

Characterizing 

Protein-Adjuvant 

Interactions in 

Vaccines

Carrie Stemple

Mentored by Dr. Amy Xu

1
Overview

 Project 

 What is in a Vaccine

 Protein Adjuvant Interactions

 Effects of Freeze Thaw Cycles on Adjuvants

 Effects of Freeze Thaw Cycles on Microstructure of 

Adjuvant and Complexes

 Summary

 Future Research/ Acknowledgements

2
Project

 Define the interactions between proteins and adjuvants –

Binding Isotherm 

 Determine the effects of freeze thaw cycles on adjuvants 

and their interactions – Binding Isotherms, Microscopy, 

and Dynamic Light Scattering

 Develop understanding of how freeze thaw cycles affect 

microstructures of proteins, adjuvants, and complexes –

Small Angle Neutron Scattering

3
Vaccines

 Purpose

 Components:

 Antigen

 Adjuvant

 Excipients

4

https://www.brenntag.com/biosector/en/products/adju-phos/index.jsp http://www.haoranbio.com/pic/product/2015-03/20150320024234346506.jpg https://www.indiatvnews.com/lifestyle/news-new-vaccine-developed-to-cure-ebola-387386
Protein Adjuvant Interactions

 Experiment

5

https://www.thermofisher.com/order/catalog/product/ND-2000 https://www.starlabgroup.com/GB-en/consumables/tubes_WebPSub-155856/15-ml-crystal-clear-microcentrifuge-tube_SLE1415-1500.html https://www.wikihow.com/Use-a-Centrifuge
6 Protein Adjuvant Interactions: Binding Isotherm

Aluminum Hydroxide + 

Ovalbumin 

Aluminum Phosphate + 

Ovalbumin
7 Protein Adjuvant Interactions: Binding Isotherm

Aluminum Hydroxide + 

Lysozyme 

Aluminum Phosphate + 

Lysozyme
8 Protein Adjuvant Interactions: Binding Isotherm

Aluminum Hydroxide + 

Lysozyme in 

Phosphate Buffer

Aluminum Hydroxide + 

Lysozyme in Sodium 

Chloride Buffer
Effects of Freeze Thaw Cycles on the Adjuvants

 Importance of Freeze Thaw Cycles

 Experiments

9
Effects of Freeze Thaw Cycles on Adjuvants: 

Binding Isotherms

10

FT Aluminum Hydroxide & 

Ovalbumin Complex

Fresh Aluminum Hydroxide 

& Ovalbumin Complex

FT Aluminum Hydroxide + 

Ovalbumin
Effects of Freeze Thaw Cycles on Adjuvants : 

Binding Isotherms

11

FT Aluminum Phosphate 

& Lysozyme Complex

Fresh Aluminum 

Phosphate & Lysozyme 

Complex

FT Aluminum Phosphate 

+ Lysozyme
Effects of Freeze Thaw Cycles on Adjuvants: Microscopy

40x Magnification

12

Aluminum 

Hydroxide: 

Fresh

Aluminum 

Hydroxide: 

FreezeThawed

Aluminum 

Phosphate: 

Fresh

Aluminum 

Phosphate: 

FreezeThawed
Effects of Freeze Thaw Cycles on Adjuvants: 

Dynamic Light Scattering

13

Fresh Aluminum Hydroxide

FT Aluminum Hydroxide

Fresh Aluminum Phosphate

FT Aluminum Phosphate
Effects of Freeze Thaw Cycles on Microstructures

 Importance of Understanding the Effects of Freeze Thaw on 

Microstructures

 Adjuvants

 Proteins 

 Complexes

 Experiment

14
Effects of Freeze Thaw Cycles on Microstructures: 

SANS and Contrast Matching

15

https://www.sciencedaily.com/releases/2016/11/161115094633.htm

Small Angle Neutron Scattering

Contrast Matching 

Solvent

Protein

Adjuvant

Solvent

Protein

Adjuvant

Solvent

Protein

Adjuvant
Effects of Freeze Thaw Cycles on Microstructures: 

Small Angle Neutron Scattering 

16

Fresh Ovalbumin + Hydroxide

FT Ovalbumin + Hydroxide

Ovalbumin

Hydroxide

Solvent

Protein

Adjuvant
Effects of Freeze Thaw Cycles on Microstructures: 

Small Angle Neutron Scattering 

17

Fresh Ovalbumin + Phosphate

FT Ovalbumin + Phosphate

Lysozyme

Phosphate

Solvent

Protein

Adjuvant
Summary

 Interactions between proteins and adjuvants

 Electrostatic interactions

 Ligand exchange

 Effects of freeze thaw cycles on adjuvants and their interactions

 Aggregation Hydroxide > Phosphate 

 More aggregation= less binding

 Effects of freeze thaw on microstructures of proteins, 

adjuvants, and complexes

 Complexes form new structures

 Protein 3D conformation is Maintained

 Little change in adjuvant characteristics after freeze-thaw

18
Future Research/Acknowledgements

 Binding Isotherms in 

Sodium Chloride buffer

 Differential Scanning 

Calorimetry 

 Dr. Amy Xu

 NIST/NCNR

 CHRNS

 SURF Directors

 Dr. Kathryn Sarachan

 Wilson College

19
20
Experimental Conditions 

21

Ovalbumin pH7.2 Lysozyme

AlPO4 Al(OH)3

pH14