Author ORCID Identifier

https://orcid.org/0000-0002-2067-1405

Semester

Fall

Date of Graduation

2024

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

F Heath Damron

Committee Member

Tracy Liu

Committee Member

Ivan Martinez

Committee Member

Holly Cyphert

Committee Member

Brian Peppers

Abstract

The onset of the 2020 COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, issued a call to action in the fields of biomedical science and public health to implement strategies to contain and protect against the viral threat. In record times, pharmaceutical companies produced antiviral therapies and novel mRNA vaccines that were approved by the FDA for human use. With time, these vaccines were able to assist in ending the pandemic. However, time also showed the limits of mRNA vaccine induced immunity. Despite antibody responses being initially robust, titers dramatically wane with time and are limited in cross-neutralizing ability. As SARS-CoV-2 viral variants of concern continued to emerge across the globe with mutations that enabled increased pathogenicity as well as greater transmission rates, strategies to improve the protective capacity of vaccines became urgently needed. Due to differences between the mouse and human ACE2 receptors, authentic SARS-CoV-2 virus causes limited disease in mice. To model viral pathogenesis in preclinical small animal models, the K18-hACE2 transgenic mouse in which human ACE2 is expressed under the cytokeratin 18 promoter, was returned to research use after its initial implementation for modeling SARS-CoV infection. We worked to characterize the pathology and disease phenotypes of emerging variant of concern (VOC) strains to prepare translatable preclinical models for the evaluation of new vaccination strategies. The Alpha (B.1.1.7) and Beta (B.1.351) variants caused differential phenotypes compared to the ancestral variant and lowered the lethal dose of virus in K18-hACE2 mice. Here, we describe the efforts to characterize the mouse challenge model for the SARS-CoV-2 Delta variant (B.1.617). In a comparison against the Alpha variant administered at the same dose, Delta caused delayed onset of morbidity phenotypes. Despite this, Delta-challenged animals showed equal viral burden in the lung and nasal wash to Alpha animals which were approaching humane endpoint criteria. Interestingly, the lung tissues of Delta-challenged animals showed increased inflammation by histopathological analysis, and high expression of proinflammatory genes in transcriptomic analyses. With these data, we determined that the K18-hACE2 mouse model of Delta challenge develops high pulmonary inflammation compared to prior models. With an understanding of the severe Delta challenge model, we aimed to develop another preclinical mouse model to better understand a prominent COVID-19 comorbidity: obesity. Individuals with obesity and metabolic disease were determined early in the pandemic to be at greater risk of developing severe disease. With the understanding that obesity predisposes to more severe infections due to dysregulated inflammatory responses, and clinical reports that persons with high body mass index (BMI) experience more dramatic antibody waning, establishing a model to evaluate vaccines in an immunodeficient host was critical. We used the diet-induced obesity (DIO) approach to accomplish metabolic disease phenotypes in K18-hACE2 animals and observed that DIO animals after SARS-CoV-2 Alpha challenge experience a shortened time to morbidity. DIO female animals had higher lung viral burden than lean animals post-challenge. Female DIO animals also showed greater expression of antiviral genes and interferon gamma in the lungs. Male DIO animals showed a decrease in antibody related genes compared to lean animals. With an understanding of the unique immune responses in healthy and comorbid hosts, we moved to use them to evaluate the correlates of protection of known and novel vaccine strategies. Intranasal vaccines are hypothesized to generate superior immunity to muscular vaccines for respiratory pathogens due to the elicitation of immunity in the respiratory mucosa. We vaccinated K18-hACE2 mice with two doses of an intranasal virus like particle expressing RBD proteins adjuvanted with a bacterial enzymatic combinatorial chemistry adjuvant (BECC470). Compared to intramuscular mRNA vaccinated mice, intranasal mice developed higher anti-RBD IgA responses. Despite showing lower in vitro virus neutralization, nasal vaccinated animals were protected against lethal Delta variant challenge and exhibited less lung inflammation than mRNA vaccinated mice. Unfortunately, the cross protection from nasal vaccination was limited, and only mRNA vaccinated animals were protected in a follow up experiment using the Omicron variant. Next, mRNA vaccination was evaluated in the DIO K18-hACE2 comorbidity model. Despite hypotheses that DIO animals would be poorly protected after vaccination, DIO male and female animals developed high titers of anti-RBD IgG. Single cell RNA sequencing suggested differences in naïve B cell and macrophage populations between lean and DIO animals after Omicron challenge. Still, this was accompanied by limited viral burden was detected in the lung tissue. These experiments demonstrated the efficacy of mRNA vaccines against multiple viral variants and in immunocompromised individuals. Still, mRNA vaccines demonstrate limitations in immune breadth and durability. Finally, we designed experiments to characterize the immune response to mRNA vaccination to identify targets for novel immune-potentiating adjuvants. First, we identified the minimum protective dose of an mRNA COVID-19 vaccine for mice that provided protection against Omicron challenge six months after vaccination. With a known dose, we then profiled the early immunological events that occur post-vaccination and identified a correlative relationship between the chemokines CXCL10 and CXCL13, and protective antibody levels. We hypothesized that exploiting expression of these chemokines using mRNA genetic adjuvants administered with the vaccine could support better antibody responses. Cxcl13-mRNA administered with a low dose of mRNA vaccine increased antibody levels to match a higher dose of vaccine alone. These data provide exciting proof of concept to the use of mRNA genetic adjuvants that can improve vaccine immunity but may also have applications across human diseases, through tailoring of the host immune response. We hypothesize that the emergence of mRNA technology during the COVID-19 pandemic introduced a new approach to solving problems in human health and has near-boundless potential.

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