Publications by authors named "Gabriel Dagotto"

9 Publications

  • Page 1 of 1

Comparison of Subgenomic and Total RNA in SARS-CoV-2 Challenged Rhesus Macaques.

J Virol 2021 Jan 20. Epub 2021 Jan 20.

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA

Respiratory virus challenge studies involve administration of the challenge virus and sampling to assess for protection from the same anatomical locations. It can therefore be difficult to differentiate actively replicating virus from input challenge virus. For SARS-CoV-2, specific monitoring of actively replicating virus is critical to investigate the protective and therapeutic efficacy of vaccines, monoclonal antibodies, and antiviral drugs. We developed a SARS-CoV-2 subgenomic RNA (sgRNA) RT-PCR assay to differentiate productive infection from inactivated or neutralized virus. Subgenomic RNAs are generated after cell entry and are poorly incorporate into mature virions, and thus may provide a marker for actively replicating virus. We show envelope (E) sgRNA was degraded by RNase in infected cell lysates, while genomic RNA (gRNA) was protected, presumably due to packaging into virions. To investigate the capacity of the sgRNA assay to distinguish input challenge virus from actively replicating virus , we compared the E sgRNA assay to a standard nucleoprotein (N) or E total RNA assay in convalescent rhesus macaques and in antibody-treated rhesus macaques after experimental SARS-CoV-2 challenge. In both studies, the E sgRNA assay was negative, suggesting protective efficacy, whereas the N and E total RNA assays remained positive. These data suggest the potential utility of sgRNA to monitor actively replicating virus in prophylactic and therapeutic SARS-CoV-2 studies.Developing therapeutic and prophylactic countermeasures for the SARS-CoV-2 virus is a public health priority. During challenge studies, respiratory viruses are delivered and sampled from the same anatomical location. It is therefore important to distinguish actively replicating virus from input challenge virus. The most common assay for detecting SARS-CoV-2 virus, reverse transcription polymerase chain reaction (RT-PCR) targeting nucleocapsid total RNA, cannot distinguish neutralized input virus from replicating virus. In this study, we assess SARS-CoV-2 subgenomic RNA as a potential measure of replicating virus in rhesus macaques.
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http://dx.doi.org/10.1128/JVI.02370-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8103707PMC
January 2021

Correlates of protection against SARS-CoV-2 in rhesus macaques.

Nature 2021 02 4;590(7847):630-634. Epub 2020 Dec 4.

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

Recent studies have reported the protective efficacy of both natural and vaccine-induced immunity against challenge with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in rhesus macaques. However, the importance of humoral and cellular immunity for protection against infection with SARS-CoV-2 remains to be determined. Here we show that the adoptive transfer of purified IgG from convalescent rhesus macaques (Macaca mulatta) protects naive recipient macaques against challenge with SARS-CoV-2 in a dose-dependent fashion. Depletion of CD8 T cells in convalescent macaques partially abrogated the protective efficacy of natural immunity against rechallenge with SARS-CoV-2, which suggests a role for cellular immunity in the context of waning or subprotective antibody titres. These data demonstrate that relatively low antibody titres are sufficient for protection against SARS-CoV-2 in rhesus macaques, and that cellular immune responses may contribute to protection if antibody responses are suboptimal. We also show that higher antibody titres are required for treatment of SARS-CoV-2 infection in macaques. These findings have implications for the development of SARS-CoV-2 vaccines and immune-based therapeutic agents.
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http://dx.doi.org/10.1038/s41586-020-03041-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906955PMC
February 2021

Ad26 vaccine protects against SARS-CoV-2 severe clinical disease in hamsters.

Nat Med 2020 11 3;26(11):1694-1700. Epub 2020 Sep 3.

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

Coronavirus disease 2019 (COVID-19) in humans is often a clinically mild illness, but some individuals develop severe pneumonia, respiratory failure and death. Studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in hamsters and nonhuman primates have generally reported mild clinical disease, and preclinical SARS-CoV-2 vaccine studies have demonstrated reduction of viral replication in the upper and lower respiratory tracts in nonhuman primates. Here we show that high-dose intranasal SARS-CoV-2 infection in hamsters results in severe clinical disease, including high levels of virus replication in tissues, extensive pneumonia, weight loss and mortality in a subset of animals. A single immunization with an adenovirus serotype 26 vector-based vaccine expressing a stabilized SARS-CoV-2 spike protein elicited binding and neutralizing antibody responses and protected against SARS-CoV-2-induced weight loss, pneumonia and mortality. These data demonstrate vaccine protection against SARS-CoV-2 clinical disease. This model should prove useful for preclinical studies of SARS-CoV-2 vaccines, therapeutics and pathogenesis.
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http://dx.doi.org/10.1038/s41591-020-1070-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671939PMC
November 2020

Approaches and Challenges in SARS-CoV-2 Vaccine Development.

Cell Host Microbe 2020 09 10;28(3):364-370. Epub 2020 Aug 10.

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA; Harvard Medical School, Boston, MA 02115, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA; Massachusetts Consortium on Pathogen Readiness, Boston, MA 02215, USA. Electronic address:

The explosive spread of SARS-CoV-2 suggests that a vaccine will be required to end this global pandemic. Progress in SARS-CoV-2 vaccine development to date has been faster than for any other pathogen in history. Multiple SARS-CoV-2 vaccine candidates have been evaluated in preclinical models and are currently in clinical trials. In this Perspective, we discuss three topics that are critical for SARS-CoV-2 vaccine development: antigen selection and engineering, preclinical challenge studies in non-human primate models, and immune correlates of protection.
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http://dx.doi.org/10.1016/j.chom.2020.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416703PMC
September 2020

Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques.

Nature 2020 10 30;586(7830):583-588. Epub 2020 Jul 30.

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

A safe and effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be required to end the coronavirus disease 2019 (COVID-19) pandemic. For global deployment and pandemic control, a vaccine that requires only a single immunization would be optimal. Here we show the immunogenicity and protective efficacy of a single dose of adenovirus serotype 26 (Ad26) vector-based vaccines expressing the SARS-CoV-2 spike (S) protein in non-human primates. Fifty-two rhesus macaques (Macaca mulatta) were immunized with Ad26 vectors that encoded S variants or sham control, and then challenged with SARS-CoV-2 by the intranasal and intratracheal routes. The optimal Ad26 vaccine induced robust neutralizing antibody responses and provided complete or near-complete protection in bronchoalveolar lavage and nasal swabs after SARS-CoV-2 challenge. Titres of vaccine-elicited neutralizing antibodies correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate robust single-shot vaccine protection against SARS-CoV-2 in non-human primates. The optimal Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in clinical trials.
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http://dx.doi.org/10.1038/s41586-020-2607-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581548PMC
October 2020

SARS-CoV-2 infection protects against rechallenge in rhesus macaques.

Science 2020 08 20;369(6505):812-817. Epub 2020 May 20.

Janssen Vaccines & Prevention BV, Leiden, Netherlands.

An understanding of protective immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for vaccine and public health strategies aimed at ending the global coronavirus disease 2019 (COVID-19) pandemic. A key unanswered question is whether infection with SARS-CoV-2 results in protective immunity against reexposure. We developed a rhesus macaque model of SARS-CoV-2 infection and observed that macaques had high viral loads in the upper and lower respiratory tract, humoral and cellular immune responses, and pathologic evidence of viral pneumonia. After the initial viral clearance, animals were rechallenged with SARS-CoV-2 and showed 5 log reductions in median viral loads in bronchoalveolar lavage and nasal mucosa compared with after the primary infection. Anamnestic immune responses after rechallenge suggested that protection was mediated by immunologic control. These data show that SARS-CoV-2 infection induced protective immunity against reexposure in nonhuman primates.
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http://dx.doi.org/10.1126/science.abc4776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243369PMC
August 2020

DNA vaccine protection against SARS-CoV-2 in rhesus macaques.

Science 2020 08 20;369(6505):806-811. Epub 2020 May 20.

Bioqual, Rockville, MD 20852, USA.

The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made the development of a vaccine a top biomedical priority. In this study, we developed a series of DNA vaccine candidates expressing different forms of the SARS-CoV-2 spike (S) protein and evaluated them in 35 rhesus macaques. Vaccinated animals developed humoral and cellular immune responses, including neutralizing antibody titers at levels comparable to those found in convalescent humans and macaques infected with SARS-CoV-2. After vaccination, all animals were challenged with SARS-CoV-2, and the vaccine encoding the full-length S protein resulted in >3.1 and >3.7 log reductions in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, as compared with viral loads in sham controls. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates.
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http://dx.doi.org/10.1126/science.abc6284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243363PMC
August 2020

Advancing Functional Genetics Through -Mediated Insertional Mutagenesis and CRISPR/Cas9 in the Commensal and Pathogenic Yeast .

Genetics 2019 08 26;212(4):1163-1179. Epub 2019 Jun 26.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710

encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. species have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study, we applied a recently-developed -mediated transformation protocol to perform transfer (T)-DNA random insertional mutagenesis in A total of 767 transformants were screened for sensitivity to 10 different stresses, and 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within open reading frames of genes, untranslated regions, and intergenic regions. Some T-DNA insertions generated chromosomal rearrangements while others could not be characterized. To validate the findings of our forward genetic screen, a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was developed to generate targeted deletion mutants for two genes identified in the screen: and This system is based on cotransformation of mediated by , to deliver both a -gRNA construct that induces double-strand DNA breaks and a gene replacement allele that serves as a homology-directed repair template. Targeted deletion mutants for both and were readily generated with this method. This study demonstrates the feasibility and reliability of -mediated transformation to aid in the identification of gene functions in , through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion.
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http://dx.doi.org/10.1534/genetics.119.302329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707463PMC
August 2019
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