Microbiome and Virome Models of Human Health and Disease

193585-Thumbnail Image.png
Description
The human gut microbiome is associated with health outcomes including gastrointestinal and metabolic health, autoimmune disease and cancer. However, the role of the microbiome in many disease processes, including in the preterm gastrointestinal tract and female genital tract, has yet…

The human gut microbiome is associated with health outcomes including gastrointestinal and metabolic health, autoimmune disease and cancer. However, the role of the microbiome in many disease processes, including in the preterm gastrointestinal tract and female genital tract, has yet to be defined. Further, the diverse community of viruses within the microbiome (the virome) is understudied compared to bacteria. Here, I examine the microbiome and virome in specific disease models that are poorly understood: necrotizing enterocolitis (NEC), discordant HIV shedding in women living with HIV (WHLIV), female genital tract inflammation and gammaherpesvirus infection. Specifically, I examined the gut virome longitudinally in a cohort of preterm infants at risk for NEC; the female genital tract (FGT) microbiome and virome longitudinally in a cohort of WLHIV from Lima, Peru; the FGT virome in women from Phoenix, Arizona with differing levels of genital inflammation and different microbiome compositions; and the gut microbiome in murine gammaherpesvirus 68 (MHV68) infection. Further, I contributed to research responding to the spread of SARS-CoV-2 in Arizona. I found that 1) gut virome beta diversity decreased before NEC onset in preterm infants, suggesting a role for the virome in NEC; 2) FGT microbiome instability was associated with discordant HIV shedding, while FGT virome composition changed in association with ART duration and immune recovery; 3) FGT virome composition was associated with inflammation and microbiome composition; and 4) MHV68 infection outcomes were independent of microbiome perturbation, which may reflect environmental influences. The results of this research advance understanding of the microbiome and virome in these specific disease processes, and support further investigation of the microbiome and virome in preterm infant gastrointestinal health and FGT health, as well as environmental effects in microbiome research.
Date Created
2024
Agent

DEAD-Box RNA Helicase DDX3X, an Intrinsic Host Factor, Regulates Oncolytic Myxoma Virus Replication in Human Cancer Cells

193379-Thumbnail Image.png
Description
Myxoma virus (MYXV), a Leporipoxvirus, is being developed as an oncolytic agent against various types of human cancers. It successfully infects and has oncolytic effects on cancer cells while remaining nonpathogenic to normal human cells and all other non-leporid species.…

Myxoma virus (MYXV), a Leporipoxvirus, is being developed as an oncolytic agent against various types of human cancers. It successfully infects and has oncolytic effects on cancer cells while remaining nonpathogenic to normal human cells and all other non-leporid species. To develop virus constructs and maximize their effectiveness against cancer cells, the interaction between virus and host should be well characterized. DEAD-box RNA helicase DDX3 was previously identified as an intrinsic host factor that regulates MYXV replication in human cancer cell lines. Here, it is reported that transient knockdown of DDX3 in human cancer cells significantly enhances MYXV replication and progeny virus production. In uninfected cells, DDX3 localizes throughout the cytoplasm of human cells; however, in the MYXV-infected cells, DDX3 localizes to the perinuclear region of the cells and forms granule-like particles. It is further demonstrated that DDX3 is likely enhancing the type-1 interferon (IFN) production as the expression of the cytokine is decreased when DDX3 is knocked down during MYXV virus infection. Thus, the absence of DDX3 significantly enhanced myxoma virus spread by reducing the production of type-1 IFN and IFN-mediated signaling. These results suggest that DDX3 is a potential cellular target for enhancing the oncolytic activity of MYXV in human cancers.
Date Created
2024
Agent

Longitudinal Quantification of Neutralizing Antibodies and T cell Responses to COVID-19 mRNA Vaccines

190893-Thumbnail Image.png
Description
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen of Coronavirus Disease 2019 (COVID-19). Successful vaccination aims to elicit neutralizing antibodies (NAbs) which inhibit viral infection. Traditional NAb quantification methods (neutralization assays) are labor-intensive and expensive, with limited practicality…

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen of Coronavirus Disease 2019 (COVID-19). Successful vaccination aims to elicit neutralizing antibodies (NAbs) which inhibit viral infection. Traditional NAb quantification methods (neutralization assays) are labor-intensive and expensive, with limited practicality for routine use (e.g. monitoring vaccination response). Thus, a rapid (10-minute) lateral flow assay (LFA) for quantification of SARS-CoV-2 NAbs was developed. Using the NAb LFA, an 18-month longitudinal study assessing monthly NAb titers was conducted in a cohort of over 500 COVID-19 mRNA vaccine recipients. Three NAb response groups were identified: vaccine strong responders (VSRs), moderate responders (VMRs), and poor responders (VPRs). VSRs generated high and durable NAb titers. VMRs initially generated high NAb titers but showed more rapid waning with time post-vaccination. Finally, VPRs rarely generated NAb titers ≥1:160, even after 3rd dose. Although strong humoral responses correlate with vaccine effectiveness, viral-specific CD4+ and CD8+ T cells are critical for long-term protection. Discordant phenotypes of viral-specific CD8+ and CD4+CXCR5+ T follicular helper (cTfh) cells have recently been associated with differential NAb responses. The second portion of this dissertation was to investigate whether/how SARS-CoV-2 T cell responses differ in individuals with impaired NAb titers following mRNA vaccination. Thus, phenotypic and functional characterization of T cell activation across NAb response groups was conducted. It was hypothesized that VPRs would exhibit discordant SARS-CoV-2 T cell activation and altered cTfh phenotypes. Peripheral blood mononuclear cells were isolated from VPRs, VMRs, VSRs, naturally infected, and normal donors. SARS-CoV-2 responsive T cells were characterized using in vitro activation induced marker assays, multicolor flow cytometry, and multiplex cytokine analysis. Further, CXCR5+ cTfh were examined for chemokine receptor expression (CCR6 and CXCR3). Results demonstrated that despite differential NAb responses, activation of SARS-CoV-2 responsive CD4+ and CD8+ T cells was comparable across NAb groups. However, double-positive CD4+CD8+, CD8low, and activated CD4+CXCR5+CCR6-CXCR3+ (Tfh1-like) T cells were expanded in VPRs compared to VMR and VSRs. Interestingly, a unique population of CD8+CXCR5+ T cells was also expanded in VPRs. These novel findings may aid in identification of individuals with impaired or altered immune responses to COVID-19 mRNA vaccination.
Date Created
2023
Agent

Determining the Role of Innate Immune Elements on RNA Viral Replication and Understanding Factors that Impact Adaptive T cell Anti-tumoral Activity Against Metastatic Osteosarcoma (mOS)

189343-Thumbnail Image.png
Description
The innate immune system serves as an immediate response to pathogenic infection and an informant to the adaptive immune system. The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)–RNase-L system is a component of the innate immune system induced by interferons (IFNs) and serves…

The innate immune system serves as an immediate response to pathogenic infection and an informant to the adaptive immune system. The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)–RNase-L system is a component of the innate immune system induced by interferons (IFNs) and serves to eliminate viral infections. In humans, three enzymatically active OAS proteins exist, OAS1, OAS2, and OAS3. Recent evidence suggests variations in cellular localization of OAS proteins may influence the impact and influence of those proteins on viral replication. However, viral suppression mechanisms involving specific OAS proteins are still unclear for most viruses. Here, I overexpress different isoforms of OAS and determined that though viruses within the same family have similar replication strategies, the extent to which each OAS protein impacts viral replication for Flaviviruses, and Alphaviruses varies. In contrast to the innate immune system, the adaptive immune system provides specific and long-lived immune responses. In the context of cancer, T cells have been shown to play a prominent role in tumor regression. It has previously been demonstrated that administration α-CTLA-4/α-PD-L1 immune checkpoint blockade (ICB) to mice inoculated with a K7M2 metastatic osteosarcoma (mOS) cell line resulted in ~50% survival. Here, I sought to determine biological differences among murine responders and non-responders to ICB for mOS to understand better what factors could increase ICB efficacy. A prospective culprit is a variance in circulating antibodies (Abs). I have shown that sera from mice, before inoculation with mOS or ICB, display distinct differences in Ab repertoire between responders and non-responders, suggesting the presence or absence of particular Abs may influence the outcome of ICB. Recent studies have also shown that malleable environmental factors, such as differences in microbiome composition, can yield subsequent changes in circulating Abs. Strong associations have been made between host-microbiome interactions and their effects on health. Here, I study potential associations of microbiome-mediated impacts on ICB efficacy for mOS. Additionally, I sought to determine potential changes in T-cellular response to mOS due to modulations in microbiome composition and showed that ICB efficacy can change in conjunction with microbiome composition changes in a murine model.
Date Created
2023
Agent

Serp-1 Treatment for Diffuse Alveolar Hemorrhage in Systemic Lupus Erythematosus; Analysis of Efficacy and Mechanism of Action

Description

Systemic Lupus Erythematosus (SLE) is an autoimmune disease resulting in widespread inflammation of various organ tissues including the lung, heart, kidneys, brain, joints, vasculature, and more. Systemic Lupus Erythematosus (SLE) has at present no cure and therefore, treatments focus on…

Systemic Lupus Erythematosus (SLE) is an autoimmune disease resulting in widespread inflammation of various organ tissues including the lung, heart, kidneys, brain, joints, vasculature, and more. Systemic Lupus Erythematosus (SLE) has at present no cure and therefore, treatments focus on improving quality of life by targeting flare-ups of inflammation (6). Diffuse Alveolar Hemorrhage (DAH) is a rare complication of SLE affecting 1-5% of people diagnosed with SLE. DAH is characterized by bleeding into the lung alveolar spaces and is associated with inflammation, potentially caused by infections and injuries. While the prevalence of DAH is low, the mortality rate is high at 50-80% (5). DAH has no proven effective treatment and many treatments used have severe side effects. Serp-1 is a Myxomavirus derived immune modulating serine protease inhibitor, a serpin, with proven efficacy in a wide range of inflammation-associated disorders (1). Due to the efficacy of Serp-1 in reducing arterial inflammation and lung consolidation in mouse herpes virus infections, Serp-1 treatment in a mouse model of pristane-induced DAH was investigated. Multiple groups were tested including mouse models that were not given pristane as well as mice with pristane-induced DAH treated with saline control, wild-type unmodified Serp-1, and a polyethylene glycol-modified variant of Serp-1 termed PEGSerp-1.

Date Created
2023-05
Agent

TNF-superfamily arming and leukocyte based delivery of oncolytic myxoma virus in a syngeneic murine lung metastasis model

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…

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.
Date Created
2021
Agent

The Effects of Selinexor Concentration and Time on the Myxoma Virus

164794-Thumbnail Image.png
Description
Cancer is one of the leading causes of death, globally, with an estimated 9.6 million deaths in 2018, according to the World Health Organization. However, this is not the only impact cancer has on affected individuals, as death rates only…

Cancer is one of the leading causes of death, globally, with an estimated 9.6 million deaths in 2018, according to the World Health Organization. However, this is not the only impact cancer has on affected individuals, as death rates only capture the mortality of cancer, there are still detrimental effects cancer has on quality of life. Newer therapies for cancer attempt to circumvent these unwanted detriments, such as hormone therapy, stem cell transplants, targeted therapy, etc.3. One such novel therapy being virotherapy, which is the subject of this study. This study follows the observations of the myxoma virus (MYXV), a prototypic poxvirus which belongs to the Leporipoxvirus genus of the Poxviridae family. This method allows larger particles to enter host cells through the process of overriding host cell endocytosis pathways, with a few exceptions. Interestingly, research has shown that MYXV has been able to infect multiple types of tumor cells of non-rabbit species both in vitro and in vivo, in not only humans but murine, rodent, species as well. This allows MYXV to pose as a potential virotherapy for human cancer cells. McFadden research lab has been researching the role of the exportin 1 protein (XPO1), also known as the chromosome maintenance region-1 (CRM-1). It is suspected that the XPO1 pathway may be one of the evasion mechanisms that MYXV utilizes as an antiviral response. KPT-330 (Selinexor) is a selective inhibitor of nuclear transport (SINE) drug that was designed as the first-in-human oral FDA approved cancer treatment. It has been shown effectiveness in inhibiting XPO1 in multiple lines of cancer cells, such as the Lewis Lung Carcinoma (LLC1) cells researched in this study. McFadden research lab has been examining the effects of various Selinexor concentrations along with different multiplicities of infection (MOIs) of MYXV to determine the best combination that can be used to reduce tumor size at the highest effectiveness. Overall, Selinexor is not increasing cell killing through a synergistic means, but rather simply by increasing the ability of MYXV to infect and spread in LLC1 cells. This then causes increased cell killing given that more LLC1 cells are penetrated by the OV and “suffocated” by the prevention of exporting essential proteins from the nucleus to their respectively critical final destinations in the cancer cells.
Date Created
2022-05
Agent

Comparison of Antibody Responses to Myxoma Virus Inactivated Using Different Methods

147702-Thumbnail Image.png
Description

In this study, we investigated the inactivation of wild-type vMyx-GFP (MYXV) using different methods. Assays were performed in vitro to test the following inactivation methods: heat, longwave UV only, longwave UV with psoralen (P + LWUV), and psoralen (P) only.…

In this study, we investigated the inactivation of wild-type vMyx-GFP (MYXV) using different methods. Assays were performed in vitro to test the following inactivation methods: heat, longwave UV only, longwave UV with psoralen (P + LWUV), and psoralen (P) only. In vitro assays demonstrated that the psoralen alone treatment did not cause any inactivation. These results showed that effective inactivation using psoralen was likely reliant on subsequent UV irradiation, creating a synergistic effect. Additionally, the UV and P + LWUV treatment demonstrated inactivation of MYXV, although by different mechanisms, as the UV-only treated virus demonstrated background infection, while P + LWUV treated virus did not. In mice, P + LWUV and UV treatment of MYXV demonstrated to be effective inactivation methods and likely preserved the antigenic epitopes of MYXV, allowing for the production of neutralizing antibodies in mice. More research is recommended on the heat treatment of MYXV as neutralizing antibodies were not observed, possibly due to the treatment denaturing antigenic epitopes or needing more booster injections to reach the threshold antibody concentration for protection. Furthermore, we demonstrated that the intraperitoneal (IP) injection of inactivated MYXV was superior to the subcutaneous injection in eliciting a strong immune response. The increased neutralizing antibodies observed after IP injection could be due to the advantage that the IP route has of reaching lymphoid tissue faster.

Date Created
2021-05
Agent

Comparison of Antibody Responses to Myxoma Virus Inactivated Using Different Methods

147703-Thumbnail Image.png
Description

In this study, we investigated the inactivation of wild-type vMyx-GFP (MYXV) using different methods. Assays were performed in vitro to test the following inactivation methods: heat, longwave UV only, longwave UV with psoralen (P + LWUV), and psoralen (P) only.…

In this study, we investigated the inactivation of wild-type vMyx-GFP (MYXV) using different methods. Assays were performed in vitro to test the following inactivation methods: heat, longwave UV only, longwave UV with psoralen (P + LWUV), and psoralen (P) only. In vitro assays demonstrated that the psoralen alone treatment did not cause any inactivation. These results showed that effective inactivation using psoralen was likely reliant on subsequent UV irradiation, creating a synergistic effect. Additionally, the UV and P + LWUV treatments demonstrated inactivation of MYXV, although by different mechanisms, as the UV-only treated virus demonstrated background infection, while P + LWUV treated virus did not. In mice, P + LWUV and UV treatment of MYXV demonstrated effective inactivation methods and likely preserved the antigenic epitopes of MYXV, allowing for the production of neutralizing antibodies in mice. More research may need to be conducted on the heat treatment of MYXV as neutralizing antibodies were not observed, possibly due to the treatment denaturing antigenic epitopes or needing more booster injections to reach the threshold antibody concentration for protection. Furthermore, we demonstrated that the intraperitoneal (IP) injection of inactivated MYXV was superior to the subcutaneous injection in eliciting a strong immune response. The increased neutralizing antibodies observed after IP injection could be due to the advantage that the IP route has of reaching lymphoid tissue faster.

Date Created
2021-05
Agent

Armed Oncolytic Myxoma Viruses that Eliminate Acute Myeloid Leukemia and Multiple Myeloma Cells

158412-Thumbnail Image.png
Description
Novel biological strategies for cancer therapy have recently been able to generate improved anti-tumor effects in the clinic. Of these new advancements, oncolytic virotherapy is a promising strategy through a dual mechanism of oncolysis and stimulation of tumor immunogenicity against…

Novel biological strategies for cancer therapy have recently been able to generate improved anti-tumor effects in the clinic. Of these new advancements, oncolytic virotherapy is a promising strategy through a dual mechanism of oncolysis and stimulation of tumor immunogenicity against the target cancer cells. Myxoma virus (MYXV) is an oncolytic poxvirus that has a natural tropism for Leporids, being nonpathogenic in humans and all other known vertebrates. MYXV is able to infect cancer cells due to mutations and defects in many innate signaling pathways, such as those involved in anti-viral responses. While MYXV alone infects and kills many classes of human cancer cells, recombinant techniques allow for the implementation of therapeutic transgenes, which have the potential of ‘arming’ the virus to enhance its potential as an oncolytic virus. The implementation of certain transgenes allows improved cancer cell killing and/or promotion of more robust anti-tumor immune responses. To investigate the potential of immune-inducing transgenes in MYXV, in vitro screening experiments were performed with several single transgene-armed recombinant MYXVs. As recent studies have shown the ability of MYXV to uniquely target malignant human hematopoietic stem cells, the potential of oncolytic MYXV armed with individual immune-enhancing transgenes was investigated through in vitro killing analysis using human acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines. Additionally, in vitro experiments were performed using primary bone marrow (BM) cells obtained from human patients diagnosed with MM. Furthermore, the action of an engineered bispecific killer engager (huBIKE) was investigated through co-culture studies between the CD138 surface marker of target MM cells and the CD16 surface marker of primary effector peripheral blood mononuclear cells (PBMCs), particularly NK cells and neutrophils. In this study, several of the test armed MYXV-infected human AML and MM cell lines resulted in increased cell death compared to unarmed MYXV-infected cells. Additionally, increased killing of CD138+ MM cells from primary human BM samples was observed following infection with huBIKE-armed MYXV relative to infection with unarmed MYXV. Furthermore, analysis of co-culture studies performed suggests enhanced killing of target MM cells via engagement of NK cells with U266 MM cells by huBIKE.
Date Created
2020
Agent
Subscribe to McFadden, Grant