Neuraminidase-based recombinant virus-like particles protect against lethal avian influenza A(H5N1) virus infection in ferrets

Abstract

Avian influenza A (H5N1) viruses represent a growing threat for an influenza pandemic. The presence of widespread avian influenza virus infections further emphasizes the need for vaccine strategies for control of pre-pandemic H5N1 and other avian influenza subtypes. Influenza neuraminidase (NA) vaccines represent a potential strategy for improving vaccines against avian influenza H5N1 viruses. To evaluate a strategy for NA vaccination, we generated a recombinant influenza virus-like particle (VLP) vaccine comprised of the NA protein of A/Indonesia/05/2005 (H5N1) virus. Ferrets vaccinated with influenza N1 NA VLPs elicited high-titer serum NA-inhibition (NI) antibody titers and were protected from lethal challenge with A/Indonesia/05/2005 virus. Moreover, N1-immune ferrets shed less infectious virus than similarly challenged control animals. In contrast, ferrets administered control N2 NA VLPs were not protected against H5N1 virus challenge. These results provide support for continued development of NA-based vaccines against influenza H5N1 viruses.

Keywords: Ferret; H5N1 influenza; Neuraminidase (NA); VLP vaccine.

Fig. 1. Cloning and characterization of NA recombinant influenza virus-like particle (VLP)

VLPs were constructed in a baculovirus genetic background with the sequences of the genes for HA and NA sequences of A/Indonesia/05/2005 (H5N1) or A/Brisbane/10/2007 (H3N2) virus and produced in recombinant baculovirus infected Spodoptera frugiperda (Sf9) cells. (A) Influenza HA, NA and M1 genes were cloned into pFastBac1 baculovirus transfer vector in a tandem manner with each gene under a polyhedron promoter. The H5, N1, and M1 genes were from A/Indonesia/05/2005 (H5N1); and H3 and N2 genes were from A/Brisbane/10/2007 (H3N2). (B) Purified VLPs were analyzed using SDS-PAGE (left panel) and a Western blot against a mixture of sheep anti-H5, rabbit anti-N1 and mouse anti-influenza A M1 serum (right panel). The identity of protein bands are labelled for influenza protein including H3 and H5 HA, N1 NA and M1, and baculovirus proteins gp64, p37 and p6.9. VLP samples were loaded at the same total protein levels. The sample in lane 1 represents only 2 proteins (N1 and M1) compared to samples in lanes 2, 3, 4, which express 3 proteins (HA, M1, N1). (C) Total protein concentration (BCA), SRID values for H5 and H3 HA; N1 and N2 NA activity by MUNANA assay. ND, not done.

Fig. 2. Selected transmission electron microscope (TEM) micrographs of NA VLPs

VLPs containing NA were negatively stained with 1% phosphotungstic acid, and examined directly by TEM using a Hitachi H-7600. Bar, 100 nm.

Fig. 3. Protective efficacy of NA VLP vaccines to A/Indonesia/05/2005 (H5N1) virus challenge in ferrets

Ferrets (4/vaccine group) received two intramuscular inoculations of VLP vaccines in a 0.5 ml volume. Six ferrets received PBS in place of vaccine. Vaccine groups are as follows: H5/N1/M1 (▼), H3/N1/M1 (■), H3/N2/M1 (●), N1/M1 (▲), and mock (PBS) (◆). Ferrets were challenged intranasally (5 weeks following boost) with 10⁶ PFU of A/Indonesia/05/2005 (H5N1) virus and observed daily for body temperatures (A), and weight loss for 14 days (B). Virus shedding was measured on 4 and 6 days post-challenge and is expressed as the log₁₀ EID₅₀/ml + SD (C). The weight loss among groups was analyzed by one-way ANOVA based on calculated Area Under the Curve (AUC) by day 6 post-challenge. Nasal wash titers among vaccine groups were analyzed by two-way ANOVA with GraphPad Prism software. ***p < 0.001, *p < 0.01, compared to PBS group.

Fig. 4. Weight loss and viral shedding in NA-immune and control ferrets following H5N1 virus challenge

Ferrets (4–5/vaccine group) received two intramuscular inoculations of VLP vaccines in a 0.5 ml volume and were challenged with 10⁶ pfu of A/Indonesia/05/2005 (H5N1) virus five weeks after final vaccine boost. (A) Mean weight loss of ferrets following H5N1 virus challenge are shown at various days post challenge (p.c.). (B) Nasal washes were collected on indicated days p.c. and titrated for virus and are expressed as the log₁₀ EID₅₀/ml + SD. Weight loss by day 5 post challenge was analyzed by one-way ANOVA based on calculated AUC. Differences in viral titers of nasal washes between groups were analyzed by two-way ANOVA with GraphPad Prism software. ***p < 0.001, **p < 0.01, *p < 0.05 compared to control group.