Location: Zoonotic and Emerging Disease Research
Project Number: 3022-32000-027-003-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 22, 2022
End Date: Apr 30, 2027
Overarching Objective: Recent outbreaks of Ebola, Lassa, Zika, and SARS-CoV-2 have illustrated how vulnerable the world is to RNA viral threats. These and other circulating pathogens, including Henipavirus (Nipah), Hendra, and Crimean Congo Hemorrhagic Fever (CCHF), have exposed the urgent need for improved infrastructure, scientific knowledge, and diagnostic tools in high-risk regions, and the need for proactive pathogen surveillance, detection, and discovery. We propose three main activities: 1: Harness technological advancements in genomics and proteomics to identify new viral targets for vaccine development. 2: Use single-cell technologies to study the pathology of emerging hemorrhagic fever viruses. 3: Pilot a holistic surveillance system for detecting and characterizing circulating pathogens. Activity 1 Objective: We recently showed that non-canonical Open Reading Frames (ORFs) in the SARS-CoV-2 genome are a major source for T cell epitopes, with some non-canonical peptides eliciting greater T cell responses than canonical epitopes.1 We now aim to apply a novel synthetic high-throughput method (in development) for the discovery of non-canonical ORFs to hundreds of pathogenic viruses, including Nipah, Hendra, and CCHF. We will combine these experiments with untargeted mass spectrometry to identify new viral targets that are presented on class I human leukocyte antigen complex (HLA-I) to killer T cells. Insights will enhance our understanding of virus recognition and elimination by T cells and provide thousands of new targets for vaccine design. Activity 2 Objective: Single-cell studies of viral infection show that characterization of the pathogen and host response within single cells allows a more qualitative and quantitative examination of infection and its progression. Our work in single-cell transcriptomics (scRNA-seq) has been used to investigate the pathology and cellular targets of Ebola virus in a non-human primate model.2 We now aim to adapt and optimize these technologies to study emerging hemorrhagic fever viruses such as Nipah, Hendra, and CCHF. We will implement this in an in-vitro model, a natural history non-human primate (NHP) experiment, and potentially on human samples. We will also perform genome-wide CRISPR screens in cell lines and validate selected factors with Perturb-seq. Activity 3 Objective: West Africa has a high burden of infectious disease while lacking a suitable system for surveillance and outbreak response to emerging and existing pathogens.4 By leveraging our long-standing West African partnerships in Nigeria, Sierra Leone, and Senegal, we aim to pilot a comprehensive surveillance system to address the urgent need for enhanced surveillance of SARS-CoV-2, Nipah, CCHF, Lassa, and other pathogenic threats. To do so, we will pilot mCARMEN (microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic Acids), alongside powerful analytics to identify and characterize circulating pathogens. Developing the appropriate infrastructure to detect pathogens will impact the region’s future ability to preempt outbreaks and prevent pandemics.
Aim 1.1: 1. Discover and characterize non-canonical ORFs in hundreds of viruses, aiming to include all known viruses pathogenic to humans. 2. Measure translated regions in cell lines infected with Nipah virus, Hendra virus, and CCHF in collaboration with the lab of Robert Cross. Aim 1.2: 1. Interrogate the contribution of non-canonical ORFs to antigen presentation on HLA-I to killer T cells. 2. Use HLA-I prediction algorithms to assess HLA-I presentation of peptides derived from all the ORFs we identify in our high-throughput assay. 3. Experimentally profile the HLA-I peptidome in cells infected with high priority viruses including Nipah virus, Hendra virus and CCHF in collaboration with the lab of Robert Cross. 4. Infect cells with high multiplicity of infection (MOI), immunoprecipitate the HLA-I complex from cells lysate, and identify the bound peptides using mass spectrometry. Aim 2.1: 1. We will develop and deploy a non-polyadenylated inclusive scRNA-seq technology that can be applied to different types of tissues and samples. 2. We will apply this scRNAseq on a time course infection of Nipah virus, CCHF, and Hendra virus in cell lines in collaboration with the laboratory of Robert Cross. 3. We will perform scRNAseq on multiple organs obtained from a NHP (African green monkey) serial euthanasia study of Nipah virus to characterize the natural disease history. 4. If samples become available through Aim 3, we will perform scRNAseq from clinical samples taken from patients. Aim 2.2: 1. We will perform genome-wide CRISPR screens with Nipah, Hendra, and CCHF in the African green monkey cell line, Vero-E6, and human cell lines HEK-293. 2. We will evaluate putative host factors using Perturb-seq on multiple cell lines. Aim 3.1: 1. We will integrate Nipah onto our respiratory panel. 2. We will integrate CCHF and monkeypox onto our West African panel. 3. We will support the procurement and transfer of mCARMEN reagents for multi-pathogen identification and surveillance based on our respiratory virus panel and West African febrile illness panel. 4. We will train staff in West Africa on the use of mCARMEN’s respiratory panel and West African panel, as well as generate mCARMEN data on at least 1000 patient specimens with respiratory or febrile illness. Aim 3.2: 1. We will develop plans to integrate Lookout with CommCare and Scout for enhanced data gathering capabilities, as well as visualization and analysis of transmission patterns and existing trends. 2. We will connect with the national communication and data modeling teams in West Africa to train on use of these platforms and integrate with existing systems. 3. We will pilot the use of Lookout alongside mCARMEN.