The 2009 pandemic H1N1 virus induces anti-neuraminidase (NA) antibodies that cross-react with the NA of H5N1 viruses in ferrets

Abstract

A miniaturized neuraminidase inhibition (NI) assay using HA-mismatched H6 reassortant viruses was performed to examine the neuraminidase (NA)-specific antibody response in ferrets immunized with live-attenuated influenza vaccine (LAIV) strains. The strains tested possessed different NAs derived from seasonal H1N1 and H3N2, 2009 pandemic H1N1, and the highly pathogenic influenza H5N1 virus. The anti-NA antibodies from the 2009 pandemic strain (A/California/7/2009) immunized ferrets cross-reacted with the NA of H5N1 but not with the NA of seasonal H1N1 viruses. The plaque size reduction assay confirmed the cross-reactivity between the NAs of A/California/7/2009 and the H5N1 virus. Sequence and structural analyses of these N1 NA proteins showed that the NA of the 2009 pandemic H1N1 strain shared at least 22 more amino acids in the head domain with the NAs of the avian H5N1 strains than with the NAs of seasonal human H1N1 viruses. Our data demonstrated LAIV-induced NA antibody responses in ferrets and cross-reactive NA antibodies induced by 2009 pandemic H1N1 and H5N1 LAIV viruses.

Figure 1

Detection of ferret anti-NA antibodies against different NA antigens by the miniaturized colorimetric NI assay. One serum sample from each of the pre-immune (pre) and post-immune ferret groups (anti-CA09, anti-HK03, or anti-SD07) was serially diluted and incubated with H6 reassortants bearing NA antigens from CA09 (A), HK03 (B) or SD07(C). Fetuin substrate was added and NA activity was tested. The OD550 value compared to that of the virus control (%) at each serum dilution was determined and plotted. The data represent the average of duplicate experiments and the standard error is indicated for each data point.

Figure 2

(A) A phylogenetic tree of the N1 proteins. The NA amino acid sequences of indicated viruses were aligned and plotted using the Mega software. The bar indicates the genetic distance. (B) Molecular surface structure of the NA of A/California/7/2009 (Protein Data Bank 5NSS, one monomer shown). The locations of the amino acids in the CA09 NA that are identical to both H5N1 strains (HK03 and VN04) but different from the two seasonal H1N1 strains (NC99 and SD07) are shown in green; the amino acids of the CA09 NA that are identical to the seasonal H1N1 strains but different from the H5N1 strains are shown in red. The amino acid positions are indicated by N2 numbering. The activity cavity (AC) is indicated and the catalytic sites are shown in wheat color. The picture was generated by using PyMOL software.

Figure 3

Plaque size reduction assay. A total of approximately 50 PFU of the indicated H6 reassortants with the indicated NA segment was inoculated in each well of a 6 well plate of MDCK cells. Serial dilutions of ferret preimmune serum, anti-pH1N1 CA09 serum, anti-H5N1 HK03, anti-H1N1 SD07, and anti-H1N1 NC99 serum were added to the agarose overlay. The cells were incubated at 33°C for 4 days. The plaques were visualized by immunostaining.