Lipid Nanoparticle Development for Fluvid mRNA Vaccines Targeting Seasonal Influenza and SARS-CoV-2 Study Notes

Study Overview and Rationale for Fluvid mRNA Vaccines

• Conceptual Framework: The study focuses on the development of a bivalent "Fluvid" mRNA vaccine targeting both seasonal influenza and SARS-CoV-2.
• Advantages of mRNA Platforms: Compared to conventional vaccines using subunit protein antigens or inactivated microorganisms, mRNA vaccines offer:
  • High potency.
  • Expedited development.
  • Low-cost manufacturing.
  • Agility for rapid deployment against emerging pandemic sequences.
  • Safety: mRNA does not integrate into the genome, removing risk of insertional mutagenesis.
• Global Health Rationale:
  • Current influenza protection requires tri- or quadrivalent vaccines (H1N1, H3N2, and 1 or 2 Influenza B viruses).
  • SARS-CoV-2 is expected to remain endemic, requiring seasonal vaccination similar to the flu.
  • Influenza has caused four major pandemics in the last century: 1918 H1N1 Spanish flu, 1957 H2N2 Asian flu, 1968 H3N2 Hong Kong flu, and 2009 H1N1 swine flu.
  • Combining vaccines improves patient compliance and reduces costs.

Lipid Nanoparticle (LNP) Composition and Mechanics

• Delivery Vehicle: Lipid Nanoparticles (LNPs) are the primary delivery vehicles for mRNA, eliciting both strong T-cell responses and robust antibody production.
• Major Lipid Components: LNPs typically comprise four major lipids:
  • Ionizable Lipid: Includes pH-responsive tertiary amines that acquire a cationic charge only at low pH (below the $pKa$). This facilitates endosomal escape while remaining neutral at physiological pH to improve biocompatibility and circulation time.
  • Cholesterol: Improves stability by filling gaps between lipids and supporting membrane fusion.
  • Helper Phospholipids: Building blocks of the lipid bilayer (e.g., DSPC, DOPC, DOPE).
  • PEGylated Lipid: Stabilizes the LNP and regulates particle size by limiting fusion (e.g., DMG-PEG2000).
• Historical Context of Cationic Lipids:
  • DOTMA and DOTAP: First used in 1989; contain permanently positively charged quaternary ammonium head groups. Limitations include cytotoxicity in vivo and rapid clearance due to interactions with serum proteins.

Experimental Optimization: Buffer Formulation and Stability

• Buffer Evaluation: Three storage buffers were initially tested for LNP preparation:
  • Phosphate-based buffer: $8\,mM$ Phosphate/ $105\,mM$ NaCl, $pH\,7.4$.
  • Tris-based buffer: $20\,mM\,Tris/ 4.3\,mM$ Acetate, $pH\,7.4$.
  • PNS buffer: Proprietary buffer from Precision NanoSystems.
• Selection: Tris-based buffer was selected as it displayed the least change in particle size and Polydispersity Index (PDI) after freeze-thaw (F/T) cycles.
• Cryoprotectant Incorporation: Inclusion of $10\%\,\text{sucrose}$ in the storage buffer (TAS: $20\,mM\,Tris/ 4.3\,mM\,Acetate/ 10\%\,\text{sucrose}$, $pH\,7.4$) maintained stability for 7 days at $4\,^{\circ}C$ or through multiple freeze-thaw cycles.

Comparative Analysis of Cationic Lipids and Immunogenicity

• Screening Six Cationic Lipids: The study compared the following lipids at an N/P ratio of 6:
  1. ALC0315: $pKa = 6.09$ (Dendrimeric-like structure).
  2. SM102: $pKa = 6.75$ (Dendrimeric-like structure).
  3. DODMA: $pKa = 6.59$.
  4. Dlin-MC3-DMA: $pKa = 6.57$.
  5. DODAP: $pKa = 5.62$.
  6. DOTMA: Permanently charged cationic lipid.
• Top Performers: ALC0315 induced the highest anti-spike IgG, followed by SM102 and DODMA. The other lipids (DOTMA, DODAP, MC3) failed to induce significant IgG activity by day 14 post-prime.
• Physical Characteristics: Particle sizes ranged from $63\,nm$ to $149\,nm$. Encapsulation efficiency was consistently high at $>88\%$.

Optimization of Helper Lipids and mRNA Modifications

• Phospholipid Comparison (DSPC vs. DOPE):
  • DOPE was tested with DODMA to see if its fusogenic properties improved endosomal escape.
  • Findings: DODMA/DSPC formulations significantly outperformed DODMA/DOPE in eliciting anti-spike IgG. DSPC likely provides greater lipid bilayer stability requisite for immune responses.
• Nucleoside Modifications: Tested the ability of three uridine types to trigger immune responses:
  1. Unmodified Uridine: Successfully induced robust antigen-specific antibody responses.
  2. $N1$-methyl pseudouridine ($m1\Psi$)-modified: Successfully induced robust responses.
  3. Methoxy uridine ($5MoU$)-modified: Failed to induce anti-spike IgG.

Development and Evaluation of the Bivalent Fluvid Vaccine

• Design: A bivalent vaccine was constructed using unmodified mRNA encapsulating:
  • HA mRNA: Encoding transmembrane domain-deleted hemagglutinin from Influenza A/California/07/2009 (H1N1).
  • Spike mRNA: Encoding full-length native spike protein from SARS-CoV-2 Wuhan strain.
• LNP Formulation: ALC0315/DSPC/CHOL/DMG-PEG2000 at $50/10/38.5/1.5\,mole\%$.
• Dosing and Schedule: Mice were immunized intramuscularly with $5\,\mu g$ per mRNA on d0 and d14.
• Immunogenicity Results:
  • High HA-specific and Spike-specific IgG responses were observed.
  • Cross-reactive IgG against variant strains (BA.1, BA.2, BA.4/BA.5, BQ.1.1) was detected.
  • The bivalent formulation did not compromise the efficacy of either individual component.

Protective Efficacy and Viral Challenges

• H1N1 Challenge:
  • Carried out at ABSL2+ on day 35.
  • Lethal dose determined as $10^3\,TCID_{50}/mL$.
  • Results: 100% protective efficacy achieved for all HA-containing groups. Control groups (PBS, Spike monovalent) exhibited significant weight loss.
• SARS-CoV-2 Challenge:
  • Carried out with MA10 variant at ABSL3 on day 42.
  • Lethal dose: $10^4\,TCID_{50}/mL$.
  • Results: 100% protection in Spike-containing groups (monovalent and bivalent). Control groups (PBS, HA monovalent) lost $>15\%$ body weight.

T-cell Recall and Cytokine Analysis

• Systemic Reactogenicity: Transient weight loss ($2-9\%$) was observed in the bivalent and adjuvanted protein groups 3 hours post-prime, correlating with elevated inflammatory cytokines (IL-6, TNF-$\alpha$, MCP-1, and IFN-$\gamma$).
• Cellular Immune Response:
  • Spike antigen stimulated higher recall responses compared to HA.
  • Significant levels of IFN-$\gamma$, IL-2, and TNF-$\alpha$ were detected in Spike mRNA LNP groups, indicating a Th1-biased response.
  • IL-6 secretion was also elevated upon spike recall, promoting T-cell differentiation.
  • No significant IL-4 was elicited, further supporting Th1 polarization.

Materials and Methods Specifications

• mRNA Products: Purchased from OZ Biosciences and Aldevron (unmodified GFP).
• Lipids: Sourced from Avanti Polar Lipids and BroadPharm.
• Equipment:
  • NanoAssemblr Ignite for microfluidic mixing.
  • Zetasizer ultra for particle sizing via dynamic light scattering.
  • Cytek Aurora 5-laser flow cytometer for spectral flow cytometry and cytokine assays.
• Virus Propagation: Influenza in chicken eggs; SARS-CoV-2 in Vero E6 or Calu-3 cells.