Neuraminidase-inhibiting antibody is a correlate of cross-protection against lethal H5N1 influenza virus in ferrets immunized with seasonal influenza vaccine

Abstract

In preparing for the threat of a pandemic of avian H5N1 influenza virus, we need to consider the significant delay (4 to 6 months) necessary to produce a strain-matched vaccine. As some degree of cross-reactivity between seasonal influenza vaccines and H5N1 virus has been reported, this was further explored in the ferret model to determine the targets of protective immunity. Ferrets were vaccinated with two intramuscular inoculations of trivalent inactivated split influenza vaccine or subcomponent vaccines, with and without adjuvant, and later challenged with a lethal dose of A/Vietnam/1203/2004 (H5N1) influenza virus. We confirmed that vaccination with seasonal influenza vaccine afforded partial protection against lethal H5N1 challenge and showed that use of either AlPO(4) or Iscomatrix adjuvant with the vaccine resulted in complete protection against disease and death. The protection was due exclusively to the H1N1 vaccine component, and although the hemagglutinin contributed to protection, the dominant protective response was targeted toward the neuraminidase (NA) and correlated with sialic acid cleavage-inhibiting antibody titers. Purified heterologous NA formulated with Iscomatrix adjuvant was also protective. These results suggest that adjuvanted seasonal trivalent vaccine could be used as an interim measure to decrease morbidity and mortality from H5N1 prior to the availability of a specific vaccine. The data also highlight that an inducer of cross-protective immunity is the NA, a protein whose levels are not normally monitored in vaccines and whose capacity to induce immunity in recipients is not normally assessed.

Fig 1

Response of ferrets vaccinated with seasonal trivalent or monovalent split influenza vaccines after challenge with a lethal dose of H5N1 virus. Ferrets were immunized with Fluvax seasonal trivalent split inactivated influenza vaccine (TIV), TIV formulated with AlPO4 or Iscomatrix adjuvant (IMX), or the individual monovalent H1N1 or H3N2 components of the TIV with Iscomatrix adjuvant or PBS alone, as indicated. The vaccines contained 30 μg HA of each component (i.e., 30 μg of each strain in the case of TIV) and were delivered as two inoculations 3 weeks apart. The ferrets were challenged with 10⁶ 50% egg infectious doses of wild-type A/Vietnam/1203/04 (H5N1) virus 4 weeks after the last immunization. (A) Weights of individual ferrets on days 0, 3, 4, 5, 6, 7, and 14 after challenge. (B) Activity scores for the indicated days after challenge out to day 14. Scores are depicted for each ferret by a strip of colored squares corresponding to the activity of the animal on the different days as indicated in the legend. Squares that are half-black indicate the activity score on the day of culling at the humane endpoint. Data are for individual ferrets, each assigned a number.

Fig 2

Response of ferrets to H5N1 challenge after vaccination with monovalent split or recombinant protein vaccines and the NA composition of these vaccines. (A) Activity scores of ferrets vaccinated with two doses, 3 weeks apart, of monovalent H1N1, H3N2, or H3N1 vaccines containing 15 μg HA or 1.5 μg rNA or rNP, all of which were formulated with Iscomatrix (IMX) adjuvant. Ferrets were challenged 4 weeks later with wild-type A/Vietnam/1203/04 virus and assigned activity scores as described in the legend to Fig. 1. (B) Relative neuraminidase activity in vaccine preparations was assessed in the fetuin cleavage assay. All split virus vaccine preparations were standardized to equivalent protein concentrations of 45.5 μg/well and the rNA to 6.25 μg/well in the first well, and then serial 2-fold dilutions were performed. (C) The level of neuraminidase protein in the vaccine preparations was determined by Western blotting. Lanes were loaded with 20 μg per split virus preparation. To estimate the level of NA content, rNA was used as a standard starting at 2 μg per lane and with further 2-fold serial dilutions of this preparation. A monoclonal antibody to a conserved region of N1 NA present in all viruses examined was used to probe the blots (S. Rockman, unpublished data).

Fig 3

The presence of serum antibodies inhibiting the neuraminidase activity of H5N1 virus in prechallenge ferret sera. Sera from ferrets given monovalent vaccines, recombinant proteins, or PBS in the experiments shown in Fig. 1 and 2 were titrated for neuraminidase inhibition activity. The titers of individual ferrets that did not survive the challenge are shown as filled circles, while the survivors are shown in open circles. The geometric mean of each group is indicated by a line.

Fig 4

Reactivity of prechallenge ferret sera in the AF assay. Pre- and postvaccination sera were collected from the ferrets immunized with PBS or two of the H1N1 or H3N2 preparations containing 30 μg HA (described in the legend to Fig. 1) or from ferrets immunized with an equivalent dose of H5N1 split virus preparations formulated with Iscomatrix adjuvant for our previous study (16). The sera were tested by the AF method for reactivity on Luminex beads coated with recombinant HA from either A/Vietnam/1203/04 (H5N1), A/Brisbane/59/07 (H1N1), or A/Wisconsin/67/05 (H3N2). The normalized mean fluorescence intensity, nMFI, is plotted as the geometric mean of the group of 4 ferrets (or 3 in the case of H1N1), and the error bars represent the standard errors of the means. White bars, prevaccination serum; gray bars, postvaccination serum.