Department of Veterinary Medicine Theses and Dissertations

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HOST-PATHOGEN INTERACTION DURING CRYPTOCOCCUS NEOFORMANS CNS INFECTION
(2024) Chen, Yanli; Shi, Meiqing MS; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen widely distributed in the environment globally. C. neoformans infection initiates from the lung through inhaling the spores. While most healthy individuals can clear the fungus or contain the fungus in the granuloma, immunosuppressed patients and a small group of healthy populations fail in controlling the cryptococcal fungal pulmonary infection. In those cases, C. neoformans transmigrates from the lung to the central nervous system (CNS) and causes fatal meningoencephalitis, which accounts for 112,000 deaths each year worldwide. However, we have a very limited understanding of the transmigration of C. neoformans from the bloodstream to the brain in vivo, and the mechanism involved in the clearance of the organism in the brain remains poorly understood. In this study, we first report a novel approach to quantitatively analyze the interactions between C. neoformans and brain endothelial cells in a mouse model using flow cytometry. Using this system, we show that C. neoformans was internalized by brain endothelial cells in vivo and that mice infected with acapsular or heat-killed C. neoformans yeast cells displayed a lower frequency of brain endothelial cells containing the yeast cell compared to mice infected with wild-type or viable yeast cells, respectively. We further demonstrate that brain endothelial cells were invaded by the serotype A strain (H99 strain) at a higher rate compared to the serotype D strain (52D strain). Moreover, we found that clearance of C. neoformans in the brain correlates with accumulation and pro-inflammatory M1 polarization of Ly6Chi mononuclear phagocytes and that these phagocytes play a critical role in the clearance of C. neoformans in the brain. Notably, the accumulation of Ly6Chi mononuclear phagocytes coincides with enhanced secretions of TNF and IFN-γ in the brain. TNF receptor (TNFR) signaling, but not IFN-γ receptor (IFN-γR) signaling, mediates the recruitment of Ly6Chi mononuclear phagocytes to the brain in a cell-intrinsic manner. By contrast, IFN-γ induces M1 polarization of Ly6Chi mononuclear phagocytes. Disruption of TNFR or IFN-γR signaling enhances cryptococcal growth in the brain. Thus, Ly6Chi mononuclear phagocytes act as effector cells for cryptococcal clearance in the brain, involving TNFR as well as IFN-γR signaling. Collectively, our study established that 1) internalization of C. neoformans by brain endothelial cells occurred in vivo and offered a powerful approach to quantitatively analyze fungal migration into the brain; 2) Ly6Chi mononuclear phagocytes accumulate in the brain during brain infection with C. neoformans and function as effector cells for clearance of C. neoformans in the brain involving TNFR signaling and IFN-γ signaling.
Biological Significance of selected Ixodes scapularis Transcription Factors regulating Tick Hematophagy and Development
(2023) Antara, Kazi Rifat; Pal, Utpal Professor; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Lyme disease is one of the most prominent vector-borne diseases, which is transmitted by the Ixodes scapularis tick and related species, and the causative agent is the bacterial pathogen Borrelia burgdorferi. Besides I. scapularis, many other tick species are also prolific vectors of several bacterial, viral, or eukaryotic pathogens affecting humans and animals. I. scapularis possess a large genome of 2.26 Gbp, predominantly featuring repetitive DNA or transposomal elements. Although many orthologous genes are present in other arthropods and blood-borne vectors, the genome also encodes numerous unique tick-specific genes. Despite many advances in Ixodes biology and genomics, the molecular basis of their hematophagy and development remains unknown. During feeding on the host, a major tick organ like the gut undergoes remarkable yet poorly understood episodes of cell division and differentiation, accommodating a huge blood meal that can be up to 100-fold greater than their body weight. The gut, therefore, plays a vital role in blood meal acquisition, digestion, and storage, supporting the long-term survival of ticks during prolonged off-host periods of nutrient deprivation. Understanding the molecular mechanism of gut physiology, including cell division and differentiation, is an essential area of research. As transcription factors are central to the biology and development of metazoan organisms yet remain largely uncharacterized in ticks, the goal of this dissertation is to decipher the biological significance of representative groups of major development-associated transcription factors in I. scapularis that are expressed in the gut, especially during blood meal engorgement process. Among them, two of the highly upregulated transcription factors in the gut were chosen for further characterization. We show that both transcription factors, Immunoglobin-fold transcription factor (SuH) and POU domain transcription factor (Nubbin), play essential roles in tick physiology, as their knockdowns impart phenotypic defects, impacting tick feeding, development and life cycle. The latter part of the dissertation will highlight the molecular mechanism of their functions. A fundamental understanding of the molecular basis of tick biology, hematophagy, and development may contribute to developing novel strategies to curb the spread of tick-infection.
Development of a Multilocus Sequence Typing Scheme for Avibacterium paragallinarum
(2023) Harris, Alyssa Meihua; Ghanem, Mostafa; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Avibacterium paragallinarum (A. paragallinarum), the causative agent of the respiratory disease Infectious Coryza (IC) in chickens, has seen a rising incidence in the United States. Current strain differentiation is inadequate for detailed epidemiological analysis. The objective of this study was to develop a Multilocus sequence typing (MLST) scheme for A. paragallinarum for outbreak investigations and to offer a better tool for strain differentiation. By evaluating whole genome sequences and clinical samples, we designed PCR amplicons for eighteen gene segments, selected six genes for their nucleotide diversity and discrimination potential. The MLST was used to differentiate seventy-five samples. Our MLST showed greater discriminatory power than existing HPG2-based methods, aligning closely with adhoc core genome MLST in 75 tested sample. Our newly developed MLST scheme enables more accurate strain differentiation, allowing for better understanding of A. paragallinarum epidemiology and population structure to help prevention and control efforts worldwide.
Development of improved recombinant NDV-vectored vaccines against highly pathogenic avian influenza virus (HPAIV)
(2023) Roy Chowdhury, Ishita; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Highly pathogenic avian influenza viruses (HPAIV) are highly contagious and economically devastating poultry pathogens with a documented transmission to humans causing severe human infections with high mortality. Circulation of these viruses is of public health concern as they have the pandemic potential to mutate to increase transmissibility among humans. The diversity of zoonotic influenza viruses causing human infections is alarming and effective vaccination is needed to control these viruses. Influenza viruses particularly with H7 and H5 subtypes of HA can naturally switch to a highly pathogenic phenotype through different mechanisms. Currently available vaccines are not satisfactory as they are mostly inactivated vaccines that require labor-intensive administration methods and provide suboptimal protection of vaccinated birds. Viral vectors offer crucial advantages over traditional vaccines, including induction of outstanding antibody and cytotoxic lymphocyte responses which is important for the control of viral infections. Newcastle Disease virus (NDV) is a promising vaccine vector for HPAIV since it is highly restricted for replication in the respiratory tract of poultry, it can be easily administered, and it induces both local and systemic immune responses. H7 influenza viruses are classified into two major genetic lineages, American and Eurasian. To develop a universal anti-H7 vaccine, we generated NDV vectors expressing chimeric HA sequences covering both North American and Asian isolates. In the first project, we designed NDV-vectored vaccines against HPAI H7N8 infection. The Hemagglutinin (HA) protein of influenza viruses is responsible for virus attachment to host cell and is the major target of the humoral immune response. Accordingly, we developed vaccines against HPAIV by generating recombinant NDV vectored H7 serotype-specific vaccines expressing HA protein. We also evaluated the protective efficacy of these recombinant vaccines against highly virulent H7 challenges in both broiler chickens and turkeys and the results were promising for broiler chickens, but for turkeys the vaccination design and scheme need to be further modified. In the second part of the study, we designed some recombinant NDV-vectored vaccines with an increased level of expression of H5HA antigen. The transcriptional unit of NDV contains a major open reading frame flanked by 5’ and 3’ untranslated regions (UTRs) followed by conserved transcriptional initiation and termination control sequences. Previous studies have shown that the addition of UTRs of P, M, and F genes positively modulated foreign gene expression. Hence, we hypothesized that cognate NDV mRNA UTRs would improve the expression of a protective antigen by an NDV-vectored vaccine. We generated recombinant NDVs where the HA of the HPAIV strain H5N1 is flanked by 5’ and 3’UTRs of NDV genes and determined the growth characteristics of these recombinant viruses, their stability, the level of HA expression and their transcription and translation modulation. Both studies aimed for the advancement of NDV-vectored vaccines emphasizing the fact of better expression of the protective antigen and improved immunogenicity for avian influenza virus considering two important strains of H5 and H7.
MOLECULAR DISSECTION OF BORRELIA BURGDORFERI BB0323 PROTEIN COMPLEX SUPPORTING MICROBIAL BIOLOGY, INFECTIVITY, AND AS A NOVEL THERAPEUTIC TARGET
(2023) Bista, Sandhya; Pal, Utpal Dr.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Lyme disease (LD), also known as Lyme borreliosis, is the most common vector-borne disease in the United States, caused by the gram-negative bacteria of the Borrelia burgdorferi sensu lato group. This atypical bacterial group features distinct genomic and antigenic elements, does not possess any classical toxins, and the pathogenesis of LD is primarily due to the immune activity of the host. These multi-organotrophic spirochetes can elicit severe clinical complications in susceptible hosts, including neuroborreliosis, carditis, and arthritis. If diagnosed early, the disease can be treated with a conventional antibiotic regimen; however, persistent, or relapsing symptoms later develop in a subset of patients. Six months to a year after the antibiotic treatment, up to 20% of the patients can experience various subjective symptoms pertaining to pain, cognitive dysfunction, or other neurological complications, collectively termed Post Treatment Lyme Disease Syndrome (PTLDS). The diagnosis, etiology, and treatment of PTLDS remain currently unknown. To better understand microbial pathogenesis, we have characterized a select set of structurally unique spirochete gene products that act as novel virulence determinants and support microbial infection in mammals. The current study focused on the BB0323 protein of B. burgdorferi, a unique and multifunctional virulence determinant undergoing a complex post-translational maturation process. The maturation, stability, and functions of BB0323 require multifaceted protein-protein interaction (PPI) events involving specific B. burgdorferi proteins, such as a protease-chaperone called BbHtrA, and a membrane-associated protein of unknown function annotated as BB0238. In our current study, we have further dissected the biological significances of the protein-protein interaction complex (PPI), either involving BbHtrA: BB0323 and BB0323:BB0238. The latter PPI event was more thoroughly investigated for its role in spirochete biology and infection and as a novel target for therapeutic intervention against B. burgdorferi infection. We identified a cleavage site where BB0323 full-length protein cleaves into N and C termini by BbHtrA. Subsequently, we have introduced point mutations in the recombinant BB0323 (at the cleavage site for BbHtrA- NL residues replaced with AA), as well as generated an isogenic B. burgdorferi isolates (Bbbb0323NL) with the point mutations in native BB0323. Further analyses show that the cleavage site mutated BB0323 protein could not be processed by the recombinant BbHtrA. Notably, despite the inability of BbHtrA to process BB0323 in vitro, within Bbbb0323NL, BB0323 could indeed be processed to some degree, which yields a basal level of mature N-terminal protein. Notably, in these B. burgdorferi cells, at least two other BB0323 polypeptides of lower molecular weight (less than 27 kDa of mature N-term BB0323) were also produced, possibly due to the action of BbHtrA on non-specific sites. However, the Bbbb0323NL mutants were non-infectious in the murine host, demonstrating the importance of precise cleavage of BB0323 full-length protein and optimal production of N-terminal, which needed to form a complex with another PPI partner, BB0238. Overall, these results further underscored the event of BbHtrA and BB0323 interaction for processing the latter protein as an essential prerequisite for spirochete infection in mammals. Our previous studies have shown that BB0323 N-terminal and BB0238 interact and post-translationally stabilize each other. We used an interaction-deficient borrelial mutant, replacing the BB0323 interaction motif in BB238 (termed as bb0238 Delta Interaction Motif, or bb0238∆IM), which despite showing no growth defects in vitro or other abnormalities, is unable to infect mammalian host. We, therefore, explored the possibility of using the BB0323:BB0238 complex as a novel therapeutic target to combat B. burgdorferi infection in mammals. We first examined whether bb0238∆IM mutants (without interaction motifs) can persist in mice for a long term or could be acquired by naïve ticks. The results show that, unlike the wild type or another B. burgdorferi mutant, The bb0238∆IM could not establish the infection in mice and, as a result, could not be acquired by the ticks, suggesting blockade of BB0323:BB0238 interaction by small molecules could be a novel therapeutic approach to combat incidence of LD. An AlphaLisa assay platform was developed in our lab to monitor BB0323-BB0238 PPI on a high-throughput basis using 384-well microtiter plates, which was then miniaturized to 1536 well at the National Center for Advancing Translational Sciences (NCATS) in a collaborative effort. An AlphaLisa quantitative HTS later screened several small molecule libraries available at NCATS, which were further filtered by counter assays, and a selected set of 84 compounds was tested in a secondary, cell-based assay for cell-permeable compounds that impair BB0323-BB0238 interaction with spirochete cells. A B. burgdorferi cell-based assay comprising a dot-blot assay and regrowth assay was developed to examine the PPI inhibitory activities of the molecules inside the cells. We finally selected one of the compounds, Lomibuvir, for the in vivo studies and demonstrated its PPI inhibitory activity in an in vitro experiment. A pharmacokinetic study in mice showed an increase in the level of the compound in plasma and liver over 21 days. Additional in vivo efficacy studies of Lomibuvir to reduce B. burgdorferi infection in mice were performed using vehicle and ceftriaxone as negative and positive controls, respectively. The results showed that the bacterial load in the skin and heart of the mice was significantly lower in the Lomibuvir-treated group, as compared to the vehicle-treated animals; however, the effect was not as dramatically effective as the antibiotic (ceftriaxone) treatment groups. While future medicinal chemistry approaches could be adopted to further enhance the impact of Lomibuvir as an anti-B. burgdorferi agent, to the best of knowledge, is the first proof-of-concept study that highlights the utility of targeting borrelial PPI events as a possible therapeutic target of Lyme disease.
A NOVEL IXODES SCAPULARIS PROTEIN DICTATES TICK HEMATOPHAGY AND CUTICLE INTEGRITY, IMPACTING TICK DEVELOPMENT
(2023) DUTTA, SHRABONI; Pal, Utpal Dr.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Ticks are prevalent throughout the world and are capable of transmitting a variety of pathogens (e.g., bacteria, protozoa, and viruses) to humans. Incidence rates for tick-borne diseases (TBD) are also increasing globally, and effective vaccinations to combat tick infestations and TBD transmission remain a critical unmet need. Of the six major tick genera that spread human illnesses worldwide, Ixodes ticks are the most prevalent. Specifically, Ixodes scapularis (also known as the blacklegged or deer tick) is an obligate blood-feeding arthropod that transmits several human and animal pathogens that include Borrelia burgdorferi sensu lato complex – the causative agent for Lyme disease. Unlike many hematophagous insects and soft ticks, I. scapularis (hard ticks) remain attached to their hosts for several days and are capable of uptaking bloodmeals that are 100 times greater than their initial body weight. A large and nutrient-dense bloodmeal is essential for their sub-adult and adult development processes and fecundity. However, the molecular and cellular processes that regulate tick blood feeding (hematophagy) and development have not been extensively elucidated. Therefore, our major objective is to characterize tick molecular components that are critical in the tick parasitism and life cycle in order to develop new strategies to combat tick infestations and spread of tick-borne diseases. Herein, we describe the structural and functional properties of a newly identified I. scapularis protein isolated from the partially fed nymphal ticks. Although the protein displays minor homology to proteins of known functions, structurally, it resembles some features of arthropod Odorant Binding Proteins (OBP). Therefore, we refer to this protein as, Ixodes Gut OBP (IGOBP). We show that the knockdown of IGOBP via RNA interference in ticks results in impaired blood feeding (hematophagy) and significantly decreases their post-fed weights. In addition, systemic IGOBP knockdown gives rise to aberrant phenotypes, significantly reduces tick molting rate, and compromises the structural integrity of the cuticle, specifically the flexible alloscutum components. Notably, IGOBP knockdown has profound effects on the molting efficacy and fitness of females than males. This is likely due to the fact that female adults consume a greater volume of bloodmeal than male adults, necessitating a more pronounced expansion of the alloscutum. Subsequently, our RNA sequencing data identifies multiple genes whose expressions are regulated by IGOBP. The underlying mechanism of possible IGOBP or associated gene functions may aid in identifying future targets for anti-tick vaccines. In summary, our studies characterized a novel I. scapularis protein revealing that the protein is essential for tick hematophagy and development. To the best of our knowledge, this is the first characterization of a tick odorant-binding protein (OBP), using structural and functional genomic tools that unearthed the unique and possibly multifunctional role of IGOBP in vector biology and parasitism. We anticipate that the presented data will enhance our fundamental understanding of tick biology and contribute to the development of potential anti-tick measures.
Immune Modulations of a Helminth Derived Protein
(2023) Mekhemadhom Rajendrakumar, Arunraj; Zhu, Xiaoping XZ; Tuo, Wenbin WT; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The immune responses at the gastrointestinal mucosa modulate nematode parasite infection, initially characterized by the production of epithelium-derived, robust T helper 2 (Th2) type alarmin cytokines, such as interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP). Subsequently, the immune responses are mediated by releasing the lymphoid cell-derived Th2 immune cytokines, such as IL-13, IL-4, IL-5, IL-9, and parasite-specific antibodies. Studies have shown that parasitic nematode worms can establish a chronic infection in the intestine, even in a robust immune response. This evidence leads us to hypothesize that the nematode evolves to evade or regulate intestinal immunity through specific modulatory mechanisms that interfere with initial intestinal immune responses, allowing the nematode to survive. We used a model nematode, Heligmosomoides polygyrus bakeri (Hpb), to identify nematode-derived proteins with regulatory effects on Th2 immune cytokines during chronic infection. Through high throughput analysis, we found that a Hpb-derived protein could precisely modulate mouse immune response. The presence of the Hpb-derived protein was essential for the parasite's survival as the vaccine conferred a sterilizing immunity. As Th2 cytokines are directly associated with the pathogenesis of several inflammatory and autoimmune diseases, we are understanding how this protein regulates the function of the Th2 cytokines in vitro and in vivo and explore whether the protein could use to treat inflammatory diseases and serve as a vaccine target to control nematode infections.
STRUCTURE-FUNCTION ANALYSES OF AN ESSENTIAL VIRULENCE DETERMINANT OF THE LYME DISEASE PATHOGEN
(2022) Foor, Shelby Dimity; Pal, Utpal; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Lyme Disease (LD) is a tick-borne disease caused by a group of gram-negative-like spirochetal pathogens called Borrelia burgdorferi sensu lato. The number of cases reported in the United States have dramatically increased with CDC estimating 476,000 cases annually. This multifaceted infection can spread throughout the entire body, causing clinical complications of the central nervous system, joint and heart. Early antibiotic treatment is available and effective; however, untreated patients can develop chronic symptoms, and even after antibiotics, symptoms of unknown etiology and pathogenesis can develop into post-treatment Lyme disease syndrome (PTLDS). The enzootic life cycle of B. burgdorferi is maintained typically between a small rodent and the Ixodes tick vector, where transmission occurs during tick feeding on a host. Infection establishes after B. burgdorferi is deposited in the dermis and undergoes the required shift in its protein expression profile necessary to support spirochete persistence and pathology, often highlighting protein targets for development of diagnostic, therapeutic, and preventative measures. Two such proteins identified, BB0238 and BB0323, serve as novel virulence determinants and are essential for mammalian infection. These two proteins directly interact, mutually stabilize each other post-translationally, and form an essential complex required for infection; however, their precise functions remain undetermined. In collaborative efforts, we predicted a two-domain structure of BB0238. The N-terminal domain was predicted by AI methods to harbor an antiparallel helix-turn-helix motif (HTH) followed by a third helix and a low-confidence predicted meandering segment. The C-terminal domain structure was determined by X-ray crystallography as well as predicted with high confidence to adopt an α+β fold that resembles closely that of the nuclear transport factor 2 (NTF2) superfamily. While full-length BB0238 lacks homology to singular proteins of known functions, the individual N- and C-terminal regions display structural homology to non-bacterial proteins, particularly to eukaryotic sorting, or transport proteins, suggesting that BB0238 supports an unconventional function in spirochetes. We discovered that BB0238 binds another borrelial protein annotated as BB0108, orthologs of two bacterial chaperones and foldases, the extracellular membrane anchored PrsA, and the periplasmic SurA. This identified interaction requires further investigation, however, may be important for BB0238 protein stability or assist with the novel BB0238 function discovered herein, which regulates proteolytic processing of BB0323. Furthermore, We show that key amino acid residues within the HTH stabilize BB0238 in an environment-specific manner, influence its oligomerization properties, and facilitate tick-to-mouse transmission by aiding spirochete evasion of host cellular immunity, underscoring BB0238’s ability to support microbial establishment during early mammalian infection. Together, these studies highlight the divergent evolution of multidomain spirochete proteins involved in multiplex protein-protein interactions, possibly facilitating multiple functions, which support pathogen survival and thus, represent novel targets for vaccine and therapeutic development against Lyme disease.
DEVELOPMENT OF AVIAN PARAMYXOVIRUS VECTORED VACCINES
(2022) Elbehairy, Mohamed Adel; Belov, George A; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Avian Avulaviruses (formerly Avian Paramyxoviruses, APMV) are important pathogens of avian species and have been used as viral vectors for more than two decades. Among all APMVs, Newcastle disease virus (NDV or APMV-1) has been most extensively used as a vaccine vector for protection against avian and animal diseases, and as an oncolytic agent. For poultry vaccination, the preexisting maternal antibodies against NDV can neutralize APMV-1 vectors resulting in vaccination failure. Hence, there is a need to develop new vaccine vectors that would escape neutralization by the maternal antibodies. In the first part of my study, I created a reverse genetics system for Avian paramyxovirus-3 strain Wisconsin (APMV-3 Wisc.) which was proven to be nonpathogenic for day-old chicks and embryonated chicken eggs. The virus was used as a vector to express the enhanced green fluorescent protein (GFP) as a heterologous antigen. The recombinant APMV-3 Wisc. expressing GFP was compared with similarly constructed APMV-1 strain LaSota and APMV-3 strain Netherlands-based vectors for GFP expression and growth kinetics in vitro, and for immunogenicity, safety, and tissue tropism in day-old specific pathogen-free (SPF) chicks. APMV-3 strain Netherlands (APMV-3 Neth.) showed the highest growth rate and GFP expression in chicken fibroblast DF-1 cells, followed by APMV-1 LaSota and APMV-3 Wisconsin. In day-old chicks, APMV-3 Neth. spread to different organs, decreased feed intake and caused stunted growth. APMV-3 Wisc. and APMV-1 LaSota were confined to the respiratory tract and did not induce any pathogenic effects. All three constructs induced seroconversion of the vaccinated chicks for the vector antigens. Thus, the reverse genetics system created in this study for APMV-3 Wisc. allows the development of safe APMV vector antigenically different from NDV that can be used for day-old chicks vaccination. In addition, it provides a tool to study the molecular basis of APMV3 pathogenesis. In the second part of my study, I explored a novel approach for the expression of a foreign gene as an uninterrupted open reading frame (ORF) with a cognate gene of NDV vector. This approach is expected to promote the foreign gene expression stability. Avian influenza virus (AIV) hemagglutinin (HA) protein-coding sequence was fused in-frame with various proteins of NDV vector, with a 2A self-cleaving peptide, a furin cleavage site, or both, placed between the AIV and NDV sequences for separation of the two proteins. Among different constructs tested, we only recovered viable viruses with AIV HA fused C-terminally to the NDV HN gene. These viruses demonstrated a higher expression level of AIV HA than the vector constructed according to a traditional scheme of expressing the transgene as a separate transcriptional unit. Also, they showed increased stability of the transgene expression over multiple passages in embryonated chicken eggs. Our results demonstrate the advantages and limitations of this novel method of foreign gene expression that need to be considered for the development of NDV-based vaccine or therapeutic vectors.
Host Immune Responses to Fungal Pathogens
(2022) Strickland, Ashley Brenda; Shi, Meiqing; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Fungal infections are an increasing threat to global public health. Worldwide, more than one billion fungal infections are diagnosed each year and result in 1.5 million deaths annually. This number is expected to rise as advances in medical care for immunocompromised patients continue to be made. Furthermore, limited antifungal treatment options, coupled with the emergence of new and resistant fungal species, is anticipated to exacerbate this issue. For these reasons, it is essential to understand the dynamic interactions between host and fungus that contribute to fungal pathogenesis. The following work examined the role of IL-27 on T cell regulation in the lungs during infection with Aspergillus fumigatus, and in the brain during infection with Cryptococcus neoformans. In contrast to most infection settings demonstrating that IL-27 is anti-inflammatory, we report that this cytokine is proinflammatory in both the lung and the brain during infection with A. fumigatus and C. neoformans respectively. Genetic ablation of IL-27Rα in mice resulted in higher fungal burdens in the lung during Aspergillus infection and in the brain during Cryptococcus infection. These were associated with reduced IFN-γ production, as well as a fewer CD4+ T cells in target organs. In the case of C. neoformans infection, IL-27 signaling enhanced T cell accumulation by promoting T cell proliferation early during infection, and inhibiting T cell death at later time points. These suggest that IL-27 promotes T cell responses required for fungal clearance during infection with A. fumigatus or C. neoformans. In addition, the work presented here also characterized lung resident macrophage responses during infection with C. neoformans. Here, we report that cryptococcal infection induced the accumulation of CD68hi macrophages in the lungs of infected mice. Approximately 10% of these cells were alveolar macrophages, while nearly 90% were interstitial macrophages. Both of these populations were observed to interact with fungi and upregulated their expression of arginase 1. The absence of either macrophage in transgenic mice, or following pharmacological depletion, significantly reduced fungal burdens in the lungs of mice, indicating that these cells enhance fungal growth and may represent attractive targets aimed at limiting the pathogenesis of C. neoformans.
Identification and functional characterization of the GBF1-controlled network of host proteins supporting enterovirus replication
(2022) Moghimi, Seyedehmahsa; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The genus Enterovirus of the Picornaviridae family contains many established and emerging pathogens. However, licensed vaccines are currently available only against poliovirus and enterovirus A71. No therapeutics have been officially approved to treat any enterovirus infections, although some are being developed. To find suitable targets for antivirals and control the infections, we need to understand the virus's life cycle better and identify the cellular factors involved in virus infection. Enterovirus genome replication occurs on the unique membranes known as replication organelles (ROs). A Golgi resident protein, GBF1, is recruited to the ROs by a viral protein 3A. GBF1 activates small GTPases Arf, which are critical regulators of the cellular secretory pathway. Here, we investigated the mechanistic details of GBF1-dependent Arf activation during enterovirus replication and characterized the proteome of the ROs in the vicinity of GBF1. We showed that Arf1 appeared to be the first to associate with the ROs, followed by other Arfs. Once activated and recruited to the ROs, all Arfs except Arf3 were no longer sensitive to inhibition of GBF1, suggesting that they do not actively cycle between GTP- and GDP-bound states in infected cells. siRNA depletion studies demonstrated an increased sensitivity of polio replication to inhibition of GBF1 in Arf1-, and to a lesser extent, Arf6-depleted cells, indicating the importance of GBF1-mediated activation of these Arfs for the viral replication. Taking advantage of the GBF1 recruitment to the ROs and GBF1’s essential role in enterovirus replication, we used a GBF1 construct fused to APEX2 peroxidase to explore the proteome of the ROs by proximity biotinylation. Among the proteins biotinylated in infected cells were the known cellular factors recruited to the ROs, including PI4KIII, OSBP, and ACBD3, indicating that these proteins are localized close to GBF1. Among the viral proteins, the intermediate products of the polyprotein processing were overrepresented, suggesting that GBF1 is localized close to the sites of active polyprotein processing. About 85% of the proteins identified by MS have not been previously associated with enterovirus infection. Gene ontology analysis revealed a significant enrichment of RNA binding and mRNA metabolic processes, suggesting a close localization of GBF1 to the RNA replication complexes. siRNA knockdown functional analysis of the selected proteins showed the recruitment of both proviral and antiviral factors to the ROs. Collectively, our work revealed important details about the involvement of Arfs in the replication process, introduced a highly efficient system to investigate the proteome of the enterovirus ROs, and provided novel data about the protein composition of the GBF1-enriched environment in the replication sites.
Development of avian paramyxovirus 3 as a vaccine vector against infectious bursal disease in one-day-old specific pathogen free chickens
(2021) Varghese, Berin Parambethu; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
ABSTRACTInfectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive disease affecting young chickens, resulting in substantial economic losses to the poultry industry worldwide. Although strict hygienic measures and various vaccination strategies have been adopted, IBD remains a major problem for the poultry industry. The economic significance of this disease is exhibited in two ways. First, the disease can cause high morbidity and mortality. Second, severe prolonged immunosuppression of chickens when infected at an early age leads to susceptibility to other diseases and vaccination failure. Therefore, chicks should be protected from early infection, usually achieved by vaccinating breeder flocks and active immunization of the newly hatched chicks. Vaccines include live-attenuated, viral vectored recombinant, subunit, and inactivated vaccines currently available to control infectious bursal disease (IBD). The major flaws of available vaccines are the reversion to virulence, the generation of new variant viruses, inability to replicate in the presence of maternal antibodies, lack of proper delivery systems, recombination and integration into the host genome, and inadequate systemic immune response. To overcome these obstacles, we have evaluated avian paramyxoviruses (APMVs) as vaccine vectors to generate vaccines against IBD. In the present study, we constructed recombinant Newcastle disease virus (NDV) strain LaSota (rLaSota/VP2) and recombinant avian paramyxovirus-3 (APMV-3) strain Netherlands expressing VP2 protein (rAPMV-3/VP2), the immunogenic protein of IBDV, and to evaluate their protective efficacies following immunization of one-day-old specific pathogen-free (SPF) chicks. Our results showed that both recombinant viruses stably express the VP2 protein, and their in vitro growth characteristics were almost similar to their respective parental viruses. Immunization of one-day-old SPF chicks demonstrated that rAPMV-3/VP2 protein elicited IBDV specific neutralizing antibodies and provided complete protection against the IBDV STC challenge. In addition, the rAPMV-3/VP2 protects chickens from clinical signs, gross lesions, and histopathology even at lower vaccine doses. Moreover, rAPMV-3/VP2 provides slightly better protection than the commercial vaccine from histopathology lesion against IBD at four weeks of age. This study suggests that recombinant APMV-3 expressing VP2 protein could be used as a potential vaccine against IBD in field conditions where maternal antibodies exist.
HEPATITIS E VIRUS MODULATES HOST FACTORS TO GENERATE A CONDUCIVE ENVIRONMENT FOR REPLICATION
(2020) lin, shaoli; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV infection mainly presents as acute and self-limiting hepatitis in young adults. However, it can be exacerbated to fulminant hepatitis in HEV-infected pregnant women, resulting in up to 30% case fatality. Besides, chronic HEV infection with rapid progression in immunocompromised patients has been a challenge in many countries since it was reported years ago. HEV infection is zoonotic, and human HEV strains are grouped into four major genotypes in the genus Orthohepevirus A, the family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. Due to the lack of an effective cell culture system and a proper animal model, HEV biology, virus-cell interactions, and pathogenesis are understudied. HEV is known to inhibit the innate immune response by targeting type I interferon (IFN) signaling pathway via its ORF1 products. Nevertheless, it remains largely unknown how the virus manipulates host factors to facilitate its replication. The objective of these studies was to elucidate the mechanism of HEV manipulation of host factors to generate a conducive environment for replication. Our results show that the capsid protein of HEV inhibits the IFN production to dampen the antiviral response through its N-terminal arginine-rich motif. In addition to the impairment of innate immunity, HEV proliferation requires the presence of other host factors: DDX3, an RNA helicase, and oxysterol-binding protein (OSBP), a lipid transporter. The knockdown of these two factors led to a significant reduction of HEV replication, whereas the reconstitution of these two genes restores the HEV proliferation level. The capsid protein was found to interact with the C-terminal domain of DDX3. The HEV helicase was shown to interact with OSBP and block its translocation to the Golgi apparatus. These results indicate that HEV employs multiple strategies including blocking antiviral response and recruiting host factors for its invasion and proliferation. Our data provide insights into the HEV-cell interactions and may facilitate the development of novel antiviral strategies.
AVIAN PARAMYXOVIRUS-VECTORED VACCINES AGAINST INFECTIOUS BRONCHITIS VIRUS AND HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS
(2019) Shirvani, Edris; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Highly pathogenic avian influenza (HPAI), infectious bronchitis (IB), and Newcastle disease (ND) are highly contagious and economically important diseases in poultry. Vaccination is the major strategy which is implemented to combat highly pathogenic avian influenza virus (HPAIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV), worldwide. However, among these viruses, some NDV strains are naturally avirulent and have been used as highly safe vaccines for more than 60 years. Live attenuated IBV vaccines that are produced by passaging virulent strains in eggs have safety concerns and are genetically unstable. Inactivated IBV and HPAIV vaccines also are less efficacious and affordable. Therefore, development of alternative vaccines against IBV and HPAIV is highly needed. In this multistep study, we have employed NDV vector and other novel avian paramyxovirus (APMV) vectors to develop improved IBV and HPAIV vaccines. Firstly, we conducted a study to investigate the contributions of the S1, S2, and S proteins of IBV in protection against virulent IBV, and to develop a safe and efficacious recombinant NDV-vectored IBV vaccine. We generated recombinant (rNDV) strain LaSota viruses expressing S1, S2 or S protein of IBV using reverse genetics. We evaluated the protective efficacies of rNDVs against virulent IBV and NDV challenges. Our results showed that the S protein, which contains the S1 and S2 neutralizing epitopes in correct confirmation is the best protective antigen of IBV. These results suggest that the rNDV expressing the S protein of IBV is a safe and effective bivalent vaccine candidate for both IBV and NDV. Secondly, besides rNDV strain LaSota vector, we employed a novel chimeric rNDV/avian paramyxovirus serotype-2 (rNDV/APMV-2) vector that replicates less efficiently and a modified NDV strain LaSota (rLaSota-527) vector that replicates more efficiently to develop a likely improved viral vectored vaccine against IBV. We generated rNDV/APMV-2 or rLaSota-527 virus expressing the best protective protein of IBV (S protein), which was found in the first study. The protective efficacies of rNDV/APMV-2 or rLaSota-527 virus expressing the S protein was evaluated against IBV in chickens. Our results showed that immunization of chickens with either chimeric rNDV/APMV-2 expressing the S protein, which is a better candidate for in ovo vaccination, or rLaSota virus expressing the S protein provided protection against IBV. Most importantly, compared to prime-boost vaccination or vaccination with rLaSota-527 virus expressing the S protein, single immunization of chickens with rLaSota virus expressing the S protein induced better immune responses against IBV. Thirdly, we conducted a study to evaluate the contributions of HA1 and HA2 subunits of HPAIV HA protein in the induction of neutralizing antibodies and protection in chickens, using rNDV strain LaSota vector. Our results showed that the HA1 and HA2 subunits when expressed separately, neither provided protection nor induced neutralizing antibodies. To be effective the HA protein must be incorporated into a vaccine as an intact protein. These results also highlight the importance of using chickens in HPAIV vaccine studies as they are susceptible natural hosts. Finally, we employed APMV-3 strain Netherlands as a vaccine vector, for its high efficiency replication in multiorgans of host, to generate an improved vaccine against HPAIV. Our results showed that immunization of chickens with either rAPMV-3 expressing the HA protein (rAPMV-3/HA) or rNDV expressing the HA protein (rNDV/HA) provided complete protection against HPAIV challenge. However, the immunization of chickens with rAPMV-3/HA induced higher levels of neutralizing antibodies than that induced by rNDV/HA. These results suggest that mass-vaccination with a rAPMV-3/HA might provide better protection against H5N1 HPAIV in field conditions. In conclusion, the individual subunits of the S protein of IBV or the HA protein of HPAIV when expressed separately, neither provided protection nor induced neutralizing antibodies. To provide protective efficacy, the intact HA or S protein must be incorporated into vaccine. The rNDV expressing the S protein is a safe and efficacious bivalent vaccine against IBV and NDV. Other than rNDV strain LaSota, rNDV/APMV-2 and rAPMV-3 are promising vaccine vectors for development vaccines against IBV and HPAIV, respectively.
IDENTIFICATION AND ENGINEERING BACTERIOPHAGE ENDOLYSINS FOR INACTIVATION OF GRAM-POSITIVE SPORE-FORMING BACILLI
(2018) Etobayeva, Irina V.; Nelson, Daniel C.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
This dissertation concentrates on the study of the antibacterial potential of bacteriophage-encoded endolysins derived from phages that infect the Gram-positive Bacillus cereus sensu lato group. Bacteriophage-encoded endolysins are peptidoglycan hydrolases that have been identified as important factors in the phage life cycle. Endolysins are encoded by phage late genes during an intracellular infection cycle to lyse the bacterial cell wall from within and allow phage progeny release. Endolysins derived from phages of Gram-positive bacterial hosts are equipped with an enzymatic domain that hydrolyzes conserved covalent bonds in bacterial peptidoglycan, and a cell wall binding domain that ensures proper attachment of endolysins to bacilli. In this study three novel endolysins, PlyP56, PlyN74, and PlyTB40 have been discovered and identified. The biochemical analysis shows that all three endolysins have relatively broad antimicrobial activity against organisms of the B. cereus group with the optimal lytic activity at physiological pH (pH 7.0–8.0), over a broad temperature range (4–55°C), and at low concentrations of NaCl (<50 mM). The domain shuffling engineering studies were undertaken to observe enhancements of bacteriolytic properties of chimeric lysins that retained their specificity to B. cereus species. Finally, these studies have identified a new development in lysis of peptidoglycan of Gram-positive B. cereus group of organisms by phage-encoded endolysins. When grown to stationary phase, bacilli, especially, in overnight cultures become more resistant to lysis despite the evidence that the cell wall domains bind the bacterial surface. In light of these findings, I hypothesize that B. cereus group of species have evolved complex behaviors to interact with phage by modulating surface associated secondary polymers throughout the maturation of the bacilli in order to render them more resistant to the lytic action of phage encoded endolysins, which, contributes to bacterial survival from phage infection.
INITIATION AND PROGRESSION OF BRAF/NRAS WILDTYPE MELANOMA IN UV-INDUCED MOUSE MODELS OF CUTANEOUS MELANOMA
(2018) Michael, Helen Thompson; Samal, Siba; Merlino, Glenn; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Melanoma is the deadliest skin cancer and is responsible for nearly 60,000 deaths worldwide each year. At least some melanomas are believed to arise from stepwise progression from normal melanocytes through a benign nevus stage to malignant melanoma and finally metastatic disease. Approximately 20-50% of melanomas have evidence of a pre-existing nevus, indicating that progression is an important route of melanomagenesis. Ultraviolet radiation exposure is believed to play an important role in nevus and melanoma formation, although the mechanisms of this remain unclear. Childhood sunburn and intermittent sun exposure are epidemiologically linked to increased melanoma risk. While most melanomas have activation of the mitogen activated protein kinase pathway, often due to mutations in BRAF or RAS genes, nearly 15% of cutaneous melanomas do not have an identified strong driver. Despite targeted therapies and immunotherapy, the death rate from melanoma has remained nearly static for several decades, so there is a need to identify additional genes and pathways to provide novel therapeutic targets. We hypothesized that progression of melanocytic lesions from benign to malignant is associated with the acquisition of additional genomic mutations. Unlike wildtype mice, hepatocyte growth factor (HGF) transgenic mice have “humanized” distribution of melanocytes along the dermal-epidermal junction. Following a single dose of UV at 3 days of age, HGF mice develop melanocytic nevi and melanomas. In this project, two HGF models were used to generate melanocytic lesions. The first model, on an albino FVB background had a tumor incidence of only 10% and used melanocyte-specific green fluorescent protein expression to identify early nevi and melanomas. The second model, on a C57BL/6 had a high tumor incidence (80%), and 60% of tumor-bearing mice have metastatic lesions. Sequencing of melanocytic lesions at different stages revealed a variety of driver mutations, including Nf1, Gnaq, and Gna11, as well as genes and pathways with less established roles in melanoma development. Our data provide a broad overview of genes and pathways involved in progression of non-BRAF, non-NRAS melanoma. Additionally, we present the first potential germline variants that may increase metastatic susceptibility for melanoma patients. These genes suggest potential biomarkers for progression of melanocytic lesions.
The immunoregulation of interleukin-27 in African trypanosome infection
(2018) Liu, Gongguan; Shi, Meiqing; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Interleukin (IL)-27 is a cytokine with diverse impacts on regulation of vertebrate T helper Type 1 (Th1) responses. Initially, it was predicted as a promoter of Th1 responses. However, it was lately identified as a potent negative regulator of T cell responses in a variety of disease models, including infection with viruses, bacteria, and intracellular parasites. The extracellular protozoan parasites, African trypanosomes, cause a chronic debilitating disease associated with persistent inflammation. Using this infection model, we aim to identify novel immunoregulatory functions of IL-27 on innate and adaptive immunity. Here we demonstrate that IL-27 receptor deficient (IL-27R-/-) mice infected with African trypanosomes display excessive production of IFN-γ by CD4+ T cells, exacerbated liver pathology, and dramatically shortened survival as compared with infected wild-type mice. Depletion of CD4+ T cells or neutralization of IFN-γ ameliorates the liver pathology and extends the survival of infected IL-27R-/- mice. Our further interest is in deciphering the mechanisms of how CD4+ T cells and IFN-γ shape the monocyte-featured innate immunity in African trypanosome infected IL-27R-/- mice. Blood monocytes typically consist of a heterogenous population of Ly6C+ and Ly6C- monocytes. Ly6C+ monocytes can give rise to inflammatory TNF-α/iNOS producing dendritic cells (Tip-DCs) and anti-inflammatory macrophages. Here we find that IL-27R-/- mice exhibit a higher frequency of Ly6C+ monocytes recruitment to the liver, where they preferentially differentiate into Tip-DCs. This is coincided with impaired development of Ly6C- monocytes and macrophages in the liver. Depletion of CD4+ T cells or neutralization of IFN-γ in infected IL-27R-/- mice diminishes the recruitment of Ly6C+ monocytes, and their differentiation into Tip-DCs in the liver. This is accompanied by the greatly enhanced counts of Ly6C- monocytes and macrophages following antibody treatments. Further evidences show that 1) IFN-γ produced by CD4+ T cells induces cell death of Ly6C- monocytes which perturb the development of Tip-DCs in infected IL-27R-/- mice and 2) cell intrinsic IFN-γ signaling drives Ly6C+ monocytes to differentiate into Tip-DCs in infected IL-27R-/- mice. Thus, our data identify IL-27 signaling as a novel immunoregulator to prevent Ly6C+ monocytes from differentiation into Tip-DCs through suppressing CD4+ T cells to secrete IFN-γ.
Antagonizing JAK-STAT signaling by porcine reproductive and respiratory syndrome virus
(2018) Yang, Liping; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway is activated by numerous cytokines. JAK-STAT pathways involve in regulation of cell growth, proliferation, differentiation, apoptosis, angiogenesis, immunity and inflammatory response. Because of their significance in immune response, they are often targeted by pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV causes reproductive failure in sows and severe respiratory disease in pigs of all ages. A typical feature of the immune response to PRRSV infection in pigs is delayed production and low titer of virus neutralizing antibodies, and weak cell-mediated immune response. One possible reason for the weak protective immune response is that PRRSV interferes with innate immunity and modulates cytokine signaling, including JAK-STAT pathways. The objective of this project was to elucidate the mechanisms of PRRSV interference with JAK-STAT2 and JAK-STAT3 signaling. This study demonstrates that PRRSV antagonizes interferon (IFN)-activated JAK-STAT2 signaling and oncostatin M (OSM)-activated JAK-STAT3 pathway via inducing STAT2 and STAT3 degradation. Mechanistically, PRRSV non-structural protein 11 (nsp11) and nsp5 induce the degradation of STAT2 and STAT3, respectively, via the ubiquitin-proteasome pathway. Notably, PRRSV manipulates karyopherin alpha 6 (KPNA6), an importin that is responsible for STAT3 nuclear translocation in the JAK-STAT signaling, to facilitate viral replication. Knockdown of KPNA6 expression led to significant reduction in PRRSV replication. These data demonstrate that PRRSV interferes with different JAK-STAT pathways to evade host antiviral response while harnessing cellular factors for its own replication. These findings provide new insights into PRRSV-cell interactions and its molecular pathogenesis in interference with the host immune response, and facilitate the development of novel antiviral therapeutics.
INTRAVASCULAR CLEARANCE OF DISSEMINATING CRYPTOCOCCUS NEOFORMANS
(2018) Sun, Donglei; Shi, Meiqing; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen causing fatal cryptococcal meningoencephalitis in humans. The initial infection organ of C. neoformans is the lung; however, lung infection is commonly seen in healthy individuals and does not always have symptoms. Under certain conditions, the fungi may disseminate out of the lung and enter blood circulation. The fungi, once trapped in the brain vasculature, will breach the blood-brain barrier and proliferate rapidly leading to fatal outcomes. Considering the weak immune responses in the brain, it is important to limit the possibility for fungi to arrive at the brain. The current dissertation focused on the interactions of C. neoformans with host immune cells after pulmonary dissemination, seeking mechanisms that can clear the fungi from circulation. It is identified that neutrophils have the ability to remove fungi from the brain vasculature. This effective fungicidal cell kills C. neoformans via C5a-C5aR and CD11b axis. The C5aR signaling provides navigation and alerts neutrophils by up-regulating CD11b surface expression, CD11b as complement receptor is essential for subsequent uptake and killing of fungi by neutrophils. In vivo, neutrophil clearance of C. neoformans occurs in the brain vasculature but the effect is more efficient in the lung vasculature because lung is able to recruit massive amount of neutrophils into the vasculature through complement activation and C5aR signaling, which induce neutrophil actin polymerization and increase their retention. Neutrophils once activated by C. neoformans can further augment self-recruitment through the release of leukotriene B4. Directing neutrophils to the brain can help with fungal clearance in the brain. Moreover, using intravital microscopy, we reported that liver Kupffer cells can filter disseminating C. neoformans. This filtering requires C3b deposition on the fungal surface to provide the holding force and CRIg on the Kupffer cells. Without the holding force, fungi in the liver have higher tendency to be flushed back into circulation. The Kupffer cells rapidly phagocytize the captured fungi and suppress their proliferation in an IFN-γ independent way. Collectively, the results suggest that neutrophils and liver Kupffer cells are critically involved during intravascular clearance of C. neoformans.
VIRAL IMMUNE EVASION OF FCRN FUNCTIONS
(2018) LIU, XIAOYANG; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Human Cytomegalovirus (HCMV) is known to evade host immunity, allowing it to persistently infect humans. Although the strategies of HCMV to evade cellular immunity is well studied, there is limited understanding on how HCMV antagonizes humoral immunity. The neonatal Fc receptor (FcRn), an MHC class I-related FcγR, plays a critical role in IgG-mediated humoral immunity. Through screening the HCMV proteome, we discovered that US11 specifically captured FcRn in both virally-infected and US11-expressing cells. US11 selectively inhibited the assembly of FcRn with β2m, impaired FcRn IgG binding capacity and blocked FcRn trafficking to the endosome by retention of FcRn in ER. Furthermore, US11 recruited Derlin-1 and E3 ubiquitin ligase TMEM129, to induce degradation of FcRn in US11+ or HCMV-infected cells. This complex led to the dislocation of FcRn from the ER to the cytosol and facilitated its degradation in an ubiquitination and proteasome-dependent manner. The cytosolic interaction between FcRn and Derlin-1 was shown necessary for degrading FcRn. FcRn is widely expressed in most cell susceptible to HCMV infection, including epithelial, endothelial and macrophage. Our data showed that either HCMV infection or recombinant US11 expression significantly inhibited human IgG transcytosis across polarized human primary intestinal epithelial Caco-2 cells, Vascular endothelial HMEC-1 cells and placental trophoblast BeWo cells, and facilitated considerable IgG degradation inside endothelial HMEC-1 cells. Hence, our results show that HCMV exploits the Derlin-1/TMEM129 pathway through US11 to disable FcRn, revealing a novel strategy for viral evasion from antibody mediated-immunity. We also studied whether HCMV viral FcγRs (gp34 and gp68) and US11 work together to facilitate IgG degradation. HCMV vFcγRs has been reported to internalize IgG via endocytosis. Interestingly, we found that in acidic pH (6.0) condition, the IgG binding capacity of gp34 was largely impaired while the IgG binding capacity of gp68 had minimal change. Consequently, in the presence of FcRn, gp34 did not enhance IgG degradation whereas gp68 significantly promoted the IgG degradation. Furthermore, the presence of US11 induced more gp34 and gp68-mediated IgG degradation in FcRn+ cells.

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