Department of Veterinary Medicine
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Item 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.Item 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.Item 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.Item 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.Item IMPROVED NEWCASTLE DISEASE VIRUS VACCINES AND VECTORS(2017) Manoharan, Vinoth Kumar; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Newcastle disease (ND) is an economically important disease of poultry worldwide. The use of vaccines to control ND is necessary because of frequent outbreaks of the disease in enzootic countries. The fusion (F) and hemagglutinin-neuraminidase (HN) proteins of Newcastle disease virus (NDV) are multifunctional proteins that play critical roles during infection. The F protein of NDV is a type I membrane glycoprotein that mediates the fusion of viral envelope to the host cell membrane. The F protein activation initiates a series of conformational changes in the F protein leading to virus-cell membrane fusion, which occurs at the cell surface at neutral pH thus modulating NDV entry and spread. In the present study, we investigated the role of tyrosine to alanine mutation at amino acid position 524 and 527 in the F protein cytoplasmic tail (CT) of NDV strain LaSota by using reverse genetic techniques. Our results suggest that tyrosine residues at 524 and 527 position of F protein CT domain play a major role in fusogenicity and in replication thus modulating NDV infectivity. The F protein is synthesized as an inactive precursor, F0, which is functionally activated after cleavage by host cell proteases into F1 and F2 polypeptides, linked by disulfide bonds. The amino acid sequence surrounding the F protein cleavage site determines the virulence of NDV. We also studied the role of other avian paramyxovirus fusion protein cleavage site sequences in F protein cleavage of NDV strain Banjarmasin. This study has helped us to understand the requirement of F protein cleavage site in proteolytic processing, plaque formation and virus infectivity. Further, the role of these F cleavage site mutant viruses as genotype-matched vaccines for virulent NDV infection has been explored. Reverse genetics has also been used to develop NDV strains as a potential vaccine vectors for various human and animal pathogens, such as highly pathogenic avian influenza (H5N1), human immunodeficiency virus, severe acute respiratory syndrome coronavirus, ebola virus, respiratory syncytial virus and human parainfluenza virus type 3. NDV has several characteristics that makes it a suitable candidate for vaccine vector development. It is safe in humans and animals due to natural host range restriction, expresses foreign protein abundantly, infects via intranasal route, produces both humoral and mucosal immune responses, is antigenically distinct from human and animal pathogens, and lack of preexisting immunity to NDV in humans and animals. In one vaccine trial with non-human primates, the mesogenic NDV strain Beaudette C (BC) replicated to a high titer and induced a substantially higher antibody response compared to the lentogenic strain LaSota, and thus appeared to be more effective. However, NDV strains that have a polybasic cleavage site in the F protein and an intracerebral pathogenicity index (ICPI) >0.7 have been classified as Select Agents. Most mesogenic strains, including strain BC, fall into this category and therefore cannot be handled in BSL-2 conditions. In this study, we constructed a series of recombinant (rNDV) vectors containing the cleavage site sequence of avirulent strain LaSota and other avian paramyxoviruses, together with various regions of the F protein exchanged between NDV strains AKO-18 and BC. We used these modified rNDV vectors to express SIV gp160 envelope protein and immunized guinea pigs. Our results showed that rNDV/SIV vaccines were immunogenic and effectively neutralized SIV mac251 strain in vitro. These results support the idea of the use of NDV as a vaccine vector for expression of SIV immunogens capable of inducing neutralizing antibodies against diverse SIV strains, thus providing an improved vaccine vector platform for ultimately testing the NDV vectored vaccines in non-human primates and humans.Item EXPLORING IL-7R-ALPHA DEVELOPMENTAL EFFECTS AND ONCOGENIC COLLABORATIONS(2017) Cramer, Sarah Delia; Samal, Siba; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Acute lymphoblastic leukemia is the most common cancer of children. Individual cases of leukemia may have multiple genetic lesions, and identifying those that drive leukemogenesis will be important in the development of targeted therapy. Approximately 10% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) cases have a mutation in IL-7Rα. These mutations are thought to be oncogenic, but little is known about the effects of the mutation on T-cell development. In addition, the mutation does not seem to induce leukemia in the absence of other genetic lesions, suggesting that collaborative mutations are required for leukemogenesis. Based on patient data, potential collaborators include TLX3 expression, HOXA gene cluster overexpression, and NRAS mutation. Given the current state of knowledge regarding mutant IL-7Rα, this project was developed with two specific aims. The first was to investigate the effects of mutant IL-7Rα gain-of-function (IL-7Rα-GOF) on T-cell development in vitro and in vivo. The second was to determine whether candidate collaborative genetic lesions would drive T-ALL formation when combined with mutated IL-7Rα. To address these aims, immature murine thymocytes were cultured on an OP9-DL4 stromal cell system, transduced with retroviral vectors, and injected into sub-lethally irradiated Rag1-/- mice. Resultant diseases were analyzed using a variety of techniques including flow cytometry, histology, immunohistochemistry, ligation-mediated PCR, TCRβ clonality assessment, RNA-sequencing, serial passage, and limiting dilution assay. Studies showed that IL-7Rα-GOF mutation caused an increase of CD8+ cells in vitro. When thymocytes transduced with IL-7Rα-GOF mutation were injected into mice, animals developed a multi-systemic inflammatory disease. This inflammation was not due to imbalance in populations of Treg and Th17 cells, as had been hypothesized. Assessing collaborations with TLX3 expression, HOXA overexpression, and NRAS mutation showed that combination of these genetic lesions with IL-7Rα-GOF mutation caused different neoplastic diseases. The combination of IL-7Rα-GOF mutation and TLX3 expression caused low-penetrance, late-onset T-cell lymphoma. Thymocytes overexpressing the HOXA gene cluster and transduced with IL-7Rα-GOF mutation caused a rapid-onset myeloid leukemia. Combination of IL-7Rα-GOF mutation with mutant NRAS yielded rapid-onset, full-penetrance T-cell lymphoblastic leukemia, suggesting that this combination of mutations was sufficient to induce T-ALL. These experimental results may help to lay the foundation for the development of targeted therapy for pediatric T-ALL.