Cross-reactive neuraminidase antibodies afford partial protection against H5N1 in mice and are present in unexposed humans

Abstract

Background: A pandemic H5N1 influenza outbreak would be facilitated by an absence of immunity to the avian-derived virus in the human population. Although this condition is likely in regard to hemagglutinin-mediated immunity, the neuraminidase (NA) of H5N1 viruses (avN1) and of endemic human H1N1 viruses (huN1) are classified in the same serotype. We hypothesized that an immune response to huN1 could mediate cross-protection against H5N1 influenza virus infection.

Methods and findings: Mice were immunized against the NA of a contemporary human H1N1 strain by DNA vaccination. They were challenged with recombinant A/Puerto Rico/8/34 (PR8) viruses bearing huN1 (PR8-huN1) or avN1 (PR8-avN1) or with H5N1 virus A/Vietnam/1203/04. Additional naïve mice were injected with sera from vaccinated mice prior to H5N1 challenge. Also, serum specimens from humans were analyzed for reactivity with avN1. Immunization elicited a serum IgG response to huN1 and robust protection against the homologous challenge virus. Immunized mice were partially protected from lethal challenge with H5N1 virus or recombinant PR8-avN1. Sera transferred from immunized mice to naïve animals conferred similar protection against H5N1 mortality. Analysis of human sera showed that antibodies able to inhibit the sialidase activity of avN1 exist in some individuals.

Conclusions: These data reveal that humoral immunity elicited by huN1 can partially protect against H5N1 infection in a mammalian host. Our results suggest that a portion of the human population could have some degree of resistance to H5N1 influenza, with the possibility that this could be induced or enhanced through immunization with seasonal influenza vaccines.

Figure 1. NA-Specific Antibody Responses to Immunization

Serum was collected from BALB/cJ mice after two injections with plasmid encoding the NA of human H1N1 influenza strain A/New Caledonia/20/99 (huN1) or saline only. Antibody reactivities with huN1 and the NA of H5N1 influenza strain A/Vietnam/1203/04 (avN1) were determined by ELISA. (A) Between the huN1 DNA and saline-only treatment groups there was a statistically significant difference in mean IgG titers against huN1 (p < 0.001). (B) IgG titers detected against avN1 were not significantly different between the treatment groups (p = 0.092). The antibody titer against each target was defined as the reciprocal of the highest serum dilution that produced an ELISA signal twice as intense as the signal from equivalently diluted naïve serum. The lower limit of detection was a titer of 20.

Figure 2. Challenge of huN1-Immunized Mice with Influenza Viruses Possessing Homologous or Heterologous NA Genes

Mice immunized twice with huN1 DNA or saline alone (control) were inoculated intranasally with PR8-huN1 (A), PR8-avN1 (B), or A/Vietnam/1203/04 (C). Mean weight change and survival data are shown. Differences in survival between huN1 DNA-vaccinated and saline control groups were statistically significant for PR8-huN1 challenge (p < 0.001) and A/Vietnam/1203/04 challenge (p = 0.016), and approached but did not reach significance for PR8-avN1 challenge (p = 0.055). Error bars represent standard deviation values. Statistical comparisons are by Fisher's exact test.

Figure 3. Sequence Dependence of huN1 DNA Vaccine Induced Protection against Lethal H5N1 Infection

Mice vaccinated at week 0 and week 3 with huN1 DNA (n = 8), saline (n = 7), or empty plasmid vector (n = 7) were challenged with 10 MLD₅₀ of A/Vietnam/1203/04 (H5N1) at week 6. Survival was monitored daily. Although the difference in mortality between saline-injected and huN1 DNA–vaccinated groups did not reach statistical significance (p = 0.18), the difference in mortality between huN1 DNA vaccinated mice and those given empty vector DNA approached significance (p = 0.051). Statistical comparison is by Fisher's exact test.

Figure 4. Cross-Protective Effects of huN1-Immune Serum against H5N1 Influenza

Mice were passively immunized with serum from mice that had recovered from H5N1 influenza virus infection (post-H5N1), were vaccinated twice with huN1 DNA (N1 DNA), or were vaccinated twice with saline alone. Eighteen hours after passive immunization, recipient mice were challenged with 10 MLD₅₀ of A/Vietnam/1203/04. Changes in mean weight (left graph) and survival (right graph) were monitored over 24 d. The difference in survival between recipients of serum from huN1 DNA-vaccinated and saline control mice was statistically significant (p = 0.037). Error bars represent standard deviation values. Statistical comparison is by Fisher's exact test.

Figure 5. Reactivity of Human Donor Sera with NA of H1N1 and H5N1 Influenza Viruses

Serum samples from human donors were analyzed by NA inhibition assay for reactivity with the NA proteins of A/New Caledonia/20/99 (H1N1), A/Hong Kong/213/03 (H5N1), and A/Vietnam/1203/04 (H5N1).