SARS-CoV-2 Variants of Concern: Biological Implications of Immunogenicity and Comorbidity
When coronavirus disease 2019 (COVID-19) emerged in human populations in late 2019, there was nothing known about the virus, SARS-CoV-2, besides that it had a high degree of similarity to severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS-CoV). The prior published research on these viruses set a strong foundation for the research approaches needed to quickly stand-up medical countermeasures for treatment of COVID-19. However, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) soon revealed a high propensity to evolve new variants that could engage host cell receptors with greater affinity and escape host immunity. With each wave of new variants of concern (VOC), scientists unveiled new mutations that granted each virus the ability to reinfect and cause disease, resulting in nearly three years of global COVID-19 infection and hospitalization that continues to this day. The pandemic created an urgent and immediate need for the development of a wide variety of in vivo and in vitro models of SARS-CoV-2 which could define the unique phenotypes of each variant so that medical countermeasures could be developed. Hence my thesis, conducted during the peak of the pandemic and after focused on two main scientific contributions toward characterizing SARS-CoV-2 in vitro and in vivo. My first research efforts sought to understand the immunity and protection conferred by challenge with an early stain of SARS-CoV-2 called Washington (or WA1) based on its origin to the later variant of concern (VOC), Alpha and Delta in a lethal mouse model. The transgenic mouse model was genetically modified to express the primary receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). As the infection of the K18 hACE2 mouse model with SARS-CoV-2 results in lethality, a dose needed to be chosen that the mouse survives in order to examine protection conferred by an initial infection. The mice that survived the SARS-CoV-2 WA1 infection were challenged three weeks later with lethal doses of SARS-CoV-2 WA1, Alpha or Delta strains. In contrast to mice that were infected and then reinfected with WA1, mice that were infected with WA1 and then infected with Alpha or Delta lost weight. In contrast the mice infected, and reinfected with WA1, continued to gain weight. Breakthrough infection was observed in the Alpha and Delta infected mice. Viral transcripts were detected by ribonucleic acid sequencing (RNA-Seq) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) in the nasal turbinates, but not in the lungs one day after reinfection. No virus was detected in any of the WA1 infected-reinfected groups suggesting protection from the first low dose (nonlethal) infection. RNASeq identified nonsynonymous amino acid changes in the viral genomes recovered from several infected mice and from one recovered from a challenged mouse. These studies provide supportive evidence that SARS-CoV-2 variants could be generated within host using the host response as selection pressure. Another dynamic that became apparent early in the pandemic was that individuals with certain comorbidities such as diabetes and heart disease were especially prone to severe COVID-19 outcomes. Diabetes was among the top represented condition of hospitalized COVID-19 patients. Not only did diabetes put an individual at an enhanced risk of developing severe symptoms with COVID-19, but COVID-19 was associated with raising an individual’s risk of developing new-onset diabetes. There is strong evidence that SARS-CoV-2 could promote diabetes through its ability to dysregulate host metabolism. Since diabetes mellitus is a disease of maligned host metabolism, I concluded that to study these mechanisms in vitro would have an impact on the field. To address the gap of available in vitro models to study SARS-CoV-2 infection, I focused the second half of my thesis on developing an in vitro transwell culture model of differentiated lung cells and using the model to characterize the effect of high glucose on viral infection and replication. I chose A549 cells, an adenocarcinoma cell line sourced from human lung tissue, since there was ample literature that described the effect of high glucose on the cell line. In my model, the cells are differentiated in low or high glucose for 21 days at an air-liquid interface. I then infected these cells at 21 dpi and evaluated the susceptibility of these cells to SARS-CoV-2 infection. High glucose supplementation did not increase the level of virus load as compared to low glucose cells. Delta infected samples had the greatest amount in recoverable infectious virus compared to the WA1 or JN.1 infected samples. Additionally, preliminary analysis of immunofluorescent stained samples reveals ACE2 and SARS-CoV-2 colocalize in the infected wells, with ACE2 not detectable above the limit of detection elsewhere. Remarkably, the model has robust infection despite a low level of ACE2. Preliminary data shows that infection and ACE2 are expressed in the same regions. A second receptor of SARS-CoV-2, transmembrane serine protease 2 (TMPRSS2) is also expressed in the differentiated, transwell A549 model. The study highlights differentiated A549 cells as a promising tool to reveal phenotypic differences between SARS-CoV-2 variants and its potential to study the effects of high glucose on viral infection and replication. In summary, my research contributed to the development of in vitro and in vivo models to study the biology of SARS-CoV-2. There remains a significant lack of insight into the mechanisms by which COVID-19 is more severe in diabetics and how COVID-19 may promote new-onset diabetes. Although case fatality rates have fallen since the height of the pandemic, COVID-19 cases continue. Hence it remains a high priority to remain vigilant in development of therapeutics that effectively combat the threat of breakthrough infection by genotypically and phenotypically different strains of SARS-CoV-2 that may continue to evolve.
coronavirus, diabetes, sars-cov-2, variants, Life Sciences, Microbiology, Virology
University of Tennessee Health Science Center
Networked Digital Library of Theses and Dissertations