Polyclonal epitope mapping reveals temporal dynamics and diversity of human antibody responses to H5N1 vaccination

Novel influenza A virus (IAV) strains elicit recall immune responses to conserved epitopes, making them favorable antigenic choices for universal influenza virus vaccines. Evaluating these immunogens requires a thorough understanding of the antigenic sites targeted by the polyclonal antibody (pAb) response, which single-particle electron microscopy (EM) can sensitively detect. In this study, we employ EM polyclonal epitope mapping (EMPEM) to extensively characterize the pAb response to hemagglutinin (HA) after H5N1 immunization in humans. Cross-reactive pAbs originating from memory B cells immediately bound the stem of HA and persisted for more than a year after vaccination. In contrast, de novo pAb responses to multiple sites on the head of HA, targeting previously determined key neutralizing sites on H5 HA, expanded after the second immunization and waned quickly. Thus, EMPEM provides a robust tool for comprehensively tracking the specificity and durability of immune responses elicited by novel universal influenza vaccine candidates

A vaccine displaying a trimeric influenza-A HA stem protein on capsid-like particles elicits potent and long-lasting protection in mice

Due to constant antigenic drift and shift, current influenza-A vaccines need to be redesigned and administered annually. A universal flu vaccine (UFV) that provides long-lasting protection against both seasonal and emerging pandemic influenza strains is thus urgently needed. The hemagglutinin (HA) stem antigen is a promising target for such a vaccine as it contains neutralizing epitopes, known to induce cross-protective IgG responses against a wide variety of influenza subtypes. In this study, we describe the development of a UFV candidate consisting of a HAstem trimer displayed on the surface of rigid capsid-like particles (CLP). Compared to soluble unconjugated HAstem trimer, the CLP-HAstem particles induced a more potent, long-lasting immune response and were able to protect mice against both homologous and heterologous H1N1 influenza challenge, even after a single dose

Host Determinants of Influenza Virus Replication

Influenza viruses are constantly evolving targets. During their intracellular life cycle, influenza viruses usurp host pathways, and pandemic strains must be able to gain access to these pathways in new organisms during cross-species transmission. Therefore, uncovering the molecular basis of pandemic influenza A virus strains is central to our preparedness against pandemics. Throughout my thesis work, I have thoroughly explored the virus:host interaction network of the potentially pandemic strain H5N1 through a CRISPR genome-wide screen, identifying and characterizing host factors, including CIC as a novel regulator of cell intrinsic immunity, and defining new roles for already known host factors, including Rab11a. These findings elicit druggable host factors for influenza viruses, augmenting our potential antiviral stockpile. Moreover, this work enhances our understanding of central pillars of the immune response, broadening the scope of this research to the control of multiple viruses and potentially other disease states

Rab11a mediates cell-cell spread and reassortment of influenza A virus genomes via tunneling nanotubes

Influenza A virus [IAV] genomes comprise eight negative strand RNAs packaged into virions in the form of viral ribonucleoproteins [vRNPs]. Rab11a plays a crucial role in the transport of vRNPs from the nucleus to the plasma membrane via microtubules, allowing assembly and virus production. Here, we identify a novel function for Rab11a in the inter-cellular transport of IAV vRNPs using tunneling nanotubes [TNTs]as molecular highways. TNTs are F-Actin rich tubules that link the cytoplasm of nearby cells. In IAV-infected cells, Rab11a was visualized together with vRNPs in these actin-rich intercellular connections. To better examine viral spread via TNTs, we devised an infection system in which conventional, virion-mediated, spread was not possible. Namely, we generated HA-deficient reporter viruses which are unable to produce progeny virions but whose genomes can be replicated and trafficked. In this system, vRNP transfer to neighboring cells was observed and this transfer was found to be dependent on both actin and Rab11a. Generation of infectious virus via TNT transfer was confirmed using donor cells infected with HA-deficient virus and recipient cells stably expressing HA protein. Mixing donor cells infected with genetically distinct IAVs furthermore revealed the potential for Rab11a and TNTs to serve as a conduit for genome mixing and reassortment in IAV infections. These data therefore reveal a novel role for Rab11a in the IAV life cycle, which could have significant implications for within-host spread, genome reassortment and immune evasion.</p

An investigation into chopsticks (disposable versus reusable)

This report aims to determine whether or not disposable or reusable chopsticks are suitable for student usage in the New Student Union Building (SUB) at UBC. Every year, approximately 350,000 pairs of disposable chopsticks are thrown away on UBC campus. This is a problem in the long run and the aim of the new SUB is to reduce the carbon footprint of students in the new SUB, the aim of this report is to minimize that from disposable utensils such as disposable chopsticks. An analysis is conducted on the two products based on the environmental, economical, and social impacts of disposable and reusable chopsticks. Reusable chopsticks are usually made out of plastic and metal whereas disposable chopsticks are wood and bamboo. Even though Canada is abundant with forests, it is important to acknowledge that it is not an unlimited source of material. After surveying a small sample population within users of the SUB, we have found that more than 80% of the participants are willing to bring their own reusable chopsticks if they were given a discount of some sort when purchasing their meals. This report concludes that reusable chopsticks are a much more sustainable option than disposable chopsticks as UBC strives to be a global leader in sustainability. By implementing cheap vending machines in the new SUB providing cheap reusable chopsticks will help raise awareness of the issues regarding disposable utensils. This will also encourage students and staff members not only to bring disposable utensils to school, but even to other places in Vancouver. It is recommended for washing stations to be implemented around the new SUB if reusable chopsticks are replacing disposable chopsticks. Disclaimer: “UBC SEEDS provides students with the opportunity to share the findings of their studies, as well as their opinions, conclusions and recommendations with the UBC community. The reader should bear in mind that this is a student project/report and is not an official document of UBC. Furthermore readers should bear in mind that these reports may not reflect the current status of activities at UBC. We urge you to contact the research persons mentioned in a report or the SEEDS Coordinator about the current status of the subject matter of a project/report.”Applied Science, Faculty ofUnreviewedUndergraduat

The Nrf1 transcription factor is induced by patulin and protects against patulin cytotoxicity.

Patulin is a mycotoxin produced by a variety of molds that is found in various food products. The adverse health effects associated with exposure to patulin has led to many investigations into the biological basis driving the toxicity of patulin. Nevertheless, the mechanisms through which mammalian cells resists patulin-mediated toxicity is poorly understood. Here, we show that loss of the Nrf1 transcription factor renders cells sensitive to the acute cytotoxic effects of patulin. Nrf1 deficiency leads to accumulation of ubiquitinated proteins and protein aggregates in response to patulin exposure. Nrf1 expression is induced by patulin, and activation of proteasome genes by patulin is Nrf1-dependent. These findings suggest the Nrf1 transcription factor plays a crucial role in modulating cellular stress response against patulin cytotoxicity

The p150 Isoform of ADAR1 Blocks Sustained RLR signaling and Apoptosis during Influenza Virus Infection

Signaling through retinoic acid inducible gene I (RIG-I) like receptors (RLRs) is tightly regulated, with activation occurring upon sensing of viral nucleic acids, and suppression mediated by negative regulators. Under homeostatic conditions aberrant activation of melanoma differentiation-associated protein-5 (MDA5) is prevented through editing of endogenous dsRNA by RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1). In addition, ADAR1 is postulated to play pro-viral and antiviral roles during viral infections that are dependent or independent of RNA editing activity. Here, we investigated the importance of ADAR1 isoforms in modulating influenza A virus (IAV) replication and revealed the opposing roles for ADAR1 isoforms, with the nuclear p110 isoform restricting versus the cytoplasmic p150 isoform promoting IAV replication. Importantly, we demonstrate that p150 is critical for preventing sustained RIG-I signaling, as p150 deficient cells showed increased IFN-β expression and apoptosis during IAV infection, independent of RNA editing activity. Taken together, the p150 isoform of ADAR1 is important for preventing sustained RIG-I induced IFN-β expression and apoptosis during viral infection.</p

Genome-wide CRISPR/Cas9 Screen Identifies Host Factors Essential for Influenza Virus Replication

Summary: The emergence of influenza A viruses (IAVs) from zoonotic reservoirs poses a great threat to human health. As seasonal vaccines are ineffective against zoonotic strains, and newly transmitted viruses can quickly acquire drug resistance, there remains a need for host-directed therapeutics against IAVs. Here, we performed a genome-scale CRISPR/Cas9 knockout screen in human lung epithelial cells with a human isolate of an avian H5N1 strain. Several genes involved in sialic acid biosynthesis and related glycosylation pathways were highly enriched post-H5N1 selection, including SLC35A1, a sialic acid transporter essential for IAV receptor expression and thus viral entry. Importantly, we have identified capicua (CIC) as a negative regulator of cell-intrinsic immunity, as loss of CIC resulted in heightened antiviral responses and restricted replication of multiple viruses. Therefore, our study demonstrates that the CRISPR/Cas9 system can be utilized for the discovery of host factors critical for the replication of intracellular pathogens. : Using a genome-wide CRISPR/Cas9 screen, Han et al. demonstrate that the major hit, the sialic acid transporter SLC35A1, is an essential host factor for IAV entry. In addition, they identify the DNA-binding transcriptional repressor CIC as a negative regulator of cell-intrinsic immunity. Keywords: CRISPR/Cas9 screen, GeCKO, influenza virus, host factors, sialic acid pathway, SLC35A1, Capicua, CIC, cell-intrinsic immunity, H5N

The Pre-Existing Human Antibody Repertoire to Computationally Optimized Influenza H1 Hemagglutinin Vaccines.

Computationally optimized broadly reactive Ag (COBRA) hemagglutinin (HA) immunogens have previously been generated for several influenza subtypes to improve vaccine-elicited Ab breadth. As nearly all individuals have pre-existing immunity to influenza viruses, influenza-specific memory B cells will likely be recalled upon COBRA HA vaccination. We determined the epitope specificity and repertoire characteristics of pre-existing human B cells to H1 COBRA HA Ags. Cross-reactivity between wild-type HA and H1 COBRA HA proteins P1, X6, and Y2 were observed for isolated mAbs. The mAbs bound five distinct epitopes on the pandemic A/California/04/2009 HA head and stem domains, and most mAbs had hemagglutination inhibition and neutralizing activity against 2009 pandemic H1 strains. Two head-directed mAbs, CA09-26 and CA09-45, had hemagglutination inhibition and neutralizing activity against a prepandemic H1 strain. One mAb, P1-05, targeted the stem region of H1 HA, but did not compete with a known stem-targeting H1 mAb. We determined that mAb P1-05 recognizes a recently discovered HA epitope, the anchor epitope, and we identified similar mAbs using B cell repertoire sequencing. In addition, the trimerization domain distance from HA was critical to recognition of this epitope by mAb P1-05, suggesting the importance of protein design for vaccine formulations. Overall, these data indicate that seasonally vaccinated individuals possess a population of functional H1 COBRA HA-reactive B cells that target head, central stalk, and anchor epitopes, and they demonstrate the importance of structure-based assessment of subunit protein vaccine candidates to ensure accessibility of optimal protein epitopes