Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies

Abstract

Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process constrained by dated technology and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here we use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited haemagglutination inhibition antibody titres more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin-nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.

Figures

Figure 1.. Molecular design and characterization of ferritin nanoparticles displaying influenza virus HA.

a, A subunit of H. pylori nonheme ferritin (PDB: 3bve) (left). The NH2- and COOH-termini are labeled as N and C, respectively. Three subunits surrounding a three-fold axis are shown (middle) and the Asp5 is colored in red. An assembled ferritin nanoparticle and an HA trimer (PDB: 3sm5) (viewed from membrane proximal end) (right). A triangle connecting the Asp 5 residues at the three-fold axis is shown in red. The same triangle is drawn on the HA trimer (right). A schematic representation of the HA-ferritin fusion protein is shown (bottom). b, Negatively stained TEM images of nanoparticles (np) (left and middle). Computational models and observed TEM image (right, top and bottom panels) representing octahedral 2-, 3- and 4-fold symmetries of HA-nanoparticles are shown as indicated. Visible HA spikes are numbered in the images.

Figure 2.. Immune responses in HA-nanoparticle-immunized mice.

a, HAI (left), IC90 neutralization (middle), and anti-HA ab endpoint titers (right) after two immunizations of TIV or HA-nanoparticles with or without (−) Ribi. b, HAI (left) and IC90 (right) titers after two immunizations of TIV with MF59 or HA-nanoparticles with or without (−) MF59. Two of five mice immunized with MF59-adjuvanted HA-nanoparticles exhibited IC90 titer >51,200 and were plotted as 51,200. The data are presented as box-and-whiskers plots (boxed from lower to upper quartile with whiskers from minimum to maximum) with lines at the mean (n=5). c, Neutralization breadth of the immune sera (with Ribi). IC50 titers against a panel of H1N1 pseudotyped viruses were determined. Heat map is colored in gradient from green to yellow to red reflecting the neutralization strength. d, Cellular (left and middle) and humoral (right) immune responses against H. pylori and mouse ferritins. Cells expressing IFN-γ, TNFα, or IL-2 upon stimulation with peptides covering H. pylori or mouse ferritins were combined and plotted as cytokine+. The data are presented as box-and-whiskers plots with lines at the mean (n=5).

Figure 3.. Protective immunity induced in ferrets immunized with the HA-nanoparticles.

a, HAI (left), IC90 (middle), and anti-HA ab endpoint titers (right) against 1999 NC HA. Immune sera were collected after the first (1) and second (2) immunizations. The data are presented as box-and-whiskers plots with lines at the mean (n=6). b, Protection of immunized ferrets from 2007 Bris virus challenge. Challenge was performed with 106.5 EID50 via intranasal inoculation. Virus titers in the nasal washes were determined by TCID50 assay (left). The mean viral loads with s.d. at each time point were plotted (n=6). Change in body weight after virus challenge was monitored (right). Each data point represents the mean percent change in body weight from day 0 (pre-challenge) with s.e.m. (n=6).

Figure 4.. Improved neutralization breadth and detection of stem- and RBS-directed abs.

a, Breadth of serum neutralization in immune ferrets. IC50 titers against a panel of H1N1 pseudotyped viruses (left) and HAI titers against 1934 PR8 and 2007 Bris H1N1 viruses (right) were determined. Heat map is colored as in Figure 2. HAI titers are presented as box-and-whiskers plots with lines at the mean (n=6). b, Stem- and RBS-directed abs elicited by HA-nanoparticle immunization. Immune sera were pre-absorbed with ΔStem (left) and ΔRBS (middle) HA-expressing cells and analyzed for their binding to WT and a respective mutant (Δ) HA. The mean endpoint titers were plotted with s.d. (n=6). Binding of ΔStem HA pre-absorbed immune sera to HA pre-incubated with a control or CR6261 mAbs (right). Each symbol represents the titer of an individual ferret (n=6). c, Neutralization competition with WT, ΔStem or ΔRBS HA (left). The neutralization of HA-nanoparticle-immune sera was measured in the presence of indicated competitor proteins. Percent neutralizations at serum dilution 1/200 (1986 Sing and 2007 Bris), 1/800 (1995 Beijing) or 1/3,200 (1999 NC) were plotted. Each symbol represents an individual ferret and mean is indicated as a red line with s.d. (n=6 except for 2007 Bris (n=3)). The relative contributions of stem- and RBS-directed neutralization were calculated and plotted as mean percent (n=6).