Publications by authors named "Thomas Geisbert"

208 Publications

LY-CoV1404 potently neutralizes SARS-CoV-2 variants.

bioRxiv 2021 May 4. Epub 2021 May 4.

LY-CoV1404 is a highly potent, neutralizing, SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody identified from a convalescent COVID-19 patient approximately 60 days after symptom onset. In pseudovirus studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.427/B.1.429, P.1, and B.1.526 and binds to these variants in the presence of their underlying RBD mutations (which include K417N, L452R, E484K, and N501Y). LY-CoV1404 also neutralizes authentic SARS-CoV-2 in two different assays against multiple isolates. The RBD positions comprising the LY-CoV1404 epitope are highly conserved, with the exception of N439 and N501; notably the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of variant binding, potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 is one in a panel of well-characterized, clinically developable antibodies that could be deployed rapidly to address current and emerging variants. New variant-resistant treatments such as LY-CoV1404 are desperately needed, given that some of the existing therapeutic antibodies are less effective or ineffective against certain variants and the impact of variants on vaccine efficacy is still poorly understood.

In Brief: LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated.

Highlights: LY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and all known variants of concernNo loss of potency against current variantsBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID databaseBreadth of neutralizing activity and potency supports clinical development.
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http://dx.doi.org/10.1101/2021.04.30.442182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109210PMC
May 2021

Broadly neutralizing antibody cocktails targeting Nipah virus and Hendra virus fusion glycoproteins.

Nat Struct Mol Biol 2021 Apr 29. Epub 2021 Apr 29.

Department of Biochemistry, University of Washington, Seattle, WA, USA.

Hendra virus (HeV) and Nipah virus (NiV) are henipaviruses (HNVs) causing respiratory illness and severe encephalitis in humans, with fatality rates of 50-100%. There are no licensed therapeutics or vaccines to protect humans. HeV and NiV use a receptor-binding glycoprotein (G) and a fusion glycoprotein (F) to enter host cells. HNV F and G are the main targets of the humoral immune response, and the presence of neutralizing antibodies is a correlate of protection against NiV and HeV in experimentally infected animals. We describe here two cross-reactive F-specific antibodies, 1F5 and 12B2, that neutralize NiV and HeV through inhibition of membrane fusion. Cryo-electron microscopy structures reveal that 1F5 and 12B2 recognize distinct prefusion-specific, conserved quaternary epitopes and lock F in its prefusion conformation. We provide proof-of-concept for using antibody cocktails for neutralizing NiV and HeV and define a roadmap for developing effective countermeasures against these highly pathogenic viruses.
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http://dx.doi.org/10.1038/s41594-021-00584-8DOI Listing
April 2021

Therapy for Argentine hemorrhagic fever in nonhuman primates with a humanized monoclonal antibody.

Proc Natl Acad Sci U S A 2021 03;118(11)

Galveston National Laboratory, University of Texas Medical Branch, Galveston, TX 77555.

The COVID-19 pandemic has reemphasized the need to identify safe and scalable therapeutics to slow or reverse symptoms of disease caused by newly emerging and reemerging viral pathogens. Recent clinical successes of monoclonal antibodies (mAbs) in therapy for viral infections demonstrate that mAbs offer a solution for these emerging biothreats. We have explored this with respect to Junin virus (JUNV), an arenavirus classified as a category A high-priority agent and the causative agent of Argentine hemorrhagic fever (AHF). There are currently no Food and Drug Administration-approved drugs available for preventing or treating AHF, although immune plasma from convalescent patients is used routinely to treat active infections. However, immune plasma is severely limited in quantity, highly variable in quality, and poses significant safety risks including the transmission of transfusion-borne diseases. mAbs offer a highly specific and consistently potent alternative to immune plasma that can be manufactured at large scale. We previously described a chimeric mAb, cJ199, that provided protection in a guinea pig model of AHF. To adapt this mAb to a format more suitable for clinical use, we humanized the mAb (hu199) and evaluated it in a cynomolgus monkey model of AHF with two JUNV isolates, Romero and Espindola. While untreated control animals experienced 100% lethality, all animals treated with hu199 at 6 d postinoculation (dpi) survived, and 50% of animals treated at 8 dpi survived. mAbs like hu199 may offer a safer, scalable, and more reproducible alternative to immune plasma for rare viral diseases that have epidemic potential.
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http://dx.doi.org/10.1073/pnas.2023332118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980402PMC
March 2021

Ultrasensitive point-of-care immunoassay for secreted glycoprotein detects Ebola infection earlier than PCR.

Sci Transl Med 2021 Apr;13(588)

Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.

Ebola virus (EBOV) hemorrhagic fever outbreaks have been challenging to deter due to the lack of health care infrastructure in disease-endemic countries and a corresponding inability to diagnose and contain the disease at an early stage. EBOV vaccines and therapies have improved disease outcomes, but the advent of an affordable, easily accessed, mass-produced rapid diagnostic test (RDT) that matches the performance of more resource-intensive polymerase chain reaction (PCR) assays would be invaluable in containing future outbreaks. Here, we developed and demonstrated the performance of a new ultrasensitive point-of-care immunoassay, the EBOV D4 assay, which targets the secreted glycoprotein of EBOV. The EBOV D4 assay is 1000-fold more sensitive than the U.S. Food and Drug Administration-approved RDTs and detected EBOV infection earlier than PCR in a standard nonhuman primate model. The EBOV D4 assay is suitable for low-resource settings and may facilitate earlier detection, containment, and treatment during outbreaks of the disease.
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http://dx.doi.org/10.1126/scitranslmed.abd9696DOI Listing
April 2021

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in non-human primates.

Sci Transl Med 2021 Apr 5. Epub 2021 Apr 5.

Lilly Biotechnology Center, Eli Lilly and Company, San Diego, CA, 92121, USA.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics which may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days, and clearance of 0.22 mL/hr/kg, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study Day 6 following viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.
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http://dx.doi.org/10.1126/scitranslmed.abf1906DOI Listing
April 2021

Combination therapy protects macaques against advanced Marburg virus disease.

Nat Commun 2021 03 25;12(1):1891. Epub 2021 Mar 25.

Galveston National Laboratory, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, USA.

Monoclonal antibodies (mAbs) and remdesivir, a small-molecule antiviral, are promising monotherapies for many viruses, including members of the genera Marburgvirus and Ebolavirus (family Filoviridae), and more recently, SARS-CoV-2. One of the major challenges of acute viral infections is the treatment of advanced disease. Thus, extending the window of therapeutic intervention is critical. Here, we explore the benefit of combination therapy with a mAb and remdesivir in a non-human primate model of Marburg virus (MARV) disease. While rhesus monkeys are protected against lethal infection when treatment with either a human mAb (MR186-YTE; 100%), or remdesivir (80%), is initiated 5 days post-inoculation (dpi) with MARV, no animals survive when either treatment is initiated alone beginning 6 dpi. However, by combining MR186-YTE with remdesivir beginning 6 dpi, significant protection (80%) is achieved, thereby extending the therapeutic window. These results suggest value in exploring combination therapy in patients presenting with advanced filovirus disease.
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http://dx.doi.org/10.1038/s41467-021-22132-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994808PMC
March 2021

Use of convalescent serum reduces severity of COVID-19 in nonhuman primates.

Cell Rep 2021 03 23;34(10):108837. Epub 2021 Feb 23.

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA; Galveston National Laboratory, University of Texas Medical Branch, Galveston, TX 77555, USA. Electronic address:

Passive transfer of convalescent plasma or serum is a time-honored strategy for treating infectious diseases. Human convalescent plasma containing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently being used to treat patients with coronavirus disease 2019 where clinical efficacy trials are ongoing. Here, we assess therapeutic passive transfer in groups of SARS-CoV-2-infected African green monkeys with convalescent sera containing either high or low anti-SARS-CoV-2 neutralizing antibody titers. Differences in viral load and pathology are minimal between monkeys that receive the lower titer convalescent sera and untreated controls. However, lower levels of SARS-CoV-2 in respiratory compartments, reduced severity of virus-associated lung pathology, and reductions in coagulopathy and inflammatory processes are observed in monkeys that receive high titer sera versus untreated controls. Our data indicate that convalescent plasma therapy in humans may be an effective strategy provided that donor sera contain high anti-SARS-CoV-2 neutralizing titers given in early stages of the disease.
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http://dx.doi.org/10.1016/j.celrep.2021.108837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901292PMC
March 2021

A single dose investigational subunit vaccine for human use against Nipah virus and Hendra virus.

NPJ Vaccines 2021 Feb 8;6(1):23. Epub 2021 Feb 8.

Profectus BioSciences, Inc., Baltimore, MD, USA.

Nipah and Hendra viruses are highly pathogenic bat-borne paramyxoviruses recently included in the WHO Blueprint priority diseases list. A fully registered horse anti-Hendra virus subunit vaccine has been in use in Australia since 2012. Based on the same immunogen, the Hendra virus attachment glycoprotein ectodomain, a subunit vaccine formulation for use in people is now in a Phase I clinical trial. We report that a single dose vaccination regimen of this human vaccine formulation protects against otherwise lethal challenges of either Hendra or Nipah virus in a nonhuman primate model. The protection against the Nipah Bangladesh strain begins as soon as 7 days post immunization with low dose of 0.1 mg protein subunit. Our data suggest this human vaccine could be utilized as efficient emergency vaccine to disrupt potential spreading of Nipah disease in an outbreak setting.
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http://dx.doi.org/10.1038/s41541-021-00284-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870971PMC
February 2021

Prominent Neutralizing Antibody Response Targeting the Glycoprotein Subunit Interface Elicited by Immunization.

J Virol 2021 Feb 3. Epub 2021 Feb 3.

Institute for Bioscience and Biotechnology Research, Rockville, MD

The severe death toll caused by the recent outbreak of Ebola virus disease reinforces the importance of developing ebolavirus prevention and treatment strategies. Here, we have explored the immunogenicity of a novel immunization regimen priming with vesicular stomatitis virus particles bearing Sudan Ebola virus (SUDV) glycoprotein (GP) that consists of GP1 & GP2 subunits and boosting with soluble SUDV GP in macaques, which developed robust neutralizing antibody (nAb) responses following immunizations. Moreover, EB46, a protective nAb isolated from one of the immune macaques, is found to target the GP1/GP2 interface, with GP-binding mode and neutralization mechanism similar to a number of ebolavirus nAbs from human and mouse, indicating that the ebolavirus GP1/GP2 interface is a common immunological target in different species. Importantly, selected immune macaque polyclonal sera showed nAb specificity similar to EB46 at substantial titers, suggesting that the GP1/GP2 interface region is a viable target for ebolavirus vaccine. The elicitation of sustained neutralizing antibody (nAb) responses against diverse ebolavirus strains remains as a high priority for the vaccine field. The most clinically advanced rVSV-ZEBOV vaccine could elicit moderate nAb responses against only one ebolavirus strain, EBOV, among the five ebolavirus strains, which last less than 6 months. Boost immunization strategies are desirable to effectively recall the rVSV vector-primed nAb responses to prevent infections in prospective epidemics, while an in-depth understanding of the specificity of immunization-elicited nAb responses is essential for improving vaccine performance. Here, using non-human primate animal model, we demonstrated that booster immunization with a stabilized trimeric soluble form of recombinant glycoprotein derived from the ebolavirus Sudan strain following the priming rVSV vector immunization led to robust nAb responses that substantially map to the subunit interface of ebolavirus glycoprotein, a common B cell repertoire target of multiple species including primates and rodents.
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http://dx.doi.org/10.1128/JVI.01907-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8103683PMC
February 2021

Transcriptional Analysis of Lymphoid Tissues from Infected Nonhuman Primates Reveals the Basis for Attenuation and Immunogenicity of an Ebola Virus Encoding a Mutant VP35 Protein.

J Virol 2021 02 24;95(6). Epub 2021 Feb 24.

Department of Molecular Biology and Biochemistry, College of Biological Sciences, University of California, Irvine, Irvine, California, USA

Infection with (EBOV), a member of the family, causes a disease characterized by high levels of viremia, aberrant inflammation, coagulopathy, and lymphopenia. EBOV initially replicates in lymphoid tissues and disseminates via dendritic cells (DCs) and monocytes to liver, spleen, adrenal gland, and other secondary organs. EBOV protein VP35 is a critical immune evasion factor that inhibits type I interferon signaling and DC maturation. Nonhuman primates (NHPs) immunized with a high dose (5 × 10 PFU) of recombinant EBOV containing a mutated VP35 (VP35m) are protected from challenge with wild-type EBOV (wtEBOV). This protection is accompanied by a transcriptional response in the peripheral blood reflecting a regulated innate immune response and a robust induction of adaptive immune genes. However, the host transcriptional response to VP35m in lymphoid tissues has not been evaluated. Therefore, we conducted a transcriptional analysis of axillary and inguinal lymph nodes and spleen tissues of NHPs infected with a low dose (2 × 10 PFU) of VP35m and then back-challenged with a lethal dose of wtEBOV. VP35m induced early transcriptional responses in lymphoid tissues that are distinct from those observed in wtEBOV challenge. Specifically, we detected robust antiviral innate and adaptive responses and fewer transcriptional changes in genes with roles in angiogenesis, apoptosis, and inflammation. Two of three macaques survived wtEBOV back-challenge, with only the nonsurvivor displaying a transcriptional response reflecting Ebola virus disease. These data suggest that VP35 is a key modulator of early host responses in lymphoid tissues, thereby regulating disease progression and severity following EBOV challenge. Zaire Ebola virus (EBOV) infection causes a severe and often fatal disease characterized by inflammation, coagulation defects, and organ failure driven by a defective host immune response. Lymphoid tissues are key sites of EBOV pathogenesis and the generation of an effective immune response to infection. A recent study demonstrated that infection with an EBOV encoding a mutant VP35, a viral protein that antagonizes host immunity, can protect nonhuman primates (NHPs) against lethal EBOV challenge. However, no studies have examined the response to this mutant EBOV in lymphoid tissues. Here, we characterize gene expression in lymphoid tissues from NHPs challenged with the mutant EBOV and subsequently with wild-type EBOV to identify signatures of a protective host response. Our findings are critical for elucidating viral pathogenesis, mechanisms of host antagonism, and the role of lymphoid organs in protective responses to EBOV to improve the development of antivirals and vaccines against EBOV.
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http://dx.doi.org/10.1128/JVI.01995-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8094945PMC
February 2021

Potent Henipavirus Neutralization by Antibodies Recognizing Diverse Sites on Hendra and Nipah Virus Receptor Binding Protein.

Cell 2020 12;183(6):1536-1550.e17

The Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN 37232, USA; Department of Microbiology & Immunology, The University of Texas Medical Branch, Galveston, TX 77555, USA; Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232, USA. Electronic address:

Hendra (HeV) and Nipah (NiV) viruses are emerging zoonotic pathogens in the Henipavirus genus causing outbreaks of disease with very high case fatality rates. Here, we report the first naturally occurring human monoclonal antibodies (mAbs) against HeV receptor binding protein (RBP). All isolated mAbs neutralized HeV, and some also neutralized NiV. Epitope binning experiments identified five major antigenic sites on HeV-RBP. Animal studies demonstrated that the most potent cross-reactive neutralizing mAbs, HENV-26 and HENV-32, protected ferrets in lethal models of infection with NiV Bangladesh 3 days after exposure. We solved the crystal structures of mAb HENV-26 in complex with both HeV-RBP and NiV-RBP and of mAb HENV-32 in complex with HeV-RBP. The studies reveal diverse sites of vulnerability on RBP recognized by potent human mAbs that inhibit virus by multiple mechanisms. These studies identify promising prophylactic antibodies and define protective epitopes that can be used in rational vaccine design.
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http://dx.doi.org/10.1016/j.cell.2020.11.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771633PMC
December 2020

Establishment of an African green monkey model for COVID-19 and protection against re-infection.

Nat Immunol 2021 01 24;22(1):86-98. Epub 2020 Nov 24.

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for an unprecedented global pandemic of COVID-19. Animal models are urgently needed to study the pathogenesis of COVID-19 and to screen vaccines and treatments. We show that African green monkeys (AGMs) support robust SARS-CoV-2 replication and develop pronounced respiratory disease, which may more accurately reflect human COVID-19 cases than other nonhuman primate species. SARS-CoV-2 was detected in mucosal samples, including rectal swabs, as late as 15 days after exposure. Marked inflammation and coagulopathy in blood and tissues were prominent features. Transcriptome analysis demonstrated stimulation of interferon and interleukin-6 pathways in bronchoalveolar lavage samples and repression of natural killer cell- and T cell-associated transcripts in peripheral blood. Despite a slight waning in antibody titers after primary challenge, enhanced antibody and cellular responses contributed to rapid clearance after re-challenge with an identical strain. These data support the utility of AGM for studying COVID-19 pathogenesis and testing medical countermeasures.
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http://dx.doi.org/10.1038/s41590-020-00835-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790436PMC
January 2021

Endotheliopathy and Platelet Dysfunction as Hallmarks of Fatal Lassa Fever.

Emerg Infect Dis 2020 11;26(11):2625-2637

Lassa fever (LF) causes multisystem disease and has a fatality rate <70%. Severe cases exhibit abnormal coagulation, endothelial barrier disruption, and dysfunctional platelet aggregation but the underlying mechanisms remain poorly understood. In Sierra Leone during 2015-2018, we assessed LF patients' day-of-admission plasma samples for levels of proteins necessary for coagulation, fibrinolysis, and platelet function. P-selectin, soluble endothelial protein C receptor, soluble thrombomodulin, plasminogen activator inhibitor 1, ADAMTS-13, von Willebrand factor, tissue factor, soluble intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 were more elevated in LF patients than in controls. Endothelial protein C receptor, thrombomodulin, intercellular adhesion molecule 1, plasminogen activator inhibitor 1, D-dimer, and hepatocyte growth factor were higher in fatal than nonfatal LF cases. Platelet disaggregation occurred only in samples from fatal LF cases. The impaired homeostasis and platelet dysfunction implicate alterations in the protein C pathway, which might contribute to the loss of endothelial barrier function in fatal infections.
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http://dx.doi.org/10.3201/eid2611.191694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588510PMC
November 2020

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

bioRxiv 2020 Oct 1. Epub 2020 Oct 1.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection.

One Sentence Summary: LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2020.09.30.318972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536866PMC
October 2020

Antibodies from Sierra Leonean and Nigerian Lassa fever survivors cross-react with recombinant proteins representing Lassa viruses of divergent lineages.

Sci Rep 2020 09 29;10(1):16030. Epub 2020 Sep 29.

Ahmadu Bello University, Zaria, Nigeria.

Lassa virus (LASV) is the causative agent of Lassa fever, an often-fatal hemorrhagic disease that is endemic in West Africa. Seven genetically distinct LASV lineages have been identified. As part of CEPI's (Coalition for Epidemic Preparedness Innovations) Lassa vaccine development program, we assessed the potential of the human immune system to mount cross-reactive and cross-protective humoral immune responses to antigens from the most prevalent LASV lineages, which are lineages II and III in Nigeria and lineage IV in Sierra Leone. IgG and IgM present in the blood of Lassa fever survivors from Nigeria or Sierra Leone exhibited substantial cross-reactivity for binding to LASV nucleoprotein and two engineered (linked and prefusion) versions of the glycoproteins (GP) of lineages II-IV. There was less cross-reactivity for the Zinc protein. Serum or plasma from Nigerian Lassa fever survivors neutralized LASV pseudoviruses expressing lineage II GP better than they neutralized lineage III or IV GP expressing pseudoviruses. Sierra Leonean survivors did not exhibit a lineage bias. Neutralization titres determined using LASV pseudovirus assays showed significant correlation with titres determined by plaque reduction with infectious LASV. These studies provide guidance for comparison of humoral immunity to LASV of distinct lineages following natural infection or immunization.
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http://dx.doi.org/10.1038/s41598-020-72539-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7525497PMC
September 2020

Intranasal exposure of African green monkeys to SARS-CoV-2 results in acute phase pneumonia with shedding and lung injury still present in the early convalescence phase.

Res Sq 2020 Aug 13. Epub 2020 Aug 13.

University of Texas Medical Branch.

We recently reported the development of the first African green monkey (AGM) model for COVID-19 based on a combined liquid intranasal (i.n.) and intratracheal (i.t.) exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we followed up on this work by assessing an i.n. particle only route of exposure using the LMA mucosal atomization device (MAD). Six AGMs were infected with SARS-CoV-2; three animals were euthanized near the peak stage of virus replication (day 5) and three animals were euthanized during the early convalescence period (day 34). All six AGMs supported robust SARS-CoV-2 replication and developed respiratory disease. Evidence of coagulation dysfunction as noted by a transient increases in aPTT and circulating levels of fibrinogen was observed in all AGMs. The level of SARS-CoV-2 replication and lung pathology was not quite as pronounced as previously reported with AGMs exposed by the combined i.n. and i.t. routes; however, SARS-CoV-2 RNA was detected in nasal swabs of some animals as late as day 15 and rectal swabs as late as day 28 after virus challenge. Of particular importance to this study, all three AGMs that were followed until the early convalescence stage of COVID-19 showed substantial lung pathology at necropsy as evidenced by multifocal chronic interstitial pneumonia and increased collagen deposition in alveolar walls despite the absence of detectable SARS-CoV-2 in any of the lungs of these animals. These findings are consistent with human COVID-19 further demonstrating that the AGM faithfully reproduces the human condition.
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http://dx.doi.org/10.21203/rs.3.rs-50023/v2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430587PMC
August 2020

In plain sight: the role of alpha-1-antitrypsin in COVID-19 pathogenesis and therapeutics.

bioRxiv 2020 Aug 15. Epub 2020 Aug 15.

Entry of SARS-CoV-2 is facilitated by endogenous and exogenous proteases. These proteases proteolytically activate the SARS-CoV-2 spike glycoprotein and are key modulators of virus tropism. We show that SARS-CoV-2 naïve serum exhibits significant inhibition of SARS-CoV-2 entry. We identify alpha-1-antitrypsin (AAT), and to a lesser degree, alpha-2-macroglobulin (A2M) as highly abundant serum protease inhibitors that potently restrict protease-mediated entry of SARS-CoV-2. AAT inhibition of protease-mediated SARS-CoV-2 entry in vitro occurs at concentrations far below what is present in serum and bronchoalveolar tissues, suggesting that AAT effects are physiologically relevant. Moreover, AAT mutations that have been characterized to affect abundance or function are highly prevalent. In addition to the effects that AAT may have on viral entry itself, we argue that the anti-inflammatory and coagulation regulatory activity of AAT have implications for coronavirus disease 2019 (COVID-19) pathogenicity, SARS-CoV-2 tissue restriction, convalescent plasma therapies, and even potentially AAT therapy.
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http://dx.doi.org/10.1101/2020.08.14.248880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430570PMC
August 2020

Intranasal exposure of African green monkeys to SARS-CoV-2 results in acute phase pneumonia with shedding and lung injury still present in the early convalescence phase.

Virol J 2020 08 18;17(1):125. Epub 2020 Aug 18.

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, 77555, USA.

We recently reported the development of the first African green monkey (AGM) model for COVID-19 based on a combined liquid intranasal (i.n.) and intratracheal (i.t.) exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we followed up on this work by assessing an i.n. particle only route of exposure using the LMA mucosal atomization device (MAD). Six AGMs were infected with SARS-CoV-2; three animals were euthanized near the peak stage of virus replication (day 5) and three animals were euthanized during the early convalescence period (day 34). All six AGMs supported robust SARS-CoV-2 replication and developed respiratory disease. Evidence of coagulation dysfunction as noted by a transient increases in aPTT and circulating levels of fibrinogen was observed in all AGMs. The level of SARS-CoV-2 replication and lung pathology was not quite as pronounced as previously reported with AGMs exposed by the combined i.n. and i.t. routes; however, SARS-CoV-2 RNA was detected in nasal swabs of some animals as late as day 15 and rectal swabs as late as day 28 after virus challenge. Of particular importance to this study, all three AGMs that were followed until the early convalescence stage of COVID-19 showed substantial lung pathology at necropsy as evidenced by multifocal chronic interstitial pneumonia and increased collagen deposition in alveolar walls despite the absence of detectable SARS-CoV-2 in any of the lungs of these animals. These findings are consistent with human COVID-19 further demonstrating that the AGM faithfully reproduces the human condition.
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http://dx.doi.org/10.1186/s12985-020-01396-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431901PMC
August 2020

Crimean-Congo hemorrhagic fever virus strains Hoti and Afghanistan cause viremia and mild clinical disease in cynomolgus monkeys.

PLoS Negl Trop Dis 2020 08 13;14(8):e0008637. Epub 2020 Aug 13.

Galveston National Laboratory, University of Texas Medical Branch, Galveston, Texas, United States of America.

Background: Development of vaccines and therapies against Crimean-Congo hemorrhagic fever virus (CCHFV) have been hindered by the lack of immunocompetent animal models. Recently, a lethal nonhuman primate model based on the CCHFV Hoti strain was reported. CCHFV Hoti caused severe disease in cynomolgus monkeys with 75% lethality when given by the intravenous (i.v.) route.

Methodology/principal Findings: In a series of experiments, eleven cynomologus monkeys were exposed i.v. to CCHFV Hoti and four macaques were exposed i.v. to CCHFV Afghanistan. Despite transient viremia and changes in clinical pathology such as leukopenia and thrombocytopenia developing in all 15 animals, all macaques survived to the study endpoint without developing severe disease.

Conclusions/significance: We were unable to attribute differences in the results of our study versus the previous report to differences in the CCHFV Hoti stock, challenge dose, origin, or age of the macaques. The observed differences are most likely the result of the outbred nature of macaques and low animal numbers often used by necessity and for ethical considerations in BSL-4 studies. Nonetheless, while we were unable to achieve severe disease or lethality, the CCHFV Hoti and Afghanistan macaque models are useful for screening medical countermeasures using biomarkers including viremia and clinical pathology to assess efficacy.
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http://dx.doi.org/10.1371/journal.pntd.0008637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447009PMC
August 2020

Prior vaccination with rVSV-ZEBOV does not interfere with but improves efficacy of postexposure antibody treatment.

Nat Commun 2020 07 27;11(1):3736. Epub 2020 Jul 27.

Galveston National Laboratory, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555-0610, USA.

A replication-competent vesicular stomatitis virus vaccine expressing the Ebola virus (EBOV) glycoprotein (GP) (rVSV-ZEBOV) was successfully used during the 2013-16 EBOV epidemic. Additionally, chimeric and human monoclonal antibodies (mAb) against the EBOV GP have shown promise in animals and humans when administered therapeutically. Uncertainty exists regarding the efficacy of postexposure antibody treatments in the event of a known exposure of a recent rVSV-ZEBOV vaccinee. Here, we model a worst-case scenario using rhesus monkeys vaccinated or unvaccinated with the rVSV-ZEBOV vaccine. We demonstrate that animals challenged with a uniformly lethal dose of EBOV one day following vaccination, and then treated with the anti-EBOV GP mAb MIL77 starting 3 days postexposure show no evidence of clinical illness and survive challenge. In contrast, animals receiving only vaccination or only mAb-based therapy become ill, with decreased survival compared to animals vaccinated and subsequently treated with MIL77. These results suggest that rVSV-ZEBOV augments immunotherapy.
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http://dx.doi.org/10.1038/s41467-020-17446-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385100PMC
July 2020

Establishment of an African green monkey model for COVID-19.

bioRxiv 2020 May 17. Epub 2020 May 17.

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for an unprecedented global pandemic of COVID-19. Animal models are urgently needed to study the pathogenesis of COVID-19 and to screen candidate vaccines and treatments. Nonhuman primates (NHP) are considered the gold standard model for many infectious pathogens as they usually best reflect the human condition. Here, we show that African green monkeys support a high level of SARS-CoV-2 replication and develop pronounced respiratory disease that may be more substantial than reported for other NHP species including cynomolgus and rhesus macaques. In addition, SARS-CoV-2 was detected in mucosal samples of all animals including feces of several animals as late as 15 days after virus exposure. Importantly, we show that virus replication and respiratory disease can be produced in African green monkeys using a much lower and more natural dose of SARS-CoV-2 than has been employed in other NHP studies.
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http://dx.doi.org/10.1101/2020.05.17.100289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263506PMC
May 2020

Ebola.

N Engl J Med 2020 05;382(19):1832-1842

From the Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, MT (H.F.); Médecins sans Frontières, Brussels (A.S.); and the Department of Microbiology and Immunology and Galveston National Laboratory, University of Texas Medical Branch at Galveston, Galveston (T.W.G.).

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http://dx.doi.org/10.1056/NEJMra1901594DOI Listing
May 2020

Early Transcriptional Changes within Liver, Adrenal Gland, and Lymphoid Tissues Significantly Contribute to Ebola Virus Pathogenesis in Cynomolgus Macaques.

J Virol 2020 05 18;94(11). Epub 2020 May 18.

Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California, Irvine, Irvine, California, USA

Ebola virus (EBOV) continues to pose a significant threat to human health, as evidenced by the 2013-2016 epidemic in West Africa and the ongoing outbreak in the Democratic Republic of the Congo. EBOV causes hemorrhagic fever, organ damage, and shock culminating in death, with case fatality rates as high as 90%. This high lethality combined with the paucity of licensed medical countermeasures makes EBOV a critical human pathogen. Although EBOV infection results in significant damage to the liver and the adrenal glands, little is known about the molecular signatures of injury in these organs. Moreover, while changes in peripheral blood cells are becoming increasingly understood, the host responses within organs and lymphoid tissues remain poorly characterized. To address this knowledge gap, we tracked longitudinal transcriptional changes in tissues collected from EBOV-Makona-infected cynomolgus macaques. Following infection, both liver and adrenal glands exhibited significant and early downregulation of genes involved in metabolism, coagulation, hormone synthesis, and angiogenesis; upregulated genes were associated with inflammation. Analysis of lymphoid tissues showed early upregulation of genes that play a role in innate immunity and inflammation and downregulation of genes associated with cell cycle and adaptive immunity. Moreover, transient activation of innate immune responses and downregulation of humoral immune responses in lymphoid tissues were confirmed with flow cytometry. Together, these data suggest that the liver, adrenal gland, and lymphatic organs are important sites of EBOV infection and that dysregulating the function of these vital organs contributes to the development of Ebola virus disease. Ebola virus (EBOV) remains a high-priority pathogen since it continues to cause outbreaks with high case fatality rates. Although it is well established that EBOV results in severe organ damage, our understanding of tissue injury in the liver, adrenal glands, and lymphoid tissues remains limited. We begin to address this knowledge gap by conducting longitudinal gene expression studies in these tissues, which were collected from EBOV-infected cynomolgus macaques. We report robust and early gene expression changes within these tissues, indicating they are primary sites of EBOV infection. Furthermore, genes involved in metabolism, coagulation, and adaptive immunity were downregulated, while inflammation-related genes were upregulated. These results indicate significant tissue damage consistent with the development of hemorrhagic fever and lymphopenia. Our study provides novel insight into EBOV-host interactions and elucidates how host responses within the liver, adrenal glands, and lymphoid tissues contribute to EBOV pathogenesis.
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http://dx.doi.org/10.1128/JVI.00250-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269430PMC
May 2020

Immune correlates of postexposure vaccine protection against Marburg virus.

Sci Rep 2020 02 20;10(1):3071. Epub 2020 Feb 20.

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, 77555, USA.

Postexposure immunization can prevent disease and reduce transmission following pathogen exposure. The rapid immunostimulatory properties of recombinant vesicular stomatitis virus (rVSV)-based vaccines make them suitable postexposure treatments against the filoviruses Ebola virus and Marburg virus (MARV); however, the mechanisms that drive this protection are undefined. Previously, we reported 60-75% survival of rhesus macaques treated with rVSV vectors expressing MARV glycoprotein (GP) 20-30 minutes after a low dose exposure to the most pathogenic variant of MARV, Angola. Survival in this model was linked to production of GP-specific antibodies and lower viral load. To confirm these results and potentially identify novel correlates of postexposure protection, we performed a similar experiment, but analyzed plasma cytokine levels, frequencies of immune cell subsets, and the transcriptional response to infection in peripheral blood. In surviving macaques (80-89%), we observed induction of genes mapping to antiviral and interferon-related pathways early after treatment and a higher percentage of T helper 1 (Th1) and NK cells. In contrast, the response of non-surviving macaques was characterized by hypercytokinemia; a T helper 2 signature; recruitment of low HLA-DR expressing monocytes and regulatory T-cells; and transcription of immune checkpoint (e.g., PD-1, LAG3) genes. These results suggest dysregulated immunoregulation is associated with poor prognosis, whereas early innate signaling and Th1-skewed immunity are important for survival.
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http://dx.doi.org/10.1038/s41598-020-59976-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033120PMC
February 2020

Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization.

Immunity 2020 02 4;52(2):388-403.e12. Epub 2020 Feb 4.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.
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http://dx.doi.org/10.1016/j.immuni.2020.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111202PMC
February 2020

Resistance of Cynomolgus Monkeys to Nipah and Hendra Virus Disease Is Associated With Cell-Mediated and Humoral Immunity.

J Infect Dis 2020 05;221(Suppl 4):S436-S447

Galveston National Laboratory, University of Texas Medical Branch, Galveston.

Background: The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are capable of causing severe and often lethal respiratory and/or neurologic disease in animals and humans. Given the sporadic nature of henipavirus outbreaks, licensure of vaccines and therapeutics for human use will likely require demonstration of efficacy in animal models that faithfully reproduce the human condition. Currently, the African green monkey (AGM) best mimics human henipavirus-induced disease.

Methods: The pathogenic potential of HeV and both strains of NiV (Malaysia, Bangladesh) was assessed in cynomolgus monkeys and compared with henipavirus-infected historical control AGMs. Multiplex gene and protein expression assays were used to compare host responses.

Results: In contrast to AGMs, in which henipaviruses cause severe and usually lethal disease, HeV and NiVs caused only mild or asymptomatic infections in macaques. All henipaviruses replicated in macaques with similar kinetics as in AGMs. Infection in macaques was associated with activation and predicted recruitment of cytotoxic CD8+ T cells, Th1 cells, IgM+ B cells, and plasma cells. Conversely, fatal outcome in AGMs was associated with aberrant innate immune signaling, complement dysregulation, Th2 skewing, and increased secretion of MCP-1.

Conclusion: The restriction factors identified in macaques can be harnessed for development of effective countermeasures against henipavirus disease.
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http://dx.doi.org/10.1093/infdis/jiz613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213570PMC
May 2020

Structure and Characterization of Crimean-Congo Hemorrhagic Fever Virus GP38.

J Virol 2020 03 31;94(8). Epub 2020 Mar 31.

Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here, we present the crystal structure of GP38 at a resolution of 2.5 Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a β-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn (a structural glycoprotein in CCHFV), suggestive of a gene duplication event. Analysis of convalescent-phase sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with subnanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle. Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function.
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http://dx.doi.org/10.1128/JVI.02005-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108853PMC
March 2020

Rational design of universal immunotherapy for TfR1-tropic arenaviruses.

Nat Commun 2020 01 3;11(1):67. Epub 2020 Jan 3.

Department of Structural Biology, Weizmann Institute of Science, Rehovot, 7610001, Israel.

Certain arenaviruses that circulate in rodent populations can cause life-threatening hemorrhagic fevers when they infect humans. Due to their efficient transmission, arenaviruses pose a severe risk for outbreaks and might be exploited as biological weapons. Effective countermeasures against these viruses are highly desired. Ideally, a single remedy would be effective against many or even all the pathogenic viruses in this family. However, despite the fact that all pathogenic arenaviruses from South America utilize transferrin receptor 1 (TfR1) as a cellular receptor, their viral glycoproteins are highly diversified, impeding efforts to isolate cross-neutralizing antibodies. Here we address this problem using a rational design approach to target TfR1-tropic arenaviruses with high potency and breadth. The pan-reactive molecule is highly effective against all arenaviruses that were tested, offering a universal therapeutic approach. Our design scheme avoids the shortcomings of previous immunoadhesins and can be used to combat other zoonotic pathogens.
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http://dx.doi.org/10.1038/s41467-019-13924-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941993PMC
January 2020