Single immunization with an influenza hemagglutinin nanoparticle-based vaccine elicits durable protective immunity

Abstract

Vaccination is the most effective strategy to combat influenza. Ideally, potent and persistent vaccine effects would be induced with a single vaccine dose. Here, we designed a virus-like particle (VLP)-based vaccine presenting multiple copies of the influenza hemagglutinin (HA) from A/Puerto Rico/8/1934 (PR8HA-VLP) and examined its immunogenicity and protective efficacy in ferrets. Serum-neutralizing antibodies were effectively induced against the homologous virus at 3-week post-vaccination with a single dose of PR8HA-VLP with or without adjuvants. When the single-immunized ferrets were challenged with the homologous virus, virus replication in the nasal mucosa was significantly reduced. Long-term monitoring of serum titers revealed that after adjuvanted vaccination with PR8HA-VLP, neutralizing antibodies were retained at similar levels 20- to 183-week post-vaccination, although a 4- to 8-fold titer decline was observed from 3- to 20-week post-vaccination. Boost immunization at 183 weeks after the first immunization elicited higher neutralizing antibody titers than those at 3 weeks after the initial immunization in most of the animals. These results confirm that nanoparticle-based vaccines are a promising approach to effectively elicit durable multiyear neutralizing antibody responses against influenza viruses.

Keywords: VLP‐based vaccine; adjuvant; duration of immunity; ferret; influenza A virus; neutralizing antibody titers; virus‐like particles (VLPs).

FIGURE 1

Preparation of PR8HA‐VLP vaccine. (a) Schematic representation of PR8HA‐VLP (MS2‐biotin = dark blue, PDB: 2MS2; SA = green, PDB: 6J6J; PR8HA = red; PDB: 1RU7). (b) SDS‐PAGE characterization of VLP and PR8HA proteins. The unprocessed gel is shown in Supporting Information S1: Figure 2. (c) Characterization of PR8HA (red), VLP control (green), and PR8HA‐VLP (blue) by dynamic light scattering. Curves are an average of five measurements. (d) Size exclusion chromatography traces for PR8HA‐VLP (blue) and PR8HA (red). The dotted gray line represents the elution volume of the molecular weight standard thyroglobulin (660 kDa). The column void volume is 7.2 mL. (e) Characterization of the binding of stalk‐binding antibody CR6261 and head‐binding antibody H28‐E23 (Sb epitope) to PR8HA, PR8HA‐VLP, VLP control, and bovine serum albumin (BSA) (mean ± SD, n = 3: one independent assay with three technical replicates). SA, streptavidin; VLP, virus‐like particle.

FIGURE 2

Immunogenicity of PR8HA‐VLP in ferrets. (a) Timeline of the immunization/virus challenge study. Groups of ferrets (N = 3/group) were subcutaneously inoculated with MS2‐SA nanoparticles presenting PR8HA (PR8HA‐VLP; HA content: 45 μg) adjuvanted with or without AddaVax (500 μL), Quil‐A (30 μg), or poly(I:C) (3 μg), or inoculated with the same amount of VLP‐only as a control. (b) Serum‐neutralizing antibody titers at 3‐week post‐immunization were analyzed against PR8 virus in micro‐neutralization assays. Data shown are the geometric means of duplicates. Dashed lines represent the detection limit of the assay. Bars show the median of the groups. (c, d) At 3‐week post‐immunization, the animals were intranasally (i.n.) challenged with 10⁶ pfu of homologous PR8 virus. Virus titers in nasal swabs (c) and body temperature increase as a clinical symptom (d) were monitored daily for 7 days post‐challenge. (c) Bars show the median of the groups. (d) Data represent the means and SD of each group (N = 3). Statistical analyses were performed by using a one‐way analysis of variance and corrected for multi‐group comparison by using Dunnett's test. SA, streptavidin; VLP, virus‐like particle. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 3

Duration of neutralizing antibody titers after single PR8HA‐VLP immunization. Ferrets were subcutaneously vaccinated with PR8HA‐VLP (HA amount: 45 μg) adjuvanted with AddaVax (500 μL), Quil‐A (30 μg), or poly(I:C) (3 μg). Serum‐neutralizing antibody titers were then monitored. At 183‐week post‐immunization, the individual animals were boosted with the identical vaccination regimen, and the neutralization titer at 3‐week post‐boost (i.e., 186 weeks after the first immunization) was analyzed. Data points show the values of individual animals connected by lines for each time point. Dashed lines represent the detection limit of the assay. VLP, virus‐like particle.