The aim of the present study was to determine whether immunity to huN1 provides sufficient cross-reactive immunity to avN1 to confer resistance to H5N1 influenza virus. To this end, we vaccinated female BALB/cJ mice twice with a plasmid encoding the NA of A/New Caledonia/20/99 (H1N1) to induce NA-specific immunity. A/New Caledonia/20/99 is the source of H1N1 antigen for currently available trivalent influenza vaccines. Readily detectable antibody responses to the homologous huN1 antigen were induced in nearly all mice, as shown by ELISA (A). In equivalent sera from control mice, titers of antibodies to huN1 were essentially undetectable, with a mean significantly different from huN1-immunized mice (p < 0.001). Using an equivalent ELISA, we also tested samples for antibody reactivity with NA of A/Vietnam/1203/04 (H5N1), which shares 80% amino acid identity with NA of A/New Caledonia/20/99. Reactivity with the heterologous avN1 was detected only in a small proportion of serum samples of the huN1 DNA vaccinated group (4/32) or the control mice (2/30) (B), and the difference in mean titers was statistically insignificant (p = 0.092). Sera from three mice in the huN1 DNA vaccinated group had avN1-specific titers greater than 100, whereas no sample from the control group possessed this level of reactivity.
NA-Specific Antibody Responses to Immunization
Mice vaccinated with huN1 DNA demonstrated robust resistance to challenge with 10 MLD50 of PR8-huN1 (A). Although all control mice lost a substantial amount of weight and died from this challenge infection, all of the vaccinated animals survived and largely recovered the weight they lost early in the infection. Protection of vaccinated mice against mortality from the homologous virus was highly significant (p < 0.001), which demonstrates the vaccine's potency. Challenge with 10 MLD50 of PR8-avN1 was also 100% lethal to control mice, whose disease course was similar to that in mice infected with PR8-huN1 (B). In addition, challenge with PR8-avN1 at this dose caused greater than 20% mean weight loss by day 7 in the mice vaccinated with huN1 DNA. However, a subset of vaccinated mice (4/11) regained weight and survived, evidence that partial cross-protection resulted from the immune response to huN1. The difference in mortality between immunized and control groups upon heterologous PR8-avN1 challenge approached but did not reach statistical significance (p = 0.055). All mice vaccinated with huN1 DNA were protected from mortality upon a 100 MLD50 challenge dose of PR8-huN1 (p < 0.0001), but cross-protection against heterologous PR8-avN1 at this 10-fold higher dose was weak (2/10 survival, p = 0.26, unpublished data).
Challenge of huN1-Immunized Mice with Influenza Viruses Possessing Homologous or Heterologous NA Genes
The third viral challenge with H5N1 strain A/Vietnam/1203/04 was highly lethal, as previously reported [11
]. Infection with 10 MLD50
of A/Vietnam/1203/04 killed all control mice (C). Disease resulting from infection with this H5N1 virus was more prolonged than that caused by the PR8-based challenge viruses, and the infected naïve mice showed a somewhat biphasic pattern of weight loss often accompanied or followed by hind leg paralysis. In contrast, mice vaccinated against huN1 typically had more moderate weight loss and fewer of them showed neurological signs. Half (5/10) of the vaccinated mice recovered from challenge with the H5N1 influenza virus, which demonstrates statistically significant protection from mortality (p
= 0.016). Against a 10-fold higher challenge dose of H5N1 virus (100 MLD50
) vaccination with huN1 DNA failed to protect against mortality (unpublished data).
In an additional experiment we addressed the possibility that the protection conferred by huN1 DNA against heterologous challenge was a result of nonspecific stimulation of the innate immune system, such as by CpG DNA motifs. Along with mice receiving huN1 DNA recombinant plasmid, a group of control mice received only the backbone plasmid without a gene insert. Vaccination with the empty vector failed to protect any animals against the lethal effects of A/Vietnam/1203/04 (). Owing to small treatment groups in this experiment, the difference in mortality between mice receiving huN1 DNA (4/8) versus saline (1/7) was not statistically significant (p = 0.18), but the difference between huN1 DNA and empty vector treatment (0/7) approached significance (p = 0.051). Thus, we exclude nonspecific immune stimulation by plasmid DNA as the mechanism for cross-subtype protection.
Sequence Dependence of huN1 DNA Vaccine Induced Protection against Lethal H5N1 Infection
We hypothesized that protection from H5N1 influenza by huN1 immunization is mediated by the humoral immune response. To test this hypothesis, we pooled sera from mice injected twice with huN1 DNA (prechallenge), from mice injected with saline only, or from mice that had recovered from infection with H5N1 influenza virus, and we transferred the sera intraperitoneally to naïve mice before challenging them with 10 MLD50 of A/Vietnam/1203/04. Serum from survivors of infection protected all recipient mice from severe disease and death upon homologous challenge, whereas mice that received serum from saline-injected animals were highly susceptible to this challenge (1/13 survival) (). In comparison, serum from huN1 DNA–vaccinated mice was partially protective: 46% (6/13) of recipient mice survived challenge with A/Vietnam/1203/04, which represents a statistically significant difference from naïve serum (p = 0.037). This outcome closely mirrors that of the vaccinated animals themselves. This set of results demonstrates that huN1-induced immunity against H5N1 virus is mediated, at least in large part, by a humoral response. The considerable weight loss of the passively immunized mice that survived challenge in this experiment might reflect a contribution by cell-mediated immunity to cross-lineage protection. Alternatively, the discrepancy in recovery may be attributable to the dilution of immune serum in passively immunized mice, the lack of memory B cells in these mice, or stress resulting from the serum transfer procedure.
Cross-Protective Effects of huN1-Immune Serum against H5N1 Influenza
We also tested serum samples from human volunteers for reactivity with avN1. Analysis of these samples by NA inhibition assay demonstrated reactivity with H1N1 influenza virus A/New Caledonia/20/99 in 31 of 38 individuals tested (). NA inhibition titers in these samples ranged from less than 20 to 320 or higher. We detected low inhibitory activity (titers between 20 and 80) against the NA of A/Hong Kong/213/03 in eight individuals and against the NA of A/Vietnam/1203/03 in nine individuals. Of these H5N1–reactive samples seven had reactivity to both H5N1 viruses. Although this human donor sample size is small, the results establish that some individuals have functionally significant levels of avN1-reactive antibodies.
Reactivity of Human Donor Sera with NA of H1N1 and H5N1 Influenza Viruses