In our evaluation of the effectiveness of a seasonal trivalent split inactivated influenza vaccine (Fluvax) to protect ferrets against lethal H5N1 influenza challenge, we have confirmed the results of other studies demonstrating partial protection (9
). However, in this study we have shown that the addition of an adjuvant (either Iscomatrix adjuvant or AlPO4
) to the seasonal influenza vaccine affords complete protection in ferrets against severe disease and death due to the highly pathogenic H5N1 virus A/Vietnam/1203/04. Protection was induced by the H1N1 and not the H3N2 component of the trivalent vaccine and is therefore unlikely to be due to cross-reactive responses to viral antigens other than HA and NA, as these are derived from PR8 virus in both cases. This would tend to rule out certain targets, such as the M2 protein or conserved epitopes for T cells on internal proteins, as the dominant inducers of protection in this model and implicate the H1 HA and/or N1 NA.
Extensive analysis using a variety of classical assay approaches (HI, VN, ELISA, Western blotting) and a newly developed sensitive assay (AF) failed to reveal antibodies in prechallenge sera from ferrets that cross-reacted with the challenge virus. This is consistent with our previous studies that have shown protection from a lethal homologous H5N1 challenge could be induced in the absence of a classical HI and VN serological response (16
). Furthermore, our findings with seasonal adjuvanted vaccines are consistent with those from other studies where cross-protection was demonstrated in the absence of detectable cross-reactive antibodies (10
). Nevertheless, we show here that antibodies to the H1N1 NA could inhibit the cleavage of fetuin by H5N1 virus, and the levels of these antibodies provided a strong correlate of protection against severe disease and death following H5N1 challenge. This implies that the N1 NAs of seasonal H1N1 and avian H5N1 viruses have one or more epitopes in common in the region affecting the enzyme-active site. The cross-reactivity with H5N1 NA was induced by a broad range of NAs, illustrated here by NA from the A/Brevig Mission/1/1918, A/New Caledonia/20/99, and A/Brisbane/59/2007 viruses. Alignment of the amino acid sequences of the NAs of these H1N1 strains with the sequence of the NA from the A/Vietnam/1203/2004 strain demonstrated overall identities in the protein sequence of 89.8% (Brevig Mission/1/18), 84.0% (A/Brisbane/59/2007), and 84.7% (A/New Caledonia/20/99) sequence and 77%, 53%, and 56%, respectively, within the putative antigenic sites (25
). It would be important to extend this panel of viruses in future studies, which may aid in the definition of the common antigenic site by sequence and structural comparisons.
Responses to the H1 HA appeared to play an additional role in cross-protection, but the nature of these responses remains elusive. Compared on an equivalent weight basis, the recombinant N1 NA appeared to be slightly more cross-protective than the purified H1 HA, but more animals would need to be tested to be confident in this conclusion. Despite the finding that the recombinant NA was essentially devoid of sialidase activity, sufficient structural integrity of the critical epitope(s) around the cleavage site appears to have been maintained, in order to induce antibodies that inhibit the sialidase activity of H5N1 virus. Whether these NI antibodies provide the effector mechanism for protection is unknown, but this is certainly a possibility. NI antibodies, like NA inhibitor antiviral drugs, may function by reducing the efficiency of release of nascent virus from the infected host cell. This occurs by blocking the ability of NA to remove sialic acid from receptors on the cell that would otherwise trap the virus during exit. Furthermore, the carbohydrate side chains of newly formed NA and HA glycoproteins have terminal sialic acids that need to be removed in order to prevent clumping of the virus during budding. Additional but untested mechanisms by which NI antibodies may contribute to the protection against severe disease and death may include binding to NA expressed on the surface of infected cells (26
), resulting in cytotoxicity via either complement-mediated or antibody-dependent cellular cytotoxicity (ADCC) mechanisms (27
), thereby reducing the overall viral load on the host. In turn this may allow the developing H5N1-specific immune responses to achieve clearance more easily. Alternately, the antibodies could bind to virus particles and promote their opsonization, also leading to reduced viral loads.
Our findings are supported by those of other researchers using different modes of delivery of NA in experimental vaccines. Easterbrook et al. (28
) showed that mice could be protected from a lethal H5N1 infection by intranasal vaccination with virus-like particles (VLPs) containing pandemic H1N1/09 NA (A/California/04/2009) and matrix proteins from a seasonal H1N1 virus (A/New York/312/2001); however, if the NA was replaced with a seasonal N1 (A/Bethesda/NIH50/2009), this protection was lost. Sandbulte et al. (13
) used DNA vaccines encoding the NA from A/New Caledonia/20/99 (the same viral antigen used in this study) to protect 50% of mice from lethal H5N1 infection, even though detectable N1 antibody was only present in 12.5% of mice. Similar studies were reported with cross-reactive NA antibodies using live attenuated vaccines in ferrets (29
). These studies demonstrate that the degree of homology of the NA used in the vaccine compared to that of the NA in the challenge virus may also be an important determinant in protection or survival.
In the initial challenge study, ferrets received two vaccine doses (21 days apart), each containing 30 μg of HA in order to maximize the likelihood of an outcome in naive animals. Having demonstrated protection using this level of antigen, all subsequent challenge experiments were undertaken using vaccines containing 15 μg of HA. Although we have not performed a formal dosing study, the level of NA-specific antibodies induced by the vaccine appears to be critical to achieving heterologous protection in this model. Without adjuvant, only half the ferrets vaccinated with seasonal trivalent vaccine survived challenge, and these showed reduced activity. The same vaccine formulated with either AlPO4
or Iscomatrix adjuvant, both of which are known to boost antibody responses (30
), led to survival of all animals, with only a very slight reduction in activity. This infers that cross-reactive antibodies to NA may well be generated by routine seasonal vaccination of humans, but their levels cannot be assumed to be sufficient to ensure protection against a possible H5N1 challenge. The levels of cross-reactive NI antibody in humans and the ability to boost this by infection or seasonal vaccination warrant further investigation.
The context of the NA in the vaccine in relation to the other antigens present may also be important. In the monovalent H1N1 vaccine based on A/Brisbane/59/2007, protection against death and severe disease was seen with vaccines with NA content as low as 0.34 μg (and 15 μg HA), but the monovalent H3N1 vaccine given at a dose that resulted in a similar NA content of 0.55 μg (and 330 μg HA) gave only 41% survival. This may be due solely to the added benefit of the H1 HA. However, we postulate that an additional consideration is the low NA/HA ratio in the H3N1 virus, which can arise in these reassortants (J. Cobbin, S. Rockman, and L. E. Brown, unpublished data) to restore the correct balance of activities between HA and NA during replication (31
). We showed that the sialidase activity of H3N1 virus was approximately 12-fold less than that of the H1N1 virus, and this was due to the presence of correspondingly less NA in the H3N1 virion. The dominance of the HA in terms of amount is thought to lead to its immunodominance in wild-type viruses (32
), and this may become even more pronounced by the further reduction of the NA/HA ratio in the reassortant, resulting in even less antibodies to the NA being produced. Thus, the context of the NA in the vaccine, in terms of the subtype and/or amount of the HA with which it is paired, may well be important.
Unlike previous studies, this study has investigated the subcomponents of seasonal influenza vaccines and concluded that the cross-protection against H5N1 observed with trivalent seasonal or H1N1 preparations formulated in adjuvant can be attributed to both NA and HA, but the production of neuraminidase-inhibiting antibodies was a strong correlate for protection in this model. These findings suggest that consideration should be given to the development of adjuvanted NA-based or seasonal influenza vaccines to provide ongoing additional immunity to reduce the mortality and morbidity should a highly pathogenic H5N1 virus become the next pandemic. Given that ferrets in this study were protected from disease but not infection by the challenge virus, the benefit afforded by these vaccines may be a decrease in overall disease burden, which would potentially provide a window for the development of strain-specific immunity to the new virus, but they are unlikely to stop transmission. Our observations also strengthen the case for better monitoring of the NA content of vaccines and of the NA-specific responses that are induced by them. However, prior to doing so, it will be important to develop and validate appropriate assays that can be used globally so that results can be compared in a meaningful manner.