UMD Theses and Dissertations
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Item ZIKA VIRUS RECRUITS CELLULAR PROTEINS TO SUPPORT ITS REPLICATION(2024) Chang, Peixi; Zhang, Yanjin YJ; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Zika virus (ZIKV) is a mosquito-borne pathogen with a massive impact on global public health due to its association with severe neurological complications, including microcephaly in newborns and Guillain-Barré syndrome in adults. The ZIKV epidemic in the Americas in 2015-2016 and its continuing spread in tropical regions have highlighted the urgent need to understand the molecular mechanisms of viral replication to develop effective antiviral strategies. However, many aspects of how ZIKV interacts with host cells remain unclear. This study identifies and characterizes host factors contributing to ZIKV replication. First, karyopherin alpha 6 (KPNA6) contributes to ZIKV replication by interacting with the ZIKV non-structural protein NS2B. Characterization and mutational analyses identified two essential amino acid residues within NS2B that are critical for interacting with KPNA6. The substitution of these two residues of NS2B in an infectious ZIKV cDNA clone resulted in a significant reduction in viral replication, suggesting that the NS2B-KPNA6 interaction plays a vital role in the viral life cycle. Further studies found that KPNA6 contributes to ZIKV RNA synthesis. Mass spectrometry analysis of the KPNA6 interactome showed that KPNA6 interacts with proteins involved in RNA synthesis, suggesting that ZIKV recruits these factors by promoting KPNA6-binding. Second, this study developed an effective method to isolate the ZIKV replication complex, a membranous structure where viral RNA is synthesized. Proteomic analysis of the isolated complex led to identifying numerous host proteins associated with the viral replication machinery. Among these proteins, human replication factor C subunit 2 (RFC2), an accessory factor involved in DNA replication and repair, was discovered to facilitate ZIKV replication, making it a potential target for therapeutic interventions. In conclusion, this study reveals crucial host factors essential for ZIKV infection and replication and provides insights into the ZIKV-cell interactions. These findings offer new possibilities for developing novel antiviral strategies for controlling future viral outbreaks.Item Characterization of the GBF1-Arf1 axis in enterovirus RNA replication(2024) Gabaglio Velazquez, Samuel Maria; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Enterovirus genus includes many known and emerging pathogens, such as poliovirus, enteroviruses A71 and D68, rhinoviruses, and others. Enterovirus infection induces the massive remodeling of intracellular membranes and the development of specialized domains harboring viral replication complexes, called replication organelles. The cellular protein Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) is essential for the replication of enteroviruses, but its molecular role in the replication process is unclear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including the functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and other enteroviruses directly interact with and recruits GBF1 to the replication organelles. Moreover, enterovirus infection induces the massive recruitment of all isoforms of the small cellular Arf GTPases to the replication organelles, but the mechanistic role of these proteins in the replication process is not understood either. Here, we sought to characterize the role of the GBF1-Arf1 axis in enterovirus replication. First, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrated that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results showed that the Arf-activating property, but not the primary structure of the Sec7 catalytic domain is essential for viral replication. They also suggest a redundant mechanism for recruiting GBF1 to the replication sites. This mechanism depends not only on the direct interaction of the protein with the viral protein 3A but also on elements located in the noncatalytic C-terminal domains of GBF1. Next, we investigated the distribution of viral proteins and Arf1 on the replication organelles and their biochemical environment. Pulse-labeling of viral RNA with 5-ethynyl uridine showed that active RNA replication is associated with Arf1-enriched membranes. We observed that Arf1 forms isolated microdomains in the replication organelles and that viral antigens are localized in both Arf1-depleted and Arf1-enriched microdomains. We investigated the viral protein composition of the Arf1-enriched membranes using peroxidase-based proximity biotinylation. Viral protein biotinylation was detected as early as 3 h.p.i., and the non-cleaved fragments of the viral polyprotein were overrepresented in the Arf1-enriched domains. Furthermore, we show that after 4 h.p.i. viral proteins could be efficiently biotinylated only upon digitonin permeabilization of the replication organelle membranes, while such permeabilization inhibited the Arf1 biotinylation signal at the Golgi in non-infected cells. Together, these data support a model that recruitment of GBF1 to the replication organelles generates foci of activated Arfs on the membranes, which further differentiate into specific microdomains through the recruitment of a specific complex of viral proteins and cellular Arf effectors likely needed to establish the lipid and protein composition required for viral replication.Item TRANSLATION, REPLICATION AND TRANSCRIPTOMICS OF THE SIMPLEST PLUS-STRAND RNA PLANT VIRUSES(2024) Johnson, Philip Zhao; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Plus (+)-strand RNA viruses are among the most common pathogens of plants and animals. Furthermore, they present model systems for the study of basic biological processes, including protein translation and RNA replication, and shed light on the versatile roles that RNA structures play in these processes. After cell entry, the next step in the (+)-strand RNA viral life cycle is translation of the viral genome to produce the viral RNA-dependent RNA polymerase (RdRp) and associated replication proteins necessary for viral replication to occur. For many (+)-strand RNA viruses lacking a 5´cap and 3´ poly(A) tail, translation depends upon RNA structural elements within their genomes capable of hijacking the host translation machinery, which for plant viruses are commonly located in their 3´ proximal regions and are termed 3´ cap-independent translation enhancer (CITE) elements. In Chapter 2, I report upon my work characterizing a new subclass of panicum mosaic virus-like translation enhancer (PTE) elements, which bind and co-opt for viral use the host translation initiation factor 4E (eIF4E) – the translation initiation factor normally responsible for binding and recognition of mRNA 5´caps during canonical eukaryotic translation initiation. Thus, PTE 3´CITEs present a novel mechanism for co-opting the critical host factor eIF4E. My work characterizing a new subclass of PTE 3´CITEs further revealed characteristics common among all PTE 3´CITEs pertaining to their mechanism of binding eIF4E.After translation of the necessary viral replication proteins, replication of the viral RNA occurs, which again is in large part mediated by RNA structural elements within the viral genome that can bind to the viral RdRp and/or host factors involved in viral replication. Indeed, RNA structural elements often serve dual roles in viral translation and replication and/or are located proximal to RNA structural elements involved in the alternate function. In Chapter 3, I discuss my work characterizing novel replication elements in the 3´ terminal regions of umbraviruses (family Tombusviridae). The uncovered replication elements appear to be specific to umbraviruses and are located immediately upstream of replication/translation elements that are common throughout the Tombusviridae, lending greater complexity to the already complex 3´ proximal structures of umbraviruses. While the study of (+)-strand RNA viruses has historically focused on their protein-coding transcripts, (+)-strand RNA viruses also commonly produce additional non-coding transcripts, including recombinant defective RNAs, typically containing 5´ and 3´ co-terminal viral genome segments, and (+/-)-foldback RNAs, composed of complementary (+)- and (-)-strand viral sequences joined together. Long non-coding RNAs that accumulate to high levels have also been reported for plant and animal (+)-strand RNA viruses in recent years, and truncations of viral transcripts also commonly arise due to host nuclease activity and/or premature termination of replication elongation by the viral RdRp. The rise of long-read high-throughput sequencing technologies such as nanopore sequencing presents an opportunity to fully map the complexity of (+)-strand RNA viral transcriptomes. In Chapter 4, I present my work performing this analysis, employing direct RNA nanopore sequencing, in which the transcripts present in an RNA sample of interest are directly sequenced. This analysis revealed for the umbra-like virus citrus yellow vein-associated virus (CY1): (i) three novel 5´ co-terminal long non-coding RNAs; (ii) D-RNA population dynamics; (iii) a common 3´ terminal truncation of 61 nt among (+)-strand viral transcripts; (iv) missing 3´ terminal CCC-OH motif in virtually all (-)-strand reads; (v) major timepoint- and tissue-specific differences; and (vi) an abundance of (+/-)-foldback RNAs at later infection timepoints in leaf tissues. This work also sheds light on the current shortcomings of direct RNA nanopore sequencing as a technique. Finally, the importance of RNA structural biology in the study of (+)-strand RNA viruses presents the need for specialized RNA structure drawing software with functionality to easily control the layout of nucleobases, edit base-pairs, and annotate/color the nucleobases and bonds in a drawing. It is through the visual exploration of RNA structures that RNA biologists routinely improve upon the outputs of RNA structure prediction programs and perform crucial phylogenetic analyses among related RNA structures. Large RNA structures, such as whole viral genomes thousands of nucleotides long, can only be studied in their entirety with the aid of RNA structure visualization tools. To this end, I have developed over the course of my doctoral education the 2D RNA structure drawing application RNAcanvas, which is available as a web app and has grown popular among the RNA biology community. RNAcanvas emphasizes graphical mouse-based interaction with RNA structure drawings and has special functionality well suited for the drawing and exploration of large RNA structures, such as automatic layout adjustment and maintenance, complementary sequence highlighting, motif finding, and performance optimizations. Large viral structures such as that of the 2.7 kb CY1 genomic RNA could not have been characterized without the aid of RNAcanvas. In Chapter 5, I present my work developing RNAcanvas.Item Characterization of chromatin assembly dynamics mediated by the histone H3.3 chaperone HIRA and implications of innate immunity during Human Papillomavirus infection(2024) Della Fera, Ashley Nichole; Scull, Margaret A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The circular double-stranded DNA genome of Human papillomavirus (HPV) is chromatinized throughout its viral lifecycle and relies on numerous host chromatin assembly processes, epigenetic modifications, and immune evasion to ensure genomic stability and productive infection. Despite its chromatinization, the HPV genome remains susceptible to innate immune pathways that sense and respond to foreign DNA. In this work next generation sequencing (RNAseq) was utilized to profile changes in the host transcriptome following cellular differentiation and HPV infection in keratinocyte cell lines. Global alterations in keratinocyte differentiation were observed upon HPV infection, and unexpectedly, upregulation of innate immune signaling upon differentiation. Recent findings indicate that packaged HPV genomes are enriched in histone H3.3. Notably, the replication-independent histone H3.3 chaperone HIRA has been implicated in several pro- and anti-viral responses, but its function during HPV infection has yet to be elucidated. Using in-situ approaches, the role of HIRA during the late phase of the HPV lifecycle was evaluated, which showed that HIRA and other chromatin assembly factors localize to sites of HPV replication. Here the requirements for this localization were further characterized, and the impacts of HIRA on HPV genome amplification and viral transcription during the late stage of the HPV life cycle were assessed. Moreover, histone H3.3 phosphorylated at serine 31 was shown to be highly associated with HPV replication factories. HIRA, in part through association with the PML nuclear body associated protein Sp100, has also been reported to promote innate immune responses following infection with other DNA viruses. Here, HIRA localization to PML-NBs was identified to increase following stimulation with IFN in an Sp100-dependent manner. However, while Sp100 is required for localization of HIRA at PML-NBs, it was not required for HIRA localization at sites of HPV replication. In summary, this work highlights the broad changes in the host transcriptome following cellular differentiation and HPV infection, elucidates a previously undescribed role for histone H3.3 chaperone HIRA during the late phase of the HPV life cycle, and further characterizes the relationship between HIRA and Sp100 at PML-NBs.Item BIOMATERIAL BASED STRATEGIES FOR VIRAL AEROSOL CAPTURE AND PREVENTION OF RESPIRATORY INFECTIONS(2024) Doski, Shadin; Duncan, Gregg; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In the 2022-2023 flu season, the Center for Disease Control (CDC) estimated 21,000 deaths and 31 million symptomatic illnesses in the United States. Current FDA approved antivirals for influenza are grouped into three categories, matrix protein 2 (M2) inhibitors, neuraminidase inhibitors (NAI) and polymerase acidic protein cap-dependent endonuclease (CEN) inhibitors. However, limitations of these treatments have been evident. For example, NAI inhibitors require early treatment to be efficacious and some influenza strains can develop resistance to both NAI and CEN inhibitors. Thus, there is a need for new classes of antivirals as well as better understanding of influenza transmission and monitoring of influenza to inform development of efficacious interventions. In chapter 2 we describe how we design biomaterials inspired by the physiological characteristics of mucus to capture and trap pathogens. We performed studies to establish this material as a suitable substrate for viral capture and release after collection using advanced aerosol capture technology. In chapter 3, we formulate an antiviral based around polyinosinic polycytidylylic acid (polyIC). PolyIC is commonly used in research as an adjuvant in vaccine delivery through its targeting of Toll like receptor 3 (TLR3). This pathway also results in type 1 and 3 interferon production, which in turn stimulate a range of antiviral mechanisms. Because of this, it has also been investigated as a prophylactic or treatment to various viruses, including hepatitis B virus, human immunodeficiency virus and rhinovirus. However, due to stability and toxicity concerns, it has not been implemented as an inhaled treatment to induce local immunity in the lungs at the site of infection. Towards this end, we used polyethylene imine-polyethylene glycol (PEI-PEG) copolymer to condense PolyIC into nanoparticles to enhance their bioavailability in target cells. By combining the two, we can utilize the antiviral capabilities of Poly(IC) while minimizing the dosage concentration to therapeutic levels.Item INVESTIGATION OF A NOVEL O-GLCNAC MODIFICATION OF A VACCINIA VIRUS CORE PROTEIN(2024) Zhang, Yunliang; Scull, Margaret; Moss, Bernard; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Vaccinia virus (VACV) is a large, complex, enveloped virus that is the prototypic member of the genus Orthopoxvirus of the Poxviridae family and is well known as the live-virus vaccine that eradicated smallpox. It has a linear, double-stranded DNA genome of approximately 190 kbp that encodes about 200 proteins some of which undergo various post-translational modifications. These modifications are crucial for regulating protein function and influencing the virus behavior within the vertebrate and insect cells. Among these, O-GlcNAcylation is notable for its reversible modulation of protein function, like phosphorylation. Although over 5,000 human proteins have been documented as O-GlcNAcylated, the prevalence and function of this modification in viral proteins remain underexplored.Early studies from the Moss laboratory demonstrated the presence of a 40-kDa protein that contained N-acetylglucosamine in purified virions. The small size of the pronase-digestion product and the absence of other sugars suggested one or few glucosamines. The current study advances this understanding by pinpointing the novel O-linked β-N-acetylglucosamine (O-GlcNAc)-modified protein in VACV infectious particles. Enzymatic labeling of purified virions was performed using the mutant β-1,4-galactosyltransferase (GalT1 (Y289L)) to specifically transfer azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAc residues. Following copper catalyzed azide-alkyne cycloaddition (CuAAC) of biotin or an infrared dye, the candidate O-GlcNAc proteins were detected by SDS-polyacrylamide gel electrophoresis and identified by mass spectrometry (MS). Then using strain-promoted cycloaddition (SPACC) chemistry to attach a polyethylene glycol mass tag of 10 kDa to the O-GlcNAc protein, a significant shift in the electrophoretic mobility of the VACV A4 protein was documented by western blotting. The presence of O-GlcNAc in A4 was confirmed by MS and by binding to specific antibodies. Multiple modification sites were pinpointed using higher-energy collisional dissociation induced electron-transfer dissociation in MS. Further evidence linking cellular protein O-GlcNAc transferase (OGT) to the modification of A4 was derived from experiments conducted with an A4-expressing cell line. Disruption of OGT activity, either through chemical inhibition or knock-down techniques, reduced A4 O-GlcNAc modification without impairing VACV infectivity. This finding suggests that the O-GlcNAc modification of A4 does not play an essential role in VACV infectivity, which is not correlated with the A4 deletion phenotype. Therefore, the specific effects of O-GlcNAc modification on the VACV lifecycle remain elusive, indicating further studies are required to determine the potentially subtle effects of O-GlcNAcylated A4 on the VACV life cycle.Item Engineering physiologically-relevant model systems to understand the requirements of rhinovirus C infection(2023) Goldstein, Monty Eli; Scull, Margaret A; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Rhinovirus (RV) is the most prevalent etiologic agent of the common cold, and infections by RV species C (RV-C) are often associated with more severe illness, and have been strongly correlated with childhood development of asthma. Due to lack of in vitro and in vivo model systems capable of supporting the RV-C life cycle, few details of RV-C biology are understood about this recently discovered, clinically-relevant respiratory pathogen. To reveal the nature of virus-host interactions and study viral pathogenesis, the application of physiologically-relevant model systems that capture relevant cell types, differentiation states, and microenvironmental cues is essential. Applying these principles to our investigations of RV-C, I engineered in vitro and in vivo model systems to better understand the requirement of specific host factors for RV-C replication in human and mouse cells. Specifically, I utilized a pseudostratified in vitro model of human airway epithelium (HAE) to study RV-C replication, and applied CRISPR/Cas9 technology in these cultures to assess the specific role for stimulator of interferon genes (STING) in promoting viral replication. Since RV-C species tropism is highly restricted, I then applied our knowledge of RV-C replication in HAE cultures towards building an improved RV-C mouse model. Here, I first characterized RV-C replication in mouse lung cells in vitro, and demonstrated that human STING expression enhanced viral replication; second, I applied these findings in vivo, where I generated a transgenic mouse expressing the human ortholog of the RV-C receptor, cadherin-related family member 3 (CDHR3), along with human STING. While these mice lack overt symptoms typically associated with viral infection, they exhibited significantly increased viral replication 24 hours-post infection. Finally, to support ongoing efforts to further develop these mice as a robust small animal model of RV-C, I developed several novel cell lines which represent important tools to interrogate the impacts of other host factors on RV-C replication in mouse cells, which upon validation, can be re-engineered into these transgenic mice.Item 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.Item PROBING BIOPHYSICAL INTERACTIONS TO UNDERSTAND VIRAL DIFFUSION AND PARTICLE FATE IN THE AIRWAY MUCOSAL BARRIER(2023) Kaler, Logan; Duncan, Gregg A; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The mucus barrier in the airway is the first line of defense against inhaled particulates and pathogens. Within the mucus barrier, large, heavily glycosylated gel-forming mucin proteins form a network to trap particles for removal. Influenza A virus (IAV) must first cross the mucus barrier before reaching the underlying airway epithelial cells to cause infection. On the IAV envelope, hemagglutinin (HA) binds sialic acid on the surface of the cell to initiate viral entry. However, HA preferentially binds sialic acid attached to galactose by either an ⍺2,3 or ⍺2,6 linkage. In addition to the cell surface, sialic acid is found on mucins and is thought to act as a decoy receptor to entrap the IAV within the mucus layer. However, neuraminidase (NA) on the envelope of IAV cleaves the bond between HA and sialic acid, releasing the virus. While the mechanism of IAV infection has been characterized, the interplay between mucus biophysical properties and the binding of IAV within the mucus network prior to infection requires further investigation. The overall objective of this dissertation is to understand how IAV moves through the mucosal barrier to subsequently cause infection. We hypothesize the structural features of the mucus gel network are responsible for the changes in IAV movement, rather than the binding and unbinding of the virus. To investigate this, we first analyzed the movement of IAV in ex vivo mucus from human endotracheal tubes. In order to further analyze this movement, we developed a novel analysis to calculate the dissociation constant of IAV-mucus binding in a 3D gel network environment. Using this data, we established a pipeline for estimating the passage of particles, including IAV, through the airway mucosal barrier. A machine learning-based trajectory analysis was employed to classify individual trajectories in order to calculate the percentage of particles able to cross the mucus barrier within a physiologically relevant time frame. Lastly, we investigated the effect of sialic acid binding preference on diffusion of IAV through mucus collected from different in vitro human airway epithelial cell cultures. The combined results of these studies confirmed our hypothesis that the mucus microstructure rather than the adhesive interactions of IAV to the mucins was responsible for the differences in IAV diffusion. This work provides further insight into role of the mucosal barrier in IAV infection and identifies the mucus gel network microstructure as a target for the development of therapeutics against IAV.Item Development and characterization of Epstein-Barr virus (EBV) antibodies and testing their efficacy in a humanized mouse model(2022) Kim, JungHyun Rachel; Zhu, Xiaoping; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Epstein-Barr virus (EBV) is one of the most prevalent human viruses with more than 90% of the adult population infected with EBV. EBV is well-known to cause infectious mononucleosis, but also is associated with several B cell lymphomas and epithelial cell carcinomas. In bone marrow or solid organ transplant patients and individuals with X-linked lymphoproliferative disease, primary EBV infection can lead to life-threatening complications. There is no licensed vaccine and even if one is available, these immunocompromised individuals might not respond well to the vaccine. Thus, an effective vaccine and therapeutic are both needed. Here, I have evaluated the efficacy of (a) EBV hyperimmune globulin isolated from healthy plasma donors with high EBV gp350 antibody and EBV B cell neutralizing titers, (b) several EBV gp350, gH/gL and gp42 monoclonal antibodies (mAbs) isolated from human plasma or vaccinated monkeys, (c) a bispecific antibody targeting both gH/gL and gp42, and (d) gH/gL and gH/gL/gp42 nanoparticle vaccines in a humanized mouse model. Animals that received hyperimmune globulin showed protection from EBV infection at a similar level as that seen in animals receiving intravenous immunoglobulin. However, humanized mice that received gH/gL mAb B10 or gp42 mAb A10 showed increased survival and reduced viremia compared to animals that received other gH/gL or gp42 mAbs. These two mAbs also demonstrated protection from development of EBV B cell lymphomas in animals. Humanized mice that received bispecific antibody derived from gH/gL mAb B10 and gp42 mAb A10 showed similar protection against EBV as animals that received the combination of the two antibodies. Passive transfer of IgG isolated from mice immunized with a gH/gL or gH/gL/gp42 nanoparticle vaccine showed reduction in viremia and no development of EBV lymphomas compared to mice that received IgG from naïve mice. These findings suggest that development of vaccines or therapeutics targeting gH/gL and/or gp42 may provide protection in healthy individuals and severely immunocompromised individuals from EBV infection and B cell lymphoma.