About

Brook Heaton is an Associate Research Professor of Molecular Genetics and Microbiology at Duke University and a member of the Duke Human Vaccine Institute. His research focuses on molecular genetics and microbiology, contributing to the understanding of microbial interactions and host-microbial dynamics. Heaton's work is integral to advancing knowledge in these fields, supporting the development of vaccines and therapeutic strategies through his role at Duke.

Research topics

• Virology
• Medicine
• Biology
• Pharmacology
• Internal medicine
• Bioinformatics
• Biotechnology
• Chemistry
• Immunology
• Cancer research
• Cell biology
• Computational biology

Selected publications

• Validation of prototype virus inactivation from seven virus families of pandemic potential with a novel low-cost, field-deployable RNA extraction and storage method

  Journal of Virological Methods · 2025-10-27

  articleOpen accessSenior authorCorresponding

  The Centers for Research in Emerging Infectious Diseases (CREID) was established to enhance pandemic preparedness by studying emerging/reemerging pathogens, especially in resource-limited regions. To overcome infrastructure challenges, a low-cost, field-deployable method for extracting total nucleic acids is essential, eliminating reliance on expensive equipment, power, and cold chain systems used in traditional extraction techniques. To address this challenge, we developed an RNA extraction and storage method (RNAES) that meets these criteria. Herein, we report RNAES inactivation efficacy against nine prototype viruses (Middle Eastern respiratory syndrome coronavirus, Japanese encephalitis virus, West Nile virus, Hantaan virus, measles virus, Heartland virus, enterovirus A71, chikungunya virus, and Western equine encephalitis virus) representing seven pandemic potential virus families. We compare the RNAES method to the Qiagen QIAamp kit across various viral loads and field sample types. The presence of infectious virus in RNA samples was quantified using plaque assays. Successful inactivation of viruses was demonstrated for six enveloped virus families spiked into matrices routinely collected at field sites. The seventh family tested (Picornaviridae) was not completely inactivated, likely due to non-enveloped viruses being differentially susceptible to the lysis chemistry of the RNAES kit. The commercial comparator inactivated all viruses tested. Specialized biosafety facilities, specific detailed permits, and comprehensive logistics are required to ensure safety when handling and shipping potentially infectious samples. Inactivating pathogens at the point of collection reduces risks and simplifies sample transfer for critical outbreak research. Confidently ensuring that an isolated nucleic acid sample is non-infectious using RNAES will enable safer, and more efficient downstream analysis.

  PublisherDOI

• The coronavirus nsp14 exoribonuclease interface with the cofactor nsp10 is essential for efficient virus replication and enzymatic activity

  Journal of Virology · 2025-01-10 · 7 citations

  articleOpen access

  ABSTRACT Coronaviruses (CoVs) encode non-structural proteins (nsp’s) 1–16, which assemble to form replication-transcription complexes that function in viral RNA synthesis. All CoVs encode a proofreading 3′−5′ exoribonuclease in non-structural protein 14 (nsp14-ExoN) that mediates proofreading and high-fidelity replication and is critical for other roles in replication and pathogenesis. The in vitro enzymatic activity of nsp14-ExoN is enhanced in the presence of the cofactor nsp10. We introduced alanine substitutions in nsp14 of murine hepatitis virus (MHV) at the nsp14-nsp10 interface and recovered mutant viruses with a range of impairments in replication and in vitro biochemical exonuclease activity. Two of these substitutions, nsp14 K7A and D8A, had impairments intermediate between wild type-MHV nsp14 and the known ExoN(-) D89A/E91A nsp14 catalytic inactivation mutant. All introduced nsp14-nsp10 interface alanine substitutions impaired in vitro exonuclease activity. Passage of the K7A and D8A mutant viruses selected second-site non-synonymous mutations in nsp14 associated with improved mutant virus replication and exonuclease activity. These results confirm the essential role of the nsp14-nsp10 interaction for efficient enzymatic activity and virus replication, identify proximal and long-distance determinants of nsp14-nsp10 interaction, and support targeting the nsp14-nsp10 interface for viral inhibition and attenuation. IMPORTANCE Coronavirus replication requires assembly of a replication transcription complex composed of nsp’s, including polymerase, helicase, exonuclease, capping enzymes, and non-enzymatic cofactors. The coronavirus nsp14 exoribonuclease mediates several functions in the viral life cycle including genomic and subgenomic RNA synthesis, RNA recombination, RNA proofreading and high-fidelity replication, and native resistance to many nucleoside analogs. The nsp-14 exonuclease activity in vitro requires the non-enzymatic cofactor nsp10, but the determinants and importance of the nsp14-nsp10 interactions during viral replication have not been defined. Here we show that for the coronavirus murine hepatitis virus, nsp14 residues at the nsp14-nsp10 interface are essential for efficient viral replication and in vitro exonuclease activity. These results shed new light on the requirements for protein interactions within the coronavirus replication transcription complex, and they may reveal novel non-active-site targets for virus inhibition and attenuation.

  PublisherDOI

• Development of DNA and mRNA-LNP vaccines against an H5N1 clade 2.3.4.4b influenza virus

  Journal of Virology · 2025-07-16 · 6 citations

  articleOpen access

  Effective vaccines are an important public health tool which may be needed to combat the emerging, highly pathogenic H5N1 avian influenza viruses currently circulating in cattle and poultry in the United States. While nucleic acid-based vaccines such as mRNA-lipid nanoparticles (LNPs) have several potential advantages during a viral epidemic compared to traditional seasonal influenza vaccines, their utility and efficacy against H5N1 viruses remain incompletely defined. Here, we developed novel DNA- and mRNA-LNP-based vaccines encoding both hemagglutinin (HA) and neuraminidase (NA) proteins from the human-isolated highly pathogenic avian influenza H5N1 strain, A/Texas/37/2024, in a single open reading frame. This dual-antigen expression approach elicited strong protective immune responses targeting both the HA and NA proteins and provided complete protection against lethal viral challenges in a murine model. The pre-clinical data described in this work suggest that these multi-valent, adaptable, and scalable vaccine approaches may represent practical and rapid solutions to mediate robust protection from emerging zoonotic influenza virus threats. IMPORTANCE: Vaccines capable of protecting from infection with the H5N1 influenza viruses actively circulating in dairy cattle could be deployed to protect livestock and potentially also be used to protect human health. Here, we describe the development of protective DNA and mRNA-lipid nanoparticle vaccines targeting hemagglutinin and neuraminidase proteins from the highly pathogenic avian influenza (HPAI) H5N1 A/Texas/37/2024 virus and show that they are both protective against severe morbidity and mortality in a mouse model. Thus, the vaccines described in this work represent effective approaches to limit the current circulation of H5N1 viruses in animals and may represent practical solutions for human vaccination in the event of sustained human transmission of HPAI H5N1 viruses.

  PublisherDOI

• Headless hemagglutinin-containing influenza viral particles direct immune responses toward more conserved epitopes

  Journal of Virology · 2024-09-26 · 2 citations

  articleOpen access

  Seasonal influenza vaccines provide mostly strain-specific protection due to the elicitation of antibody responses focused on evolutionarily plastic antigenic sites in the hemagglutinin head domain. To direct the humoral response toward more conserved epitopes, we generated an influenza virus particle where the full-length hemagglutinin protein was replaced with a membrane-anchored, "headless" variant while retaining the normal complement of other viral structural proteins such as the neuraminidase as well as viral RNAs. We found that a single administration of a headless virus particle-based vaccine elicited high titers of antibodies that recognized more conserved epitopes on the major viral glycoproteins. Furthermore, the vaccine could elicit these responses even in the presence of pre-existing, hemagglutinin (HA) head-focused influenza immunity. Importantly, these antibody responses mediated protective, but non-neutralizing functions such as neuraminidase inhibition and antibody-dependent cellular cytotoxicity. Additionally, we show the vaccine can provide protection from homologous and heterologous challenges in mouse models of severe influenza without any measurable HA head-directed antibody responses. Thus, headless hemagglutinin containing viral particles may represent a tool to drive the types of antibody responses predicted to increase influenza vaccine breadth and durability.IMPORTANCECurrent seasonal influenza vaccines provide incomplete protection from disease. This is partially the result of the antibody response being directed toward parts of the virus that are tolerant of mutations. Redirecting the immune response to more conserved regions of the virus has been a central strategy of next-generation vaccine designs and approaches. Here, we develop and test a vaccine based on a modified influenza virus particle that expresses a partially deleted hemagglutinin protein along with the other viral structural proteins. We demonstrate this vaccine elicits antibodies that recognize the more conserved viral epitopes of the hemagglutinin stalk and neuraminidase protein to facilitate protection against influenza viruses despite a lack of classical viral neutralization activity.

  PublisherOA PDFDOI

• The coronavirus nsp14 exoribonuclease interface with the cofactor nsp10 is essential for efficient virus replication and enzymatic activity

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-09-26

  preprintOpen access

  ABSTRACT Coronaviruses (CoVs) encode nonstructural proteins (nsps) 1-16, which assemble to form replication-transcription complexes that function in viral RNA synthesis. All CoVs encode a proofreading 3’-5’ exoribonuclease (ExoN) in nsp14 (nsp14-ExoN) that mediates proofreading and high-fidelity replication and is critical for other roles in replication and pathogenesis. The in vitro enzymatic activity of nsp14 ExoN is enhanced in the presence of the cofactor nsp10. We introduced alanine substitutions in nsp14 of murine hepatitis virus (MHV) at the nsp14-10 interface and recovered mutant viruses with a range of impairments in replication and in vitro biochemical exonuclease activity. Two of these substitutions, nsp14 K7A and D8A, had impairments intermediate between WT-MHV nsp14 and the known ExoN(-) D89A/E91A nsp14 catalytic inactivation mutant. All introduced nsp14-10 interface alanine substitutions impaired in vitro exonuclease activity. Passage of the K7A and D8A mutant viruses selected second-site non-synonymous mutations in nsp14 associated with improved mutant virus replication and exonuclease activity. These results confirm the essential role of the nsp14-nsp10 interaction for efficient enzymatic activity and virus replication, identify proximal and long-distance determinants of nsp14-nsp10 interaction, and support targeting the nsp14-10 interface for viral inhibition and attenuation. IMPORTANCE Coronavirus replication requires assembly of a replication transcription complex composed of nonstructural proteins (nsp), including polymerase, helicase, exonuclease, capping enzymes, and non-enzymatic cofactors. The coronavirus nsp14 exoribonuclease mediates several functions in the viral life cycle including genomic and subgenomic RNA synthesis, RNA recombination, RNA proofreading and high-fidelity replication, and native resistance to many nucleoside analogs. The nsp-14 exonuclease activity in vitro requires the non-enzymatic co-factor nsp10, but the determinants and importance the nsp14-10 interactions during viral replication have not been defined. Here we show that for the coronavirus murine hepatitis virus, nsp14 residues at the nsp14-10 interface are essential for efficient viral replication and in vitro exonuclease activity. These results shed new light on the requirements for protein interactions within the coronavirus replication transcription complex, and they may reveal novel non active-site targets for virus inhibition and attenuation.

  PublisherOA PDFDOI

• Fluorescent and bioluminescent bovine H5N1 influenza viruses for evaluation of antiviral interventions

  Journal of Virology · 2024-10-10 · 2 citations

  letterOpen access

  In early 2024, a clade 2.3.4.4b high pathogenic H5N1 avian influenza virus was detected in dairy cows and humans in the United States. Since then, it has spread to herds in at least 13 states and caused symptomatic disease in at least fifteen people. To facilitate rapid testing of existing and novel countermeasures, here, we report the development of an H5N1 viral reverse genetic system, its use to produce fluorescent and bioluminescent variant strains, and their utility in high-throughput evaluation of antiviral interventions.

  PublisherOA PDFDOI

• A low-background, fluorescent assay to evaluate inhibitors of diverse viral proteases

  Journal of Virology · 2023-08-14 · 4 citations

  articleOpen access

  ABSTRACT Multiple coronaviruses (CoVs) can cause respiratory diseases in humans. While prophylactic vaccines designed to prevent infection are available for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), incomplete vaccine efficacy, vaccine hesitancy, and the threat of other pathogenic CoVs for which vaccines do not exist have highlighted the need for effective antiviral therapies. While antiviral compounds targeting the viral polymerase and protease are already in clinical use, their sensitivity to potential resistance mutations as well as their breadth against the full range of human and preemergent CoVs remain incompletely defined. To begin to fill that gap in knowledge, we report here the development of an improved, noninfectious, cell-based fluorescent assay with high sensitivity and low background that reports on the activity of viral proteases, which are key drug targets. We demonstrate that the assay is compatible with not only the SARS-CoV-2 M pro protein but also orthologues from a range of human and nonhuman CoVs as well as clinically reported SARS-CoV-2 drug-resistant M pro variants. We then use this assay to define the breadth of activity of two clinically used protease inhibitors, nirmatrelvir and ensitrelvir. Continued use of this assay will help define the strengths and limitations of current therapies and may also facilitate the development of next-generation protease inhibitors that are broadly active against both currently circulating and preemergent CoVs. IMPORTANCE Coronaviruses (CoVs) are important human pathogens with the ability to cause global pandemics. Working in concert with vaccines, antivirals specifically limit viral disease in people who are actively infected. Antiviral compounds that target CoV proteases are already in clinical use; their efficacy against variant proteases and preemergent zoonotic CoVs, however, remains incompletely defined. Here, we report an improved, noninfectious, and highly sensitive fluorescent method of defining the sensitivity of CoV proteases to small molecule inhibitors. We use this approach to assay the activity of current antiviral therapies against clinically reported SARS-CoV-2 protease mutants and a panel of highly diverse CoV proteases. Additionally, we show this system is adaptable to other structurally nonrelated viral proteases. In the future, this assay can be used to not only better define the strengths and limitations of current therapies but also help develop new, broadly acting inhibitors that more broadly target viral families.

  PublisherOA PDFDOI

• Rapid tissue prototyping with micro-organospheres

  Stem Cell Reports · 2022-08-18 · 49 citations

  articleOpen access

  In vitro tissue models hold great promise for modeling diseases and drug responses. Here, we used emulsion microfluidics to form micro-organospheres (MOSs), which are droplet-encapsulated miniature three-dimensional (3D) tissue models that can be established rapidly from patient tissues or cells. MOSs retain key biological features and responses to chemo-, targeted, and radiation therapies compared with organoids. The small size and large surface-to-volume ratio of MOSs enable various applications including quantitative assessment of nutrient dependence, pathogen-host interaction for anti-viral drug screening, and a rapid potency assay for chimeric antigen receptor (CAR)-T therapy. An automated MOS imaging pipeline combined with machine learning overcomes plating variation, distinguishes tumorspheres from stroma, differentiates cytostatic versus cytotoxic drug effects, and captures resistant clones and heterogeneity in drug response. This pipeline is capable of robust assessments of drug response at individual-tumorsphere resolution and provides a rapid and high-throughput therapeutic profiling platform for precision medicine.

  PublisherDOI

• A Virion-Based Combination Vaccine Protects against Influenza and SARS-CoV-2 Disease in Mice

  Journal of Virology · 2022-07-12 · 24 citations

  articleOpen accessSenior authorCorresponding

  The rapid emergence of SARS-CoV-2 variants since the onset of the pandemic has highlighted the need for both periodic vaccination "boosts" and a platform that can be rapidly reformulated to manufacture new vaccines. In this work, we report an approach that can utilize current influenza vaccine manufacturing infrastructure to generate combination vaccines capable of protecting from both influenza virus- and SARS-CoV-2-induced disease. The production of a combined influenza/SARS-CoV-2 vaccine may represent a practical solution to boost immunity to these important respiratory viruses without the increased cost and administration burden of multiple independent vaccines.

  PublisherOA PDFDOI

• Host protein kinases required for SARS-CoV-2 nucleocapsid phosphorylation and viral replication

  Science Signaling · 2022-10-25 · 97 citations

  articleOpen accessCorresponding

  Multiple coronaviruses have emerged independently in the past 20 years that cause lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated or who experience breakthrough infections. Understanding the common host factors necessary for the life cycles of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that coordinately phosphorylate a cluster of serine and threonine residues in the viral N protein, which is required for viral replication. We also showed that loss or inhibition of SRPK1/2, which we propose initiates the N protein phosphorylation cascade, compromised the viral replication cycle. Because these phosphorylation sites are highly conserved across coronaviruses, inhibitors of these protein kinases not only may have therapeutic potential against COVID-19 but also may be broadly useful against coronavirus-mediated diseases.

  PublisherDOI

Frequent coauthors

• Nicholas S. Heaton

  19 shared

• Tomer M. Yaron

  Columbia University

  11 shared

• Alfred T. Harding

  Massachusetts Institute of Technology

  9 shared

• Gad Getz

  7 shared

• Cait E. Hamele

  University of Virginia

  6 shared

• Ting-Yu Lin

  Tunghai University

  6 shared

• Katarina Liberatore

  Weill Cornell Medicine

  6 shared

• Ryan R. Chaparian

  Duke University

  6 shared