Biomedical Sciences Dissertations

Inflammatory bowel disease (IBD) is a multifactorial, chronic disease that affects approximately 1.5 million people in the United States [1]. It presents with inflammation of the intestine with unknown etiology. Its two main forms are Crohn’s disease (CD), which can affect any part of the GI tract, and ulcerative colitis (UC), which impacts primarily the colon. Several important factors are implicated in the pathogenesis of IBD, one factor being dysregulation of the immune system. This dysregulation results in the accumulation and stimulation of innate and adaptive immune cells and subsequent release of soluble factors, including pro-inflammatory cytokines. One of these cytokines is a member of the IL-36 cytokine family, IL-36γ, which is overexpressed in human IBD and experimental models of colitis. In this study, we explored the role of IL-36γ in promoting CD4+ T cell activation and cytokine secretion. We found that IL-36γ stimulation of naïve CD4+ T cells significantly induced IFNγ expression in vitro and was associated with augmented intestinal inflammation in vivo using the T cell transfer model of colitis. Using IFNγ-/- naive T cells, we observed a dramatic decrease in the ability of these cells to produce TNFα and IL-12. Moreover, the transfer of these cell did not cause robust colitis. These data not only suggest that IL-36γ is a master regulator of a pro-inflammatory cytokine network involving IFNγ, TNFα and IL-12, but also highlight the importance of targeting IL-36y and IFNy as therapeutic approaches. Our studies have broad implications in relation to targeting of specific cytokines in human IBD.

Aims: Valosin-containing protein (VCP) has recently been identified as a novel mediator of mitochondrial respiration and cell survival in the heart, in which increased inducible nitric oxide synthase (iNOS) expression and activity is considered an essential mechanistic link in the cardioprotection conferred by VCP. iNOS is one of the three isoforms of nitric oxide synthase (NOS) that generates nitric oxide (NO) from L-arginine, which can then react with cysteine residues in proteins to form protein S-nitrosothiols (SNOs). The study aimed to investigate whether VCP directly mediates protein S-nitrosylation in the heart through the iNOS/NO/SNO pathway. We hypothesized that VCP plays a crucial role in mediating mitochondrial protein S-nitrosylation through an iNOS-dependent mechanism in the heart. To test this hypothesis, we utilized four distinct transgenic (TG) mouse models: cardiac-specific VCP TG mice, bigenic iNOS knockout (KO) with VCP overexpression mice (VCP TG/iNOS KO^−/−), cardiac-specific dominant-negative (DN) VCP TG mice, and cardiac-specific VCP KO mice.

Methods and results: To investigate the potential impact of VCP on both overall and specific protein S-nitrosylation in mouse heart tissues, we utilized a biotin switch assay combined with streptavidin purification. Our results showed that VCP overexpression increased S-nitrosylation of both VCP and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the heart, which was diminished by genetic iNOS deletion. Conversely, function inhibition of VCP resulted in a decrease in the S-nitrosylation levels of VCP and the mitochondrial respiration complex I, but did not affect the S-nitrosylation level of GAPDH in the heart.

Conclusion: Taken collectively, these data provide compelling evidence that VCP could serve as a novel mediator of cardiac protein S-nitrosylation through an iNOS-dependent mechanism.

The host's innate immune response begins with the identification of molecules known as pathogen-associated molecular patterns by a series of germline-encoded pattern-recognition receptors. Retinoic acid-inducible gene I (RIG-I) is one such innate immune receptor that detects viral RNA in the cytosol of cells and is crucial to mount an effective antiviral immune response in the course of viral infections. RIG-I with the adaptor protein mitochondrial antiviral signaling protein (MAVS) leads to the activation of the transcription factors including interferon-regulatory factor 3 (IRF3), IRF7 and nuclear factor-κB (NF-κB), which induce type I and III interferon (IFN), and proinflammatory cytokines. Many studies have demonstrated the crucial role of RIG-I in viral infections such as influenza A virus (IAV) and respiratory syncytial virus (RSV) infections. However, as airway viral infection is frequently followed by bacterial co-infections, the role of RIG-I and how it is regulated in airway epithelium inflammation in mixed viral and bacterial infection remains undefined. Despite the well-known role of viruses in bacterial co-infections, the impact of bacterial infections on viral co-infections remains largely unknown. Therefore, understanding the mechanism of airway bacterial pathogen Nontypeable Haemophilus influenzae (NTHi)-induced RIG-I will help to understand the complex inflammatory signaling pathway and further contribute to the development of new therapies.

In the present study, we showed that NTHi induced RIG-I upregulation in airway epithelial cells. We also demonstrated that NTHi induced RIG-I upregulation by activation of TNF receptor associated factor 6 (TRAF6)-inhibitor of nuclear factor-κB kinase subunit β (IKKβ)-p65 pathway. Interestingly, interleukin-1 receptor-associated kinase M (IRAK-M) negatively regulated NTHi-induced up-regulation of RIG-I via inhibiting the IKKβ-p65 pathway and inflammation. We also confirmed that IKKβ activation is sufficient for RIG-I up-regulation. We showed that p65 phosphorylation at S276 and S536 residues is likely critical for RIG-I up-regulation. We also investigated the role of histone deacetylases (HDACs) as a positive regulator for NTHi-induced RIG-I up-regulation. Along with NTHi-induced RIG-I upregulation, we also confirmed the functional significance of RIG-I by confirming the CXCL10 expression mediated via RIG-I. Thus, our study provides new insights into the regulation of NTHi-induced RIG-I upregulation, which may contribute to the development of a new therapeutic strategy for controlling inflammation in mixed airway infections.

Inflammatory bowel disease (IBD), comprised of Crohn’s disease (CD) and ulcerative colitis (UC), is chronic inflammation affecting more than 1.5 million people in the United States and 2.2 million people in Europe. CD can affect any portion of the gastrointestinal (GI) tract and is commonly patchy with a transmural inflammation pattern. UC is limited to the large intestine and has a continuous inflammatory pattern that involves only the mucosa. A commonality is a dysregulated immune response in the intestine. IBD develops in genetically susceptible individuals due to complex interactions between environmental stimuli, genome, microbiome, and an inappropriate mucosa immune response. However, the current treatment is not effective enough to show significant improvement in many patients. Here, we developed two therapeutic approaches to treat intestinal mucosal wounds found in ulcerative colitis in vitro and in vivo.

In the first section, I studied fecal metabolites derived from genetic knockout (KO) of peptide transporter-1 (PepT1) mice which are known to show resistance to acute colitis and colitis-associated cancer (CAC). The previous publication provided evidence that the PepT1 KO microbiota is sufficient to protect against colitis and CAC. Given that PepT1 KO alters the gut microbiome and thereby changes the intestinal metabolites that are ultimately reflected in the fecal samples. Therefore, we investigated the fecal metabolites of PepT1 KO mice using a liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics technique. We found that the specific fecal metabolite, tuberonic acid (TA), increased seven times higher in KO mouse fecal samples when compared to wild-type (WT) mouse feces. Accordingly, we performed research on whether the increased TA could have an anti-inflammatory effect. In vitro study discovered that TA not only prevented lipopolysaccharides (LPS)-induced inflammation in macrophages but also accelerated the epithelial cell healing processes.

In the next section, we studied mRNA-based therapeutics using lipid nanoparticle encapsulation. Inspired by the colon-targeting ability of ginger-derived nanoparticles (GDNPs) from our laboratory, we reversely engineered lipid nanoparticles (LNPs) that comprise the most abundant lipids in GDNPs, including phosphatidic acid (PA), monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) at 5:2:3 ratio. We encapsulated IL-22 mRNA within the newly generated lipid nanoparticles (nLNPs) as enhanced IL-22 expression in the colon is known to have a potent anti-inflammatory effect against ulcerative colitis (UC). The IL-22 mRNA-loaded nLNPs were shown to be approximately 200 nm in diameter and have a zeta potential of -18 mV. Oral administration of the IL-22/nLNPs complex increased the protein expression level in the colonic mucosa. During acute colitis, mice fed with the IL-22/nLNPs experienced an accelerated healing process, as indicated by the recovery of higher body weight and longer colon length as well as reduction of colonic MPO activity, fecal lipocalin concentration, histological index, and mRNA expression level of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β). These results indicate that the reversely engineered nLNPs are an effective mRNA delivery system to treat ulcerative colitis.

The last two sections elaborate on two different protocols to generate and characterize novel, natural, non-toxic ginger-derived nanoparticles (GDNPs) and ginger-derived lipid vesicles (GDLVs) that can be utilized as well-defined drug vehicles for treating inflamed intestinal mucosa. The first method uses sucrose gradient ultracentrifugation with extracted ginger juice to purify GDNPs. The concentration of obtained GDNPs was measured by a protein quantification assay kit and microplate reader at an absorbance of 750 nm. The second method uses liquid-liquid extraction to isolate lipid vesicles from GDNPs from the first protocol. Ginger-derived lipid vesicles (GDLVs) were generated by thin-film hydration and dissolved therapeutic agent, siRNA, by brief suspension by sonication. This GDLVs/siRNA complex was measured for its size and zeta potential, then its TEM and AFM images were acquired. In addition, in vivo test was performed to ensure its delivery functionality, bio-distribution, and efficiency. These two protocols provide a novel and crucial method to develop an efficient and safe drug delivery system that causes no immunotoxicity when orally administered. Furthermore, the nanostructure of GDNPs is the primary source of reversely engineered lipid nanoparticles for the mRNA-loaded delivery system (Page 41).

In conclusion, these therapeutic studies prove that the two different approaches of treating fecal metabolite and IL-22-loaded lipid nanoparticles to accelerate the healing process of intestinal inflammation are effective both in vitro and in vivo. Metabolites have emerged as essential effectors in mediating commensal microbiomes in human physiology. Positive results to show the anti-inflammatory effect of the specific metabolite derived from genetic knockout of PepT1 could be the groundwork of novel metabolites-based therapies. Safe and efficient RNA-based gene therapy using lipid nanoparticles was propelled into the spotlight due to the pandemic outbreak. The short-term oral administration of IL-22 mRNA protected by nLNPs successfully demonstrated a powerful mucosal healing effect. This may provide a potent gene therapy to treat ulcerative colitis.

Neisseria gonorrhoeae causes the eponymous sexually transmitted infection gonorrhea, a global disease that afflicts millions of individuals worldwide per year. To date, no effective vaccine to prevent gonococcal infection has been produced, and the pathogen rapidly acquires and maintains mechanisms for antimicrobial drug resistance. Further, natural infection by N. gonorrhoeae elicits no protective immune response, making reinfection a common outcome. During infection, the gonococcus employs an arsenal of TonB-dependent outer-membrane transporters (TdTs) which facilitate the acquisition of essential nutrients such as iron and zinc from the human host, which itself goes to great lengths to restrict these metals’ availability. The TdTs are well conserved, surface accessible, and play critical roles in gonococcal pathogenesis, making them promising targets for therapeutic and/or vaccination efforts. One TdT, called TdfJ, allows the gonococcus to acquire zinc from S100A7, an innate immunity protein that typically suppresses bacterial growth via zinc sequestration. TdfJ contains an α-helix motif in its extracellular loop 3 (L3H), a conserved structure which has been shown to participate in the binding of and subsequent iron extraction from transferrin by another TdT, TbpA. In this report, we generated a series of mutations in the TdfJ L3H and assessed their impacts on S100A7 binding and utilization. We found that proline insertions at residues K261 and R262 fully abrogated the interaction between the two proteins, highlighting the importance of this motif in metal acquisition. We went on to explore the functionality of TdfJ as a vaccine antigen via two approaches. In one approach, we grafted extracellular loop sections of TdfJ onto a soluble lipoprotein scaffold and immunized mice, whose post-immune sera and vaginal secretions were screened for TdfJ-reactive antibodies. In another, we formulated recombinant TdfJ with multiple adjuvants to assess the duration of gonococcal colonization in mice and whether adjuvant type affected efficacy. These two approaches demonstrated that TdfJ as a vaccine antigen is protective in a mouse model of infection, and that antibodies generated during upon vaccination may inhibit gonococcal ability to utilize S100A7. Taken together, these data may direct the path towards future prevention of gonococcal infections.

Regulatory T cells (Tregs) are an essential subtype of immune cells that controls self-tolerance, inflammatory responses, and tissue homeostasis. In tumor immunity, Treg cells are involved in tumor development and progression by inhibiting antitumor immunity. Therapeutically targeting Treg cells in inflammation-related diseases and cancers will therefore require the identification of context-specific mechanisms that guide their functions. The AMP-activated protein kinase (AMPK) functions as a master sensor and modulator of cellular energy and redox homeostasis, both of which are vital to various immune cells including Tregs. However, whether and how AMPK plays its roles in Tregs was unknown. My dissertation aims to elucidate the contributions of AMPK in the regulation of Tregs function in different mouse disease status.

In the first part of this dissertation, I established the essential roles of AMPK in the regulation of immune homeostasis from AMPKα1 global knock-out mice. By using both bone marrow transplantation and the adoptive Treg transfer experiment, I discovered that both Treg dysfunction and the autoimmune diseases occurred in AMPKα1 global knock-out mice. By using Treg-specific AMPKα1 knock-out mice, I further uncovered the indispensable roles of AMPK in the regulation of Treg functions, as the Treg-specific AMPKα1 knock-out mice develops autoimmune disease in livers. Mechanistically, I found that AMPK regulates normal functions of Treg by maintaining the protein stability of Foxp3, a critical transcriptional factor of Tregs.

In the second part, I studied the roles of AMPK in Tregs in tumor growth and metastasis. Treg-specific AMPKα1 knock-out mice exhibited delayed tumor progression and enhanced antitumor T cell immunity. Further experiments showed that AMPKα1 maintains the functional integrity of Treg cells and prevents interferon-γ production in tumor-infiltrating Tregs cells, indicating that AMPK promotes tumor growth by maintaining high levels of Foxp3 in infiltrating Tregs in tumor tissues. Mechanistically, AMPKα1 maintains the protein stability of FOXP3 in Tregs cells by downregulating the expression of E3 ligase CHIP (STUB1). Our results suggest that selective inhibition of AMPK in Tregs cells might be an effective anti-tumor therapy.

In the third part of the dissertation, I interrogated the role of AMPK in Tregs senescence and aging, as a decline of AMPK has been widely described in aged animals and humans. Compared to non-senescent Tregs, senescent Tregs showed fewer protective effects on arterial inflammation and atherosclerosis development. Treg-specific AMPKα1 knock-out mice displayed accelerated Treg senescence and arterial inflammation. Hypercholesterolemia, an established risk factor of cardiovascular diseases, promoted Treg senescence, resulting in the formation of plasticity Tregs with uncontrolled production of interferon-γ and impaired suppressive function. Selective activation of AMPK in Tregs restrained Treg senescence, downregulated the levels of reactive oxygen species (ROS) in aortic walls, and suppressed high fat diets-induced atherosclerosis in mice in vivo. Taken together, my results demonstrates that AMPK activation in Tregs might be a valid target for treating aging related diseases including atherosclerosis.

In conclusion, in this dissertation I have established essential roles of AMPK in maintaining Tregs functions and deregulated AMPK with consequent Tregs dysfunctions plays causative roles in the initiations and progressions of autoimmune liver diseases, cancer, and atherosclerosis-related cardiovascular diseases.

Inflammatory bowel disease (IBD) is marked by inflammation mediated epithelial-mucosal damage. The intestinal epithelial forms a tight barrier displaying two contrasting functions: restricting the entry of potentially harmful substances while, on the other hand allowing the selective passage of nutrients. The damaged epithelial-mucosal barrier causes the exposure of mucosa layers to luminal inflammatory contents. This eventually leads to the leaky epithelium, exposing the immune cells, release of various cytokines, and results in loss of epithelial homeostasis. Therefore, maintenance of healthy epithelial- mucosal lining is critical during IBD recovery. Notch signaling is an evolutionarily conserved molecular pathway crucial for the development and homeostasis of most tissues. Notably, the deregulation of Notch signaling is involved in IBD (Ulcerative colitis and Crohn’s disease). Notch signaling also plays a vital role in wound healing and tissue repair. Here, we investigated the role of Notch-1 signaling in wound healing and regeneration of colonic epithelium during colitis recovery phase by using conditional deletion of Notch-1 in colonic epithelium of mice. We used colonic carcinoma cell line HCT116 transiently transfected with Notch1 intracellular domain (NICD) to support in vivo data. We observed that deletion of Notch1 among mice was associated with compromised healing after colitis. Therefore, targeting the Notch-1 pathway might provide a novel therapeutic strategy for the patients recovering from colitis.

Conventional hemagglutinin (HA) based inactivated influenza vaccines provide insufficient cross-protection against antigenically distant influenza strains, especially in older adults. The major goal of my dissertation research projects was to study influenza vaccination strategies to improve cross-protection by inducing extra immunity to neuraminidase (NA) and M2 ion channel ectodomain (M2e) proteins.

In chapter one, to address the issue of poor influenza vaccine efficacy in the elderly population, I employed a supplementation vaccination strategy, where I combined bivalent inactivated split vaccines (H1N1+H3N2) with a virus-like particle (VLP) expressing consensus NA (N1+ N2 + B NA) plus M2e repeat (5xM2e of human, swine and avian M2e sequences), referred as NA-M2e here, in 17-month-old-mice by a 2-dose immunication regimen. Vaccination with split plus NA-M2e induced protective IgG antibodies towards T-helper type 1 and effector T cell responses in aged mice, conferring enhanced protection against homologous and heterologous viruses compared to split vaccine . These findings suggest a promising strategy to overcome aging-related declines in vaccine efficacy and improve immune responses and vaccine efficacy in the elderly.

In chapter two, I investigated the protection efficacy of split plus NA-M2e vaccination in young adult mice and the adjuvant effects of supplementing NA-M2e on improving the immunogenicity and efficacy of split vaccine. Vaccination with combined split and NA-M2e was more effective in conferring enhanced cross-protection than either vaccine alone in young adult mice. Injection of NA-M2e VLP and combined split and NA-M2e VLP vaccines resulted in recruiting more activated monocytes, macrophages, and dendritic cell subsets in the lymphoid tissues within a day. Young adult mice, when co-immunized with split plus NA-M2e vaccines, could induce stronger humoral and cellular immune responses compared to their aged counterparts. These findings suggest the combination of split plus NA-M2e vaccines is more effective in conferring the high efficacy of homo and cross-protection, probably via activating both innate and adaptive immune responses.

Most approved vaccines provide robust systemic immunity via intramuscular immunization (IM) but not effective mucosal immunity in protecting against respiratory pathogens. In Chapter 3, I investigated the immunogenicity and efficacy of NA-M2e VLP vaccine after delivery via the intranasal route versus intramuscular route. Intranasal immunization with NA-M2e induced comparable systemic IgG antibodies, elevated IgA antibodies, and increased populations of effector T cells, memory B cells, and antigen-presenting cells in the mucosal site compared to intramuscular immunization in mice. Intranasal delivery of NA-M2e VLP vaccine induced more effective cross-protection against antigenically variant influenza strains than intramuscular injection. These findings suggest that intranasal delivery of NA-M2e vaccine can be an effective route in inducing humoral and cellular immune responses at local mucosal sites, contributing to cross-protection.

Neisseria gonorrhoeae and its obligate human host require transition metals for biological functions including cell signaling and metabolism, gene regulation, enzymatic processing, and oxidative stress resistance. During infection, the host employs two innate immunity mechanisms: either starve the pathogen of metals or overload the pathogen with intoxicating amounts of metals, either resulting in bacterial growth inhibition. The mechanisms by which the host starves or overloads the pathogen with metal nutrients are termed nutritional immunity and metal overload, respectively. In response to nutritional immunity and metal overload, N. gonorrhoeae differentially expresses metal transport systems that allow it to maintain homeostatic metal concentrations within the cytoplasm. Such transporters include the high-affinity zinc importer, ZnuABC, which is regulated by the zinc uptake regulator Zur, and the manganese exporter, MntX. As the gonococcus acquires and maintains antibiotic resistance mechanisms, the necessity to develop novel therapeutics and treatments becomes more urgent. Metal transporters are attractive therapeutic targets as they are often required for survival and virulence. However, the precise mechanism by which zinc and manganese are transported, sensed by Zur, and impact the transcriptional response to maintain metal homeostasis have not yet been elucidated. Investigating the mechanism by which zinc and manganese are transported and sensed is crucial to characterizing gonococcal metal homeostasis in the face of host-employed nutritional immunity and metal overload. In this work, I hypothesized that Zur mounts zinc- and manganese-dependent transcriptional responses to metal limitation and metal overload and that this response maintains internal metal concentrations at homeostatic levels. RNA-sequencing, RT-qPCR showed that Zur is a zinc-dependent regulator of the genes encoding ZnuABC and that manganese-dependent regulation by Zur is strain-specific. ICP-MS, growth assays, and transporter complementation experiments showed that internal homeostatic manganese levels differ between gonococcal strains and that this difference can be attributed to the manganese exporter, MntX. Therefore, novel treatment strategies that target metal transporters as a means of starving or overloading and subsequently killing N. gonorrhoeae should be informed by the metal environment sensed by N. gonorrhoeae and the intracellular metal pools maintained by different gonococcal strains.

This dissertation focuses on enhancing our understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV), measles virus (MeV), and Nipah virus (NiV). These viruses represent significant public health challenges, with the potential to cause long-term health issues and substantial economic impacts. By developing effective treatments that interrupt viral transmission, exploring how past infections influence current susceptibility, pinpointing drug targets, advancing our structural knowledge of viral proteins, and gaining deeper mechanistic insights into pathogen replication, we can reduce disease-related complications and healthcare costs.

The therapeutic efficacy of several treatments was evaluated using the ferret and Roborovski dwarf hamster models of severe COVID-19-like lung disease. These treatments included molnupiravir, Paxlovid-like nirmatrelvir/ritonavir, GS-621763 (an oral prodrug of remdesivir), 4’-fluorouridine (4’-FlU), EDP-235, and BioBlock (a neutralizing antibody targeting the spike protein delivered via nasal spray). In the ferret model, molnupiravir, GS-621763, EDP- 235, and BioBlock effectively prevented SARS-CoV-2 transmission between ferrets, while Paxlovid-like nirmatrelvir/ritonavir did not fully inhibit transmission. Additionally, 4’-FlU demonstrated strong antiviral activity against SARS-CoV-2 Wuhan lineage A and variants of concern (VOCs) alpha, delta, and gamma in ferrets, as well against RSV A in mice. In Roborovski dwarf hamsters, molnupiravir significantly reduced severe lung injury and viral lung titers for VOCs delta, gamma, and omicron. Similarly, Paxlovid-like nirmatrelvir/ritonavir reduced lung titers for VOCs delta and omicron in these hamsters.

This thesis also highlights the therapeutic potential of GHP-88309, a broad-spectrum paramyxovirus polymerase inhibitor, which can effectively counteract measles-like immune amnesia. It identifies an immune-priming mechanism that may explain the occurrence of bacterial superinfections following measles virus infection, offering insights that could reshape future treatment approaches for measles. Additionally, the thesis thoroughly evaluates the use of allosteric polymerase inhibitors as chemical probes to enhance the structural resolution of essential viral proteins. Furthermore, it expands our understanding of Nipah virus replication dynamics, revealing a promising target site for drug development.

Chapter1: Broad cross-protection by recombinant live attenuated influenza H3N2 seasonal virus expressing conserved M2 extracellular domain in a chimeric hemagglutinin.

The objective of project 1 was to determine whether intranasal immunization with live recombinant H3N2 virus expressing chimeric 4xM2e-HA would induce broadly cross-protective immunity against different subtypes of influenza A viruses in a mouse model. In recent years, antigenic drifts have severely limited the effectiveness of the H3N2 component of seasonal influenza vaccines. Here, using the reverse genetic (rg) technique, we generated reassortant seasonal influenza rgH3N2 4xM2e virus containing chimeric 4xM2e-HA in which the HA and NA genes were derived from A/Switzerland/9715293/2013 (H3N2) and the remaining 6 genes from the A/PR8 backbone. Reassortant rgH3N2 4xM2e virus containing chimeric 4xM2e-HA was found to retain comparable growth properties but displayed highly attenuated phenotypes in mice. Cross-protective efficacy against different subtypes (H1N1, H3N2, H5N1, H7N9, H9N2) of influenza A virus was tested in intranasally immunized BALB/c mice with rgH3N2 4xM2e. This study implicates a strategy of improving cross-protection by utilizing currently licensed recombinant influenza vaccine platforms.

Chapter2: Enhanced cross-protection by hetero prime-boost vaccination with recombinant influenza viruses containing chimeric hemagglutinin-M2e epitopes.

The goal of project 2 was to test whether a strategy of hetero prime-boost vaccination with recombinant influenza viruses expressing chimeric 4xM2e-HA would induce more effective cross-protection than homologous prime-boost vaccination. The impact of heterosubtypic vaccination with recombinant 4xM2e-HA influenza virus vaccines and pre-existing immunity on cross-protection against influenza viruses remains unknown. In this study, I investigated the efficacy of cross-protection by heterosubtypic prime- boost vaccination with live recombinant 4xM2e-HA H1N1 and H3N2 influenza virus vaccines in C57BL/6 mice known to be a low responder to immune-subdominant conserved epitopes. The experimental outcomes of project 2 demonstrated that hetero prime-boost strategies using recombinant 4xM2e-HA influenza virus vaccination induced more effective cross-protection against antigenically different viruses than homologous repeat vaccination in C57BL/6 mice. The roles of M2e and stalk immunity in conferring cross-protection were explored and discussed in this study.

Chapter 3: Hemagglutinin virus-like particle is immunogenic and provides heterologous protection against influenza virus in young adult and aged mice.

The goal of project 3 was to investigate immune responses and homo and cross-protective efficacy in aged mice after vaccination with a platform of virus-like particles (VLP) presenting H1 HA with and without molecularly anchored cytokine adjuvants incorporated, in comparison with those in young adult mice. Vaccine effectiveness is inferior in the aged population at high risk of severe illness from influenza virus infection. For the elderly, safe and highly immunogenic vaccines need to be developed. In project 3, host immune responses and homo and cross-protective efficacy was determined, after vaccination with a VLP vaccine platform which expresses H1 HA from A/PR8/34 (PR8 HA VLP) in young adults and aged (18-month-old) BALB/c mice. In addition, I investigated the adjuvant impact of cytokines (GM-CSF and IL-12) engineered to be incorporated into HA VLP vaccines on inducing IgG antibodies, hemagglutination inhibition titers, and homo and hetero protection in aged mice, compared to those in young adult mice. Higher doses of vaccination with H1 HA VLP and cytokines incorporated onto H1 HA VLP were found to be more effective in inducing protective immunity against homo and hetero viruses in aged mice.

Influenza remains a persistent global health challenge. One of the weaknesses of current seasonal flu vaccines is their limited efficacy against drifted influenza strains. Our study focused on developing a novel self-adjuvanted double-layered protein nanoparticle vaccine that will be intranasally delivered and cross-protective. These nanoparticles consisted of an influenza nucleoprotein (NP) core encapsulated by hemagglutinin (HA) and a truncated form of bacterial flagellin (tFliC). Immunizations with these double-layered nanoparticles that included tFliC as a mucosal adjuvant, which activates toll-like receptor 5 (TLR5), induced significant mucosal and systemic immune responses, conferring cross-protection against influenza in mice. Compared to traditional vaccines, the double-layered nanoparticles induced higher levels of antigen-specific IgA and IgG in mucosal samples and serum and robust T-cell responses. In addition, the nanoparticles demonstrated robust immune responses in pre-infected mice, demonstrating that prior exposure to a heterologous influenza strain synergizes the vaccine's efficacy. To further optimize the vaccine's effectiveness, we employed a slow delivery method for the prime dose, spreading the administration over several days. This strategy markedly enhanced germinal center reactions and T-cell activation in lung-draining lymph nodes, resulting in superior protective efficacy against homologous and heterologous H3N2 influenza challenges. Our results demonstrate that the tFliC-adjuvanted, double-layered protein nanoparticles can be developed into a highly effective universal influenza vaccine. This novel intranasal vaccine formulation provides robust and broad protection and highlights a promising approach to improving influenza vaccine efficacy through simplified intranasal immunization, such as nasal drops.

Gonorrhea poses a significant public health issue due to the lack of immunity post-infection, increasing antibiotic resistance, and the absence of an effective vaccine. This study focuses on the gonococcal TonB-dependent transporter (TdT), TdfH, and its interaction with human calprotectin (hCP), which sequesters essential metals like zinc to inhibit bacterial growth. Findings highlight the critical regions of TdfH involved in zinc acquisition, making them potential targets for therapeutic interventions. TdfH is highly conserved among pathogenic gonococcal strains and surface-exposed, making it a promising vaccine target.

This study found that specific mutations in TdfH affect its ability to bind hCP and facilitate zinc uptake. Mutations in loop 2 inhibited hCP binding and growth significantly when hCP was provided as the only zinc source This suggests that drugs targeting critical hCP binding residues of TdfH could limit bacterial growth and serve as an effective therapeutic strategy. Additionally, investigating the structure and role of the hCP C-terminal tail in interacting with TdfH could reveal new targets for vaccines and therapies. Mutating the hCP tail resulted in the notable outcome that N. gonorrhoeae (Ngo) expressing TdfH failed to grow with or bind to the tailless hCP provided as the zinc source. This underscores the crucial role of the HCP tail in facilitating zinc uptake. In the third phase of the study, evaluations using hybrid antigens fused to a lipoprotein scaffold generated TdfH specific antigens with diverse functional capabilities. Some antibodies against TdfH loops inhibited HCP utilization, indicating a potential therapeutic or preventative mechanism against Ngo infection.

Ultimately, research suggests that interfering with TdTs binding function can effectively starve pathogens. This concept, known as "Starve and Kill," can be demonstrated by applying monoclonal antibodies (mAbs). Developing mAbs to disrupt the ability of TdfH to bind to hCP presents a promising prophylactic approach for gonorrhea treatment and a proof-of-concept for using TdfH as part of a vaccine cocktail. Subsequent studies will test this hypothesis in various mouse models of gonorrhea infection, using human transgenes to produce hCP and other TdTs ligands. These pre-clinical studies will inform the next steps in developing new therapies and vaccines against this challenging human pathogen.

ABSTRACT

The goal of this thesis project was to understand how fecal microbiota transplant treats Clostridioides difficile (C. difficile) infection, potentially inspiring more effective noninvasive therapies to restore the microbiome as a means of mitigating the disease burden caused by this pathogen. Accordingly, I sought to identify taxa that provide colonization resistance against C. difficile. I devised a gnotobiotic ASF (Altered Schaedler Flora) mouse model of CDI that provided a tractable and defined platform for studying microbiota’s role in CDI. Challenge of ASF mice with 10⁵ spores of hypervirulent C. difficile strain VPI10463 (acute challenge) led to complete mortality within three days; challenge with 10² spores resulted in low mortality and mice that remained chronically infected by C. difficile (chronic CDI model) and, concomitantly, chronic CDI symptoms. Furthermore, I found a previously described Clostridia preparation (Kim et al., 2017) protected ASF mice from acute challenge, and led to cessation of VPI10563 shedding and resolution of CDI symptoms in chronically infected ASF mice. Sequencing-based microbiome analysis revealed an association between Lachnospiraceae microbes and recovery. Bulk culture of the Clostridia preparation was used to generate a Lachnospiraceae consortium that protected mice against acute and chronic CDI. Deep sequencing found that recovery of chronic CDI mice strongly corelated with the appearance of a Lachnospiraceae species, namely Uncultured Bacteria and Archaea (UBA) 3401 a bacterium, which, as its name implies, was only known to exist as from databases of shotgun sequencing. While iterative removal of non-UBA3401 microbes from the Lachnospiraceae consortium enabled in vitro isolation of PCR positive-UBA3401 colonies, subculture of such colonies failed to propagate UBA3401. Thus, I performed iterative limiting dilution fecal transplants of the Lachnospiraceae consortium resulting in generation of a consortium predominated by UBA3401, albeit accompanied by a few other microbes (UBA3401 consortium). Inoculation of mice with the UBA3401 consortium was tracked longitudinally. Sequencing and PCR testing showed UBA3401 became detectable 3 days post inoculation and peaked in absolute and relative abundance at 18 days post inoculation. The UBA3401 consortium-colonized mice and age matched ASF controls underwent acute challenge with VPI10463. Severe CDI followed resulting in complete mortality of the control group, however UBA3401-consortium mice suffered no mortality and demonstrated minimal illness, showing that despite the simple, defined composition of the UBA3401 consortium, significant protection was retained. A concentrated chloroform extract of UBA3401 mouse feces was shown to result in reduced in-vitro growth of VPI10463 compared to ASF feces chloroform extract. Genomic sequencing of UBA3401 uncovered a probable biosynthetic gene cluster (BSC) responsible for production of a Thiopeptide bacteriocin. Canonic thiopeptide BSC genes, including the critical YCAO enzyme, were predicted along with adjacent genes for transport and localization (Vinogradov & Suga, 2020). The UBA3401 genome was predicted to encode many genes involved with quorum sensing, potentially regulating bacteriocin production (M. Kleerebezem et al., 1997), suggesting that a threshold of UBA3401 is required to induce thiopeptide production. Collectively, my results provide a more tractable model to study CDI and yield UBA3401 as potential means of providing microbiota-mediated CDI resistance.

Inflammatory bowel disease (IBD) continues to affect millions worldwide, with an increasing prevalence that highlights the urgent need for deeper understanding of its underlying immune mechanisms. The cytokine interactions, especially those mediated by cells from the TH1 and TH17 lymphocyte subsets, are crucial in orchestrating the immune landscape of IBD. TH1 cells are well known for producing TNF-α and IFNγ, which have been extensively studied for their roles in conjunction with each other within the context of IBD (Fish, 1999). TH17 cells secrete IL-22 and IL-17, with existing studies primarily focusing on IL-22’s interaction with IL-17 rather than its interplay with other cytokines such as IFNγ. Our study focuses on the co-stimulatory effects of IL-22 and IFNγ using organoids derived from mouse small intestines to model epithelial interactions. We found that IFNγ interferes with the capacity of IL-22 to up-regulate antimicrobial peptides, which is essential in mucosal defense. Additionally, higher concentrations of IL-22 enhance IFNγ's ability to stimulate TNF-α gene expression and CXCL10 protein production, indicating a dose-dependent relationship. This co-stimulation also led to an increased rate of cell death, influenced partly by TNF-α. These findings suggest that IL-22, typically seen as an anti-inflammatory agent, can assume a pro-inflammatory role when combined with IFNγ, complicating its effects on epithelial cells. This study highlights the need to consider specific cytokine interactions in developing more effective IBD treatments.

Gonorrhea is a sexually transmitted infection, caused by the bacterial pathogen Neisseria gonorrhea (Ngo) and affects millions of individuals of all age groups across the globe every year. Infection with Ngo does not result in protection and no effective vaccine has been developed, leaving antibiotics as the only treatment option. With the emergence of strains showing high levels of antibiotic resistance, there is an urgent need for development of novel therapeutics for disease prevention. During pathogenesis the host employs nutritional immunity, to restrict important transition metals such as zinc away from Ngo. This process is counteracted in Ngo by the production of highly efficient zinc import TonB-dependent Transporters (TDTs) which are promising vaccine antigens and zinc shuttle ABC transporters found to be important for intracellular survival. In Ngo zinc homeostasis and transport proteins are regulated by the Zinc uptake regulator (Zur) which represses transcription in the presence of zinc and activates transcription in the absence of zinc. In this study, we performed RNA sequencing to identify the global profile of genes in Ngo under the control of Zur and found that it differentially regulates 26 genes in response to zinc levels. We report the activity of Zur activity as a global regulator, able to both repress and activate gene expression in the presence of zinc and identified a consensus region on their promoters. We went on to further characterize the promoter elements of the zinc import TDT, tdfJ, which results in dual regulation by zinc and iron. We characterize specificity and binding affinities for regulation of tdfJ by a second regulator, Ferric uptake regulator (Fur) in response to iron. The response of tdfJ to both iron and zinc and its potential to be an important invasin, makes it an attractive candidate to investigate female genital tract infections. The female genital tract is a conglomerate of these conditions and infections here are often asymptomatic. Taken together, this research provides important knowledge on the regulation of virulence mechanisms in response to zinc, which will aid in the development of therapeutics and an efficacious vaccine against a gonococcal infection.

Cancer has been one of the leading causes for decades. Conventional cancer treatments present several shortcomings, emphasizing the urgent need for more advanced therapies. This thesis aims to provide innovative strategies targeting different types of cancer. Focusing on pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC), we com- bined oncolytic virus (OV) and immune checkpoint inhibitors (ICIs) to increase the efficacy of cancer treatment. In another approach, we also revealed how the inhibitor MA242 binds to the oncoprotein MDM2, generating valuable information for future drug design improve- ments. First, we investigate the efficacy of a novel genetically modified OV, vesicular stom- atitis virus expressing Smac protein (VSV-S), combining with ICIs. Through comprehensive in vitro and in vivo analyses, we demonstrate that the tumor microenvironment (TME) has been altered by VSV-S infection. Following with the programmed cell death protein-1 (PD- 1) antibody treatment, a more significant antitumor effect was observed. Next, we expand our combination therapy to NSCLC, which is traditionally considered resistant to anti-PD1 treatment due to mutations in oncogenic drivers such as EGFR, ALK, BRAF. We explored the transformative potential of VSV-S in sensitizing immunosuppressive TME to ICI treat- ment by eliciting inflammatory responses. The anticancer effect of combination is robust and TME is shifting towards ‘hot’ tumor phenotype. The combination remarkably inhibits tumor growth. Additionally, we delve into the molecular mechanism underlying the Mouse double minute 2 homolog (MDM2) inhibitor MA242. Through structural analysis and functional assays, we elucidated the inhibitory mechanism of MA242 on MDM2-mediated ubiquitina- tion. This study offered deeper insights of the mechanism of MA242 action, contributing to future structure improvement. In conclusion, the integration of OVs and immunotherapy, and the targeted molecular inhibitors represent advanced approaches in cancer therapy, and hold promises for improvement of cancer treatment.

Gonorrhea, a prevalent sexually transmitted infection affecting millions annually worldwide, is caused by Neisseria gonorrhoeae (Ngo), a "superbug" resistant to all antibiotic classes. Compounding the challenge, the absence of protective immunity upon infection allows for reinfection, and a viable vaccine against gonococcal infection remains elusive.

In response to infection, the human host deploys nutritional immunity, sequestering essential metals like iron from invading bacteria, using metal binding proteins. To overcome this, Ngo employs outer-membrane TonB-dependent transporters (TdTs), like HpuAB, to acquire iron from host metal binding proteins, such as hemoglobin (Hb).

Part of our study focused on HpuA, the lipoprotein component of the HpuAB system. Mutations targeting hydrophobic residues crucial for Hb interaction were studied. Results demonstrated that without HpuB, strains failed to grow, emphasizing HpuB's role in iron internalization. Notably, when HpuB is produced, deletion and insertion mutations in loop 2 of HpuA affected growth and binding to Hb.

Further investigations into HpuB, the transmembrane protein of the HpuAB system, uncovered essential loop regions for binding and growth on Hb as a sole iron source. Deletion mutations in loops 2, 3, and 4 facilitated binding and growth independently of HpuA production. Intriguingly, mutations in loop 7 abrogated binding and impaired growth in the absence of HpuA, but partial growth and full binding recovery occurred when HpuA was present. This highlighted the importance of loop 7 in iron acquisition and suggested a potential role for both HpuA and HpuB in the binding Hb.

As a combination of non-binding TdT mutants is hypothesized to have the potential to improve vaccine efficacy and provide protection, identifying non-binding HpuB mutants could be important. In summary, this research sheds light on the intricacies of the HpuAB system, contributing valuable insights that could inform the development of an effective gonorrhea vaccine.

Innumerable future deaths and hospitalizations could be avoided through improved pandemic preparedness and the use of personalized medicine. Here, various animal models have been used to improve an understanding of the pathogenicity and therapeutic treatment potential of three viral pathogens that have a significant impact on public health: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV), and influenza virus.

Roborovski dwarf hamsters were developed as a novel animal model to study SARS-CoV-2-induced acute lower respiratory tract injury. Dwarf hamsters recapitulate the pathogenicity of SARS-CoV-2 variants of concern (VOC) and demonstrate that the effect size of molnupiravir and paxlovid-like nirmatrelvir/ritonavir is VOC-dependent. Additionally, the ferret model of upper respiratory disease was used to study human-to-human-like transmission of SARS-CoV-2 and define a therapeutic window and human effect-size equivalent dose (HESED) for the approved drugs molnupiravir and paxlovid. Therapeutic administration of molnupiravir successfully prevented contact transmission, whereas paxlovid-like nirmatrelvir/ritonavir only partially reduced upper respiratory viral shedding. Moreover, the oral prodrug of remdesivir parent GS-441524 was shown to be efficacious against SARS-CoV-2 in ferrets.

A broad-spectrum polymerase inhibitor, 4’-Fluorouridine (4’-FlU), was identified and characterized, revealing potent activity against influenza virus, RSV, and SARS-CoV-2. This pre-clinical candidate is orally available and was shown to block replication of RSV in mice and SARS-CoV-2 in ferrets. In the mouse and ferret model, 4’-FlU mitigated lethal infection with pandemic human and highly pathogenic avian influenza. 4’-FlU could overcome moderate resistance, making this candidate a promising first line defense broad-spectrum antiviral. In parallel, an allosteric orally efficacious AVG-inhibitor series, which targets a dynamic interface in the RSV polymerase, was further developed to proof-of-concept in vivo efficacy testing.

To explore a relationship between the gut microbiota and the host response to respiratory viral infections, the impact of the intestinal microbiome on influenza virus infection outcome was investigated. Colonization with segmented filamentous bacteria (SFB) in the gut stably and broadly altered the phenotype of alveolar macrophages (AM) enabling these cells to better protect their hosts from lethal respiratory viral infections. Moreover, gut microbiota-derived metabolites were discovered which conferred protection against SARS-CoV-2.

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