Versatile and Scalable Nanoparticle Vaccine as a Scaffold Against Newly Emerging Influenza Viruses

Abstract

Influenza remains a major health threat due to its high contagiousness and global spread, affecting not only humans but also agricultural livestock and wild animals through transmission via migratory birds. Despite over 70 years of vaccination, influenza still creates epidemics and pandemics, and the ongoing use of vaccination is an essential but currently insufficient strategy. In this study, we assessed the immunogenicity and efficacy of an AP205 virus-like particle (VLP) carrying the HA head domain of the A/PR8/H1N1 strain, administered intranasally and subcutaneously in mice. For this purpose, the entire head region of A/PR8/H1N1 was genetically integrated into a sterically improved version of AP205, which exhibits capsid monomers fused into a dimer, thereby offering inexpensive and scalable production processes. The vaccine induced strong systemic anti-HA IgG and IgA antibodies via both routes, with no significant difference in the levels of IgG. Both immunisation strategies induced protection against a lethal challenge with H1PR8 mouse-adapted influenza virus. The findings demonstrate the potential of the AP205 VLP platform for HA1-based influenza vaccines and its applicability for controlling influenza in both humans and livestock.

Keywords: influenza; public health; vaccine; virus-like particle.

Figure 1

Design, production, and verification of the HA1–AP205dim fusion vaccine. (a) Schematic representations of the generation of the HA1–AP205dim vaccine. In a first step, the HA1 head domain of A/Puerto Rico/8/1934/H1N1 was genetically fused to the N-terminus of dimerised capsid protein. The capsid protein consists of a dimerised AP205dim coat protein. (b) SDS-PAGE analysis (12%) of the purified HA1–AP205dim fusion protein, showing a prominent band at the expected molecular weight. M, protein marker; lanes 1–3, purified HA1–AP205dim. (c) Western blot conducted with α-AP205 antibody, with AP205 as control VLP. M: protein marker; 1: HA1–AP205dim; and 2: AP205 control. The Western blot confirms the correct size of the HA1 to AP205dim fusion (Red Star). (d) Transmission electron microscopy (TEM) image of the purified HA1–AP205dim VLPs, scale bar is set to 200 nm. Figure (a) was created with Biorender.com (accessed on Q1–Q2 2025).

Figure 2

Subcutaneous and intranasal immunisation with the HA1–AP205dim vaccine induced strong specific antibodies in sera. (a) Schematic of the vaccination and sampling timeline. Mice were immunised on day 0 and boosted on day 28 with 40 μg of HA1–AP205dim either subcutaneously (s.c.) or intranasally (i.n.). Sera were collected weekly and analysed on day 49. After day 49, mice were challenged with a 2 × LD50 dosage of A/PR8/H1N1 mouse-adapted influenza virus and monitored for two weeks. (b,c) Serum IgG responses to recombinant HA protein (A/Puerto Rico/8/1934(H1N1)) measured via ELISA. Log-transformed OD50 values were calculated from dilution curves. (d,e) Specific serum IgA responses to HA, assessed via endpoint titre. (f) IgG subclasses in blood sera. HA1–A/PR8/H1N1-specific IgG-subclass titres on day 49 measured via ELISA Log₁₀ (OD₄₅₀) shown in either vaccinated s.c. or i.n. (g) Avidity index of HA-specific IgG in serum. Statistical analysis (mean ± SEM) using Student’s t-test. p ≤ 0.01 (**). One representative graph is shown. Vaccine and control groups n = 6. Figure (a) was created with Biorender.com (accessed on Q1–Q2 2025).

Figure 3

Subcutaneous and intranasal immunisation with HA1–AP205dim induces strong specific antibody in the bronchoalveolar fluid (BAL). (a,b) HA–A/PR8/H1N1-specific IgG in BAL on day 49 measured via ELISA in either vaccinated s.c. or i.n. AP205dim was used as a control vaccination, both s.c. and i.n. (c,d) HA-specific IgA endpoint titre in BAL. IgA was detected only in i.n.-immunised mice. (e) IgG subclasses in BAL. HA1–A/PR8/H1N1-specific IgG-subclass titres on day 49 measured via ELISA Log₁₀ (OD₄₅₀) shown in either vaccinated s.c. or i.n. Statistical analysis (mean ± SEM) using Student’s t-test. p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***). Control group n = 3 (s.c., i.n.), vaccine group n = 6 (s.c., i.n.). For (e) vaccine group n = 5 (i.n.) and n = 6 (s.c.) (BAL).

Figure 4

HA1–AP205dim-induced antibodies can bind efficiently to the HA protein and can neutralise (H1N1/A/PR8/1934) virus in vitro. (a,b) Average BLI sensorgrams generated from six individual replicates showing binding of sera from mice immunised intranasally (i.n.) or subcutaneously (s.c.) with HA1–AP205dim to recombinant HA protein (A/Puerto Rico/8/1934(H1N1)). A monoclonal antibody against the HA1 domain was used as a positive control with four individual replicates. Sera from AP205dim-immunised mice served as negative controls (n = 6). (c) Comparison of dissociation rate constants between sera from s.c.- and i.n.-immunised mice and a monoclonal antibody. The decrease in signal was recorded during the dissociation phase in the buffer. (d) Virus neutralisation test (VNT) against A/Puerto Rico/8/1934(H1N1) virus. Neutralisation titres are reported as the serum dilution that achieves 50% neutralisation (ND₅₀). One representative graph is shown. Statistical analysis (mean ± SEM) using one-way ANOVA. p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.0001 (****). Control group n = 6 (s.c., i.n., sera), vaccine group n = 6 (s.c., i.n., sera), and n = 4 (positive control, mAb).

Figure 5

Immunised mice with HA1–AP205dim are protected against A/PR8/H1N1 live virus challenge. (a) Survival rate of intranasally challenged mice. Mice were challenged with 2 × LD₅₀ of live virus for 14 days. (b) Body weight difference from day 0 until day 14 and survival rate. We showed that s.c. and i.n. immunizations of mice with HA1–AP205dim vaccines prevent body weight loss and confer protection against a lethal challenge with A/PR8/H1N1 virus. Body weight change (%) following viral challenge over 14 days, last point carried forward. One representative graph is shown. Data represent mean ± SEM. Survival was analysed via the Mantel–Cox log-rank test, p values are denoted as follows: p ≤ 0.01 (**), p ≤ 0.001 (***). Control group n = 6 (s.c., i.n.); vaccine group n = 6 (s.c., i.n.).