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- Creators: Mor, Tsafrir
- Creators: Arizona State University. School of Life Sciences. Center for Biology and Society. Embryo Project Encyclopedia.
Vaccines are one of the most effective ways of combating infectious diseases and developing vaccine platforms that can be used to produce vaccines can greatly assist in combating global public health threats. This dissertation focuses on the development and pre-clinical testing of vaccine platforms that are highly immunogenic, easily modifiable, economically viable to produce, and stable. These criteria are met by the recombinant immune complex (RIC) universal vaccine platform when produced in plants. The RIC platform is modeled after naturally occurring immune complexes that form when an antibody, a component of the immune system that recognizes protein structures or sequences, binds to its specific antigen, a molecule that causes an immune response. In the RIC platform, a well-characterized antibody is linked via its heavy chain, to an antigen tagged with the antibody-specific epitope. The RIC antibody binds to the epitope tags on other RIC molecules and forms highly immunogenic complexes. My research has primarily focused on the optimization of the RIC platform. First, I altered the RIC platform to enable an N-terminal antigenic fusion instead of the previous C-terminal fusion strategy. This allowed the platform to be used with antigens that require an accessible N-terminus. A mouse immunization study with a model antigen showed that the fusion location, either N-terminal or C-terminal, did not impact the immune response. Next, I studied a synergistic response that was seen upon co-delivery of RIC with virus-like particles (VLP) and showed that the synergistic response could be produced with either N-terminal or C-terminal RIC co-delivered with VLP. Since RICs are inherently insoluble due to their ability to form complexes, I also examined ways to increase RIC solubility by characterizing a panel of modified RICs and antibody-fusions. The outcome was the identification of a modified RIC that had increased solubility while retaining high immunogenicity. Finally, I modified the RIC platform to contain multiple antigenic insertion sites and explored the use of bioinformatic tools to guide the design of a broadly protective vaccine.
Karl Landsteiner studied blood types in Europe and in the United States in the late nineteenth and early twentieth centuries. Landsteiner won the Nobel Prize in Physiology or Medicine in 1930 for detailing immunological reactions in the ABO blood group system. The ABO blood group system divides human blood into one of four types based on the antibodies that are present on each cell. Landsteiner's work with blood types led physicians to safely perform blood transfusions and organ transplants. Additionally, Landsteiner researched the Rh blood factor, a protein marker on the surface of blood cells and that can impact pregnancy.
Emil von Behring researched treatments for the common childhood disease diphtheria in Germany in the 1890s and early 1900s. Diphtheria is a lethal disease that infected approximately 40,000 people in Germany between 1886 and 1888 with a general mortality rate of twenty-five percent. Behring investigated treatment of diphtheria using serum therapy, which is an alternative to vaccination that uses protective agents from other people’s blood to defend a patient against disease. Behring termed those protective agents antitoxins. He received the first Nobel Prize in Physiology or Medicine for his work on serum therapy, which was one of the first Nobel Prizes given in the field of immunology. Additionally, Behring researched active vaccination as another way to protect patients from diphtheria. Behring’s studies lowered the mortality rate of diphtheria in Germany through serum therapy and vaccination, especially since vaccination confers protection to both mother and infant during pregnancy and after birth.
In 2014, Flor M. Munoz and colleagues published “Safety and Immunogenicity of Tetanus Diphtheria and Acellular Pertussis (Tdap) Immunization During Pregnancy in Mothers and Infants: A Randomized Clinical Trial,” hereafter “Tdap Immunization During Pregnancy,” in the Journal of the American Medical Association. The authors conducted a study to determine how Tdap immunization affected the mother and infant’s immune response to the common childhood diseases tetanus, diphtheria, and pertussis. They found that Tdap immunization did not lead to an increased risk of adverse health events. Furthermore, maternal Tdap immunization provided the infant with protective levels of pertussis antibodies after delivery and did not affect the infant differently from the DTaP vaccination series, which is the version of Tdap for young children. The authors’ findings in “Tdap Immunization During Pregnancy” supported the United States Centers for Disease Control and Prevention’s, or CDC’s, recommendation for pregnant women to receive the Tdap vaccine to prevent disease in mother and infant.
In 2014, Flor M. Munoz and colleagues published “Safety and Immunogenicity of Tetanus Diphtheria and Acellular Pertussis (Tdap) Immunization During Pregnancy in Mothers and Infants: A Randomized Clinical Trial,” hereafter “Tdap Immunization During Pregnancy,” in the Journal of the American Medical Association. The authors conducted a study to determine how Tdap immunization affected the mother and infant’s immune response to the common childhood diseases tetanus, diphtheria, and pertussis. They found that Tdap immunization did not lead to an increased risk of adverse health events. Furthermore, maternal Tdap immunization provided the infant with protective levels of pertussis antibodies after delivery and did not affect the infant differently from the DTaP vaccination series, which is the version of Tdap for young children. The authors’ findings in “Tdap Immunization During Pregnancy” supported the United States Centers for Disease Control and Prevention’s, or CDC’s, recommendation for pregnant women to receive the Tdap vaccine to prevent disease in mother and infant.
ano-crystals have been established for the different variants of the fusion protein. Diffraction patterns were collected by using both conventional and serial femto-second crystallography techniques. The two crystallography techniques showed very interesting differences in both the crystal packing and unit cell dimensions of the same CTB-MPR construct. Although information has been gathered on CTB-MPR, the intact structure of fusion protein was not solved as the MPR region showed only weak electron density or was cleaved during crystallization of macroscopic crystals. The MPR region is present in micro
ano-crystals, but due to the severe limitation of the Free Electron Laser beamtime, only a partial data set was obtained and is insufficient for structure determination. However, the work of this thesis has established methods to purify large quantities of CTB-MPR and has established procedures to grow crystals for X-ray structure analysis. This has set the foundation for future structure determination experiments as well as immunization studies.
In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression.