Fig 1.
Nanoparticle design integrating the conserved helix-A of hemagglutinin (HA) onto a nanoparticle scaffold.
(A) Structure of influenza H1 HA ectodomain (PDBID 3LZG). HA1 is shown in red and HA2 in blue. (B) HA2 with the HA1 region computationally removed. The conserved helix-A is shown in green within the HA2 stem region. (C) Computationally extracted helix-A segments are shown. (D) The scaffold, hepatitis B virus (HBV) capsid dimer, showing the alpha helical fold of the protein. The immunodominant loop at the tip of the dimeric spike is denoted by an asterisk, which is also referred to as the c1 epitope. (E) Homology model of the designed H1-nanoparticle dimeric unit. The protein design consists of a capsid monomer with two copies of helix-A (green) inserted into the tip of the loop of the capsid protein (gray). Scale bar is 10 nm. (F) Structure of the scaffold (HBV capsid (PDBID 1QGT)), which has T = 4 icosahedral symmetry and 240 dimer subunits per capsid. (G) Homology model for the H1-nanoparticle with icosahedral symmetry. The surface has the helix-A stem epitopes of HA (green) on the surface with the capsid scaffold (gray) forming the base core of the nanoparticle. Scale bars 5 nm. (H) Analysis of purified H1-nanoparticle protein (H1-Nano) by SDS-PAGE with standards (std). (I, J) Images from negative-staining (panel I) and cryo-electron microscopy (panel J) of the purified H1-nanoparticles. Scale bars 50 nm. Arrows in Panel J denote particles and protein is black. (K) Reference-free 2D class averages of the H1-nanoparticle. Protein is white. Scale bar 5 nm. (L) Central-slice through the 3D reconstruction of the H1-nanoparticle. Black arrows denote surface spikes and epitope insertion points, where lower electron density is observed. Protein is black. Scale bar 5 nm.
Fig 2.
Probing the effect of temperature on H1 nanoparticle integrity and immunogenicity and storage effects on H1 and H7 nanoparticles.
(A, B, C) Negative-stained electron microscopy images of nanoparticles incubated at 4, 40, and 90°C. Particles were incubated for 60 min and then equilibrated to 25°C. Scale bars 100 nm. There are observed differences in stain penetration of the nanoparticles with a central cavity observed for 4°C (panel A) while there is less stain penetration and smoother looking particles for 40°C (panel B). Some aggregated particles appear in 90°C (panel C). (D) Schedule for immunization of mice with H1-nanoparticles with day 35 sera used in ELISA. (E) Comparison of sera reactivity to full-length recombinant H1 HA protein from mice immunized with temperature-treated H1-nanoparticles (H1-Nano) via ELISA with serially diluted sera. (F) Comparing the endpoint titer levels of different sera for reactivity to scaffold, H1-nanoparticle (H1-Nano) and full-length recombinant H1 HA protein (H1 rHA). There were four groups of sera tested consisting of PBS (used to determine threshold, not displayed) and H1-nanoparticle exposed to three temperatures (H1-Nano 4°C, 40°C, 90°C, displayed). ELISAs for panels E and F are independent experiments. (G, H, I) Negative-stained electron microscopy images of H1 and H7 nanoparticles stored at 4°C for three years. (G) H1 nanoparticles present intact particles (black arrows) and some broken particles (white arrows). (H) A different image area of H1 nanoparticles at a higher magnification. (I) H7 nanoparticles present intact particles (black arrows) and background protein (white arrows). Scale bars 100 nm. The symbol α is an abbreviation for “anti-”.
Fig 3.
Immunogenicity of H1 nanoparticle and analysis of sera reactivity to different proteins and assessment of H1 nanoparticle efficacy in mice.
(A) ELISA binding analysis of day 35 sera from mice immunized with H1-nanoparticles binding to different recombinant H1 HA proteins: H1 rHA full-length, H1 rHA ectodomain and H1 rHA1 head domain (blue bars). Controls were a mouse monoclonal stem antibody (C179, white bars) and a rabbit polyclonal serum to H1 HA (green bars). (B) Reactivity analysis via western blot of sera from mice immunized with H1-nanoparticle alone to H1-nanoparticle (H1-Nano), scaffold, recombinant H1 HA (H1 rHA), and (C) to recombinant H1 rHA1 head and H1 rHA2 stem proteins. (D) Evaluation of endpoint titer levels from immunized mice for days 14 and 35. There were three groups of sera tested, which consisted of saline (used to determine threshold, not displayed), H1-nanoparticle without adjuvant (blue bar), and H1-nanoparticle with adjuvant (red bar). Sera was tested for reactivity by ELISA to scaffold (Scaf), H1-nanoparticle (H1-Nano) and full-length recombinant H1 HA protein (H1 rHA). Recombinant proteins were from H1 HA of influenza (A/California/07/09) H1N1, and adjuvant was Sigma Adjuvant System (oil-in-water emulsion). H1 rHA stem ectodomain protein was based on a group 1 HA 1 stalk (stem) construct #4900, (Impagliazzo 2015). (E, top) Schedule for mouse immunization with H1-nanoparticle and challenge with H1N1 influenza virus. Groups of mice (N = 5 per group) received one of three types of intramuscular injections: PBS, H1-nanoparticle without adjuvant or H1-nanoparticle with adjuvant on day 0 and 21. Adjuvant was Sigma Adjuvant System. Mice were challenged with 10x MLD50 (50% Mouse Lethal Dose) of H1N1 (A/California/07/2009) virus on day 42. (E, bottom) Survival curves for mice immunized with PBS (black), H1-nanoparticle (blue), or H1-nanoparticle with adjuvant (red) after challenging with virus. (F) Weight-loss curves for challenged mice that were immunized with PBS control (black), H1-nanoparticle (blue), or H1-nanoparticle with adjuvant (red).
Fig 4.
Analysis of a library of helix-A nanoparticle constructs representing group 1 and group 2 hemagglutinin (HA) subtypes for expression and H7 and H5 nanoparticle immunogenicity.
(A) Maximum Likelihood (ML) phylogenetic tree of full-length sequences of influenza type A HA subtypes (H1-H18) classified into group 1 (blue) and group 2 (magenta) HAs. Scale bar indicates number of substitutions per site. (B) Assessment by western blots of expression of designed helix-A HA-nanoparticle proteins representing different HA subtypes (H1-H16) from a nanoparticle library as indicated. Antibody 10E11 which binds an endogenous epitope tag in the scaffold was used as primary antibody. Each HA subtype is denoted as H1, H2, etc., and asterisks denote detected bands. HA subtypes H4 and H14 have the same helix-A sequence and HA subtypes H10 and H15 have the same helix-A sequence and two panels represent the H4/H14 and H10/H15 helix-A nanoparticle constructs, respectively. (C, D) Purified H7-nanoparticles (panel C) and H5-nanoparitcles (panel D) observed by negative-staining electron microscopy. Scale bar, 50 nm. (E) Western blot displaying reactivity of H7-nanoparticle mice sera to recombinant H7 HA proteins from H7N9 (H7 rHA Anhui), and H7N7 (H7 rHA Netherland [Neth]) viruses. (F) Western blot displaying reactivity of H5 nanoparticle mice sera to recombinant H5 HA protein A/Vietnam/1203/2004 (H5N1) (H5 HA Viet) influenza virus.
Fig 5.
Probing group 1 (H1) and group 2 (H7) helix-A nanoparticle sera for homosubtypic and heterosubtypic antibody reactivity to recombinant HA proteins and solvent accessibility of helix-A residues.
(A, B, C) Western blot displaying reactivity of sera from mice immunization with either H1 Nanoparticle (panel A), H7 Nanoparticle (panel B), or saline (panel C) to recombinant H1 HA proteins from H1N1 (H1 rHA CA/04, A/California/04/09) and (H1 rHA CA/07, A/California/07/09), recombinant H1 rHA1 head and H1 rHA2 stem proteins, and recombinant H7 HA proteins from A/Anhui/1/2013 (H7N9) (H7 rHA Anhui), and A/Netherlands/219/2003 (H7N7) (H7 rHA Neth). (D) ELISA binding of sera from mice immunized with different nanoparticles (i.e., H1 nanoparticle, H7 nanoparticle, scaffold, and saline) to recombinant H7 HA protein (H7 rHA). (E) ELISA binding of sera from mice immunized with the different nanoparticles to recombinant H1 HA protein (H1 rHA). (F, top) Sequence alignment of helix-A sequences from H1 HA and H7 HA with different chemical categories of amino acids color-coded based on Clustal X. (F, bottom) Identity and similarity percentages for H1 and H7 HA sequences arranged from highest to lowest as helix-A, HA2, HA0, and HA1 regions. (G, H) Distribution of solvent inaccessible (black), solvent accessible (green) and partially solvent accessible (gray) residues on the helix-A regions for H1 (PDBID:3lzg) (panel G) and H7 (PDBID:4kol) (panel H) ectodomain trimers. HA1 is shown in red and HA2 in blue. The conserved helix-A is shown in green within the HA2 stem region. (I) Sequence alignment of helix-A sequences from H1 HA and H7 HA with different solvent accessibility denoted via color key below. Solvent accessible/partially accessible residues are underlined. Solvent accessibility of residues of coordinates were determined using a water probe with a radius of 1.4 angstroms by the online server GETAREA (https://curie.utmb.edu/getarea.html).
Fig 6.
Probing group 1 (H1, H5) and group 2 (H7) helix-A nanoparticle sera for homosubtypic and heterosubtypic binding to HAs from potential pandemic influenza viruses and for pseudovirus microneutralization activities.
(A, B, C) Analysis of binding by ELISA of recombinant HA proteins H5 (panel A), H2 (panel B), H15 (panel C) by different mice sera: H1-nanoparticle (H1-Nano), H7-nanoparticle (H7-nano), H5-nanoparticle (H5-nano), scaffolding, and saline. The color key is shown with the different nanoparticle sera and scaffold and saline sera. (D, E, F) Western blots displaying reactivity of different mouse sera from immunization with (D) H5 Nanoparticle, (E) H1 Nanoparticle, (F) H7 Nanoparticle. (G). Maximum Likelihood (ML) phylogenetic tree of helix-A sequences from influenza type A HA subtypes (H1-H18) classified into group 1 (blue) and group 2 (magenta) HAs. Ovals denote HA subtypes that displayed homosubtypic and heterosubtypic binding by helix-A nanoparticle sera. For the H4 and H14 sequences, the helix-A sequences are identical. (H) H1 CA09 and (I) H7 Anhui pseudoviruses, in microneutralization assays with sera from mice immunized with individual nanoparticles for H1 (CA/09), H7 (Anhui/13), H5 (VN/04), scaffold, and saline. Dotted lines represent background baseline. The symbol α is an abbreviation for “anti-”.
Fig 7.
Isolation and characterization of cross-reactive H1 and H7 monoclonal antibodies via sorting of B-cells from nanoparticle immunized mice and analysis of binding and microneutralization.
(A) B-cells from groups of mice immunized with H1 and H7 helix-A nanoparticles sorted into H7+, H1+ and H1H7++ cell populations. Each group received only one type of nanoparticle. Sorted cells are indicated in black. (B) Summary of sorting of B cells from mice immunized with H1 helix-A nanoparticles (H1-nano), H7 helix-A nanoparticles (H7-nano) and a mice immunized with H7 nanoparticles that survived H1N1 challenge (H7-nano/H1). (C) SDS-PAGE of monoclonal antibody mAb-11 under reducing (+DTT, dithiothreitol) and non-reducing conditions (-DTT). The molecular weights of the standards (std) are denoted. Arrows denote separated heavy (H) and light (L) chains under reducing conditions and disulfide linked chains (H+L) under non-reducing conditions. (D) ELISA used to probe the binding of monoclonals antibodies mAb-11, mAb-49, mAb-63, mAb-67 and mAb-85 to recombinant H1 and H7 proteins. (E, F) Analysis of monoclonals antibodies to neutralize H1(CA09) and H7 (Anhui/13) psuedoviruses in a microneutralization assay. Each antibody is in a different color and the color key is shown. The positive control is MEDI8852 which is a human broadly reactive antibody that recognizes all influenza HA subtypes.
Fig 8.
Passive-transfer of monoclonal antibodies (mab) and protection from H1N1 challenge.
(A) Survival curves for mice that were intranasally challenged with H1N1 virus 24 hours after intraperitoneally (IP) transfer of mAb-11, mAb-49, mAb-63, mAb-67 and mAb-85 at doses of 17.5 mg/kg. (B) The corresponding weight-lost curves for panel A. (C) Survival curve after challenge for mAb-11 that was administered before challenge via passive transfer at an increased dose of 26.2 mg/kg. (D) The corresponding mAb-11 weight-lost curves for panel C. (E) Survival curve after challenge for mAb-49 that was administered before challenge via passive transfer at a decreased dose of 4.3 mg/kg. (F) The corresponding mAb-49 weight-lost curves for panel C. Each antibody is in a different color with a saline control and the color key is shown in panel B.