CryoTEM in Preclinical Vaccine Development: Characterizing the lead candidate adjuvant formulation
Mayuresh M. Abhyankar1, Mark T. Orr2,3†, Robert Kinsey2, Sandra Sivananthan2, Andrew J. Nafziger1, David N. Oakland1, Mary K. Young1, Laura Farr1, Md Jashim Uddin1, Jhansi L. Leslie1, Stacey L. Burgess1, Hong Liang2†, Ines De Lima2, Elise Larson2, Jeffrey A. Guderian2, Susan Lin2, Aaron Kahn2, Prakash Ghosh2†, Sierra Reed2, Mark A. Tomai4, Karl Pedersen5, William A. Petri Jr.1 and Christopher B. Fox2,3*
1Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia Health System, Charlottesville, VA, United States
2Infectious Disease Research Institute (IDRI), Seattle, WA, United States
3Department of Global Health, University of Washington, Seattle, WA, United States
43M Corporate Research Materials Laboratory, 3M Center, St Paul, MN, United States
5TECHLAB, Inc., Blacksburg, VA, United States
Front. Immunol., 25 June 2021 | https://doi.org/10.3389/fimmu.2021.683157
Amebiasis is a neglected tropical disease caused by Entamoeba histolytica that is estimated to account for approximately 55,000 deaths and millions of infections globally per year. There are currently no licensed vaccines for prevention of amebiasis.
A liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhances antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen, has been developed. In this paper, the authors, in partnership with NIS, use cryo-TEM as part of a comprehensive analytical toolkit, to optimize the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. The approach described in the paper led to the identification of a lead candidate composition that demonstrates immune response durability and protective efficacy in a mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection.
The paper, published in Frontiers in Immunology, concludes:
- Efficient dose and formulation optimization approaches employing tools such as Design of Experiments (DOE) or desirability functions are only rarely reported in vaccine development. Nevertheless, such approaches offer the benefit of objective multifactorial analysis while reducing the number of experimental subjects, such as animals, and the number of tests that would otherwise be required. This paper shows that CryoEM is a valuable part of the optimization toolkit.
- Cryo-transmission electron microscopy was performed by NanoImaging Services, Inc.
- Samples were imaged undiluted by applying 3-µl drop on cleaned grid consisting of holey carbon films on 400-mesh copper grid, blotting with filter paper, and immediate vitrification in liquid ethane.
- Imaging was performed on an FEI Technai T12 electron microscope at 120 keV with FEI Eagle 4k x 4k CCD camera. The cryostage maintained the grid below -170°C. Images were acquired at 52,000x (0.21 nm/pixel) using electron doses of ~10-25 e-/Å.
- Formulation morphology of the three lead candidate adjuvant formulations was assessed by cryo-transmission electron microscopy and compared to the morphology of the proof-of-concept formulation manufactured at the same time as the lead candidate compositions.
- The cryoEM images indicated the expected morphology for the proof-of-concept formulation based on previous experience, consisting of unilamellar vesicles <100 nm in diameter with some disk-like structures.
- The three lead candidate adjuvant formulations also contained unilamellar vesicles generally smaller than 100 nm as expected; however, extensive linear striated structures of irregular lengths up to ~500 nm were also present. These structures appeared most prevalent in lead candidate #3 ‘SS’.
- The different morphology apparent in the lead candidates compared to the proof-of-concept formulation was hypothesized to be due to their increased TLR ligand:phospholipid ratio which could change the preferred packing orientation and curvature of the lipid structures, or the addition of α-tocopherol may have played a role.
- The complexity of vaccine adjuvant formulation and evaluation requires a robust experimental design methodology to meaningfully interrogate the effects of multiple factors on multiple immunological and physicochemical stability readouts.
- Together, these findings have resulted in a lead candidate vaccine adjuvant composition suitable for advanced preclinical development.