Design and proof-of-concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain

Abstract

Development of effective vaccines against Coronavirus Disease 2019 (COVID-19) is a global imperative. Rapid immunization of the world human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and many different vaccine approaches are being pursued to meet this task. Engineered filamentous bacteriophage (phage) have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the design, development, and initial evaluation of targeted phage-based vaccination approaches against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) by using dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. Towards a unique phage- and AAVP-based dual-display candidate approach, we first performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein for display on the recombinant major capsid coat protein pVIII. Targeted phage particles carrying one of these epitopes induced a strong and specific humoral response. In an initial experimental approach, when these targeted phage particles were further genetically engineered to simultaneously display a ligand peptide (CAKSMGDIVC) on the minor capsid protein pIII, which enables receptor-mediated transport of phage particles from the lung epithelium into the systemic circulation (termed "dual-display"), they enhanced a systemic and specific spike (S) protein-specific antibody response upon aerosolization into the lungs of mice. In a second line of investigation, we engineered targeted AAVP particles to deliver the entire S protein gene under the control of a constitutive cytomegalovirus (CMV) promoter, which induced tissue-specific transgene expression stimulating a systemic S protein-specific antibody response. As proof-of-concept preclinical experiments, we show that targeted phage- and AAVP-based particles serve as robust yet versatile enabling platforms for ligand-directed immunization and promptly yield COVID-19 vaccine prototypes for further translational development.

Significance: The ongoing COVID-19 global pandemic has accounted for over 2.5 million deaths and an unprecedented impact on the health of mankind worldwide. Over the past several months, while a few COVID-19 vaccines have received Emergency Use Authorization and are currently being administered to the entire human population, the demand for prompt global immunization has created enormous logistical challenges--including but not limited to supply, access, and distribution--that justify and reinforce the research for additional strategic alternatives. Phage are viruses that only infect bacteria and have been safely administered to humans as antibiotics for decades. As experimental proof-of-concept, we demonstrated that aerosol pulmonary vaccination with lung-targeted phage particles that display short epitopes of the S protein on the capsid as well as preclinical vaccination with targeted AAVP particles carrying the S protein gene elicit a systemic and specific immune response against SARS-CoV-2 in immunocompetent mice. Given that targeted phage- and AAVP-based viral particles are sturdy yet simple to genetically engineer, cost-effective for rapid large-scale production in clinical grade, and relatively stable at room temperature, such unique attributes might perhaps become additional tools towards COVID-19 vaccine design and development for immediate and future unmet needs.

Fig 1.. Schematic representation of the phage- and AAVP-based vaccine candidates.

The scheme represents the approach used for the conception, design, and application of two strategies of immunization against SARS-CoV-2 S protein using phage particles. Step 1: Structural analysis, selection of structurally-defined epitopes, and cloning steps for the generation of dual-display phage particles and AAVP encoding the full-length S protein. Step 2: Molecular engineering of single- and dual-display phage particles, and AAVP S constructs. Step 3: Functional validation and vaccination studies in vivo in mice.

Fig 2.. Identification of structural epitopes on S protein trimer.

(A) Six epitopes (orange) spanning the SARS-CoV-2 S protein were selected for display on rpVIII protein. Four epitopes are located within the S1 subunit: epitope 1 (aa 336–361), epitope 2 (aa 379–391), epitope 3 (aa 480–488), epitope 4 (aa 662–671); and two within the S2 subunit: epitope 5 (aa 738–760), and epitope 6 (aa 1032–1043). These epitopes are solvent-exposed in the surface representation of the predominantly closed-state conformation of the S protein trimer (gray, cornflower blue, and rosy brown) (PDB ID: 6ZP0) (60). Only epitope 1 (aa 336–361) contains a site for glycosylation (at N343) (purple). (B) All of the epitopes (orange) maintain a cyclic conformation in the ribbon representation of a S protein protomer (gray); disulfide bridges (yellow) are present between the flanking cysteine residues of all epitopes except on epitope 2 (aa 379–391). The open-state conformation of the S protein trimer with one RBD erect displays a change in orientation of epitopes 1, 2, and 3, though all remain solvent-exposed (PDB ID: 6ZGG) (60).

Fig 3.. Explicit-solvent simulations reveal the near-native conformation of epitope 4 relative to the full-length S protein.

(A) RMSD from the S protein conformation for each epitope within the S1 subunit: epitope 1 (aa 336–361), epitope 2 (aa 379–391), epitope 3 (aa 480–488) and epitope 4 (aa 662–671). Epitope 4 frequently samples low values (~2Å), while the other epitopes undergo substantial conformational rearrangements (RMSD<4Å). (B) Probability as a function of RMSD shows that epitope 4 is most likely to sample conformations that are similar (RMSD ~2.5Å) to the S protein conformation. (C) RMSD as a function of time for the epitopes within the S2 subunit: epitope 5 (aa 738–760) and epitope 6 (aa 1032–1043). Both epitopes rapidly adopt and maintain large RMSD values (D).

Fig 4.. Immunogenicity of S protein epitopes on single-display phage particles.

Five-week-old female Swiss Webster mice were immunized via subcutaneous injection with single-display phage constructs containing each of the six different epitopes expressed on rpVIII protein or the control insertless phage. Animals received a boost injection three weeks after the first administration. (A) S protein-specific IgG antibodies and (B) phage-specific IgG antibodies were evaluated in sera of mice after two- and five-weeks by ELISA (n=3 mice per group). (C) Five-week-old female BALB/c mice were immunized via intratracheal administration with the epitope 4/CAKSMGDIVC dual-display phage particles, epitope 4 single-display phage particles, or the control insertless phage. Animals received a boost three weeks after the first administration. S protein-specific IgG antibodies were weekly evaluated by ELISA (n=10 mice per group). Data represent ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001).

Fig 5.. Immunogenicity of the RGD4C-AAVP S in mice.

Schematic representation of the AAVP-based vaccine candidate. (A) The modified, synthetic SARS-CoV-2 S protein gene was excised from the pUC57 and cloned into the RGD4C-AAVP -TNFΔEcoRI829. Expression of the S protein transgene cassette is driven by the cytomegalovirus (CMV) promoter and flanked by AAV ITRs. (B) Schematic representation of the RGD4C-AAVP S and the control RGD4C-AAVP empty vector (RGD4C AAVP S-null). (C) S protein-specific IgG antibody response in the sera of mice immunized with RGD4C-AAVP S via different routes of administration (n=5 mice per group) by ELISA. (D) S protein-specific IgG antibodies in sera of mice weekly immunized with RGD4C-AAVP S or the control RGD4C-AAVP S-null (n=12 mice per group) via subcutaneous administration. Data represent ± SEM (** P < 0.01). (E) Tissue-specific expression of the S protein transgene in mice immunized with AAVP S five weeks after the first immunization. Data represent ± SEM (*** P< 0.001). (F) Phage-specific IgG antibody response in the sera of mice immunized with RGD4C-AAVP S via different routes of administration (n=5 mice per group). Phage-specific IgG antibody response in mice immunized with RGD4C-AAVP S (G) or RGD4C-AAVP S-null (H) at two and five weeks after the first immunization. Phage-specific IgG antibody response was evaluated by ELISA in 96-well plates coated with 10¹⁰ AAVP particles per well. Tet^R, tetracycline resistance gene. Amp^R, ampicillin resistance gene. Ori origin of replication.