to conserved epitopes of HA or matrix protein (M2),36
or the possible minor induction of IFN-γ
T cells or CD8+
T cells. In humans, the H1N1 antibody response is likely boosted or primed by seasonal influenza infection or vaccination, but does not offer complete protection because of high variability in the antigenic regions of HA and NA.
Mice immunized with 1976 swH1N1 or 2009 pH1N1 vaccines induced MN titers (>150) against 1918, whereas administration of the 2009 TIV or vehicle alone did not neutralize against 1918. Protection against viral replication, weight loss, and death was evident in mice that had measurable MN titers against the 1918 virus, but no detectable or very low (<40 GMT) HI or NI titers, supporting MN as a means to evaluate vaccine efficacy. Such protective antibodies may be detected by MN assay, but not HI or NI assays, because they bind to HA epitopes that are not closely associated with the receptor-binding domain, have specificity for conserved epitopes on antigens other than HA, or have low affinity for HA or NA. The laboratory-produced whole virus vaccines were made as similarly as possible to the commercial vaccines, including the use of vaccine strains when available (1976 swH1N1) and inactivation with β-propiolactone, but HA quantification was performed by densitometry to determine the percentage of HA of total protein in the laboratory vaccines and by single radial immunodiffusion (SRID) analysis for the commercial vaccines, so discrepancies in the amount of HA administered may be possible.
Virus neutralization was due primarily to cross-reactive antibodies to HA that were elicited by vaccination. Neuraminidase, though to a lesser extent than HA, also may contribute to cross-protection among related influenza viruses. Recombinant NA protein, in the absence of other influenza virus proteins, can induce NA-specific antibodies, reduce the replication of both homologous and heterovariant virus, and suppress weight loss in mice by reducing virus release from cells.26
Additionally, high levels of population immunity to the 1957 pandemic N2, as well as the N2 of subsequent seasonal strains, may have lessened the severity of the 1968 pandemic in which the HA, but not NA, underwent reassortment.37
In our experiments, mice that received the 1976 vaccine had lower titers of virus in the lungs than mock-vaccinated mice throughout infection, whereas mice that received the 2009 pH1N1 vaccine had less virus in the lungs that was limited to early infection at 2 dpi. It is possible that this may be because of a reduced ability of the virus to be released from cells and spread efficiently by cross-reactive NA-inhibiting antibodies elicited by vaccination with the 1976 vaccine that were not evident following 2009 pH1N1 vaccination. The 2009 pH1N1 and 2009 seasonal TIV vaccines were commercially derived and elicited low or no antibody responses to the NA of the appropriate homologous virus. This likely reflects steps in the manufacturing process that do not retain a stable immunogenic form of NA, while NA activity itself was maintained. The role of NA in cross-protection is often overlooked38
and should be recognized in vaccine development and evaluation of population immunity. Future studies will examine the role of NA in protection against influenza infection and whether NI titers from a laboratory-produced 2009 pH1N1 vaccine that possesses an immunogenic NA can inhibit the activity of the 1918 NA.
Mice infected with a lethal dose of 1918 have necrotizing bronchitis and bronchiolitis and moderate to severe alveolitis that is composed of neutrophils, lymphocytes, and macrophages, with accompanying edema and hemorrhage and viral antigen is distributed throughout the lungs.39,40
Mock and 2009 TIV-vaccinated mice demonstrated similar pathology following infection with 1918, while immunization with 2009 pH1N1 or 1976 vaccines significantly reduced the extent of pathology and the amount of viral antigen in the lungs. These data correlated with survival data and the amount of infectious virus in the lungs as determined by plaque assay, thus reinforcing that the amount of 1918 virus in the lungs directly correlates with pathology.
The 1976 vaccination campaign may have helped protect some of the normally high-risk older population during the 2009 pH1N1 outbreak, as well as any potential future exposure to a 1918-like H1N1. Similarly, exposure to the 2009 pH1N1 and the associated vaccination campaign also may contribute to protection against an accidental release or zoonotic re-introduction of 1918 or 1918-like influenza. Vaccination is an effective method of protection against influenza infection and the associated morbidity and mortality, but vaccine development should optimize the range and duration of protection. Vaccines should not only elicit antibodies against HA but also induce other protective responses, such as antibodies against other surface antigens, including NA and possibly M2, as well as innate, antiviral, and T-cell responses. Thus, neutralization of influenza infection may be a better correlate of protection for vaccines than the more limited and currently accepted measurement of the ability to inhibit HA activity, as expressed by HI titers.