COMPARATIVE INFECTION PHENOTYPES OF WILD-TYPE AND CpG-RECODED ZIKA VIRUS VARIANTS IN VITRO AND IN VIVO
Date
2022-04-28
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0002-5611-2779
Type
Thesis
Degree Level
Doctoral
Abstract
Viruses contain genetic material in DNA or RNA strands, consisting of building blocks—nucleotides. Each nucleotide is represented by a letter; for RNA, these are A (adenine), C (cytosine), G (guanine), and U (uracil). Two linear nucleotides linked by a phosphate (p) molecule, e.g., CpG, are called dinucleotides. Increasing CpG dinucleotide numbers in RNA viral genomes while preserving the original amino acid composition leads to attenuated infection. Beneficially, the attenuated infection may lead to robust antiviral host immunity providing a cutting-edge approach for vaccines. While CpG recoding is an emerging and promising vaccine approach, little is known about infection phenotypes caused by recoded viruses in primary cells and their safety/efficacy in animal models.
My overreaching goal was to compare infection phenotypes of wild-type and CpG-recoded Zika virus variants/vaccine candidates and test their safety and protective efficacy in mouse and pig models.
First, I compared in vivo infection phenotypes of wild-type Zika virus strains from different lineages, specifically Asian and African strains. Emerged Asian strains caused the recent Zika epidemic; epidemic potential and pathogenicity of historical African strains' were not well-known. I showed that the historical African Zika virus strain caused more severe fetal infection than the epidemic Asian strain in the fetal pig transmission model. This was the first comparative study of Asian and African Zika virus strains in the immunocompetent fetal model. All recent efforts on vaccine development against Zika virus were focused on epidemic Asian strains. But my study and studies from other groups showed that future vaccine development should account for Zika virus genomic heterogeneity and the epidemiological potential of more pathogenic African strains. Accordingly, a recent study from Brazil identified emerging African strain-specific mutations in wild non-human primate Alouatta guariba. Also, these findings informed my vaccine efficacy experiments where I used highly pathogenic heterologous African strain for the challenge. Second, we generated Zika virus vaccine candidates with increased CpG content, and I studied their stability, safety, and efficacy. I found that increased CpG content in recoded viruses was stable in vitro and in vivo. In comparison to the wild-type virus, recoded variants caused impaired infection in vitro as well as in neonatal and adult pregnant mice. This was the first study that addressed interactions of CpG-recoded viruses with dendritic cells and demonstrated the safety in pregnant animals. Despite impaired infection, recoded Asian viruses evoked robust cellular and humoral immune responses comparable to immunity evoked by wild-type Zika virus and complete protection against lethal challenge with heterologous African strain. Third, to better understand potential safety risks of CpG recoded live vaccines, I studied infection phenotypes of the recoded neurotropic and congenital virus—Zika virus, directly in target tissues—brain and placenta after uttermost challenge. I used direct intracerebral or in utero injection of wild-type and recoded virus variants. While overall infection kinetics were comparable between wild-type and recoded variants, I found convergent phenotypical differences characterized by reduced pathology in the mouse brain and reduced replication of CpG variants in fetal lymph nodes.
Altogether, my studies show that CpG recoding is the promising approach for vaccines against emerging flavivirus—Zika virus. The approach utilizes swift gene synthesis and reverse genetics techniques and can be rapidly applied for vaccine development during epidemics and rapidly adapted to different emerging strains. However, more efforts are needed to understand mechanisms of CpG-mediated virus attenuation, fine-tuning of the CpG-recoding technology for better vaccine safety, and multisided testing of recoded viruses in different physiological groups. The knowledge generated in my thesis will contribute to future theories and applications of the emerging vaccine technology and potentially may be translated to vaccines against other flaviviruses and viruses from different families.
Description
Keywords
CpG, Zika virus, In vitro, In vivo, Vaccine, Infection, Pig, Mouse.
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
School of Public Health
Program
Vaccinology and Immunotherapeutics