Browsing by Subject "Virology"
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Item Open Access A Cross-Sectional Study Comparing Torque Teno Virus Infection and Bushmeat Exposure Among Pneumonia Patients: Sarawak, Malaysia(2017) Berkhouse, Hudson TaylorBackground: Torque Teno Virus (TTV) is ubiquitous, possibly zoonotic, and has potential for clinical and global health research application. The goals of the current study were to determine prevalence of TTV among pneumonia patients in two Malaysian hospitals, compare results from qPCR and conventional PCR detection methods, and to compare TTV infection against self-reported exposure to bushmeat products.
Methods: Medical officers obtained plasma, PBMC, and NP swab samples, along with bushmeat exposure information from 34 pneumonia patients in Sibu and Kapit hospitals. Samples were tested for TTV using qPCR. Results were corroborated using conventional PCR. Cornfield’s and McNemar’s exact methods were used to analyze infection by exposure, and agreement between PCR results respectively. Diagnostic abilities of the qPCR test were analyzed using conventional PCR as the gold standard.
Results: Conventional PCR reported 17.65% TTV prevalence, while qPCR reported 91.18%. Household size (OR=0.00, 95% CI=0.00, 0.96) and gender (OR=infinity, 95% CI=1.39, infinity) had statistically significant impacts on odds of infection. The qPCR method showed 100% sensitivity, but 7.69% specificity.
Conclusions: Results suggest low prevalence of TTV within the study population, and reaffirm findings by other researchers that smaller household size and being female are associated with decreased odds of TTV infection. Though statistically insignificant, indications that exposure to bushmeat in general increases odds of TTV infection should be investigated further. Researchers must be aware of differences in diagnostic capabilities between PCR methods for TTV detection when designing their own diagnostic studies or conducting TTV related literature reviews.
Item Open Access An Exploratory Search for Novel Coronaviruses in Sarawak, Malaysia(2017) Fatima, HibaBackground: In recent years, emerging zoonotic microbes are gaining more attention from the public and policy makers. Explosive outbreaks such as those from avian influenza viruses, severe acute respiratory syndrome (SARS) virus, swine influenza viruses, Hendra virus, Nipah virus, and Middle East respiratory syndrome (MERS) have had tremendous international economic and social impact. In particular, livestock workers have been found to be at increased infection risk and some of the first impacted by a novel pathogen. One of the main obstacles in averting outbreaks of novel microbes is detecting it when it first begins to cross species from animals to man and may not cause severe disease. Often routine diagnostics will fail to detect a new pathogen. The purpose of this research was to evaluate diagnostics for emerging coronaviruses that would be missed with routine diagnostics.
Methods: In 2016, I learned how to run new diagnostics adapted at Duke University to detect novel coronaviruses. I took this molecular technology to Sarawak, Malaysia, where I applied the assays against a panel of human clinical specimens from patients seen at three hospitals for respiratory illnesses. Our collaborators in Sarawak had previously examined these specimens with other assays against human coronaviruses but did not tell me of their results.
Results: In my hands, the new pan-species coronavirus assay detected only one coronavirus among 88 clinical specimens. After I finished my assay work, I learned from our collaborators that 27of the 88 specimens had been positive for at least one previously recognized human coronavirus. Hence, the sensitivity of the new assay in my hands found to be 3.70% (95% confidence interval 0% - 11.91%). However, the assay accurately showed negative results with a specificity of 100%
Conclusion: While this low sensitivity may have been real, it may also been influenced by a number of confounding factors such as specimen nucleic acid degradation with numerous freeze-thaw cycles, imprecise adaptation of an assay to new equipment in a new laboratory, or my or our collaborators’ operator error. It is difficult to precisely identify the cause of the discordance. Nevertheless, I learned a great deal about global health in conducting this research in Sarawak and have chronicled some of these lessons in this report.
Item Open Access Assessing Neurocognitive Impairment in HIV-positive Patients: The Sensitivity and Specificity of the CogState Brief Battery(2012) Yechoor, NirupamaObjective: The prevalence of cognitive impairment in HIV-positive patients is estimated to be 39% in Uganda [1]. Despite the high prevalence of impairment, routine HIV management in Uganda does not include neuropsychological assessment. The objective of this study was to compare performance on the gold standard neuropsychological exam and on the CogState computerized exam. We hypothesized that there would be a high degree of correlation between performance on the two exams.
Methods: This cross-sectional survey was conducted from August to October 2011. Each participant completed the standard neuropsychological exam, which consisted of 10 tasks across 7 cognitive ability domains. Patients also completed CogState, which consisted of 4 tasks using playing cards. Performance for each exam was standardized using normative data from HIV seronegative controls to produce z-scores. The primary outcome measures were average z-scores of performance for each exam.
Results: Out of 181 patients tested, 130 (72%) were classified as impaired on the gold standard neuropsychological exam, while 104 (57%) were classified as impaired on CogState. The sensitivity of CogState compared to the gold standard was 65% (95% CI = 56% - 73%) and the specificity was 63% (95% CI = 48% - 73%). The Pearson's correlation of cumulative performance between the exams is 0.552, which is significant at the 0.01 level. Finally, those patients with normal cognition performed the best on CogState, while those with the most severe impairment performed the worst.
Conclusions: Our findings suggest that CogState is a feasible and useful tool to screen for and monitor impairment in HIV-positive patients, especially in resource-limited settings. Future studies are needed to examine how individual performance on CogState changes over time.
Item Open Access Characterization of Host Factors and Anti-viral Compounds for Diverse Mosquito-borne Flaviviruses(2016) Barrows, Nicholas J.Our ability to convert basic knowledge into robust anti-viral therapeutics requires discovery of novel host-virus interactions as well as an informed anti-viral discovery pipeline. We used a genome-scale RNAi-based screen followed by a chemical screen of FDA-approved therapeutics to identify scores of novel dengue virus (DENV) human host dependency factors (HDF) and identified more than 20 potential anti-Zika virus (ZIKV) therapeutics.
Two genes in particular, TTC35 and TMEM111, strongly inhibited DENV infection and, based on comparisons with published literature, implicated a larger protein, the ER Membrane Protein Complex (EMC), as a pan-flavivirus HDF. The EMC is a poorly characterized multiprotein complex that may function in ER-associated protein biogenesis and/or lipid metabolism. Based on our screen data, we hypothesized that the EMC is an uncharacterized HDF that functions through a common mechanism to promote replication of flaviviruses. We report that DENV, ZIKV, and yellow fever virus (YFV) infections were impressively inhibited, while West Nile Virus (WNV) infection was unchanged, in cell lines engineered to lack EMC subunit 4 (EMC4). Furthermore, targeted depletion of EMC subunits in live mosquitos significantly reduced DENV-2 propagation in vivo. In addition, the accumulation of DENV proteins shortly after infection in EMC4 knockout cells was significantly reduced, suggesting that the EMC promotes viral protein biogenesis.
We interrogated a library of FDA-approved drugs for their ability to block infection of human HuH-7 cells by a newly isolated ZIKV strain. Selected compounds were further validated for inhibition of ZIKV infection in human cervical, placental, and neural stem cell lines, as well as primary human amnion cells. Established anti-flaviviral drugs (e.g., bortezomib and mycophenolic acid) and others that had no previously known antiviral activity (e.g., daptomycin) were identified as inhibitors of ZIKV infection. Several drugs reduced ZIKV infection across multiple cell types.
We propose that the EMC may be exploited as a novel therapeutic target for multiple flaviviruses in the future. Also we identified drugs that could be tested in clinical studies of ZIKV infection and provides a resource of small molecules to study ZIKV pathogenesis.
Item Open Access Characterization of Influenza A Virus Infection through Analysis of Intrahost Viral Evolution and Within-host Infection Dynamics(2016) Sobel Leonard, Ashley ElizabethInfluenza A virus is a major source of morbidity and mortality, annually resulting in over 9000 deaths in the United States alone. As a segmented, RNA virus, influenza has a high mutation rate, facilitating its evolution to overcome cross protective immunity through natural selection and adapt to new host species or sources of evolutionary pressure through reassortment. The high viral mutation rate also means that these processes affect not only evolution at the population level, but also at the intrahost level. While these processes have been well characterized for population-level viral evolution, viral evolution within a single host is far less well defined. In this dissertation, I characterize influenza infection at the intrahost level with respect to viral evolution and infection dynamics. In the second chapter, I critically evaluate methods for estimating the transmission bottleneck size for influenza A virus from viral sequencing data. The transmission bottleneck describes the infecting population size, a determinant for the level of genetic diversity present at the onset of infection. I show current methods may be biased, both by the criteria used to identify sequencing variants and the presence of demographic stochasticity. In response to these biases, I introduce a new method that (1) corrects for differences in variant calling criteria and (2) accommodates demographic stochasticity. Chapters 3-5 are based on data collected from an existing human challenge study with influenza A virus. In this challenge study, volunteers were experimentally infected with a heterogeneous viral inoculum that had adapted to the conditions in which it had been generated. In chapter 3, I show that transmission was governed by a selective bottleneck and that subsequent intrahost viral evolution was dominated by purifying selection. In chapter 4, I further characterize the observed intrahost viral evolution through the reconstruction of viral haplotypes to evaluate different models of selection. These models differed by the level at which selection was acting, whether selection is focused on individual loci, multiple loci within a single gene segment, or across gene segments. Model selection favored the third model, wherein selection acted across gene segments, thereby establishing that the effective viral reassortment rate was limited in these subjects. In chapter 5, I develop a mathematical model for within-host influenza infection linking viral replication and the host immune response with the development of disease symptoms. I fit this model to experimental data collected from the challenge study. Analysis of the model fits indicated that much of the heterogeneity in the data between subjects could be explained by interindividual variation in viral infectivity. This finding echoed the results of chapters 3 and 4, that there were quantifiable differences in the infecting viral populations between the study subjects. Taken together, these observations suggest that
intrahost viral genetics may underlie the differences between the subjects’ response to infection.
Item Open Access Characterizing novel molecular regulators of antiviral gene expression(2020) McFadden, Michael JThe intracellular innate immune response to viral infection is among the first lines of defense against these pathogens. For the early establishment of an antiviral cellular state and initiation of inflammatory responses, type I interferons (IFNs) are particularly important, as they potently induce the production of hundreds of IFN-stimulated genes (ISGs), many of which have antiviral functions. The type I IFN response requires tight molecular coordination to achieve both efficient production of antiviral proteins and controlled shutoff of inflammatory responses to avoid tissue damage and autoimmunity. Despite the importance of regulation of this antiviral response, current knowledge of the molecular controls governing its activation and suppression remains incomplete. Further, although ISGs have diverse functions and are induced to differing potencies, our understanding of regulatory controls governing the expression of individual or subclasses of ISGs is limited. Current knowledge of type I IFN response regulation is predominantly centered on transcriptional and post-translational regulatory controls. However, post-transcriptional regulation of antiviral responses has begun to emerge as an important layer of control. An example of these post-transcriptional regulatory controls is the RNA base modification N6-methyladenosine (m6A), which regulates many aspects of mRNA metabolism through transcript-specific effects. m6A deposition is mediated by a cellular complex of proteins including METTL3 and METTL14 (METTL3/14) and other cofactors, and m6A can also be removed from RNA by the demethylase proteins FTO and ALKBH5. The presence of m6A on viral and host RNAs has been shown to influence the outcome of infection by diverse viruses. However, the role of m6A in the response to type I IFNs has not been explored. To investigate the role of m6A in the type I IFN response, we began by manipulating m6A levels in the transcriptome through perturbation of the expression of the cellular m6A machinery and measuring the induction of ISGs after IFN treatment. We found that depletion of the m6A methyltransferase proteins METTL3 and METTL14 (METTL3/14) resulted in less protein production of a subset of ISGs, including the antiviral genes IFITM1 and MX1, after IFN treatment. However, the expression of other ISGs and the overall activation of the IFN responses were unchanged. Using methyl RNA immunoprecipitation and sequencing (meRIP-seq), we found that the transcripts of many ISGs are modified by m6A, and these included the METTL3/14-regulated ISGs IFITM1 and MX1 that we had identified. Using polysome profiling and ribosome profiling, we determined that METTL3/14-regulated ISGs are translationally enhanced by METTL3/14. Additionally, ablation of putative m6A sites within the 3’UTR of IFITM1 decreased the translation of a reporter molecule. Overexpression of the m6A reader protein YTHDF1, which has known roles in promoting translation, enhanced the expression of IFITM1 in an m6A binding-dependent fashion. These experiments characterized METTL3/14 and m6A as novel enhancers of the type I IFN response. To determine whether m6A contributes to type I IFN-mediated viral restriction, we depleted or overexpressed METTL3/14 and pretreated cells with a low dose of IFN-β prior to infection with vesicular stomatitis virus (VSV). Interestingly, METTL3/14 depletion decreased the expression of ISGs and allowed increased VSV infection, while METTL3/14 overexpression had the opposite effect. Together, these studies demonstrate that METTL3/14 and m6A enhance the antiviral effect of type I IFN by promoting the translation of ISGs to support the establishment of an antiviral cellular state. Having discovered a role for m6A in the type I IFN response, we also investigated the role of an m6A demethylase protein, FTO. FTO polymorphisms can have profound effects on human health. Certain polymorphisms are associated with fat mass and obesity, cardiovascular disease, while others can cause growth retardation or embryonic lethality. However, the molecular functions of FTO and the cellular pathways that it affects are still not well characterized. We depleted FTO and measured the production of ISGs following IFN-β treatment and found that the production of m6A-regulated ISGs was increased, as expected. However, unexpectedly, we found that FTO depletion increased the mRNA levels of a subset of ISGs. Pulse labeling of nascent transcripts revealed that FTO suppresses the transcription of these ISGs and that FTO-depleted cells are primed for the production of certain ISGs in response to IFN. We then used cells lacking PCIF1, the writer of 2’-O-N6-dimethyladenosine (m6Am), an RNA modification that FTO can also remove, and found that FTO-mediated regulation of ISGs occurs independently of the m6Am modification. These results identify FTO as a transcriptional regulator of a subset of ISGs, which will add an important dimension to our understanding of the molecular functions of FTO and its contributions to inflammatory disease. Future research revealing the mechanisms by which FTO suppresses ISG transcription will be of great interest. Together, these data identify novel functions of m6A and its related cellular machinery in both positive and negative regulation of the type I IFN response and antiviral gene expression.
Item Open Access Combating Respiratory RNA Viruses with Adaptable Tools and Innate Host Defenses(2022) Froggatt, Heather MarieIn the 21st century, the world has already experienced two pandemics caused by respiratory RNA viruses, the 2009 H1N1 “swine-flu” pandemic, and the ongoing COVID-19 pandemic. The 2009 pandemic was, thankfully, mild in terms of casualties; however, it revealed certain failings in existing systems. As a circulating virus, we had approved, in-use antiviral treatments for influenza, but the 2009 H1N1 viruses were resistant to an entire class, adamantanes. Also, the requirement for annually updated influenza vaccines meant there were systems in place for large-scale vaccine production, yet reliance on eggs dramatically limited the ability to scale-up a new vaccine quickly. Finally, global vaccine inequities meant many countries remained relatively unvaccinated against the H1N1 virus, even as the pandemic was declared over in August 2010. The COVID-19 pandemic has been a very different story on some fronts, most notably the death toll, which exceeds 6 million globally. Also, the decade of progress in vaccine research since 2009 has been evident in the rapid development of many highly effective COVID-19 vaccines using different platforms, while vaccine distribution issues remain. Finally, an entire lack of approved coronavirus antivirals required swift action to identify existing drugs and treatments to alleviate disease. The research and clinical response to the COVID-19 pandemic took advantage of existing technologies, pharmaceuticals, and systems and adapted them to solve the present crisis. The problem of this dissertation is to extend these efforts by investing in adaptable research tools, methods, and subjects to prepare for potential future respiratory RNA virus pandemics.In Chapters 2 and 3, we generated virus-specific research tools that are rapidly adaptable to new viral strains. Chapter 2 describes a fluorescent reporter of coronavirus protease activity that may be used to screen antiviral drugs and is compatible with diverse coronavirus protease proteins. Chapter 3 discusses a novel method for generating influenza reporter viruses with wide research applications, notably this manner of introducing exogenous proteins requires minimal genome rearrangements increasing transferability to newly identified strains. These projects demonstrate that even research tools requiring updates with the emergence of new strains can be designed to prioritize rapid adaptability. In Chapters 4 and 5, we interrogate the innate immune responses that determine the outcomes of viral infections. Chapter 4 identifies ETV7 as a negative regulator of the antiviral type I interferon response using CRISPR activation screening. ETV7 was previously known to be induced by interferon, but its role during the response to viral infection remained undetermined. We found ETV7 limits transcription of interferon stimulated gene expression, influencing the antiviral state of a responding cell. Chapter 5 reviews the impact of influenza viral disease outside the site of infection, the respiratory tract, and established methods of studying these effects using animal models. Many of the circulating cytokines implicated in non-respiratory influenza disease from these models, IL-6, IFNs, and TNF-alpha, are known to play a role in influenza and COVID-19 patient disease severity. These investigations show that our understanding of how innate immunity is regulated, and dysregulated, continues to require updating even as the main pathway members and downstream effectors have been identified.
Item Open Access Competing RNA Structures and Their Effects on HDV Antigenomic RNA Self-cleavage and mRNA Processing(2010) Brown, Abigail LeighHDV antigenomic RNA is processed in two distinct pathways; it can be cleaved at the polyA site and polyadenylated to become mRNA for the delta antigens, or the RNA can be cleaved by the antigenomic ribozyme to become full-length antigenomic RNA that is used for synthesis of genomic HDV RNA. The polyA site is located just 33 nucleotides upstream of the ribozyme cleavage site. If processing occurs primarily at the upstream polyA site, there may not be enough full-length antigenomic RNA to support replication. On the other hand, ribozyme cleavage downstream of the polyA site could inhibit polyadenylation by interfering with polyadenylation complex assembly. Thus, it appears that HDV may need a mechanism to control RNA processing so that both products can be generated in the proper amounts during the infection cycle.
A model has been proposed in which the choice between ribozyme cleavage and polyadenylation is determined by alternative RNA secondary structures formed by the polyA sequence (Wadkins and Been 2002). One of the hypothetical structures, AltP2, is a pairing between part of the upstream polyA sequence and the 3' end of the ribozyme sequence. For this model, the same upstream sequence that forms AltP2 could also form a stem loop, P(-1), within the leader, by pairing with sequences located farther upstream. A processing choice is possible because AltP2 is predicted to inhibit ribozyme cleavage and favor polyadenylation resulting in mRNA production, whereas P(-1) would inhibit polyadenylation and favor ribozyme cleavage resulting in full-length replication product.
The P(-1) vs. AltP2 model was tested using an antigenomic HDV ribozyme construct with the 60-nucleotide sequence upstream of the ribozyme cleavage site. This leader sequence contains the proposed polyA sequence elements. In vitro analysis of this construct revealed that the kinetic profile of ribozyme self-cleavage was altered in two ways. Relative to the ribozyme without upstream sequences, the fraction of precursor RNA that cleaved decreased to about 50%, but the active ribozyme fraction cleaved faster. Native gel electrophoresis revealed that the active and inactive precursor RNAs adopted persistent alternative structures, and structure mapping with Ribonuclease T1 and RNase H provided evidence for structures resembling P(-1) and AltP2.
Sequence changes in the 5' leader designed to alter the relative stability of P(-1) and AltP2 increased or decreased the extent of ribozyme cleavage in a predictable way, but disrupting AltP2 did not completely restore ribozyme activity. The analysis of deletion and base change variants supported a second alternative pairing, AltP4, formed by the pyrimidine-rich sequence immediately 5' of the ribozyme cleavage site and a purine-rich sequence from the 5' side of P4. A similar approach was used to test if the effect of disrupting both AltP2 and AltP4 might be additive, and the results suggested that ribozyme precursors with 5' leader sequences could fold into multiple inactive conformations, which can include, but may not be limited to, AltP2, AltP4, or a combination of both.
Luciferase expression constructs with HDV polyA and ribozyme sequences were used to investigate the effects of RNA structure and ribozyme cleavage on polyadenylation in cells. One hypothesis was that P(-1) could inhibit polyadenylation by making the polyA sequence elements less accessible to polyA factors, but sequence changes designed to alter the stability of the stem loop had no effect on polyadenylation. The model also predicts that the ribozyme sequence downstream of the polyA site could affect polyadenylation, possibly in two different ways. Ribozyme cleavage could interfere with polyadenylation by uncoupling transcription from processing, however, the ribozyme sequence might also influence polyadenylation in a manner independent of the ribozyme cleavage activity. As such, the AltP2 structure could potentially have a positive effect on polyadenylation either by inhibiting ribozyme cleavage or by making the polyA signal sequences more accessible to the polyA factors. To distinguish between the effects of ribozyme cleavage and alternative RNA structures, luciferase expression levels from constructs with an HDV polyA sequence followed by the active wild-type ribozyme or the inactive C76u version of the ribozyme were compared. For the wild-type HDV polyA sequence, the active ribozyme reduced expression, whereas the inactive ribozyme control had no effect on expression. However, for the modified leader sequences, which were efficiently polyadenylated in the absence of ribozyme, there were changes in expression that appeared to be independent of ribozyme cleavage. Based on these findings, two alternative models are proposed. One model predicts that protein factors might affect antigenomic RNA processing, and the other model suggests that additional alternative structures, such as AltP4, might influence the choice between ribozyme cleavage and polyadenylation.
Item Open Access Cross-Species Evolution of New AAV Variants(2023) Gonzalez, Trevor JohnTherapeutic gene transfer and genome editing require effective delivery of genetic cargo to target cells and tissues. Recombinant adeno-associated viral (AAV) vectors are a promising delivery platform, but ongoing clinical trials continue to highlight a relatively narrow therapeutic window. Efforts to optimize vector dosing or engineer improved vectors are confounded, at least in part, by differences in AAV biology across animal species. Here, we present a broadly applicable, cross-species evolution approach to tackle this challenge. Specifically, I iteratively cycled AAV libraries administered intravenously and amplified isolates from CNS tissue in pigs, mice, and non-human primates to generate cross-species compatible AAVs (ccAAVs). By sequentially evolving AAV libraries in three different species, we discover a highly potent variant (AAV.cc47) that demonstrates improved attributes benchmarked against AAV serotype 9 (AAV9). Increased potency of AAV.cc47 is evidenced through robust reporter gene expression as well as Cre-mediated recombination and CRISPR/Cas9-mediated genome editing in a fluorescent reporter mouse model. Enhanced transduction efficiency of AAV.cc47 vectors is further corroborated in macaques and pigs, providing a strong rationale for potential clinical translation into human gene therapies. Lastly, we report increased expression of a therapeutic acid alpha-glucosidase (GAA) transgene in a mouse model of Pompe disease and enhanced restoration of dystrophin through CRISPR/Cas9 gene editing in the mdx mouse model of Duchenne Muscular Dystrophy using AAV.cc47 vectors. We discovered another cross-species compatible variant (AAV.cc84) with enhanced CNS transduction and de-targeted from the liver in vivo compared to AAV9. We demonstrate improved targeting of neurons in the brain following both systemic and intracerebroventricular (ICV) injection of reporter vectors in mice. Reporter gene expression and vector genome biodistribution reveal a liver detargeted phenotype with AAV.cc84 compared to AAV9. Lastly, enhanced neuronal transduction was confirmed in the pig CNS following intrathecal infusion of AAV.cc84 reporter vectors. Taken together, we envision that ccAAV vectors can potentially improve predictive modeling in preclinical studies as well as clinical translatability by broadening the therapeutic window of AAV based gene therapies.
Item Open Access Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition.(PLoS pathogens, 2018-06-04) Esher, Shannon K; Ost, Kyla S; Kohlbrenner, Maria A; Pianalto, Kaila M; Telzrow, Calla L; Campuzano, Althea; Nichols, Connie B; Munro, Carol; Wormley, Floyd L; Alspaugh, J AndrewThe human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the β-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.Item Open Access Defining the Role of Antibodies in Protection Against Cytomegalovirus Acquisition and Congenital Disease for Rational Vaccine Design(2018) Nelson, Cody ShawHuman cytomegalovirus (HCMV) is the most common cause of congenital infection worldwide, impacting 1 in 150 live-born infants. Children afflicted by congenital HCMV frequently suffer from lifelong, debilitating neurologic sequelae including microcephaly, sensorineural hearing loss, and cognitive impairment. Natural maternal immunity to HCMV reduces the frequency of congenital infection, but does not prevent the disease altogether. Thus, a vaccine to reduce the incidence and severity of infant infection is a public health priority. Employing a nonhuman primate model of congenital CMV transmission as well as clinical samples from a partially-efficacious HCMV vaccine trial, we sought to examine both the attributes of anti-HCMV immune responses that provide protective immunity as well as the impact of vaccine-elicited immunity on the in vivo HCMV viral population.
First, we used a nonhuman primate model of congenital CMV infection to investigate the ability of preexisting antibodies to protect against placental CMV transmission in the setting of primary maternal infection and subsequent viremia, which is required for placental virus exposure. Pregnant, CD4+ T cell-depleted, rhesus CMV (RhCMV)-seronegative rhesus monkeys were treated with either standardly-produced hyperimmune globulin (HIG) from RhCMV-seropositive macaques or dose-optimized, potently RhCMV-neutralizing HIG prior to intravenous challenge with an RhCMV swarm. HIG passive infusion provided complete protection against fetal loss in both groups. The dose-optimized, RhCMV-neutralizing HIG additionally inhibited placental transmission of RhCMV and reduced viral replication and diversity. Our findings suggest that the presence of durable and potently-neutralizing antibodies at the time of primary infection can prevent transmission of systemically-replicating maternal RhCMV to the developing fetus.
Next, we assessed the properties of antibody responses elicited by glycoprotein B (gB) + MF59 adjuvant subunit vaccination – the most successful HCMV vaccine tested clinicaly to-date, which demonstrated approximately 50% efficacy in preventing HCMV acquisition in multiple phase 2 trials. Plasma from 33 gB/MF59 vaccinees at peak immunogenicity was tested for gB epitope specificity as well as neutralizing and non-neutralizing anti-HCMV effector functions, and compared to an HCMV-seropositive cohort. gB/MF59 vaccination elicited IgG responses with gB-binding magnitude and avidity comparable to natural infection. Additionally, IgG subclass distribution was similar with predominant IgG1 and IgG3 responses induced by gB vaccination and HCMV infection. However, vaccine-elicited antibodies exhibited limited neutralization of the autologous virus, negligible neutralization of multiple heterologous strains, and limited binding responses against gB structural motifs targeted by neutralizing antibodies including AD-1, AD-2, and Domain I. Interestingly, vaccinees had high-magnitude IgG responses against AD-3 linear epitopes, demonstrating immunodominance against this non-neutralizing, cytosolic region. Finally, vaccine-elicited IgG robustly bound trimeric, membrane-associated gB on the surface of transfected or HCMV-infected cells and mediated virion phagocytosis, raising the possibility that non-neutralizing antibody effector functions contributed to the partial protection against HCMV acquisition observed in gB/MF59 vaccinees.
Lastly, we evaluated the impact of gB/MF59-elicited immune responses on the population of viruses acquired by trial participants. In this analysis, we employed quantitative PCR as well as two distinct next-generation sequencing strategies (short amplicon and whole gene) to interrogate genetic differences between the HCMV populations infecting gB/MF59 vaccinees and placebo recipients. For the majority of subject-specific viral populations analyzed, we identified 1 or 2 dominant viral variants, as well as a large number of minor variants present at very low frequency. This finding suggests that the intrahost viral population constitutes a heterogeneous swarm of genetically-distinct virus quasi-species. Additionally, we identified several distinctions between the viral populations of acutely-infected vaccinees and placebo recipients. First, there was reduced magnitude viral shedding in the saliva of gB vaccinees compared to placebo. Furthermore, we noted evidence of genetic compartmentalization at the gB locus in 3 of 4 vaccinees, though only in 1 of 7 placebo recipients. Finally, we observed an enrichment of gB1 genotype HCMV variants among placebo recipients compared to vaccinees, and hypothesize that the gB1 genotype vaccine immunogen might have elicited genotype-specific protection that accounts for the efficacy observed in clinical trial.
Thus, we have made several observations that will inform rational design of the next generation of HCMV vaccines. First, our data suggests that preexisting antibodies can protect against congenital CMV transmission in a rhesus monkey model, and thus that antibodies could be a primary target of vaccines to eliminate congenital HCMV infection. Secondly, our analysis of antibody responses elicited by gB/MF59 vaccination indicates that non-neutralizing antibody functions contributed to the observed 50% vaccine protection and therefore should be a consideration in future vaccine design. Finally, our examination of viral populations in gB/MF59 vaccinees indicates that gB-elicited antibodies had a measurable impact on viral intrahost population dynamics and that gB immunogen strain-specific responses may have defined vaccine protection, suggesting that immunogen strain breadth may be an important factor to consider for future vaccine design.
Item Open Access Dengue Virus Host Factors(2009) Sessions, October MichaelDengue fever and dengue hemorrhagic fever are estimated to afflict 50-100 million people annually and are caused by one of the four serotypes of dengue virus. Dengue virus is carried and transmitted to humans by mosquitoes of the Aedes genus. Given the broad geographic distribution of Aedes mosquitoes, it has been estimated that nearly half the world's population is at risk of contracting the disease. Currently, no vaccine or specific antiviral treatment is available to combat this emerging menace.
A greater understanding of how dengue virus interacts with its insect and human hosts will facilitate the intelligent design of specific antivirals to combat the disease and enable the selective breeding of mosquitoes resistant to the virus. Although the genomes of the two primary mosquito vectors have been sequenced, the molecular tools necessary for conducting a systematic genetic analysis of host factors required for DEN infection are not yet available. These tools do however exist in the closely related fruit fly, Drosophila melanogaster. By using a strain of dengue virus that was adapted to propagate in fruit fly cells, we completed a full genetic screen for host factors required for efficient dengue virus propagation. When homologues of these host factors were assayed in a human cell line, over half were also shown to be required for efficient viral propagation. This indicates that while the virus is utilizing many of the same pathways in both of its hosts, the interaction with the insect vector has unique features that may contribute to the observed lack of pathogenesis in mosquitoes.
Item Open Access Diverse Strategies Deployed by Poliovirus to Cope with Host Antiviral Responses(2020) Kastan, JonathanIn the following document, I will describe two distinct strategies that poliovirus
(PV) deploys to manage host antiviral responses. In the first section, I report on a role of
the constitutive repressor of eIF2α phosphorylation (CReP) in translation of PV and the
endoplasmic reticulum (ER)-resident chaperone binding immunoglobulin protein (BiP)
at the ER. Functional, proximity-dependent labeling and cell fractionation studies
revealed that CReP, through binding of the eukaryotic translation initiation factor eIF2α,
anchors translation initiation machinery at the ER and enables protein synthesis in this
compartment. This ER site was protected from the suppression of cytoplasmic protein
synthesis by acute stress responses. I propose that partitioning of translation initiation
machinery at the ER enables cells to maintain active translation of PV during stress.
In the second section, I report that PV 2A protease cleaves all three members of
the YTHDF protein family, cytosolic N6-methyladenosine (m6A) ‘readers’ that regulate
target mRNA fate. These cleavages occurred early during infection, and preemptive
YTHDF3 depletion enhanced viral replication. This corresponded with diminished type-
I interferon (IFN) receptor (IFNAR) expression and IFN-stimulated gene induction,
while IFN production was not significantly changed. I propose that 2A protease cleaves
YTHDF proteins, in part, to interfere with IFNAR expression and antagonize the host
antiviral response.
Item Open Access DNA Damage Response Suppresses Epstein-Barr Virus-Driven Proliferation of Primary Human B Cells(2012) Nikitin, Pavel AThe interaction of human tumor viruses with host growth suppressive pathways is a fine balance between controlled latent infection and virus-induced oncogenesis. This dissertation elucidates how Epstein-Barr virus interacts with the host growth suppressive DNA damage response signaling pathways (DDR) in order to transform infected human B lymphocytes.
Here I report that the activation of the ATM/Chk2 branch of the DDR in hyper-proliferating infected B cells results in G1/S cell cycle arrest and limits viral-mediated transformation. Similar growth arrest was found in mitogen-driven proliferating of B cells that sets the DDR as a default growth suppressive mechanism in human B cells. Hence, the viral protein EBNA3C functions to attenuate the host DDR and to promote immortalization of a small portion of infected B cells. Additionally, the pharmacological inhibition of the DDR in vitro increases viral immortalization of memory B cells that facilitates the isolation of broadly neutralizing antibodies to various infectious agents. Overall, this work defines early EBV-infected hyper-proliferating B cells as a new stage in viral infection that determines subsequent viral-mediated tumorigenesis.
Item Open Access EBV-Associated Gastric Cancer: From Initial Infection to Unique Therapeutic Approaches(2023) Stanland, Lyla JuneEpstein-Barr virus (EBV) is a ubiquitous human herpesvirus that infects over 95% of the adult population. While infection is typically asymptomatic, in some individuals, particularly the immunocompromised, EBV is the causative agent of several cancers including lymphomas and epithelial cancers. Specifically, gastric carcinoma, nasopharyngeal carcinoma, and lymphoepithelioma-like carcinoma which occurs across multiple sites in the body, most notably, in the lung. EBV-associated gastric cancer (EBVaGC) is a unique subset of gastric cancer that makes up 10% of all GCs worldwide. EBVaGCs display an 80% rate of activating PIK3CA mutation and are also the most hypermethylated of any tumor type, displaying what is known as a CpG island hypermethylator phenotype (CIMP). EBV infection of B cells can be easily modeled in vitro using primary B cells and lymphoblastoid cell lines (LCLs). However, epithelial cell infection models have proven much more difficult to develop. Given these difficulties, EBV infection and outgrowth in epithelial cells is comparatively understudied and the process of tumorigenesis in vivo is poorly understood. In this dissertation, I developed methods to generate EBV infected epithelial cell lines derived from both gastric cancer and lung adenocarcinoma using diverse strains of EBV. I used these models to complete a CRISPR/Cas9 whole genome knockout screen to identify cellular restriction factors of infection and outgrowth. Together, these data will provide novel insights into the process of EBV infection and the dynamic interplay between virus and host during tumorigenesis. Furthermore, I have used these models to explore unique therapeutic approaches for EBV+ epithelial cancers. Specifically, I have characterized the lytic reactivation potential to histone deacetylase (HDAC) inhibitors and generated preclinical data supporting the use of HDAC inhibitors and the anti-viral ganciclovir for treatment of EBV+ epithelial tumors. Lastly, I have identified modulators of the response to a PI3Ka inhibitor in PIK3CA mutant gastric cancers. I found that loss of NEDD9 or inhibition of BCL-XL rendered cells hyper-sensitive to the PI3Ka inhibitor BYL719. Additionally, I found that loss of CBFB conferred resistance to BYL719 through up- regulation of the protein kinase PIM1 and defined the clinical utility of our data in the context of PI3K inhibition more broadly. The work outlined in this dissertation contributes to the study of EBV infection and tumorigenesis in the stomach as well as provides mechanistic insights into novel therapeutic approaches for EBV+ epithelial cancers and PIK3CA mutant gastric cancers.
Item Embargo Elevated Serum Chymase as a Risk Factor for Severe Dengue(2016) Farouk, Farouk ShihabDengue Virus (DENV) is one of the major viral diseases that has a high burden in Southeast Asia and the Americas. Despite advances in supportive care for mild Dengue Fever (DF) and the more severe Dengue Hemorrhagic Fever (DHF), little is known about how to identify the progression to DHF before it occurs. The purpose of our retrospective study is to assess whether there is an association between the concentration of a serum protein, chymase, with DHF (and the symptoms, pre-existing conditions, and comorbidities associated with it).
Data from 291 dengue-confirmed patients were collected from a surveillance study conducted in Sri Lanka. We selected a series of demographic, symptom, and pre-existing condition variables to see if there was an association between these, elevated chymase levels, and a DHF diagnosis. Our results confirmed that the correlation between a DHF diagnosis and increased chymase levels was statistically significant (p = <0.001). Furthermore, the negative correlation between a DF diagnosis and increased chymase levels was significant (p = <0.001). Obesity was also significantly predictive of increased chymase levels (p = <0.001). Elevated chymase levels correlated with (and predicted) a DHF diagnosis between ages 9 and 44, with those under age 9 having the strongest correlation.
Overall, an increased chymase level was associated with a DHF diagnosis. The identification of chymase as a biomarker for severe dengue may lead to improved diagnostic and surveillance systems that can identify and treat patients at risk for developing DHF.
Item Open Access Envelope-specific IgG Responses in HIV-infected Women(2018) Martinez, David RafaelA better understanding of 1) maternal HIV Envelope (Env)-specific IgG responses that are partially protective against vertical HIV transmission, and 2) factors that mediate the transplacental transfer of maternal protective IgG is needed to improve infant health in early life, in which maternal passively-acquired IgG mediates protection against neonatal infections. To understand maternal factors and IgG characteristics that mediate transplacental IgG transfer, we examined transplacental transfer efficiency determinants of maternal HIV and standard vaccine-antigen-specific IgG in a population of HIV-infected women, which have disrupted transplacental IgG transfer. Our findings suggest that maternal health factors and maternal IgG characteristics, such as binding to placentally expressed Fc receptors, IgG subclass frequency, and Fc region glycan profiles all mediate transplacental IgG transfer efficiency. We also identified maternal linear variable loop 3 (V3)-specific IgG binding and neutralizing responses targeting the C terminal region as partially protective against vertical transmission of HIV. These novel findings provide a roadmap of maternal factors and IgG characteristics as targets that can be harnessed to improve the transplacental IgG transfer of routinely-administered maternal vaccines and benchmarks for assessing maternal HIV vaccines that target the V3 loop.
Item Open Access Epigenetic and transcriptional responses in circulating leukocytes are associated with future decompensation during SARS-CoV-2 infection.(iScience, 2024-01) McClain, Micah T; Zhbannikov, Ilya; Satterwhite, Lisa L; Henao, Ricardo; Giroux, Nicholas S; Ding, Shengli; Burke, Thomas W; Tsalik, Ephraim L; Nix, Christina; Balcazar, Jorge Prado; Petzold, Elizabeth A; Shen, Xiling; Woods, Christopher WTo elucidate host response elements that define impending decompensation during SARS-CoV-2 infection, we enrolled subjects hospitalized with COVID-19 who were matched for disease severity and comorbidities at the time of admission. We performed combined single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) on peripheral blood mononuclear cells (PBMCs) at admission and compared subjects who improved from their moderate disease with those who later clinically decompensated and required invasive mechanical ventilation or died. Chromatin accessibility and transcriptomic immune profiles were markedly altered between the two groups, with strong signals in CD4+ T cells, inflammatory T cells, dendritic cells, and NK cells. Multiomic signature scores at admission were tightly associated with future clinical deterioration (auROC 1.0). Epigenetic and transcriptional changes in PBMCs reveal early, broad immune dysregulation before typical clinical signs of decompensation are apparent and thus may act as biomarkers to predict future severity in COVID-19.Item Open Access Epstein-Barr virus infection phenocopies apoptosis regulation in germinal center B cells(2019) Dai, JoanneThe Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects more than >95% of the global adult population. In immunocompetent individuals, EBV infection is asymptomatic and takes place in the oral cavity, where EBV establishes a life-long latent infection in memory B cells by temporally regulating viral gene expression to mimic B cell maturation. In immunocompromised individuals, however, EBV infection can give rise to infectious mononucleosis, epithelial carcinomas, and lymphomas. To model EBV-mediated lymphomagenesis, infection of EBV in vitro generates growth-transformed and immortalized lymphoblastoid cell lines (LCLs), which allows for the characterization of dynamic viral and host gene expression. Our lab has found that the early phase after infection is transcriptionally distinct from the late phase when infected B cells are fully growth-transformed. We also found that apoptosis regulation in each phase of infection is uniquely regulated by a single viral nuclear protein that regulates host gene expression through epigenetic mechanisms. To determine if apoptosis regulation in EBV-infected B cells is virus-specific, I have characterized apoptosis regulation in uninfected maturing B cells and mitogen-stimulated B cells. For the upregulation of one anti-apoptotic protein, EBV infection promotes a chromatin structure resembling that in germinal center light zone B cells, indicating that EBV phenocopies germinal center chromatin regulation to promote apoptosis resistance. In addition to apoptosis regulation, EBV infection phenocopies various aspects of GC B cells and plasmablasts, where the inhibition of plasma cell differentiation increases the efficiency of immortalization and growth-transformation of B cells infected in vitro. The work outlined in this dissertation demonstrate that viral and host genes cooperate in mediating apoptosis regulation, differentiation, and ultimately fate-determination of EBV-infected B cells.
Item Embargo Evaluate Glycoprotein Complexes-Elicited Antibody Responses to Inform Congenital Cytomegalovirus Vaccine Design.(2024) Wang, Hsuan-YuanHuman CMV (HCMV), a ubiquitous β-herpesvirus, remains the most common congenital infection and infectious complication in immunocompromised patients. Despite the severe clinical impacts, there is no current approved HCMV vaccine or immunotherapy. Two major hurdles have been identified for the HCMV vaccine development. First, the HCMV genome is highly diverse and variable, i.e., a HCMV vaccine design that only includes immunogens from a single strain might not provide effective protection against other HCMV strains. Second, a large gap remains to understand what virologic determinants are essential for congenital CMV transmission.
HCMV glycoprotein B (gB) and pentameric glycoprotein complex (PC) are currently the most promising vaccine targets. gB is essential for viral entry into all host cells and was shown to elicit both neutralizing and non-nAb responses. The most successful HCMV vaccine to-date, a gB subunit vaccine adjuvanted with MF59, achieved 50% efficacy against primary HCMV infection. The gB/MF59 vaccinees were less frequently infected with HCMV gB genotype strains most similar to the vaccine strain than strains encoding genetically distinct gB genotypes, suggests that the strain-specific immunity might account for the limited efficacy. Applying the lipid nanoparticles-encapsulated nucleoside-modified mRNA (mRNA-LNP) vaccine platform, we hypothesized that vaccination with multiple HCMV gB genotypes could increase the breadth of gB-specific humoral and cellular responses, leading to broader protection.
To test our hypothesis, we intradermally immunized female rabbits with three doses of the monovalent or multivalent gB mRNA-LNP vaccines and measured the vaccine-elicited humoral and cellular responses. Compared to the monovalent vaccine, the multivalent vaccines did not demonstrate a higher magnitude or breadth of gB-specific IgG binding or functional antibody responses against multiple gB genotype. In addition, the multivalent vaccines did not elicit a stronger T cell response against variable regions among gB genotypes. Our data suggests that inclusion of multivalent gB antigens is not an effective strategy to increase the breadth of anti-HCMV gB antibody and T cell responses.
PC, composed of the subunits gH/gL/UL128/UL130/UL131A, has been demonstrated to be essential for CMV entry into non-fibroblast cells in vitro. The PC was also identified as the major target of neutralizing antibodies. As HCMV infection of most cell types found near the maternal-fetal interface depends on the PC-mediated entry in vitro, it is conceivable that this complex could be required for cross-placental CMV transmission in vivo. These findings link the PC to broad cell tropism and virus dissemination in vivo, denoting all subunits as potential targets for intervention strategies and vaccine development.
To determine the importance of the PC for congenital transmission in a translational non-human primate (NHP) model, we engineered a rhesus CMV (RhCMV) mutant lacking the homologues of UL128 and UL130 which demonstrated diminished infection of epithelial cell in vitro. Nevertheless, intravenous inoculation of immunocompetent and CD4+ T cell-depleted, RhCMV-seronegative, pregnant rhesus macaques with the PC-deficient mutant resulted in similar maternal RhCMV peak plasma viremia levels to that of PC-intact RhCMV, while virus shedding in saliva and urine was limited. Infections with the PC-intact virus induced strong IgG responses that were able to neutralize RhCMV entry into epithelial cells. These responses were reduced, but not absent, from animals infected with the PC-deficient mutant, which also induced IgG responses against gH. Most importantly, congenital infection rates determined by the viral DNA detection in amniotic fluid was identical between the PC-deficient and PC-intact RhCMV. Our data indicates that the PC is dispensable for transplacental transmission in non-human primates.
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