Structure-based vaccine design by electron microscopy

Modern vaccine design relies on multiscale, interdisciplinary efforts that take advantage of innovative technologies such as in silico identification of antigens, high throughput screening of antigen immunogenicity, and gene expression profiling to predict host immune responses. In recent years, structural analysis has played an increasingly important role in vaccine development as a means to improve antigen stability, immunogenicity and large scale production. Transmission electron microscopy (TEM), and in particular cryo-TEM, is an established and powerful imaging technique applicable to many specimens, including the three-dimensional (3D) reconstruction of macromolecules and their associated complexes to high resolution. The technique is parsimonious in its material requirements and captures the specimens in their fully hydrated state, close to their native environment. The resolution of cryo-TEM reconstructions was limited to the subnanometer range until the recent development of direct electron detectors and improvements in image processing software, which has led to a so-called “resolution revolution” in the cryo-TEM field. Several protein structures have now been solved at near atomic resolution, establishing the technique as a viable alternative to X-ray analysis for high resolution structure determination. We have determined several structures with and without bound compounds at 2.9 Å – 3.6 Å resolution, which are being integrated into drug discovery and development workflows by our clients. Here we present the 2.4Å resolution structure of apoferritin determined with our Titan Krios electron microscope as an example of the cryo-TEM services available at NIS. These services are significantly enhanced with unique access by NIS to a new instrument, Spotiton, a robotic device that dispenses picoliter-volumes of sample onto a self-blotting nanowire grid as it flies past en route to vitrification. This provides several advantages over standard vitrification methods, including more automated and reproducible preparation of specimens and reducing the deleterious effects of particles interacting with the air-water interface. While high resolution 3D structure determination by cryo-TEM is at the forefront of structural biology, averages of 2D projection images at moderate resolution in negative stain or vitreous ice can also provide a wealth of information that may be difficult to obtain using other methods. This is illustrated in a number of case studies, including (1) mapping of neutralizing epitopes on the CMV pentameric glycoprotein complex; (2) mapping of neutralizing epitopes on the HIV-1 envelope glycoprotein trimer; (3) assessment of structure and conformational stability of pre- and post-fusion RSV-F protein; (4) characterization of novel adjuvants and protein delivery systems. In summary, both the moderate resolution TEM and high resolution cryo-TEM methods are well suited to extensively characterize antigen structure-function relationships, some of which may be refractory to other experimental methods

A multifunctional human monoclonal neutralizing antibody that targets a unique conserved epitope on influenza HA

The high rate of antigenic drift in seasonal influenza viruses necessitates frequent changes in vaccine composition. Recent seasonal H3 vaccines do not protect against swine-origin H3N2 variant (H3N2v) strains that recently have caused severe human infections. Here, we report a human VH1-69 gene-encoded monoclonal antibody (mAb) designated H3v-47 that exhibits potent cross-reactive neutralization activity against human and swine H3N2 viruses that circulated since 1989. The crystal structure and electron microscopy reconstruction of H3v-47 Fab with the H3N2v hemagglutinin (HA) identify a unique epitope spanning the vestigial esterase and receptor-binding subdomains that is distinct from that of any known neutralizing antibody for influenza A H3 viruses. MAb H3v-47 functions largely by blocking viral egress from infected cells. Interestingly, H3v-47 also engages Fcγ receptor and mediates antibody dependent cellular cytotoxicity (ADCC). This newly identified conserved epitope can be used in design of novel immunogens for development of broadly protective H3 vaccines

A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response.

Broadly neutralizing antibodies targeting a highly conserved region in the hemagglutinin (HA) stem protect against influenza infection. Here, we investigate the protective efficacy of a protein (HB36.6) computationally designed to bind with high affinity to the same region in the HA stem. We show that intranasal delivery of HB36.6 affords protection in mice lethally challenged with diverse strains of influenza independent of Fc-mediated effector functions or a host antiviral immune response. This designed protein prevents infection when given as a single dose of 6.0 mg/kg up to 48 hours before viral challenge and significantly reduces disease when administered as a daily therapeutic after challenge. A single dose of 10.0 mg/kg HB36.6 administered 1-day post-challenge resulted in substantially better protection than 10 doses of oseltamivir administered twice daily for 5 days. Thus, binding of HB36.6 to the influenza HA stem region alone, independent of a host response, is sufficient to reduce viral infection and replication in vivo. These studies demonstrate the potential of computationally designed binding proteins as a new class of antivirals for influenza

Design and structure of two HIV-1 clade C SOSIP.664 trimers that increase the arsenal of native-like Env immunogens

A key challenge in the quest toward an HIV-1 vaccine is design of immunogens that can generate a broadly neutralizing antibody (bnAb) response against the enormous sequence diversity of the HIV-1 envelope glycoprotein (Env). We previously demonstrated that a recombinant, soluble, fully cleaved SOSIP.664 trimer based on the clade A BG505 sequence is a faithful antigenic and structural mimic of the native trimer in its prefusion conformation. Here, we sought clade C native-like trimers with comparable properties. We identified DU422 and ZM197M SOSIP.664 trimers as being appropriately thermostable (Tm of 63.4 °C and 62.7 °C, respectively) and predominantly native-like, as determined by negative-stain electron microscopy (EM). Size exclusion chromatography, ELISA, and surface plasmon resonance further showed that these trimers properly display epitopes for all of the major bnAb classes, including quaternary-dependent, trimer-apex (e.g., PGT145) and gp120/gp41 interface (e.g., PGT151) epitopes. A cryo-EM reconstruction of the ZM197M SOSIP.664 trimer complexed with VRC01 Fab against the CD4 binding site at subnanometer resolution revealed a striking overall similarity to its BG505 counterpart with expected local conformational differences in the gp120 V1, V2, and V4 loops. These stable clade C trimers contribute additional diversity to the pool of native-like Env immunogens as key components of strategies to induce bnAbs to HIV-

HB36.6 induces a transient cytokine response that is not required for protection.

<p>(a) Inflammatory cytokines were assayed by Bio-Plex using supernatants from lung homogenates obtained from BALB/c mice 2, 24 and 48 hours following administration with HB36.6 (6.0 mg/kg) or the scaffold protein (PDB ID 1u84) (6.0 mg/kg) (n = 10 mice per group). Fold change over naïve mice is shown. *P < 0.05. (b) Survival and weight change in SCID and MyD88-/- mice (n = 10 per group) that received 6.0 mg/kg of HB36.6, scaffold protein, or Protein Ctr (lysozyme) IN 2 hours before IN infection with 10 MLD₅₀ CA09 virus.</p

Intranasal delivery of HB36.6 affords prophylactic protection against lethal challenge by influenza virus.

<p>(a) Survival and weight change in BALB/c mice (n = 10 per group) that received 6.0 mg/kg of HB36.6 administered intranasally (IN) at 2, 24, or 48 hours before challenge with 10 MLD₅₀ CA09 virus. The Protein Control (Ctr) group received 6.0 mg/kg of lysozyme at 2 or 48 hours before challenge with 10 MLD₅₀ CA09 virus. (b) Survival and weight change in BALB/c mice (n = 5 per group) that received 0.01–1 mg/kg IN doses of HB36.6 2 hours before challenge with 10 MLD₅₀ of CA09 virus. (c) Survival and weight change in BALB/c mice (n = 10 per group) that received 3.0 mg/kg of HB36.6 IN 2 hours before IN infection with 10 MLD₅₀ of H1N1 CA09 virus, 6 MLD₅₀ H1N1 A/PR/8/34 (PR8), or 3 MLD₅₀ of H5N1 A/Duck/MN/1525/81 (MN81) virus. Mean and SEM are shown.</p

HB36.6 suppresses viral replication and inflammation in the lung.

<p>(a) Viral titers in nasal washes of untreated infected controls (Ctr) and mice that received 6.0 mg/kg HB36.6 either 1 day before (Prophylaxis, Pro) or 1 day after (Therapeutic, Ther) infection with 10 MLD₅₀ CA09 virus. Nasal washes collected on days 2, 4 and 6 post-infection were measured by determining the 50% tissue culture infectious dose (TCID₅₀) (bars indicate mean viral titer ±SD, n = 18 mice per group, three replicate experiments). (b) IHC staining of intracellular influenza NP (H1N1) of representative lung sections from uninfected (Naïve) and untreated infected controls (Control) and HB36.6-treated mice (Prophylactic and Therapeutic) at 4 days post-infection with 10 MLD₅₀ CA09 virus. Mouse lungs were not inflated with formalin and consequently resulted in lung collapse and a more hypercellular appearance in the uninfected control. Images selected show representative staining of influenza (NP) positive cells for each group. (c) Quantification of influenza positive cells in lung tissues was performed by measuring the area of positive staining compared to the total tissue on the slide (uniform random sampling of 50% lung tissue). (d) Inflammatory cytokines were assayed by Bio-Plex using supernatants from lung homogenates obtained from BALB/c mice on day 2 following infection with 10 MLD₅₀ CA09 virus (n = 8 mice per group). The fold change over naïve-uninfected mice is shown. For a, c and d, significant differences between the Pro and Ther groups to the Ctr group are shown: *P < 0.05, **P < 0.001.</p

Specificity-enhancing mutations and overall affinity-increasing substitutions using selection against different HA subtypes.

<p>(<b>a</b>) HB36.5, in complex with HA, colored by average residue enrichment in FACS sorts against 7 different HA subtypes. Final residue identity after mutagenesis and selection to obtain HB36.6 are labeled in black. Positions 54, 64, and 68 retained their HB36.5 identities. (<b>b</b>) Enrichment of substitutions at 12 key positions in HB36.5 in selections against each HA strain. Labels at the bottom indicate position in HB36.5; numbers at the top represent the different flu strains and subtypes (1: A/South Carolina/1/1918 (H1), 2: A/California/04/2009 (H1), 3: A/Vietnam/1203/2004 (H5), 4: A/Indonesia/05/2005 (H5), 5: A/Adachi/2/1957 (H2), 6: A/turkey/Wisconsin/1/1966 (H9), 7: A/duck/Alberta/60/1976 (H12)). At most positions, the enrichment profiles against the different HA strains are similar but, at several positions, they are quite distinct. At position 54, for example, arginine is highly conserved and substitutable for lysine in selections for binding against HAs 1–4, but is outcompeted by smaller charged/polar residues in selections against HAs 5–7 (red region at upper right of R54 panel). White cells indicate insufficient data (<15 sequences in the input library) and black boxes indicate the residue identities in HB36.6. (<b>c</b>) Origin of HA strain dependence of substitutions at HB36.5 position R54. Arg54 forms a hydrogen bond network with Asp and Arg residues in HAs 1–4. In HAs 5–7, the Asp is substituted by a Glu, disrupting the interface with Arg54 leading to a preference for smaller polar residues.</p