Publications by authors named "Daved H Fremont"

165 Publications

An intranasal vaccine durably protects against SARS-CoV-2 variants in mice.

Cell Rep 2021 Jul 10:109452. Epub 2021 Jul 10.

Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA; The Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

SARS-CoV-2 variants that attenuate antibody neutralization could jeopardize vaccine efficacy. We recently reported the protective activity of an intranasally administered spike protein-based chimpanzee adenovirus-vectored vaccine (ChAd-SARS-CoV-2-S) in animals, which has advanced to human trials. Here, we assessed its durability, dose response, and cross-protective activity in mice. A single intranasal dose of ChAd-SARS-CoV-2-S induced durably high neutralizing and Fc effector antibody responses in serum and S-specific IgG and IgA secreting long-lived plasma cells in the bone marrow. Protection against a historical SARS-CoV-2 strain was observed across a 100-fold vaccine dose range and over a 200-day period. At 6 weeks or 9 months after vaccination, serum antibodies neutralized SARS-CoV-2 strains with B.1.351, B.1.1.28, and B.1.617.1 spike proteins and conferred almost complete protection in the upper and lower respiratory tracts after challenge with variant viruses. Thus, in mice, intranasal immunization with ChAd-SARS-CoV-2-S provides durable protection against historical and emerging SARS-CoV-2 strains.
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http://dx.doi.org/10.1016/j.celrep.2021.109452DOI Listing
July 2021

A single intranasal or intramuscular immunization with chimpanzee adenovirus-vectored SARS-CoV-2 vaccine protects against pneumonia in hamsters.

Cell Rep 2021 Jul 29;36(3):109400. Epub 2021 Jun 29.

Department of Internal Medicine, Washington University in Saint Louis School of Medicine, St. Louis, MO 63110, USA; Department of Molecular Microbiology and Microbial Pathogenesis, Washington University in Saint Louis School of Medicine, St. Louis, MO 63110, USA; Department of Pathology and Immunology, Washington University in Saint Louis School of Medicine, St. Louis, MO 63110, USA. Electronic address:

The development of an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), is a global priority. Here, we compare the protective capacity of intranasal and intramuscular delivery of a chimpanzee adenovirus-vectored vaccine encoding a prefusion stabilized spike protein (chimpanzee adenovirus [ChAd]-SARS-CoV-2-S) in Golden Syrian hamsters. Although immunization with ChAd-SARS-CoV-2-S induces robust spike-protein-specific antibodies capable of neutralizing the virus, antibody levels in serum are higher in hamsters vaccinated by an intranasal compared to intramuscular route. Accordingly, against challenge with SARS-CoV-2, ChAd-SARS-CoV-2-S-immunized hamsters are protected against less weight loss and have reduced viral infection in nasal swabs and lungs, and reduced pathology and inflammatory gene expression in the lungs, compared to ChAd-control immunized hamsters. Intranasal immunization with ChAd-SARS-CoV-2-S provides superior protection against SARS-CoV-2 infection and inflammation in the upper respiratory tract. These findings support intranasal administration of the ChAd-SARS-CoV-2-S candidate vaccine to prevent SARS-CoV-2 infection, disease, and possibly transmission.
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http://dx.doi.org/10.1016/j.celrep.2021.109400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238649PMC
July 2021

In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains.

Nature 2021 Jun 21. Epub 2021 Jun 21.

Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN, USA.

Rapidly emerging SARS-CoV-2 variants jeopardize antibody-based countermeasures. Although cell culture experiments have demonstrated a loss of potency of several anti-spike neutralizing antibodies against variant strains of SARS-CoV-2, the in vivo importance of these results remains uncertain. Here we report the in vitro and in vivo activity of a panel of monoclonal antibodies (mAbs), which correspond to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron and Lilly, against SARS-CoV-2 variant viruses. Although some individual mAbs showed reduced or abrogated neutralizing activity in cell culture against B.1.351, B.1.1.28, B.1.617.1 and B.1.526 viruses with mutations at residue E484 of the spike protein, low prophylactic doses of mAb combinations protected against infection by many variants in K18-hACE2 transgenic mice, 129S2 immunocompetent mice and hamsters, without the emergence of resistance. Exceptions were LY-CoV555 monotherapy and LY-CoV555 and LY-CoV016 combination therapy, both of which lost all protective activity, and the combination of AbbVie 2B04 and 47D11, which showed a partial loss of activity. When administered after infection, higher doses of several mAb cocktails protected in vivo against viruses with a B.1.351 spike gene. Therefore, many-but not all-of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing variant strains of SARS-CoV-2.
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http://dx.doi.org/10.1038/s41586-021-03720-yDOI Listing
June 2021

Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets.

Immunity 2021 06 6;54(6):1290-1303.e7. Epub 2021 May 6.

Department of Medicine, Washington University School of Medicine, St Louis, MO 63130, USA.

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we used single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic human coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.
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http://dx.doi.org/10.1016/j.immuni.2021.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8101792PMC
June 2021

Broadly neutralizing monoclonal antibodies protect against multiple tick-borne flaviviruses.

J Exp Med 2021 May;218(5)

Department of Medicine, Washington University School of Medicine, St. Louis, MO.

Although Powassan virus (POWV) is an emerging tick-transmitted flavivirus that causes severe or fatal neuroinvasive disease in humans, medical countermeasures have not yet been developed. Here, we developed a panel of neutralizing anti-POWV mAbs recognizing six distinct antigenic sites. The most potent of these mAbs bind sites within domain II or III of the envelope (E) protein and inhibit postattachment viral entry steps. A subset of these mAbs cross-react with other flaviviruses. Both POWV type-specific and cross-reactive neutralizing mAbs confer protection in mice against POWV infection when given as prophylaxis or postexposure therapy. Several cross-reactive mAbs mapping to either domain II or III also protect in vivo against heterologous tick-transmitted flaviviruses including Langat and tick-borne encephalitis virus. Our experiments define structural and functional correlates of antibody protection against POWV infection and identify epitopes targeted by broadly neutralizing antibodies with therapeutic potential against multiple tick-borne flaviviruses.
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http://dx.doi.org/10.1084/jem.20210174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040518PMC
May 2021

The mechanistic basis of protection by non-neutralizing anti-alphavirus antibodies.

Cell Rep 2021 Apr;35(1):108962

Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA; The Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine. St. Louis, MO 63110, USA. Electronic address:

Although neutralizing monoclonal antibodies (mAbs) against epitopes within the alphavirus E2 protein can protect against infection, the functional significance of non-neutralizing mAbs is poorly understood. Here, we evaluate the activity of 13 non-neutralizing mAbs against Mayaro virus (MAYV), an emerging arthritogenic alphavirus. These mAbs bind to the MAYV virion and surface of infected cells but fail to neutralize infection in cell culture. Mapping studies identify six mAb binding groups that localize to discrete epitopes within or adjacent to the A domain of the E2 glycoprotein. Remarkably, passive transfer of non-neutralizing mAbs protects against MAYV infection and disease in mice, and their efficacy requires Fc effector functions. Monocytes mediate the protection of non-neutralizing mAbs in vivo, as Fcγ-receptor-expressing myeloid cells facilitate the binding, uptake, and clearance of MAYV without antibody-dependent enhancement of infection. Humoral protection against alphaviruses likely reflects contributions from non-neutralizing antibodies through Fc-dependent mechanisms that accelerate viral clearance.
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http://dx.doi.org/10.1016/j.celrep.2021.108962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055377PMC
April 2021

Potent neutralization of Rift Valley fever virus by human monoclonal antibodies through fusion inhibition.

Proc Natl Acad Sci U S A 2021 Apr;118(14)

Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232;

Rift Valley fever virus (RVFV), an emerging arboviral and zoonotic bunyavirus, causes severe disease in livestock and humans. Here, we report the isolation of a panel of monoclonal antibodies (mAbs) from the B cells of immune individuals following natural infection in Kenya or immunization with MP-12 vaccine. The B cell responses of individuals who were vaccinated or naturally infected recognized similar epitopes on both Gc and Gn proteins. The Gn-specific mAbs and two mAbs that do not recognize either monomeric Gc or Gn alone but recognized the hetero-oligomer glycoprotein complex (Gc+Gn) when Gc and Gn were coexpressed exhibited potent neutralizing activities in vitro, while Gc-specific mAbs exhibited relatively lower neutralizing capacity. The two Gc+Gn-specific mAbs and the Gn domain A-specific mAbs inhibited RVFV fusion to cells, suggesting that mAbs can inhibit the exposure of the fusion loop in Gc, a class II fusion protein, and thus prevent fusion by an indirect mechanism without direct fusion loop contact. Competition-binding analysis with coexpressed Gc/Gn and mutagenesis library screening indicated that these mAbs recognize four major antigenic sites, with two sites of vulnerability for neutralization on Gn. In experimental models of infection in mice, representative mAbs recognizing three of the antigenic sites reduced morbidity and mortality when used at a low dose in both prophylactic and therapeutic settings. This study identifies multiple candidate mAbs that may be suitable for use in humans against RVFV infection and highlights fusion inhibition against bunyaviruses as a potential contributor to potent antibody-mediated neutralization.
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http://dx.doi.org/10.1073/pnas.2025642118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040655PMC
April 2021

A single intranasal dose of chimpanzee adenovirus-vectored vaccine protects against SARS-CoV-2 infection in rhesus macaques.

Cell Rep Med 2021 Apr 18;2(4):100230. Epub 2021 Mar 18.

Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.

The deployment of a vaccine that limits transmission and disease likely will be required to end the coronavirus disease 2019 (COVID-19) pandemic. We recently described the protective activity of an intranasally administered chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike (S) protein (ChAd-SARS-CoV-2-S [chimpanzee adenovirus-severe acute respiratory syndrome-coronavirus-2-S]) in the upper and lower respiratory tracts of mice expressing the human angiotensin-converting enzyme 2 (ACE2) receptor. Here, we show the immunogenicity and protective efficacy of this vaccine in non-human primates. Rhesus macaques were immunized with ChAd-Control or ChAd-SARS-CoV-2-S and challenged 1 month later by combined intranasal and intrabronchial routes with SARS-CoV-2. A single intranasal dose of ChAd-SARS-CoV-2-S induces neutralizing antibodies and T cell responses and limits or prevents infection in the upper and lower respiratory tracts after SARS-CoV-2 challenge. As ChAd-SARS-CoV-2-S confers protection in non-human primates, it is a promising candidate for limiting SARS-CoV-2 infection and transmission in humans.
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http://dx.doi.org/10.1016/j.xcrm.2021.100230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969912PMC
April 2021

Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies.

Nat Med 2021 04 4;27(4):717-726. Epub 2021 Mar 4.

Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic. Rapidly spreading SARS-CoV-2 variants may jeopardize newly introduced antibody and vaccine countermeasures. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera and human sera from recipients of the BNT162b2 mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, chimeric strains with South African or Brazilian spike genes and isogenic recombinant viral variants. Many highly neutralizing mAbs engaging the receptor-binding domain or N-terminal domain and most convalescent sera and mRNA vaccine-induced immune sera showed reduced inhibitory activity against viruses containing an E484K spike mutation. As antibodies binding to spike receptor-binding domain and N-terminal domain demonstrate diminished neutralization potency in vitro against some emerging variants, updated mAb cocktails targeting highly conserved regions, enhancement of mAb potency or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.
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http://dx.doi.org/10.1038/s41591-021-01294-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058618PMC
April 2021

Dual role of endothelial in tumor angiogenesis and tumor immunity.

Sci Transl Med 2021 03;13(583)

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110-1093, USA.

The cross-talk between angiogenesis and immunity within the tumor microenvironment (TME) is critical for tumor prognosis. While pro-angiogenic and immunosuppressive TME promote tumor growth, anti-angiogenic and immune stimulatory TME inhibit tumor progression. Therefore, there is a great interest in achieving vascular normalization to improve drug delivery and enhance antitumor immunity. However, anti-vascular endothelial growth factor (VEGF) mechanisms to normalize tumor vessels have offered limited therapeutic efficacies for patients with cancer. Here, we report that , a direct target of ETV2, was nearly exclusively expressed in endothelial cells. In preclinical mouse tumor models, deficiency reduced angiogenesis, enhanced high endothelial venule formation, and promoted antitumor immunity, leading to restricted tumor progression. Analysis of The Cancer Genome Atlas (TCGA) datasets revealed a significant ( < 0.05) correlation between expression, angiogenesis, and antitumor immunity in human cancers, as suggested by decreased expression and increased antitumor macrophages in patients with low expression. Mechanistically, MYCT1 interacted with tight junction protein Zona Occludens 1 and regulated Rho GTPase-mediated actin cytoskeleton dynamics, thereby promoting endothelial motility in the angiogenic environment. -deficient endothelial cells facilitated trans-endothelial migration of cytotoxic T lymphocytes and polarization of M1 macrophages. targeting combined with anti-PD1 treatment significantly ( < 0.05) increased complete tumor regression and long-term survival in anti-PD1-responsive and -refractory tumor models in mice. Our data collectively support a critical role for in controlling tumor angiogenesis and reprogramming tumor immunity. -targeted vascular control, in combination with immunotherapy, may become an exciting therapeutic strategy.
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http://dx.doi.org/10.1126/scitranslmed.abb6731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8252962PMC
March 2021

Seroprevalence of SARS-CoV-2 Antibodies in Children and Adults in St. Louis, Missouri, USA.

mSphere 2021 02 3;6(1). Epub 2021 Feb 3.

Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri, USA

Reported coronavirus disease 2019 (COVID-19) case counts likely underestimate the true prevalence because mild or asymptomatic cases often go untested. Here, we use a sero-survey to estimate the seroprevalence of IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the St. Louis, MO, metropolitan area in a symptom-independent manner. Five hundred three adult and 555 pediatric serum/plasma samples were collected from patients presenting to Barnes-Jewish Hospital or St. Louis Children's Hospital between 14 April 2020 and 12 May 2020. We developed protocols for in-house enzyme-linked immunosorbent assays (ELISAs) using spike and nucleoprotein and used the assays to estimate a seroprevalence rate based on our samples. Overall IgG seropositivity was estimated to be 1.71% (95% credible interval [CI], 0.04% to 3.38%) in pediatric samples and 3.11% (95% CI, 0.92% to 5.32%) in adult samples. Seropositivity was significantly lower in children under 5 years of age than in adults, but rates between adults and children aged 5 or older were similar. Of the 176 samples tested from children under 4 years of age, none were positive. This study determined the percentages of both children and adult samples from the greater St. Louis metropolitan area who had antibodies to SARS-CoV-2 in late April to early May 2020. Approximately 1.7 to 3.1% of the tested individuals had antibodies, indicating that they had previously been infected by SARS-CoV-2. These results demonstrate that the extent of infection was about 10 times greater than the number of confirmed cases at that time. Furthermore, it demonstrated that by 5 years of age, children were infected to an extent similar to that of adults.
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http://dx.doi.org/10.1128/mSphere.01207-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7860990PMC
February 2021

Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization.

Cell Host Microbe 2021 03 27;29(3):477-488.e4. Epub 2021 Jan 27.

Department of Molecular Microbiology, School of Medicine, Washington University in St. Louis, St. Louis, MO, USA. Electronic address:

Neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of COVID-19 vaccines and have received emergency use authorization as therapeutics. However, viral escape mutants could compromise efficacy. To define immune-selected mutations in the S protein, we exposed a VSV-eGFP-SARS-CoV-2-S chimeric virus, in which the VSV glycoprotein is replaced with the S protein, to 19 neutralizing monoclonal antibodies (mAbs) against the receptor-binding domain (RBD) and generated 50 different escape mutants. Each mAb had a unique resistance profile, although many shared residues within an epitope of the RBD. Some variants (e.g., S477N) were resistant to neutralization by multiple mAbs, whereas others (e.g., E484K) escaped neutralization by convalescent sera. Additionally, sequential selection identified mutants that escape neutralization by antibody cocktails. Comparing these antibody-mediated mutations with sequence variation in circulating SARS-CoV-2 revealed substitutions that may attenuate neutralizing immune responses in some humans and thus warrant further investigation.
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http://dx.doi.org/10.1016/j.chom.2021.01.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839837PMC
March 2021

A single intranasal dose of chimpanzee adenovirus-vectored vaccine protects against SARS-CoV-2 infection in rhesus macaques.

bioRxiv 2021 Jan 26. Epub 2021 Jan 26.

The deployment of a vaccine that limits transmission and disease likely will be required to end the Coronavirus Disease 2019 (COVID-19) pandemic. We recently described the protective activity of an intranasally-administered chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike (S) protein (ChAd-SARS-CoV-2-S) in the upper and lower respiratory tract of mice expressing the human angiotensin-converting enzyme 2 (ACE2) receptor. Here, we show the immunogenicity and protective efficacy of this vaccine in non-human primates. Rhesus macaques were immunized with ChAd-Control or ChAd-SARS-CoV-2-S and challenged one month later by combined intranasal and intrabronchial routes with SARS-CoV-2. A single intranasal dose of ChAd-SARS-CoV-2-S induced neutralizing antibodies and T cell responses and limited or prevented infection in the upper and lower respiratory tract after SARS-CoV-2 challenge. As this single intranasal dose vaccine confers protection against SARS-CoV-2 in non-human primates, it is a promising candidate for limiting SARS-CoV-2 infection and transmission in humans.
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http://dx.doi.org/10.1101/2021.01.26.428251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852261PMC
January 2021

A host receptor enables type 1 pilus-mediated pathogenesis of Escherichia coli pyelonephritis.

PLoS Pathog 2021 01 29;17(1):e1009314. Epub 2021 Jan 29.

Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, United States of America.

Type 1 pili have long been considered the major virulence factor enabling colonization of the urinary bladder by uropathogenic Escherichia coli (UPEC). The molecular pathogenesis of pyelonephritis is less well characterized, due to previous limitations in preclinical modeling of kidney infection. Here, we demonstrate in a recently developed mouse model that beyond bladder infection, type 1 pili also are critical for establishment of ascending pyelonephritis. Bacterial mutants lacking the type 1 pilus adhesin (FimH) were unable to establish kidney infection in male C3H/HeN mice. We developed an in vitro model of FimH-dependent UPEC binding to renal collecting duct cells, and performed a CRISPR screen in these cells, identifying desmoglein-2 as a primary renal epithelial receptor for FimH. The mannosylated extracellular domain of human DSG2 bound directly to the lectin domain of FimH in vitro, and introduction of a mutation in the FimH mannose-binding pocket abolished binding to DSG2. In infected C3H/HeN mice, type 1-piliated UPEC and Dsg2 were co-localized within collecting ducts, and administration of mannoside FIM1033, a potent small-molecule inhibitor of FimH, significantly attenuated bacterial loads in pyelonephritis. Our results broaden the biological importance of FimH, specify the first renal FimH receptor, and indicate that FimH-targeted therapeutics will also have application in pyelonephritis.
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http://dx.doi.org/10.1371/journal.ppat.1009314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875428PMC
January 2021

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

Cell Rep 2021 Jan;34(4):108682

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2020.108682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888560PMC
January 2021

SARS-CoV-2 Infection Severity Is Linked to Superior Humoral Immunity against the Spike.

mBio 2021 01 19;12(1). Epub 2021 Jan 19.

Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, Illinois, USA

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity of the antibody response mounted against this novel virus is not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and nonstructural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike. With the ongoing pandemic, it is critical to understand how natural immunity against SARS-CoV-2 and COVID-19 develops. We have identified that subjects with more severe COVID-19 disease mount a more robust and neutralizing antibody response against SARS-CoV-2 spike protein. Subjects who mounted a larger response against the spike also mounted antibody responses against other viral antigens, including the nucleocapsid protein and ORF8. Additionally, this study reveals that subjects with more severe disease mount a larger memory B cell response against the spike. These data suggest that subjects with more severe COVID-19 disease are likely better protected from reinfection with SARS-CoV-2.
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http://dx.doi.org/10.1128/mBio.02940-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845638PMC
January 2021

Landscape analysis of escape variants identifies SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization.

bioRxiv 2020 Nov 8. Epub 2020 Nov 8.

Although neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of most COVID-19 vaccines and being developed as therapeutics, escape mutations could compromise such countermeasures. To define the immune-mediated mutational landscape in S protein, we used a VSV-eGFP-SARS-CoV-2-S chimeric virus and 19 neutralizing monoclonal antibodies (mAbs) against the receptor binding domain (RBD) to generate 48 escape mutants. These variants were mapped onto the RBD structure and evaluated for cross-resistance by convalescent human plasma. Although each mAb had unique resistance profiles, many shared residues within an epitope, as several variants were resistant to multiple mAbs. Remarkably, we identified mutants that escaped neutralization by convalescent human sera, suggesting that some humans induce a narrow repertoire of neutralizing antibodies. By comparing the antibody-mediated mutational landscape in S protein with sequence variation in circulating SARS-CoV-2 strains, we identified single amino acid substitutions that could attenuate neutralizing immune responses in some humans.
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http://dx.doi.org/10.1101/2020.11.06.372037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805447PMC
November 2020

A single intranasal or intramuscular immunization with chimpanzee adenovirus vectored SARS-CoV-2 vaccine protects against pneumonia in hamsters.

bioRxiv 2020 Dec 3. Epub 2020 Dec 3.

The development of an effective vaccine against SARS-CoV-2, the etiologic agent of COVID-19, is a global priority. Here, we compared the protective capacity of intranasal and intramuscular delivery of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in Golden Syrian hamsters. While immunization with ChAd-SARS-CoV-2-S induced robust spike protein specific antibodies capable or neutralizing the virus, antibody levels in serum were higher in hamsters immunized by an intranasal compared to intramuscular route. Accordingly, ChAd-SARS-CoV-2-S immunized hamsters were protected against a challenge with a high dose of SARS-CoV-2. After challenge, ChAd-SARS-CoV-2-S-immunized hamsters had less weight loss and showed reductions in viral RNA and infectious virus titer in both nasal swabs and lungs, and reduced pathology and inflammatory gene expression in the lungs, compared to ChAd-Control immunized hamsters. Intranasal immunization with ChAd-SARS-CoV-2-S provided superior protection against SARS-CoV-2 infection and inflammation in the upper respiratory tract. These findings support intranasal administration of the ChAd-SARS-CoV-2-S candidate vaccine to prevent SARS-CoV-2 infection, disease, and possibly transmission.
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http://dx.doi.org/10.1101/2020.12.02.408823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724659PMC
December 2020

Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion.

Proc Natl Acad Sci U S A 2020 12 25;117(50):32105-32113. Epub 2020 Nov 25.

Department of Molecular Microbiology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110;

Cholesterol 25-hydroxylase () is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.
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http://dx.doi.org/10.1073/pnas.2012197117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749331PMC
December 2020

LDLRAD3 is a receptor for Venezuelan equine encephalitis virus.

Nature 2020 12 18;588(7837):308-314. Epub 2020 Nov 18.

Department of Medicine, Washington University School of Medicine, St Louis, MO, USA.

Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans. VEEV is a biodefence concern because of its potential for aerosol spread and the current lack of sufficient countermeasures. The host factors that are required for VEEV entry and infection remain poorly characterized. Here, using a genome-wide CRISPR-Cas9-based screen, we identify low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3)-a highly conserved yet poorly characterized member of the scavenger receptor superfamily-as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation with LDLRAD3. LDLRAD3 binds directly to VEEV particles and enhances virus attachment and internalization into host cells. Genetic studies indicate that domain 1 of LDLRAD3 (LDLRAD3(D1)) is necessary and sufficient to support infection by VEEV, and both anti-LDLRAD3 antibodies and an LDLRAD3(D1)-Fc fusion protein block VEEV infection in cell culture. The pathogenesis of VEEV infection is abrogated in mice with deletions in Ldlrad3, and administration of LDLRAD3(D1)-Fc abolishes disease caused by several subtypes of VEEV, including highly virulent strains. The development of a decoy-receptor fusion protein suggests a strategy for the prevention of severe VEEV infection and associated disease in humans.
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http://dx.doi.org/10.1038/s41586-020-2915-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769003PMC
December 2020

Requisite Chromatin Remodeling for Myeloid and Erythroid Lineage Differentiation from Erythromyeloid Progenitors.

Cell Rep 2020 11;33(7):108395

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA; Graduate School of Biotechnology, Kyung Hee University, Yong In, Korea. Electronic address:

The mammalian SWitch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling BAF (BRG1/BRM-associated factor) complex plays an essential role in developmental and pathological processes. We show that the deletion of Baf155, which encodes a subunit of the BAF complex, in the Tie2(+) lineage (Baf155 (CKO) leads to defects in yolk sac myeloid and definitive erythroid (EryD) lineage differentiation from erythromyeloid progenitors (EMPs). The chromatin of myeloid gene loci in Baf155 CKO EMPs is mostly inaccessible and enriched mainly by the ETS binding motif. BAF155 interacts with PU.1 and is recruited to PU.1 target gene loci together with p300 and KDM6a. Treatment of Baf155 CKO embryos with GSK126, an H3K27me2/3 methyltransferase EZH2 inhibitor, rescues myeloid lineage gene expression. This study uncovers indispensable BAF-mediated chromatin remodeling of myeloid gene loci at the EMP stage. Future studies exploiting epigenetics in the generation and application of EMP derivatives for tissue repair, regeneration, and disease are warranted.
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http://dx.doi.org/10.1016/j.celrep.2020.108395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694876PMC
November 2020

A molecular understanding of alphavirus entry.

PLoS Pathog 2020 10 22;16(10):e1008876. Epub 2020 Oct 22.

Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America.

Alphaviruses cause severe human illnesses including persistent arthritis and fatal encephalitis. As alphavirus entry into target cells is the first step in infection, intensive research efforts have focused on elucidating aspects of this pathway, including attachment, internalization, and fusion. Herein, we review recent developments in the molecular understanding of alphavirus entry both in vitro and in vivo and how these advances might enable the design of therapeutics targeting this critical step in the alphavirus life cycle.
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http://dx.doi.org/10.1371/journal.ppat.1008876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580943PMC
October 2020

Affinity-Restricted Memory B Cells Dominate Recall Responses to Heterologous Flaviviruses.

Immunity 2020 11 2;53(5):1078-1094.e7. Epub 2020 Oct 2.

Department of Immunobiology, University of Arizona, Tucson, AZ 85724, USA. Electronic address:

Memory B cells (MBCs) can respond to heterologous antigens either by molding new specificities through secondary germinal centers (GCs) or by selecting preexisting clones without further affinity maturation. To distinguish these mechanisms in flavivirus infections and immunizations, we studied recall responses to envelope protein domain III (DIII). Conditional deletion of activation-induced cytidine deaminase (AID) between heterologous challenges of West Nile, Japanese encephalitis, Zika, and dengue viruses did not affect recall responses. DIII-specific MBCs were contained mostly within the plasma-cell-biased CD80 subset, and few GCs arose following heterologous boosters, demonstrating that recall responses are confined by preexisting clonal diversity. Measurement of monoclonal antibody (mAb) binding affinity to DIII proteins, timed AID deletion, single-cell RNA sequencing, and lineage tracing experiments point to selection of relatively low-affinity MBCs as a mechanism to promote diversity. Engineering immunogens to avoid this MBC diversity may facilitate flavivirus-type-specific vaccines with minimized potential for infection enhancement.
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http://dx.doi.org/10.1016/j.immuni.2020.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677180PMC
November 2020

Human Antibodies Targeting Influenza B Virus Neuraminidase Active Site Are Broadly Protective.

Immunity 2020 10 24;53(4):852-863.e7. Epub 2020 Sep 24.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA; The Andrew M. and Jane M. Bursky Center for Human Immunology & Immunotherapy Programs, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus in vitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.
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http://dx.doi.org/10.1016/j.immuni.2020.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572813PMC
October 2020

SARS-CoV-2 infection severity is linked to superior humoral immunity against the spike.

bioRxiv 2020 Sep 13. Epub 2020 Sep 13.

Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, IL 60637, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity and kinetics of the antibody response mounted against this novel virus are not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and non-structural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.
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http://dx.doi.org/10.1101/2020.09.12.294066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491512PMC
September 2020

A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower Respiratory Tracts against SARS-CoV-2.

Cell 2020 10 19;183(1):169-184.e13. Epub 2020 Aug 19.

Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63110, USA; The Andrew M. and Jane M. Bursky Center for Human Immunology & Immunotherapy Programs, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

The coronavirus disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a prefusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of neutralizing antibodies, promotes systemic and mucosal immunoglobulin A (IgA) and T cell responses, and almost entirely prevents SARS-CoV-2 infection in both the upper and lower respiratory tracts. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission and curtailing pandemic spread.
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http://dx.doi.org/10.1016/j.cell.2020.08.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437481PMC
October 2020

Replication-Competent Vesicular Stomatitis Virus Vaccine Vector Protects against SARS-CoV-2-Mediated Pathogenesis in Mice.

Cell Host Microbe 2020 09 30;28(3):465-474.e4. Epub 2020 Jul 30.

Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA; Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA; Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO, USA; The Andrew M. and Jane M. Bursky Center for Human Immunology & Immunotherapy Programs, Washington University School of Medicine, St. Louis, MO, USA. Electronic address:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections, and an effective vaccine is critical to mitigate coronavirus-induced disease 2019 (COVID-19). Previously, we developed a replication-competent vesicular stomatitis virus (VSV) expressing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Here, we show that vaccination with VSV-eGFP-SARS-CoV-2 generates neutralizing immune responses and protects mice from SARS-CoV-2. Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high antibody titers that neutralize SARS-CoV-2 and target the receptor binding domain that engages human angiotensin-converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice that expressed human ACE2 and were immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung, indicating protection against pneumonia. Passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals also protects naive mice from SARS-CoV-2 challenge. These data support development of VSV-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.
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http://dx.doi.org/10.1016/j.chom.2020.07.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391951PMC
September 2020

Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2.

Cell Host Microbe 2020 09 3;28(3):475-485.e5. Epub 2020 Jul 3.

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA. Electronic address:

Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, which engages with host ACE2 receptor for entry. Using an infectious molecular clone of vesicular stomatitis virus (VSV) expressing eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput-imaging-based neutralization assay at biosafety level 2. We also developed a focus-reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. Comparing the neutralizing activities of various antibodies and ACE2-Fc soluble decoy protein in both assays revealed a high degree of concordance. These assays will help define correlates of protection for antibody-based countermeasures and vaccines against SARS-CoV-2. Additionally, replication-competent VSV-eGFP-SARS-CoV-2 provides a tool for testing inhibitors of SARS-CoV-2 mediated entry under reduced biosafety containment.
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http://dx.doi.org/10.1016/j.chom.2020.06.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332453PMC
September 2020

Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2.

SSRN 2020 May 27:3606354. Epub 2020 May 27.

Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA.

Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus as to which assay should be used for such measurements. Using an infectious molecular clone of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally high degree of concordance. The two assays will help define correlates of protection for antibody-based countermeasures including therapeutic antibodies, immune γ-globulin or plasma preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-CoV-2 provides a rapid assay for testing inhibitors of SARS-CoV-2 mediated entry that can be performed in 7.5 hours under reduced biosafety containment. Funding: This study was supported by NIH contracts and grants (75N93019C00062, HHSN272201700060C and R01 AI127828, R37 AI059371 and U01 AI151810) and the Defense Advanced Research Project Agency (HR001117S0019) and gifts from Washington University in Saint Louis. J.B.C. is supported by a Helen Hay Whitney Foundation postdoctoral fellowship. Conflict of Interest: M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and on the Scientific Advisory Board of Moderna. D.C. and H.W.V. are employees of Vir Biotechnology Inc. and may hold shares in Vir Biotechnology Inc. S.P.J.W. and P.W.R. have filed a disclosure with Washington University for the recombinant VSV. Ethical Approval: This study was approved by the Mayo Clinic Institutional Review Board.
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http://dx.doi.org/10.2139/ssrn.3606354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366811PMC
May 2020