8th Annual CEND Symposium Poster Abstracts
1. Dynamics of human cytomegalovirus infection of Langerhans-type dendritic cells isolation
Presenter: Laura Hertel
Affiliation: Children’s Hospital of Oakland Research Institute
Abstract: Acquisition of human cytomegalovirus (CMV) usually occurs by contact between contaminated bodily fluids, such as urine and saliva, and host mucosal cells. Langerhans-type dendritic cells (LC) are the only type of immune cells found in the outermost layers of the oral mucosae, where they not only provide a first line of defense against CMV, but can easily be targeted by orally administered vaccines. In this work, we tracked the progress of infection in immature and mature LC (iLC and mLC) exposed to the clinical-like strain TB40-BAC4, or to the vaccine strain AD169varATCC, prior to their long-term maintenance in either immature or mature conditions. We show that maturation using lipopolysaccharide and CD40 ligand augmented and protracted expression of the viral immediate-early proteins 1 and 2 (IE1/IE2) in iLC induced to mature immediately after infection. Viral genome replication was enhanced in LC infected when already mature but not in iLC exposed to maturation stimuli after infection, and maturation did not stimulate viral progeny production or release. No difference was observed between the behavior of the clinical-like and the attenuated strain tested, and both iLC and mLC produced viral progeny, suggesting that these cells may contribute to CMV horizontal transmission in vivo.
2. Inhibition of the Proteasome by the KSHV Protein ORF68
Presenter: Matthew Gardner
Affiliation: Berkeley (Glaunsinger Lab)
Abstract: The proteasome is a large ATP-dependent protease, responsible for the majority of non-lysosomal protein degradation in mammalian cells. Inhibition of the proteasome during Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) infection disrupts the replication cycle of the virus, preventing the production of infectious virions. The recently published KSHV interactome revealed several viral proteins that interact with host protein degradation pathways. These interactions were confirmed to occur by the co-affinity purification of proteasomes with viral proteins from transfected cells. Interacting proteins were screened for alteration of proteasome-mediated degradation by a luciferase assay using a tetra-ubiquitin luciferase linear fusion. The delayed early protein ORF68 was found to inhibit protein degradation. This observation was further examined by monitoring the degradation of a model substrate by western blotting. In cells expressing ORF68, the half-life of the model substrate was increased, indicating a decrease in protein degradation. A mutant virus with a premature termination codon (PTC) in the coding region of ORF68 was produced. This virus was unable to replicate the viral genome, despite sufficiently expressing early genes. Surprisingly, ORF68 was found to be restricted to the cytoplasm during infection, whereas DNA replication takes place in the nucleus. Additionally, ORF68 was found to be unable to affect the stability of nuclear substrates by monitoring the degradation of a nuclear-restricted model substrate. These data suggest that ORF68 is suppressing the proteasome-mediated degradation of a protein in the cytoplasm that is essential for KSHV DNA replication in the nucleus.
3. Rodent Models of Respiratory Syncytial Virus (RSV) Infection
Presenter: Ken Meek
Affiliation: Aragen Biosciences
Abstract: Respiratory syncytial virus (RSV) infection is the majorcause of severe respiratory illness in infants and young children, as well as immune compromised individuals and the elderly. It causes a range of illnesses varying from mild infection to life-threatening bronchiolitis and respiratory failure. Despite over 50 years of research, disease due to RSV infection remains an unmet medical need. Rodent models for testing efficacy and safety in preclinical studies provides a critical component to the development of anti-RSV antibodies, small molecules and vaccines. Aragen Bioscience offers both mouse and cotton rat RSV infection model as well as a wide range of functional readouts to assess the efficacy of anti-RSV biologics and small molecules. These models enable the development of customized study designs that are performed by trained personnel in dedicated work areas specifically designed for infectious disease models. We have now conducted more than 60 pre-clinical studies in rodent models of RSV infection (30-130 animals per study). While BALB/c mice (which are semi-permissive to RSV infection) provide quick information on preliminary proof of concept studies, the cotton rat (Sigmodon hispidus) model represents a clinically relevant model of RSV infection that can be used to develop next generation RSV therapeutics.
4. Distinguishing the Role of EIF4A1 vs EIF4A2 during HCV Infection
Presenter: Tatsinda Spears
Affiliation: Stanford University (Sarnow Lab)
Abstract: Hepatitis C virus (HCV) is an RNA virus that causes hepatitis and can lead to cirrhosis and liver cancer. Direct-acting antiviral drugs against HCV have already been developed. The aim of this research is to define host-pathogen interactions and specific host proteins that could be alternative therapeutic targets for the treatment of HCV infection. The translation initiation factor EIF4A is a DEAD-box RNA helicase that unwinds secondary structures in the 5’-untranslated region (UTR) of mRNA, as part of the cap-binding EIF4F complex, during the initiation phase of translation. EIF4A was perceived as dispensable for the translation of HCV RNA without distinguishing the roles of the isoforms, namely EIF4A1 and EIF4A2 (A1 and A2). Recent studies have suggested functional divergences between the isoforms of EIF4A, showing that while these proteins are 90% identical, they play distinct roles in other viral infections.
Depletion (siRNA) and overexpression (tagged expression vectors) analyses of A1 and A2 were used to determine whether these EIF4A isoforms have an impact on HCV protein and RNA abundances. Western and Northern blots of relevant samples were quantified to show the extent of the impact on HCV protein and RNA. A dual luciferase assay was used to examine the impact of depletion and overexpression of A1 and A2 on HCV IRES activity. HCV replicons containing a Renilla luciferase reporter were used to determine whether A1 or A2 depletion affected replicon translation, replication or both. The data support the notion that A1 and A2 should not be considered redundant in the context of HCV infection. Depending on the siRNA used, loss of A2 negatively impacts HCV RNA and protein abundance. Preliminary studies into the mechanism of this inhibitory regulation of HCV using the replicon system suggest that A2 has a role in HCV RNA replication. Further analysis is needed to determine whether A2 has a direct or indirect role in HCV life cycle.
5. CD55 is a key complement regulatory protein that counteracts complement-mediated inactivation of Newcastle Disease Virus
Presenter: Udaya S. Rangaswamy
Affiliation: MedImmune/AZ
Abstract: Newcastle disease virus (NDV) is an avian paramyxovirus that has been evaluated as an oncolytic virus (OV) due to its selective replication in tumor cells and the lack of pre-existing immunity in humans. Similar to other enveloped viruses, NDV is sensitive to complement (C’) mediated inactivation, which could be an important consideration in selecting viral production substrate and in determining clinical dosing regimen in systemic delivery of OV . The C’ system is a complex, multi-component pathway of the innate immune response that limits the spread of virus in host. The C’ pathway is tightly regulated by species-specific proteins referred to as Regulators of Complement Activation (RCA). Some enveloped viruses incorporate host RCA proteins during egress from the host cell to protect the virion from C’-mediated inactivation. A systematic screen of animal serum from several different species confirmed the species specific nature of RCA protein function in limiting NDV infectivity. We found that NDV grown in different human cell lines or eggs exhibited different sensitivity to human serum. This difference in C’ mediated viral inactivation correlated with the levels of expression of cellular RCA proteins and incorporation of RCA proteins into virions. Specifically, HeLa-S3 cells express three membrane-bound RCA proteins (CD46, CD55, and CD59) at levels much higher than 293F cells, and HeLa-S3 cell-derived virions are more resistant to C’. By introducing each of the RCA proteins into the NDV genome, CD55 was found to be the major RCA protein conferring viral resistance to C’. This data will enable a better understanding of the biology underlying the interactions of NDV with the host immune system, informing the choice of cell substrate for viral manufacture.
6. Genetic Modification of Oncolytic Newcastle Disease Virus for Cancer Therapy
Presenter: Xing Cheng
Affiliation: MedImmune/AZ
Abstract: Clinical development of a mesogenic strain of Newcastle disease virus (NDV) as an oncolytic agent for cancer therapy has been hampered by its select agent status due to its pathogenicity in avian species. Using reverse genetics, we have generated a lead candidate oncolytic NDV based on the mesogenic NDV-73T strain that is no longer classified as a select agent for clinical development. This recombinant NDV has modification at the fusion protein (F) cleavage site to reduce the efficiency of F protein cleavage and an insertion of a 198 nucleotide sequence into the HN-L intergenic region resulting in reduced viral gene expression and viral replication in avian cells but not in mammalian cells. In mammalian cells, except for viral polymerase (L) gene expression, other viral gene expression is not negatively impacted or increased by the HN-L intergenic insertion. Furthermore, the virus can be engineered to express a foreign gene and still retain the ability to grow to high titers in cell culture. The recombinant NDV selectively replicates in and kills tumor cells and is able to drive potent tumor growth inhibition following intratumoral or intravenous administration in a mouse tumor model. The candidate is well positioned for clinical development as an oncolytic virus.
7. Expanded IgG Lineages in Lung Cancer Non-Progressors Reveal Anti-Tumor Antibodies
Presenter: Daniel Emerling
Affiliation: Atreca, Inc.
Abstract: Analyzing anti-cancer immune responses is key to understanding cancer immunotherapy mechanisms. We used Immune Repertoire Capture™ (IRC™) technology to sequence the full length variable regions of natively paired immunoglobulin heavy and light chain genes expressed by over 5000 blood plasmablasts (activated B cells) from a patient with Stage 4 lung adenocarcinoma during a period of long term non-progression (2+ years). There was extensive diversity of germline gene usage and elevated levels of somatic hypermutation (SHM) among the individual B cells. Sequences were grouped into putative clonal families based on immunoglobulin gene usage and other sequence features. Over 1500 putative antibody clonal families were identified, including families observed across blood collection time points and similar to families from another lung adenocarcinoma patient. The full length variable regions of IRC™ sequences were directly gene synthesized to generate recombinant antibodies representing over 150 large and small putative families. Antibodies showed a range of staining patterns on tumor and normal tissues, including some antibodies that bound tumor types other than lung tumor and some that bound tumor much better than normal tissue. Over one third of the antibodies bound lung cancer-derived cell lines. Some antibodies mediated ADCC killing in vitro. Human proteome arrays are being used to identify the targets of the antibodies. Analyses identified clones with differing SHM from the same putative family that bind the same target with varying potencies, indicating that the mutational differences between sibling antibodies reveal structure-activity information.
8. Metabolic Barriers to Intracellular Bacterial Pathogenesis
Presenter: Jordan Price
Affiliation: UC Berkeley (Vance Lab)
Abstract: The mammalian macrophage displays a high degree of metabolic plasticity and is the preferred host cell for many species of pathogenic intracellular bacteria. The capacity of macrophages to remodel their metabolism in response to diverse stimuli underwrites their ability to mount defenses against diverse pathogens. However, the metabolic plasticity of macrophages may also explain why these cells are exploited by enterprising intracellular bacterial pathogens as a replicative niche. To untangle the potential roles of different macrophage metabolic states during infection, we employ the facultative Gram-negative intracellular bacteria Legionella pneumophila. While Legionella naturally infects freshwater amoebae, it is an accidental pathogen of humans with the ability to cause lethal pneumonia (Legionnaires’ disease) through infection of alveolar macrophages.
Interferon gamma (IFNγ) plays a critical role in controlling Legionella infection in vitro and in vivo. Replication of Legionella is completely restricted in IFNγ-stimulated macrophages; however, the mechanism of IFNγ-dependent restriction of Legionella remains mysterious and is independent of known IFNγ-driven antimicrobial pathways, including induction of autophagy and production of reactive nitrogen. As enhanced glycolysis is a hallmark metabolic characteristic of IFNγ and toll-like receptor-stimulated macrophages, we hypothesized that changes in macrophage metabolism are required for IFNγ-mediated restriction of Legionella. Using a strain of Legionella we evolved to be resistant to the glycolysis inhibitor 2-deoxyglucose (2DG), we were able to rescue bacterial growth in IFNγ-stimulated macrophages treated with 2DG. Parallel RNA-seq and metabolomics analyses identified several metabolic pathways in IFNγ-stimulated macrophages that we suspect may be important for the restriction of intracellular bacterial pathogens.
9. Long-term effects of early-life arsenic exposure on innate immunity and Tuberculosis Risk
Presenter: Fenna Sillé
Affiliation: UCB (Smith Lab)
Abstract: In a unique study area in Chile, our research group found that relative risks of adult mortality from cancer, bronchiectasis and tuberculosis (TB) are much greater when arsenic exposure occurred only in utero /early-life, rather than later in life. Notably, these risks remain high up to 40 years after the exposures ended. This provides rare human evidence in support of the “Developmental Origins of Health and Disease” hypothesis. However, the mechanisms behind this phenomenon remain unclear. We hypothesize that early-life arsenic exposures permanently impact immune development and increase the risks of immune-related diseases later in life. Here we focus on macrophages, innate immune cells known to influence tumor progression and TB pathogenesis. First we assessed the effects of different in vitro doses of inorganic arsenic and arsenic metabolites on mouse bone-marrow-derived macrophages (BMDMs). Multiplexed cytokine/chemokine profile analysis on homeostatic and activated BMDMs revealed significant dose- and time-dependent expression changes. Several of these cytokines/chemokines are involved in pattern recognition receptor pathways that are critical for the innate immune response against TB. Metabolomics analysis of these BMDMs showed that arsenic also led to changes in pro-inflammatory and tumor-promoting signaling lipids. Correspondingly, we observed differential effects of arsenic on nitric oxide production and Mycobacterium tuberculosis infections. Our preliminary analysis of plasma samples from early-life arsenic exposed adults also shows changes in immune signaling profiles. We are currently validating our findings in vivo using animal models and ex vivo in plasma and macrophages from early-life exposed human adults. Our current results provide a model to further study how early-life exposures may alter macrophage development and contribute to an overall immunomodulatory signaling landscape that drives delayed disease pathogenesis.
Supported by NIEHS Superfund Research Program P42ES004705 (M.S.) and NIEHS K99ES024808 (F.S.)
10. Development of a STING-targeted Vaccine for Mycobacterium tuberculosis
Presenter: Kim Sogi
Affiliation: UCB (Stanley Lab)
Abstract: Not available
11. Insights into human malaria from a novel model of asymptomatic Plasmodium infection
Presenter: Mary Fontana
Affiliation: UCSF (Kim Lab)
Abstract: In humans, immunity to Plasmodium sp. generally provides protection from symptomatic malaria (‘clinical immunity’) rather than infection (‘sterilizing immunity’). In contrast, mice infected with Plasmodium develop sterilizing immunity, hindering progress in understanding the mechanistic basis of clinical immunity. Here we present a novel model in which mice persistently infected with P. chabaudi demonstrate key characteristics of clinical immunity, appearing largely healthy despite sustained parasite burdens. Persistently infected mice exhibited T cell exhaustion; however, blockade of exhaustion-associated receptors did not facilitate clearance. Strikingly, a Ly6clo nonclassical monocyte (NCM) population expressing immunosuppressive markers expanded over fifty-fold in persistently infected mice to become the most abundant blood leukocyte. NCM frequencies also increased with Plasmodium exposure in human subjects. Following drug-mediated clearance, previously persistently infected mice failed to suppress a secondary challenge, but exhibited improved health parameters upon re-infection. Both phenotypes correlated with the rapid re-expansion of NCMs, suggesting that these cells may promote the shift from sterilizing immunity to clinical immunity. This study establishes an animal model of asymptomatic, persistent Plasmodium infection that recapitulates key aspects of clinical immunity in humans. Further, it suggests a novel immunoregulatory mechanism that may limit pathology at the cost of preventing sterile protection.
12. A Novel Membrane-associated Protein, MYR1, is Crucial for Effector Transport in Toxoplasma gondii
Presenter: Nicole Blackburn-Marino
Affiliation: Stanford University (Boothroyd Lab)
Abstract: Toxoplasma gondii is an obligate intracellular parasite capable of infecting almost all warm-blooded animals, rendering it one of the most successful parasites in the world. The ability of Toxoplasma to infect and successfully manipulate its host is dependent on its ability to transport “GRA” proteins that originate in unique secretory organelles called dense granules into the host cell in which they reside; however, the essential transport machinery has not been identified. GRAs have diverse roles in Toxoplasma’s intracellular lifecycle including co-opting crucial host cell functions and proteins such as the cell cycle, c-Myc and p38 MAP kinase. To identify proteins necessary for the transit of GRAs across the parasitophorous vacuole (PV) membrane, we screened mutants defective in c-Myc upregulation for their ability to export GRA24 to the host cell nucleus as well as modulate host cell processes associated with this and other proteins. This screen led to the discovery that MYR1, a novel membrane-associated protein, plays a crucial role in translocation of a subset of GRAs into the host cell. Consistent with such a role, MYR1 was found to localize to the PV, including the membrane that separates the PV from the host cytosol. Importantly, MYR1 is not necessary for the function of two effector proteins that localize to the PVM, but is required for proteins that are transported across it. Thus, MYR1 represents the first identified component of a highly unusual process in eukaryotic biology, whereby protein effectors are secreted from an intracellular parasite and then transported across the PV membrane and into the host cytosol. The biological importance of this process is demonstrated by the attenuated phenotype of myr1- mutants in infected mice, and in my presentation I will describe recent results on how MYR1 functions.
13. Gold nanoparticle-mediated delivery of CRISPR/Cas9 ribonucleoprotein with DNA donor for efficient genome editing in vitro and in vivo
Presenter: Anirudh Rao
Affiliation: UC Berkeley (Murthy Lab)
Abstract: The CRISPR-associated enzyme Cas9 enables site-specific genome engineering by introducing double-strand breaks at DNA loci of interest that are complementary to guide RNA (gRNA). Cells repair the double-strand breaks by the non-homologous end joining (NHEJ) that generates variable insertions or deletions, or homology-directed repair (HDR) pathways that employ homologous donor DNA sequences for repair. The CRISPR/Cas9 technology has an immense potential to treat human genetic diseases. However, the therapeutic application of the CRISPR/Cas9 is dependent on the development of a safe delivery method. There is great interest in delivering gRNA and Cas9 ribonucleoprotein (RNP) because of their advantages in clinical translation: safe and effective genome editing without viral usage or random integration. There has been no effective method to deliver both gRNA/Cas9 RNP and DNA donor together to achieve efficient HDR.