Team:UCSC/Description
Description
A Need for Heat Stable Vaccines
The challenge of shipping and storing vaccines at refrigerated temperatures has caused devastating problems in most immunization programs around the world. According to a recent study, 53% of 158 immunization stakeholders in Brazil, China, India, Peru, Philippines, and Tanzania reported poor refrigerator performance and stated that they would benefit from thermostable vaccines [1] .
Humans are not the only ones with a need for heat-stable vaccines. The well-being of agricultural animals is affected by a lack of proper immunization. Failure to have refrigerated vaccines, a lack of education for farmers, and unsuccessful immunization protocols result in the spread of disease and eventual loss of viable livestock, especially in lower-middle-income nations. Newcastle disease virus (NDV) is considered the most devastating agricultural disease in rural areas and affects 80% of unprotected poultry [2] . This can result in the euthanization of an entire flock to prevent further spread of the disease [3] .
The Beauty of Intrinsic Disorder
Tardigrades are micro-animals, most renowned for their ability to survive in extreme conditions such as desiccation, radiation, and temperatures ranging between -272°C to 150°C [4] . Their ability to do so is attributed to tardigrade-specific intrinsically disordered proteins or TDPs [7] . We became interested in intrinsically disordered proteins (IDPs) and TDPs when Dr. Pamela Silver, Principal Investigator in the Silver Lab at Harvard Medical School, held a Q&A session with us and spoke about their potential use in biostasis. Further research led us to papers authored by Dr. Thomas Boothby, a scientist with extensive research in the field of tardigrades, that spoke about the physical and mechanical properties of IDPs. Boothby's papers pushed the idea to use tardigrades in an aspect of our research.
Our team talked with David Bunn, the director of the CDC, who provided us with information on agricultural practices in resource-constrained areas. He explained to us his project in Kenya, where they are trying to introduce chickens in local communities to increase health and economic stability. Through his work, he learned the significant obstacle of keeping vaccines for livestock refrigerated due to a lack of resources. After learning about the great extent many go through to ensure refrigeration of vaccines, we decided to utilize these powerful proteins to potentially increase the thermostability of vaccines.
Expression, Extraction, and Purification of TDPs
We obtained several plasmids containing IDPs from the Silver Lab and the Pielak Lab which were subsequently expressed in E. coli. IDPs were then purified through heat precipitation as they can be subjected to high temperatures without losing their structural integrity. The extraction method isolated our proteins, but to ensure and increase purity, we also performed immobilized metal affinity chromatography to attain >95% purity [6] . Only proteins that could withstand thermostability testing were chosen to assess their ability to protect the NDV vaccine.
Thermostability Testing of NDV Vaccine
To improve the thermostability of NDV vaccines, we will mix our purified TDPs with a lentogenic NDV vaccine. This mixture will undergo diafiltration to remove water from the solution. The removal of water from the TDPs is hypothesized to produce a glass-like state in a process called vitrification [7] . Piszkiewicz et al has shown evidence of TDP vitrification protecting the activity of proteins that undergo lyophilization, desiccation, and freeze/thaw cycles [3] .
Therefore, we hypothesized that diafiltration would cause the TDPs to vitrify around the vaccine and act as a protective coating. We will heat the filtered mixture to high temperatures to observe the protective properties of the vitrified TDPs. After heating, the mixture will be rehydrated and the vaccine will be extracted and titered for quantitative analysis of the vaccine’s infectability and thermostability.
We’re developing a method to improve the heat stability of vaccines by using the Newcastle disease vaccine as a model for other vaccines. The Newcastle virus is in the viral family Paramyxoviridae; human viruses such as influenza, measles, and mumps are all included in the Paramyxoviridae family. If we can successfully create a heat-stable Newcastle vaccine, then we could apply this heat-stable method to human vaccines as well.
References
1.) Kristensen, Debra D et al.
“Can thermostable vaccines help address cold-chain challenges?
Results from
stakeholder interviews in six low- and middle-income countries.”
Vaccine
vol. 34,7 (2016): 899-904. doi:10.1016/j.vaccine.2016.01.001
2.) (http://www.synergy.co.nz), Synergy International
Limited. Issg Database: Impact Information for Newcastle Disease Virus (NDV)
3.) Spickler, Anna Rovid. 2016. Newcastle Disease. Retrieved from here
4.) Roy, P. (2017). The Resolved Mystery of Tardigrades. Journal of Investigative Genomics, 4(2). doi:10.15406/jig.2017.04.00060
5.) Piszkiewicz, S., Gunn, K.H., Warmuth, O., Propst, A., Mehta, A., Nguyen, K.H., Kuhlman, E., Guseman, A.J. Stadmiller, S.S., Boothby, T.C., et al. (2019). Protecting activity of desiccated enzymes. Protein Science 28, 941–951.
6.) Popova, A.V., Hundertmark, M., Seckler, R.,
and Hincha, D.K. (2011). Structural transitions in the intrinsically disordered plant
dehydration stress protein LEA7 upon drying are modulated by the presence of membranes.
Biochimica et Biophysica Acta (BBA) - Biomembranes 1808, 1879–1887.
7.) Boothby, T.C., Tapia, H., Brozena, A.H., Piszkiewicz, S., Smith, A.E., Giovannini, I., Rebecchi, L., Pielak, G.J., Koshland, D., and Goldstein, B. (2017). Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation. Molecular Cell 65, 975-984.e5.
