Matching Items (13)
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- All Subjects: Virology
- Creators: Jacobs, Bertram
Description
Vaccination remains one of the most effective means for preventing infectious diseases. During viral infection, activated CD8 T cells differentiate into cytotoxic effector cells that directly kill infected cells and produce anti-viral cytokines. Further T cell differentiation results in a population of memory CD8 T cells that have the ability to self-renew and rapidly proliferate into effector cells during secondary infections. However during persistent viral infection, T cell differentiation is disrupted due to sustained antigen stimulation resulting in a loss of T cell effector function. Despite the development of vaccines for a wide range of viral diseases, efficacious vaccines for persistent viral infections have been challenging to design. Immunization against virus T cell epitopes has been proposed as an alternative vaccination strategy for persistent viral infections, such as HIV. However, vaccines that selectively engage T cell responses can result in inappropriate immune responses that increase, rather than prevent, disease. Quantitative models of virus infection and immune response were used to investigate how virus and immune system variables influence pathogenic versus protective T cell responses generated during persistent viral infection. It was determined that an intermediate precursor frequency of virus-specific memory CD8 T cells prior to LCMV infection resulted in maximum T cell mediated pathology. Increased pathology was independent of antigen sensitivity or the diversity of TCR in the CD8 T cell response, but was dependent on CD8 T cell production of TNF and the magnitude of initial virus exposure. The threshold for exhaustion of responding CD8 T cells ultimately influences the precursor frequency that causes enhanced disease.In addition, viral infection can occur in the context of co-infection by heterologous pathogens that modulate immune responses and/or disease. Co-infection of two unrelated viruses in their natural host, Ectromelia virus (ECTV) and Lymphocytic Choriomeningitis virus (LCMV) infection in mice, were studied. ECTV infection can be a lethal infection in mice due in part to the blockade of antiviral cytokines, including Type I Interferons (IFN-I). It was determined that ECTV/LCMV co-infection results in decreased ECTV viral load and amelioration of ECTV-induced disease, presumably due to IFN-I induction by LCMV. However, immune responses to LCMV in ECTV co-infected mice were also lower compared to mice infected with LCMV alone and biased toward effector-memory cell generation. Thus, providing evidence for bi-directional effects of viral co-infection that modulate disease and immunity. Together the results suggest heterogeneity in T cell responses during vaccination with viral vectors may be in part due to heterologous virus infection or vaccine usage and that TNF-blockade may be useful for minimizing pathology while maintaining protection during virus infection. Lastly, quantitative mathematical models of virus and T cell immunity can be useful to generate predictions regarding which molecular and cellular pathways mediate T cell protection versus pathology.
ContributorsMcAfee, Megan (Author) / Blattman, Joseph N (Thesis advisor) / Anderson, Karen (Committee member) / Jacobs, Bertram (Committee member) / Hogue, Brenda (Committee member) / Arizona State University (Publisher)
Created2015
Description
Vaccinia virus (VV) is a prototype virus of the Orthopox viruses. The large dsDNA virus composed of 200kbp genome contains approximately 200 genes and replicates entirely in the cytosol. Since its use as a live vaccine against smallpox that leads to the successful eradication of smallpox, Vaccinia has been intensely studied as a vaccine vector since the large genome allows for the insertion of multiple genes. It is also studied as a molecular tool for gene therapy and gene functional study. Despite its success as a live vaccine, the vaccination causes some mild to serious bur rare adverse events in vaccinees such as generalized Vaccinia and encepharitis. Therefore, identification of virulence genes and removal of these genes to create a safer vaccine remain an important tasks. In this study, the author seeks to elucidate the possible relationship between immune evading proteins E3 and B19. VV did not allow double deletions of E3 and B19, indicating the existence of a relationship between the two genes.
ContributorsBarclay, Shizuka (Author) / Jacobs, Bertram (Thesis director) / Ugarova, Tatiana (Committee member) / Kibler, Karen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Vaccinia virus is a cytoplasmic, double-stranded DNA orthopoxvirus. Unlike mammalian cells, vaccinia virus produces double-stranded RNA (dsRNA) during its viral life cycle. The protein kinase R, PKR, is one of the principal host defense mechanisms against orthopoxvirus infection. PKR can bind double-stranded RNA and phosphorylate eukaryotic translation initiation factor, eIF2α, shutting down protein synthesis and halting the viral life cycle. To combat host defenses, vaccinia virus encodes E3, a potent inhibitor of the cellular anti-viral eIF2α kinase, PKR. The E3 protein contains a C-terminal dsRNA-binding motif that sequesters dsRNA and inhibits PKR activation. We demonstrate that E3 also interacts with PKR by co-immunoprecipitation. This interaction is independent of the presence of dsRNA and dsRNA-binding by E3, indicating that the interaction is not due to dsRNA-bridging.
PKR interaction mapped to a region within the dsRNA-binding domain of E3 and overlapped with sequences in the C-terminus of this domain that are necessary for binding to dsRNA. Point mutants of E3 were generated and screened for PKR inhibition and direct interaction. Analysis of these mutants demonstrates that dsRNA-binding but not PKR interaction plays a critical role in the broad host range of VACV. Nonetheless, full inhibition of PKR in cells in culture requires both dsRNA-binding and PKR interaction. Because E3 is highly conserved among orthopoxviruses, understanding the mechanisms that E3 uses to inhibit PKR can give insight into host range pathogenesis of dsRNA producing viruses.
PKR interaction mapped to a region within the dsRNA-binding domain of E3 and overlapped with sequences in the C-terminus of this domain that are necessary for binding to dsRNA. Point mutants of E3 were generated and screened for PKR inhibition and direct interaction. Analysis of these mutants demonstrates that dsRNA-binding but not PKR interaction plays a critical role in the broad host range of VACV. Nonetheless, full inhibition of PKR in cells in culture requires both dsRNA-binding and PKR interaction. Because E3 is highly conserved among orthopoxviruses, understanding the mechanisms that E3 uses to inhibit PKR can give insight into host range pathogenesis of dsRNA producing viruses.
ContributorsFoster, Clayton (Co-author) / Alattar, Hamed (Co-author) / Jacobs, Bertram (Thesis director) / Blattman, Joseph (Committee member) / McFadden, Grant (Committee member) / School of Life Sciences (Contributor) / W. P. Carey School of Business (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Laboratory animals represent an invaluable, yet controversial, resource in the field of biomedical research. Animal research has been behind many influential discoveries in the field of emerging therapeutics. They provide the link between the theory of the lab bench and the functional application of medicine to influence human health. The use of animals in research is a consideration which must be heavily weighed, and the implementation must be carried out at a very high standard in order to retain research integrity and responsibility. We are in the process of conducting an experiment using laboratory mice to demonstrate cancer treatment using vaccinia (VACV) mutants as a possible oncolytic therapy for certain strains of melanoma. VACV is a double-stranded DNA poxvirus with a large and easily altered genome. This virus contains many genes dedicated to immune evasion, but has shown sensitivity to cell death by necroptosis in mouse studies (5). We have identified the absence of the kinase RIP3 which is vital in the necroptosis pathway as a potential target for oncolytic therapy using VACV mutants in specific strains of melanoma. Multiple groups of SCID Beige mice were inoculated with different melanoma cell lines and observed for tumor growth. Upon reaching 1 cm3 in volume, tumors were injected with either VACV- Δ83N, VACV- Δ54N, or PBS, and observed for regression. It was hypothesized that melanoma tumors that are RIP3-/- such as the MDA5 cell line will show regression, but melanoma tumors that are RIP3-positive and capable of necroptosis, such as the 2427 cell line, will resist viral replication and continue to proliferate. Our results so far tentatively support this hypothesis, but the data collection is ongoing. Strict and specific protocols with regard to the ethical and responsible use of mice have been implemented and upheld throughout the experiment. Animals are closely monitored, and if their quality of life becomes too poor to justify their continued use in the experiment, they are humanely euthanized, even at the expense of valuable data. The importance of commitment to a high ethical standard is pervasive throughout our work. Animals represent an invaluable contribution to research, and it is important to maintain high standards and transparency with regard to their use. Education and engagement in critical discussions about the use and care of animals in the laboratory contribute to the overall merit and legitimacy of biomedical research in the public and professional eye as a whole, and give legitimacy to the continued use of animals as models to advance science and health.
ContributorsBergamaschi, Julia (Author) / Kibler, Karen (Thesis director) / Jacobs, Bertram (Committee member) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative enteric pathogen that causes self-limiting gastroenteritis in healthy individuals and can cause systemic infections in those who are immunocompromised. During its natural lifecycle, S. Typhimurium encounters a wide variety of stresses it must sense and respond to in a dynamic and coordinated fashion to induce resistance and ensure survival. Salmonella is subjected to a series of stresses that include temperature shifts, pH variability, detergent-like bile salts, oxidative environments and changes in fluid shear levels. Previously, our lab showed that cultures of S. Typhimurium grown under physiological low fluid shear (LFS) conditions similar to those encountered in the intestinal tract during infection uniquely regulates the virulence, gene expression and pathogenesis-related stress responses of this pathogen during log phase. Interestingly, the log phase Salmonella mechanosensitive responses to LFS were independent of the master stress response sigma factor, RpoS, departing from our conventional understanding of RpoS regulation. Since RpoS is a growth phase dependent regulator with increased stability in stationary phase, the current study investigated the role of RpoS in mediating pathogenesis-related stress responses in stationary phase S. Typhimurium grown under LFS and control conditions. Specifically, stationary phase responses to acid, thermal, bile and oxidative stress were assayed. To our knowledge the results from the current study demonstrate the first report that the mechanical force of LFS globally alters the S. Typhimurium χ3339 stationary phase stress response independently of RpoS to acid and bile stressors but dependently on RpoS to oxidative and thermal stress. This indicates that fluid shear-dependent differences in acid and bile stress responses are regulated by alternative pathway(s) in S. Typhimurium, were the oxidative and thermal stress responses are regulated through RpoS in LFS conditions. Results from this study further highlight how bacterial mechanosensation may be important in promoting niche recognition and adaptation in the mammalian host during infection, and may lead to characterization of previously unidentified pathogenesis strategies.
ContributorsCrenshaw, Keith (Author) / Nickerson, Cheryl A. (Thesis advisor) / Barrila, Jennifer (Thesis advisor) / Ott, C. (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2016
Description
Coronaviruses are medically important viruses that cause respiratory and enteric infections in humans and animals. The recent emergence through interspecies transmission of severe acute respiratory syndrome coronavirus (SARS-CoV) strongly supports the need for development of vaccines and antiviral reagents. Understanding the molecular details of virus assembly is an attractive target for development of such therapeutics. Coronavirus membrane (M) proteins constitute the bulk of the viral envelope and play key roles in assembly, through M-M, M-spike (S) and M-nucleocapsid (N) interactions. M proteins have three transmembrane domains, flanked by a short amino-terminal domain and a long carboxy-terminal tail located outside and inside the virions, respectively. Two domains are apparent in the long tail - a conserved region (CD) at the amino end and a hydrophilic, charged carboxy-terminus (HD). We hypothesized that both domains play functionally important roles during assembly. A series of changes were introduced in the domains and the functional impacts were studied in the context of the virus and during virus-like particle (VLP) assembly. Positive charges in the CD gave rise to viruses with neutral residue replacements that exhibited a wild-type phenotype. Expression of the mutant proteins showed that neutral, but not positive, charges formed VLPs and coexpression with N increased output. Alanine substitutions resulted in viruses with crippled phenotypes and proteins that failed to assemble VLPs or to be rescued into the envelope. These viruses had partially compensating changes in M. Changes in the HD identified a cluster of three key positive charges. Viruses could not be recovered with negatively charged amino acid substitutions at two of the positions. While viruses were recovered with a negative charge substitution at one of the positions, these exhibited a severely crippled phenotype. Crippled mutants displayed a reduction in infectivity. Results overall provide new insight into the importance of the M tail in virus assembly. The CD is involved in fundamental M-M interactions required for envelope formation. These interactions appear to be stabilized through interactions with the N protein. Positive charges in the HD also play an important role in assembly of infectious particles.
ContributorsArndt, Ariel L (Author) / Hogue, Brenda G (Thesis advisor) / Jacobs, Bertram (Committee member) / Francisco, Wilson (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2010
Description
Host organisms have evolved multiple mechanisms to defend against a viral infection and likewise viruses have evolved multiple methods to subvert the host's anti-viral immune response. Vaccinia virus (VACV) is known to contain numerous proteins involved in blocking the cellular anti-viral immune response. The VACV E3L protein is important for inhibiting the anti-viral immune response and deletions within this gene lead to a severe attenuation. In particular, VACV containing N-terminal truncations in E3L are attenuated in animal models and fail to replicate in murine JC cells. Monkeypox virus (MPXV) F3L protein is a homologue of the VACV E3L protein, however it is predicted to contain a 37 amino acid N-terminal truncation. Despite containing an N-terminal truncation in the E3L homologue, MPXV is able to inhibit the anti-viral immune response similar to wild-type VACV and able to replicate in JC cells. This suggests that MPXV has evolved another mechanism(s) to counteract host defenses and promote replication in JC cells. MPXV produces less dsRNA than VACV during the course of an infection, which may explain why MPXV posses a phenotype similar to VACV, despite containing a truncated E3L homologue. The development of oncolytic viruses as a therapy for cancer has gained interest in recent years. Oncolytic viruses selectively replicate in and destroy cancerous cells and leave normal cells unharmed. Many tumors possess dysregulated anti-viral signaling pathways, since these pathways can also regulate cell growth. Creating a mutation in the N-terminus of the VACV-E3L protein generates an oncolytic VACV that depends on dysregulated anti-viral signaling pathways for replication allowing for direct targeting of the cancerous cells. VACV-E3Ldel54N selectively replicates in numerous cancer cells lines and not in the normal cell lines. Additionally, VACV-E3Ldel54N is safe and effective in causing tumor regression in a xenograph mouse model. Lastly, VACV-E3Ldel54N was capable of spreading from the treated tumors to the untreated tumors in both a xenograph and syngeneic mouse model. These data suggest that VACV-E3Ldel54N could be an effective oncolytic virus for the treatment of cancer.
ContributorsArndt, William D (Author) / Jacobs, Bertram (Thesis advisor) / Curtiss Iii, Roy (Committee member) / Chang, Yung (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2010
Description
Protein folding is essential in all cells, and misfolded proteins cause many diseases. In the Gram-negative bacterium Escherichia coli, protein folding must be carefully controlled during envelope biogenesis to maintain an effective permeability barrier between the cell and its environment. This study explores the relationship between envelope biogenesis and cell stress, and the return to homeostasis during envelope stress. A major player in envelope biogenesis and stress response is the periplasmic protease DegP. Work presented here explores the growth phenotypes of cells lacking degP, including temperature sensitivity and lowered cell viability. Intriguingly, these cells also accumulate novel cytosolic proteins in their envelope not present in wild-type. Association of novel proteins was found to be growth time- and temperature-dependent, and was reversible, suggesting a dynamic nature of the envelope stress response. Two-dimensional gel electrophoresis of envelopes followed by mass spectrometry identified numerous cytoplasmic proteins, including the elongation factor/chaperone TufA, illuminating a novel cytoplasmic response to envelope stress. A suppressor of temperature sensitivity was characterized which corrects the defect caused by the lack of degP. Through random Tn10 insertion analysis, aribitrarily-primed polymerase chain reaction and three-factor cross, the suppressor was identified as a novel duplication-truncation of rpoE, here called rpoE'. rpoE' serves to subtly increase RpoE levels in the cell, resulting in a slight elevation of the SigmaE stress response. It does so without significantly affecting steady-state levels of outer membrane proteins, but rather by increasing proteolysis in the envelope independently of DegP. A multicopy suppressor of temperature sensitivity in strains lacking degP and expressing mutant OmpC proteins, yfgC, was characterized. Bioinformatics suggests that YfgC is a metalloprotease, and mutation of conserved domains resulted in mislocalization of the protein. yfgC-null mutants displayed additive antibiotic sensitivity and growth defects when combined with null mutation in another periplasmic chaperone, surA, suggesting that the two act in separate pathways during envelope biogenesis. Overexpression of YfgC6his altered steady-state levels of mutant OmpC in the envelope, showing a direct relationship between it and a major constituent of the envelope. Curiously, purified YfgC6his showed an increased propensity for crosslinking in mutant, but not in a wild-type, OmpC background.
ContributorsLeiser, Owen Paul (Author) / Misra, Rajeev (Thesis advisor) / Jacobs, Bertram (Committee member) / Chang, Yung (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2010
Description
Lung metastatic cancers represent a major challenge in both basic and clinical cancer research. The ability to treat lung metastases to date has been challenging, current treatment paradigms are a mix of classic radiotherapy, chemotherapies and tumor-targeted therapies, with no one treatment that is effective for all tumors. Oncolytic viruses (OVs) represent a new therapeutic modality for hard-to-treat tumors. However, major questions still exist in the field, especially around how to therapeutically arm and deliver OVs to sites of disseminated tumors. To address this need, oncolytic myxoma viruses (MYXV) that expresses TNF superfamily member transgenes (vMYX-hTNF or vMyx-mLIGHT) were tested in an immunocompetent syngeneic lung metastatic murine osteosarcoma model. Three versions of this model were used; 1-an early intervention model, 2-an established tumor model, defined by both average tumor burden and failure of anti-PD-L1 and vMyx-TNF monotherapies, and 3-a late-stage disease model, defined by the failure the combination of vMyx-hTNF/PBMCs and anti-PD-L1 therapy. These three models were designed to test different questions about therapeutic efficacy of armed MYXV and delivery of MYXV to lung metastases. In the early intervention model, vMyx-hTNF was found to be an effective therapy, especially when delivered by leukocyte carrier cells (either bone marrow or PBMCs). Next, the combination of immune checkpoint inhibitors, including anti-PD-L1, anti-PD-1 and anti-CTLA-4, with vMyx-TNF/PBMCs were found to increase efficacy in treated mice compared to monotherapies. The established model was used to test potential synergy of vMyx-hTNF with anti-PD-L1 therapy. This model was defined by the failure of the monotherapies, however, in combination, treated mice survived significantly longer, and had lower average tumor burden throughout. This model was also used to test tumor specific delivery using ex vivo loaded PBMCs as carrier cells. Using MYXV expressing Tdtomato, PBMCs were found to deliver MYXV to tumors more effectively than free virus. In the most stringent late-stage disease model, vMyx-mLIGHT/PBMCs and vMyx-mLIGHT/PBMCs plus anti-PD-1 were tested and found to be efficacious where combination vMyx-TNF/PBMCs plus PD-1 failed. These results taken together show that TNFSF arming of MYXV, especially when delivered by autologous PBMCs, represents a new potential treatment strategy for lung metastatic tumors.
ContributorsChristie, John Douglas (Author) / McFadden, Grant (Thesis advisor) / Blattman, Joseph (Committee member) / Jacobs, Bertram (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2021
Description
Monkeypox virus (MPXV) is an orthopoxvirus that causes smallpox-like disease and has up to a 10% mortality rate, depending on the infectious strain. The global eradication of the smallpox virus has led to the decrease in smallpox vaccinations, which has led to a drastic increase in the number of human MPXV cases. MPXV has been named the most important orthopoxvirus to infect humans since the eradication of smallpox and has been the causative agent of the 2022 world-wide MPXV outbreak. Despite being highly pathogenic, MPXV contains a natural truncation at the N-terminus of its E3 homologue. Vaccinia virus (VACV) E3 protein has two domains: an N- terminus Z-form nucleic acid binding domain (Z-BD) and a C-terminus double stranded RNA binding domain (dsRBD). Both domains are required for pathogenesis, interferon (IFN) resistance, and protein kinase R (PKR) inhibition. The N-terminus is required for evasion of Z-DNA binding protein 1 (ZBP1)-dependent necroptosis. ZBP1 binding to Z- form deoxyribonucleic acid/ribonucleic acid (Z-DNA/RNA) leads to activation of receptor-interacting protein kinase 3 (RIPK3) leading to mixed lineage kinase domain- like (MLKL) phosphorylation, aggregation and cell death. This study investigated how different cell lines combat MPXV infection and how MPXV has evolved ways to circumvent the host response. MPXV is shown to inhibit necroptosis in L929 cells by degrading RIPK3 through the viral inducer of RIPK3 degradation (vIRD) and by inhibiting MLKL aggregation. Additionally, the data shows that IFN treatment efficiently inhibits MPXV replication in a ZBP1-, RIPK3-, and MLKL- dependent manner, but independent of necroptosis. Also, the data suggests that an IFN inducer with a pancaspase or proteasome inhibitor could potentially be a beneficial treatment against MPXV infections. Furthermore, it reveals a link between PKR and pathogen-induced necroptosis that has not been previously described.
ContributorsWilliams, Jacqueline (Author) / Jacobs, Bertram (Thesis advisor) / Langland, Jeffrey (Committee member) / Lake, Douglas (Committee member) / Varsani, Arvind (Committee member) / Arizona State University (Publisher)
Created2022