Publications by authors named "Randy A Albrecht"

92 Publications

Tissue-Based SARS-Cov-2 Detection in Fatal COVID-19 Infections: Sustained Direct Viral-Induced Damage is Not Necessary to Drive Disease Progression.

Hum Pathol 2021 May 4. Epub 2021 May 4.

Departments of Pathology, Molecular and Cell-Based Medicine, The Icahn School of Medicine at Mount Sinai Hospital, New York, NY. Electronic address:

Background: Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although viral infection is known to trigger inflammatory processes contributing to tissue injury and organ failure, it is unclear whether direct viral damage is needed to sustain cellular injury. An understanding of pathogenic mechanisms has been handicapped by the absence of optimized methods to visualize the presence and distribution of SARS-CoV-2 in damaged tissues.

Methods: We first developed a positive control cell line (Vero E6) to validate SARS-CoV-2 detection assays. We then evaluated multiple organs (lungs, kidneys, heart, liver, brain, intestines, lymph nodes and spleen) from fourteen COVID-19 autopsy cases using immunohistochemistry (IHC) for the spike and the nucleoprotein proteins, and RNA in-situ hybridization (RNA ISH) for the spike protein mRNA. Tissue detection assays were compared with quantitative PCR (qPCR)-based detection.

Results: SARS-CoV-2 was histologically detected in the Vero E6 positive cell line control, 1 of 14 (7%) lungs, and none (0%) of the other 59 organs. There was perfect concordance between the IHC and RNA ISH results. qPCR confirmed high viral load in the SARS-CoV-2 ISH-positive lung tissue, and absent or low viral load in all ISH-negative tissues.

Conclusions: In patients who die of COVID-19-related organ failure, SARS-CoV-2 is largely not detectable using tissue-based assays. Even in lungs showing widespread injury, SARS-CoV-2 viral RNA or proteins were detected in only a small minority of cases. This observation supports the concept that viral infection is primarily a trigger for multiple organ pathogenic pro-inflammatory responses. Direct viral tissue damage is a transient phenomenon that is generally not sustained throughout disease progression.
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http://dx.doi.org/10.1016/j.humpath.2021.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8095022PMC
May 2021

A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics.

Nat Biomed Eng 2021 May 3. Epub 2021 May 3.

Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.

The rapid repurposing of antivirals is particularly pressing during pandemics. However, rapid assays for assessing candidate drugs typically involve in vitro screens and cell lines that do not recapitulate human physiology at the tissue and organ levels. Here we show that a microfluidic bronchial-airway-on-a-chip lined by highly differentiated human bronchial-airway epithelium and pulmonary endothelium can model viral infection, strain-dependent virulence, cytokine production and the recruitment of circulating immune cells. In airway chips infected with influenza A, the co-administration of nafamostat with oseltamivir doubled the treatment-time window for oseltamivir. In chips infected with pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant doses of the antimalarial drug amodiaquine inhibited infection but clinical doses of hydroxychloroquine and other antiviral drugs that inhibit the entry of pseudotyped SARS-CoV-2 in cell lines under static conditions did not. We also show that amodiaquine showed substantial prophylactic and therapeutic activities in hamsters challenged with native SARS-CoV-2. The human airway-on-a-chip may accelerate the identification of therapeutics and prophylactics with repurposing potential.
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http://dx.doi.org/10.1038/s41551-021-00718-9DOI Listing
May 2021

TOP1 inhibition therapy protects against SARS-CoV-2-induced lethal inflammation.

Cell 2021 Mar 30. Epub 2021 Mar 30.

Department of Genetics and Genomics, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.
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http://dx.doi.org/10.1016/j.cell.2021.03.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008343PMC
March 2021

Longitudinal Metabolomics of Human Plasma Reveals Robust Prognostic Markers of COVID-19 Disease Severity.

medRxiv 2021 Feb 8. Epub 2021 Feb 8.

There is an urgent need to identify which COVID-19 patients will develop life-threatening illness so that scarce medical resources can be optimally allocated and rapid treatment can be administered early in the disease course, when clinical management is most effective. To aid in the prognostic classification of disease severity, we performed untargeted metabolomics profiling of 341 patients with plasma samples collected at six longitudinal time points. Using the temporal metabolic profiles and machine learning, we then built a predictive model of disease severity. We determined that the levels of 25 metabolites measured at the time of hospital admission successfully predict future disease severity. Through analysis of longitudinal samples, we confirmed that these prognostic markers are directly related to disease progression and that their levels are restored to baseline upon disease recovery. Finally, we validated that these metabolites are also altered in a hamster model of COVID-19. Our results indicate that metabolic changes associated with COVID-19 severity can be effectively used to stratify patients and inform resource allocation during the pandemic.
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http://dx.doi.org/10.1101/2021.02.05.21251173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872388PMC
February 2021

Chimeric Hemagglutinin-Based Live-Attenuated Vaccines Confer Durable Protective Immunity against Influenza A Viruses in a Preclinical Ferret Model.

Vaccines (Basel) 2021 Jan 11;9(1). Epub 2021 Jan 11.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Epidemic or pandemic influenza can annually cause significant morbidity and mortality in humans. We developed novel chimeric hemagglutinin (cHA)-based universal influenza virus vaccines, which contain a conserved HA stalk domain from a 2009 pandemic H1N1 (pH1N1) strain combined with globular head domains from avian influenza A viruses. Our previous reports demonstrated that prime-boost sequential immunizations induced robust antibody responses directed toward the conserved HA stalk domain in ferrets. Herein, we further followed vaccinated animals for one year to compare the efficacy and durability of these vaccines in the preclinical ferret model of influenza. Although all cHA-based immunization regimens induced durable HA stalk-specific and heterosubtypic antibody responses in ferrets, sequential immunization with live-attenuated influenza virus vaccines (LAIV-LAIV) conferred the best protection against upper respiratory tract infection by a pH1N1 influenza A virus. The findings from this study suggest that our sequential immunization strategy for a cHA-based universal influenza virus vaccine provides durable protective humoral and cellular immunity against influenza virus infection.
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http://dx.doi.org/10.3390/vaccines9010040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826668PMC
January 2021

A Newcastle Disease Virus (NDV) Expressing a Membrane-Anchored Spike as a Cost-Effective Inactivated SARS-CoV-2 Vaccine.

Vaccines (Basel) 2020 Dec 17;8(4). Epub 2020 Dec 17.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

A successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine must not only be safe and protective, but must also meet the demand on a global scale at a low cost. Using the current influenza virus vaccine production capacity to manufacture an egg-based inactivated Newcastle disease virus (NDV)/SARS-CoV-2 vaccine would meet that challenge. Here, we report pre-clinical evaluations of an inactivated NDV chimera stably expressing the membrane-anchored form of the spike (NDV-S) as a potent coronavirus disease 2019 (COVID-19) vaccine in mice and hamsters. The inactivated NDV-S vaccine was immunogenic, inducing strong binding and/or neutralizing antibodies in both animal models. More importantly, the inactivated NDV-S vaccine protected animals from SARS-CoV-2 infections. In the presence of an adjuvant, antigen-sparing could be achieved, which would further reduce the cost while maintaining the protective efficacy of the vaccine.
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http://dx.doi.org/10.3390/vaccines8040771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766959PMC
December 2020

Mass Cytometry Defines Virus-Specific CD4 T Cells in Influenza Vaccination.

Immunohorizons 2020 12 11;4(12):774-788. Epub 2020 Dec 11.

Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, CA 94305;

The antiviral response to influenza virus is complex and multifaceted, involving many immune cell subsets. There is an urgent need to understand the role of CD4 T cells, which orchestrate an effective antiviral response, to improve vaccine design strategies. In this study, we analyzed PBMCs from human participants immunized with influenza vaccine, using high-dimensional single-cell proteomic immune profiling by mass cytometry. Data were analyzed using a novel clustering algorithm, denoised ragged pruning, to define possible influenza virus-specific clusters of CD4 T cells. Denoised ragged pruning identified six clusters of cells. Among these, one cluster (Cluster 3) was found to increase in abundance following stimulation with influenza virus peptide ex vivo. A separate cluster (Cluster 4) was found to expand in abundance between days 0 and 7 postvaccination, indicating that it is vaccine responsive. We examined the expression profiles of all six clusters to characterize their lineage, functionality, and possible role in the response to influenza vaccine. Clusters 3 and 4 consisted of effector memory cells, with high CD154 expression. Cluster 3 expressed cytokines like IL-2, IFN-γ, and TNF-α, whereas Cluster 4 expressed IL-17. Interestingly, some participants had low abundance of Clusters 3 and 4, whereas others had higher abundance of one of these clusters compared with the other. Taken together, we present an approach for identifying novel influenza virus-reactive CD4 T cell subsets, a method that could help advance understanding of the immune response to influenza, predict responsiveness to vaccines, and aid in better vaccine design.
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http://dx.doi.org/10.4049/immunohorizons.1900097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891553PMC
December 2020

Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models.

bioRxiv 2020 Dec 1. Epub 2020 Dec 1.

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, and analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.
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http://dx.doi.org/10.1101/2020.12.01.404483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724667PMC
December 2020

Real-Time Investigation of a Large Nosocomial Influenza A Outbreak Informed by Genomic Epidemiology.

Clin Infect Dis 2020 Nov 30. Epub 2020 Nov 30.

Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY.

Background: Nosocomial respiratory virus outbreaks represent serious public health challenges. Rapid and precise identification of cases and tracing of transmission chains is critical to end outbreaks and to inform prevention measures.

Methods: We combined conventional surveillance with influenza A virus (IAV) genome sequencing to identify and contain a large IAV outbreak in a metropolitan healthcare system. A total of 381 individuals, including 91 inpatients and 290 health care workers (HCWs), were included in the investigation.

Results: During a 12-day period in early 2019, infection preventionists identified 89 HCWs and 18 inpatients as cases of influenza-like illness (ILI), using an amended definition without the requirement for fever. Sequencing of IAV genomes from available nasopharyngeal (NP) specimens identified 66 individuals infected with a nearly identical strain of influenza A H1N1pdm09 (43 HCWs, 17 inpatients, and 6 with unspecified affiliation). All HCWs infected with the outbreak strain had received the seasonal influenza virus vaccination. Characterization of five representative outbreak viral isolates did not show antigenic drift. In conjunction with IAV genome sequencing, mining of electronic records pinpointed the origin of the outbreak as a single patient and a few interactions in the emergency department that occurred one day prior to the index ILI cluster.

Conclusions: We used precision surveillance to delineate a large nosocomial IAV outbreak, mapping the source of the outbreak to a single patient rather than HCWs as initially assumed based on conventional epidemiology. These findings have important ramifications for more effective prevention strategies to curb nosocomial respiratory virus outbreaks.
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http://dx.doi.org/10.1093/cid/ciaa1781DOI Listing
November 2020

Animal models for COVID-19.

Nature 2020 10 23;586(7830):509-515. Epub 2020 Sep 23.

Department of Immunology, Center for Vaccine Research, University of Pittsburgh, Pittsburgh, PA, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel coronavirus into humans late in 2019 (first detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the findings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19.
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http://dx.doi.org/10.1038/s41586-020-2787-6DOI Listing
October 2020

A Newcastle disease virus (NDV) expressing membrane-anchored spike as a cost-effective inactivated SARS-CoV-2 vaccine.

bioRxiv 2020 Jul 31. Epub 2020 Jul 31.

A successful SARS-CoV-2 vaccine must be not only safe and protective but must also meet the demand on a global scale at low cost. Using the current influenza virus vaccine production capacity to manufacture an egg-based inactivated Newcastle disease virus (NDV)/SARS-CoV-2 vaccine would meet that challenge. Here, we report pre-clinical evaluations of an inactivated NDV chimera stably expressing the membrane-anchored form of the spike (NDV-S) as a potent COVID-19 vaccine in mice and hamsters. The inactivated NDV-S vaccine was immunogenic, inducing strong binding and/or neutralizing antibodies in both animal models. More importantly, the inactivated NDV-S vaccine protected animals from SARS-CoV-2 infections or significantly attenuated SARS-CoV-2 induced disease. In the presence of an adjuvant, antigen-sparing could be achieved, which would further reduce the cost while maintaining the protective efficacy of the vaccine.
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http://dx.doi.org/10.1101/2020.07.30.229120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402029PMC
July 2020

An In Vitro Microneutralization Assay for SARS-CoV-2 Serology and Drug Screening.

Curr Protoc Microbiol 2020 09;58(1):e108

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the city of Wuhan, Hubei Province, China, in late 2019. Since then, the virus has spread globally and caused a pandemic. Assays that can measure the antiviral activity of antibodies or antiviral compounds are needed for SARS-CoV-2 vaccine and drug development. Here, we describe in detail a microneutralization assay, which can be used to assess in a quantitative manner if antibodies or drugs can block entry and/or replication of SARS-CoV-2 in vitro. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Microneutralization assay to test inhibition of virus by antibodies (purified antibodies or serum/plasma) Basic Protocol 2: Screening of anti-SARS-CoV-2 compounds in vitro Support Protocol: SARS-CoV-2 propagation.
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http://dx.doi.org/10.1002/cpmc.108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7361222PMC
September 2020

Author Correction: Microbiome disturbance and resilience dynamics of the upper respiratory tract during influenza A virus infection.

Nat Commun 2020 06 16;11(1):3132. Epub 2020 Jun 16.

Departmento de Enfermedades Infecciosas e Inmunología Pediátrica, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-17020-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298031PMC
June 2020

Microbiome disturbance and resilience dynamics of the upper respiratory tract during influenza A virus infection.

Nat Commun 2020 05 21;11(1):2537. Epub 2020 May 21.

Departmento de Enfermedades Infecciosas e Inmunología Pediátrica, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile.

Infection with influenza can be aggravated by bacterial co-infections, which often results in disease exacerbation. The effects of influenza infection on the upper respiratory tract (URT) microbiome are largely unknown. Here, we report a longitudinal study to assess the temporal dynamics of the URT microbiomes of uninfected and influenza virus-infected humans and ferrets. Uninfected human patients and ferret URT microbiomes have stable healthy ecostate communities both within and between individuals. In contrast, infected patients and ferrets exhibit large changes in bacterial community composition over time and between individuals. The unhealthy ecostates of infected individuals progress towards the healthy ecostate, coinciding with viral clearance and recovery. Pseudomonadales associate statistically with the disturbed microbiomes of infected individuals. The dynamic and resilient microbiome during influenza virus infection in multiple hosts provides a compelling rationale for the maintenance of the microbiome homeostasis as a potential therapeutic target to prevent IAV associated bacterial co-infections.
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http://dx.doi.org/10.1038/s41467-020-16429-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242466PMC
May 2020

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19.

Cell 2020 05 15;181(5):1036-1045.e9. Epub 2020 May 15.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Virus Engineering Center for Therapeutics and Research, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. Electronic address:

Viral pandemics, such as the one caused by SARS-CoV-2, pose an imminent threat to humanity. Because of its recent emergence, there is a paucity of information regarding viral behavior and host response following SARS-CoV-2 infection. Here we offer an in-depth analysis of the transcriptional response to SARS-CoV-2 compared with other respiratory viruses. Cell and animal models of SARS-CoV-2 infection, in addition to transcriptional and serum profiling of COVID-19 patients, consistently revealed a unique and inappropriate inflammatory response. This response is defined by low levels of type I and III interferons juxtaposed to elevated chemokines and high expression of IL-6. We propose that reduced innate antiviral defenses coupled with exuberant inflammatory cytokine production are the defining and driving features of COVID-19.
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http://dx.doi.org/10.1016/j.cell.2020.04.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227586PMC
May 2020

Pandemic influenza virus vaccines boost hemagglutinin stalk-specific antibody responses in primed adult and pediatric cohorts.

NPJ Vaccines 2019 6;4:51. Epub 2019 Dec 6.

9GSK, Rixensart, Belgium.

Licensed influenza virus vaccines target the head domain of the hemagglutinin (HA) glycoprotein which undergoes constant antigenic drift. The highly conserved HA stalk domain is an attractive target to increase immunologic breadth required for universal influenza virus vaccines. We tested the hypothesis that immunization with a pandemic influenza virus vaccine boosts pre-existing anti-stalk antibodies. We used chimeric cH6/1, full length H2 and H18 HA antigens in an ELISA to measure anti-stalk antibodies in recipients participating in clinical trials of A/H1N1, A/H5N1 and A/H9N2 vaccines. The vaccines induced high titers of anti-H1 stalk antibodies in adults and children, with higher titers elicited by AS03-adjuvanted vaccines. We also observed cross-reactivity to H2 and H18 HAs. The A/H9N2 vaccine elicited plasmablast and memory B-cell responses. Post-vaccination serum from vaccinees protected mice against lethal challenge with cH6/1N5 and cH5/3N4 viruses. These findings support the concept of a chimeric HA stalk-based universal influenza virus vaccine. clinicaltrials.gov: NCT02415842.
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http://dx.doi.org/10.1038/s41541-019-0147-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6898674PMC
December 2019

Viral Determinants in H5N1 Influenza A Virus Enable Productive Infection of HeLa Cells.

J Virol 2020 01 31;94(4). Epub 2020 Jan 31.

Infectious and Inflammatory Diseases Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA

Influenza A virus (IAV) is a human respiratory pathogen that causes yearly global epidemics, as well as sporadic pandemics due to human adaptation of pathogenic strains. Efficient replication of IAV in different species is, in part, dictated by its ability to exploit the genetic environment of the host cell. To investigate IAV tropism in human cells, we evaluated the replication of IAV strains in a diverse subset of epithelial cell lines. HeLa cells were refractory to the growth of human H1N1 and H3N2 viruses and low-pathogenic avian influenza (LPAI) viruses. Interestingly, a human isolate of the highly pathogenic avian influenza (HPAI) H5N1 virus successfully propagated in HeLa cells to levels comparable to those in a human lung cell line. Heterokaryon cells generated by fusion of HeLa and permissive cells supported H1N1 virus growth, suggesting the absence of a host factor(s) required for the replication of H1N1, but not H5N1, viruses in HeLa cells. The absence of this factor(s) was mapped to reduced nuclear import, replication, and translation, as well as deficient viral budding. Using reassortant H1N1:H5N1 viruses, we found that the combined introduction of nucleoprotein (NP) and hemagglutinin (HA) from an H5N1 virus was necessary and sufficient to enable H1N1 virus growth. Overall, this study suggests that the absence of one or more cellular factors in HeLa cells results in abortive replication of H1N1, H3N2, and LPAI viruses, which can be circumvented upon the introduction of H5N1 virus NP and HA. Further understanding of the molecular basis of this restriction will provide important insights into the virus-host interactions that underlie IAV pathogenesis and tropism. Many zoonotic avian influenza A viruses have successfully crossed the species barrier and caused mild to life-threatening disease in humans. While human-to-human transmission is limited, there is a risk that these zoonotic viruses may acquire adaptive mutations enabling them to propagate efficiently and cause devastating human pandemics. Therefore, it is important to identify viral determinants that provide these viruses with a replicative advantage in human cells. Here, we tested the growth of influenza A virus in a subset of human cell lines and found that abortive replication of H1N1 viruses in HeLa cells can be circumvented upon the introduction of H5N1 virus HA and NP. Overall, this work leverages the genetic diversity of multiple human cell lines to highlight viral determinants that could contribute to H5N1 virus pathogenesis and tropism.
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http://dx.doi.org/10.1128/JVI.01410-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997754PMC
January 2020

Immunogenicity of chimeric haemagglutinin-based, universal influenza virus vaccine candidates: interim results of a randomised, placebo-controlled, phase 1 clinical trial.

Lancet Infect Dis 2020 01 17;20(1):80-91. Epub 2019 Oct 17.

Duke Early Phase Clinical Research Unit, Duke Clinical Research Institute, Durham, NC, USA.

Background: Influenza viruses cause substantial annual morbidity and mortality globally. Current vaccines protect against influenza only when well matched to the circulating strains. However, antigenic drift can cause considerable mismatches between vaccine and circulating strains, substantially reducing vaccine effectiveness. Moreover, current seasonal vaccines are ineffective against pandemic influenza, and production of a vaccine matched to a newly emerging virus strain takes months. Therefore, there is an unmet medical need for a broadly protective influenza virus vaccine. We aimed to test the ability of chimeric H1 haemagglutinin-based universal influenza virus vaccine candidates to induce broadly cross-reactive antibodies targeting the stalk domain of group 1 haemagglutinin-expressing influenza viruses.

Methods: We did a randomised, observer-blinded, phase 1 study in healthy adults in two centres in the USA. Participants were randomly assigned to one of three prime-boost, chimeric haemagglutinin-based vaccine regimens or one of two placebo groups. The vaccine regimens included a chimeric H8/1, intranasal, live-attenuated vaccine on day 1 followed by a non-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine on day 85; the same regimen but with the inactivated vaccine being adjuvanted with AS03; and an AS03-adjuvanted, chimeric H8/1, intramuscular, inactivated vaccine followed by an AS03-adjuvanted, chimeric H5/1, intramuscular, inactivated vaccine. In this planned interim analysis, the primary endpoints of reactogenicity and safety were assessed by blinded study group. We also assessed anti-H1 haemagglutinin stalk, anti-H2, anti-H9, and anti-H18 IgG antibody titres and plasmablast and memory B-cell responses in peripheral blood. This trial is registered with ClinicalTrials.gov, number NCT03300050.

Findings: Between Oct 10, 2017, and Nov 27, 2017, 65 participants were enrolled and randomly assigned. The adjuvanted inactivated vaccine, but not the live-attenuated vaccine, induced a substantial serum IgG antibody response after the prime immunisation, with a seven times increase in anti-H1 stalk antibody titres on day 29. After boost immunisation, all vaccine regimens induced detectable anti-H1 stalk antibody (2·2-5·6 times induction over baseline), cross-reactive serum IgG antibody, and peripheral blood plasmablast responses. An unsolicited adverse event was reported for 29 (48%) of 61 participants. Solicited local adverse events were reported in 12 (48%) of 25 participants following prime vaccination with intramuscular study product or placebo, in 12 (33%) of 36 after prime immunisation with intranasal study product or placebo, and in 18 (32%) of 56 following booster doses of study product or placebo. Solicited systemic adverse events were reported in 14 (56%) of 25 after prime immunisation with intramuscular study product or placebo, in 22 (61%) of 36 after immunisation with intranasal study product or placebo, and in 21 (38%) of 56 after booster doses of study product or placebo. Disaggregated safety data were not available at the time of this interim analysis.

Interpretation: The tested chimeric haemagglutinin-based, universal influenza virus vaccine regimens elicited cross-reactive serum IgG antibodies that targeted the conserved haemagglutinin stalk domain. This is the first proof-of-principle study to show that high anti-stalk titres can be induced by a rationally designed vaccine in humans and opens up avenues for further development of universal influenza virus vaccines. On the basis of the blinded study group, the vaccine regimens were tolerable and no safety concerns were observed.

Funding: Bill & Melinda Gates Foundation.
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http://dx.doi.org/10.1016/S1473-3099(19)30393-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928577PMC
January 2020

Vaccination With Viral Vectors Expressing Chimeric Hemagglutinin, NP and M1 Antigens Protects Ferrets Against Influenza Virus Challenge.

Front Immunol 2019 21;10:2005. Epub 2019 Aug 21.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, United States.

Seasonal influenza viruses cause significant morbidity and mortality in the global population every year. Although seasonal vaccination limits disease, mismatches between the circulating strain and the vaccine strain can severely impair vaccine effectiveness. Because of this, there is an urgent need for a universal vaccine that induces broad protection against drifted seasonal and emerging pandemic influenza viruses. Targeting the conserved stalk region of the influenza virus hemagglutinin (HA), the major glycoprotein on the surface of the virus, results in the production of broadly protective antibody responses. Furthermore, replication deficient viral vectors based on Chimpanzee Adenovirus Oxford 1 (ChAdOx1) and modified vaccinia Ankara (MVA) virus expressing the influenza virus internal antigens, the nucleoprotein (NP) and matrix 1 (M1) protein, can induce strong heterosubtypic influenza virus-specific T cell responses in vaccinated individuals. Here, we combine these two platforms to evaluate the efficacy of a viral vectored vaccination regimen in protecting ferrets from H3N2 influenza virus infection. We observed that viral vectored vaccines expressing both stalk-targeting, chimeric HA constructs, and the NP+M1 fusion protein, in a prime-boost regimen resulted in the production of antibodies toward group 2 HAs, the HA stalk, NP and M1, as well as in induction of influenza virus-specific-IFNγ responses. The immune response induced by this vaccination regime ultimately reduced viral titers in the respiratory tract of influenza virus infected ferrets. Overall, these results improve our understanding of vaccination platforms capable of harnessing both cellular and humoral immunity with the goal of developing a universal influenza virus vaccine.
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http://dx.doi.org/10.3389/fimmu.2019.02005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6712942PMC
October 2020

Innate Immune Response to Influenza Virus at Single-Cell Resolution in Human Epithelial Cells Revealed Paracrine Induction of Interferon Lambda 1.

J Virol 2019 10 30;93(20). Epub 2019 Sep 30.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA

Early interactions of influenza A virus (IAV) with respiratory epithelium might determine the outcome of infection. The study of global cellular innate immune responses often masks multiple aspects of the mechanisms by which populations of cells work as organized and heterogeneous systems to defeat virus infection, and how the virus counteracts these systems. In this study, we experimentally dissected the dynamics of IAV and human epithelial respiratory cell interaction during early infection at the single-cell level. We found that the number of viruses infecting a cell (multiplicity of infection [MOI]) influences the magnitude of virus antagonism of the host innate antiviral response. Infections performed at high MOIs resulted in increased viral gene expression per cell and stronger antagonist effect than infections at low MOIs. In addition, single-cell patterns of expression of interferons (IFN) and IFN-stimulated genes (ISGs) provided important insights into the contributions of the infected and bystander cells to the innate immune responses during infection. Specifically, the expression of multiple ISGs was lower in infected than in bystander cells. In contrast with other IFNs, IFN lambda 1 (IFNL1) showed a widespread pattern of expression, suggesting a different cell-to-cell propagation mechanism more reliant on paracrine signaling. Finally, we measured the dynamics of the antiviral response in primary human epithelial cells, which highlighted the importance of early innate immune responses at inhibiting virus spread. Influenza A virus (IAV) is a respiratory pathogen of high importance to public health. Annual epidemics of seasonal IAV infections in humans are a significant public health and economic burden. IAV also causes sporadic pandemics, which can have devastating effects. The main target cells for IAV replication are epithelial cells in the respiratory epithelium. The cellular innate immune responses induced in these cells upon infection are critical for defense against the virus, and therefore, it is important to understand the complex interactions between the virus and the host cells. In this study, we investigated the innate immune response to IAV in the respiratory epithelium at the single-cell level, providing a better understanding on how a population of epithelial cells functions as a complex system to orchestrate the response to virus infection and how the virus counteracts this system.
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http://dx.doi.org/10.1128/JVI.00559-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798124PMC
October 2019

Sequential Immunization With Live-Attenuated Chimeric Hemagglutinin-Based Vaccines Confers Heterosubtypic Immunity Against Influenza A Viruses in a Preclinical Ferret Model.

Front Immunol 2019 10;10:756. Epub 2019 Apr 10.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, United States.

Due to continuous antigenic drift and occasional antigenic shift, influenza viruses escape from human adaptive immunity resulting in significant morbidity and mortality in humans. Therefore, to avoid the need for annual reformulation and readministration of seasonal influenza virus vaccines, we are developing a novel chimeric hemagglutinin (cHA)-based universal influenza virus vaccine, which is comprised of sequential immunization with antigens containing a conserved stalk domain derived from a circulating pandemic H1N1 strain in combination with "exotic" head domains. Here, we show that this prime-boost sequential immunization strategy redirects antibody responses toward the conserved stalk region. We compared the vaccine efficacy elicited by distinct vaccination approaches in the preclinical ferret model of influenza. All ferrets immunized with cHA-based vaccines developed stalk-specific and broadly cross-reactive antibody responses. Two consecutive vaccinations with live-attenuated influenza viruses (LAIV-LAIV) conferred superior protection against pH1N1 and H6N1 challenge infection. Sequential immunization with LAIV followed by inactivated influenza vaccine (LAIV-IIV regimen) also induced robust antibody responses. Importantly, the LAIV-LAIV immunization regimen also induced HA stalk-specific CD4IFN-γ and CD8IFN-γ effector T cell responses in peripheral blood that were recalled by pH1N1 viral challenge. The findings from this preclinical study suggest that an LAIV-LAIV vaccination regimen would be more efficient in providing broadly protective immunity against influenza virus infection as compared to other approaches tested here.
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http://dx.doi.org/10.3389/fimmu.2019.00756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499175PMC
July 2020

Host-Specific NS5 Ubiquitination Determines Yellow Fever Virus Tropism.

J Virol 2019 07 28;93(14). Epub 2019 Jun 28.

Department of Microbiology and Plant Pathology, University of California, Riverside, California, USA

The recent yellow fever virus (YFV) epidemic in Brazil in 2017 and Zika virus (ZIKV) epidemic in 2015 serve to remind us of the importance of flaviviruses as emerging human pathogens. With the current global flavivirus threat, there is an urgent need for antivirals and vaccines to curb the spread of these viruses. However, the lack of suitable animal models limits the research questions that can be answered. A common trait of all flaviviruses studied thus far is their ability to antagonize interferon (IFN) signaling so as to enhance viral replication and dissemination. Previously, we reported that YFV NS5 requires the presence of type I IFN (IFN-α/β) for its engagement with human signal transducer and activator of transcription 2 (hSTAT2). In this manuscript, we report that like the NS5 proteins of ZIKV and dengue virus (DENV), YFV NS5 protein is able to bind hSTAT2 but not murine STAT2 (mSTAT2). Contrary to what has been demonstrated with ZIKV NS5 and DENV NS5, replacing mSTAT2 with hSTAT2 cannot rescue the YFV NS5-STAT2 interaction, as YFV NS5 is also unable to interact with hSTAT2 in murine cells. We show that the IFN-α/β-dependent ubiquitination of YFV NS5 that is required for STAT2 binding in human cells is absent in murine cells. In addition, we demonstrate that mSTAT2 restricts YFV replication These data serve as further impetus for the development of an immunocompetent mouse model that can serve as a disease model for multiple flaviviruses. Flaviviruses such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV) are important human pathogens. A common flavivirus trait is the antagonism of interferon (IFN) signaling to enhance viral replication and spread. We report that like ZIKV NS5 and DENV NS5, YFV NS5 binds human STAT2 (hSTAT2) but not mouse STAT2 (mSTAT2), a type I IFN (IFN-α/β) pathway component. Additionally, we show that contrary to what has been demonstrated with ZIKV NS5 and DENV NS5, YFV NS5 is unable to interact with hSTAT2 in murine cells. We demonstrate that mSTAT2 restricts YFV replication in mice and that this correlates with a lack of IFN-α/β-induced YFV NS5 ubiquitination in murine cells. The lack of suitable animal models limits flavivirus pathogenesis, vaccine, and drug research. These data serve as further impetus for the development of an immunocompetent mouse model that can serve as a disease model for multiple flaviviruses.
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http://dx.doi.org/10.1128/JVI.00151-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600188PMC
July 2019

Diminished B-Cell Response After Repeat Influenza Vaccination.

J Infect Dis 2019 04;219(10):1586-1595

Department of Medicine, Stanford University School of Medicine, Stanford.

Annual vaccination with influenza vaccines is recommended for protection against influenza in the United States. Past clinical studies and meta-analysis, however, have reported conflicting results on the benefits of annual vaccination. B-cell responses elicited following repeat influenza vaccinations over multiple seasons have not been examined in detail. We analyzed the B-cell and antibody (Ab) responses in volunteers vaccinated yearly, from 2010 or 2011 through 2014, with seasonal trivalent inactivated influenza vaccines. Statistical analyses were designed to help correct for possible bias due to reduced sample size in the later years of the study. We show that, after the second annual vaccination, the frequency of vaccine-specific plasmablasts and the binding reactivity of plasmablast-derived polyclonal Abs are reduced and do not increase in subsequent years. Similar trends are observed with the serum hemagglutination inhibition Ab response after each annual vaccination, as well as the binding reactivity of plasmablast-derived polyclonal Abs to the hemagglutinin of influenza A virus vaccine components, even with changes in the seasonal vaccine components during the study. Our findings indicate a diminished B-cell response to annual vaccination with seasonal trivalent influenza vaccine. These results emphasize the need for developing improved strategies to enhance the immunogenicity and efficacy of annual influenza vaccination.
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http://dx.doi.org/10.1093/infdis/jiy685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6473172PMC
April 2019

Antigenic sites in influenza H1 hemagglutinin display species-specific immunodominance.

J Clin Invest 2018 11 8;128(11):4992-4996. Epub 2018 Oct 8.

Department of Microbiology.

Hemagglutination inhibition (HI) titers are a major correlate of protection for influenza-related illness. The influenza virus hemagglutinin possesses antigenic sites that are the targets of HI active antibodies. Here, a panel of mutant viruses each lacking a classically defined antigenic site was created to compare the species-specific immunodominance of the antigenic sites in a clinically relevant hemagglutinin. HI active antibodies of antisera from influenza virus-infected mice targeted sites Sb and Ca2. HI active antibodies of guinea pigs were not directed against any specific antigenic site, although trends were observed toward Sb, Ca2, and Sa. HI titers of antisera from infected ferrets were significantly affected by site Sa. HI active antibodies of adult humans followed yet another immunodominance pattern, in which sites Sb and Sa were immunodominant. When comparing the HI profiles among different species by antigenic cartography, animals and humans grouped separately. This study provides characterizations of the antibody-mediated immune responses against the head domain of a recent H1 hemagglutinin in animals and humans.
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http://dx.doi.org/10.1172/JCI122895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205383PMC
November 2018

Analyses of Cellular Immune Responses in Ferrets Following Influenza Virus Infection.

Methods Mol Biol 2018 ;1836:513-530

Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY, USA.

Ferrets are an ideal animal model in which to study the transmission of respiratory viruses as well as disease progression and vaccine efficacy because of their close anatomical and physiological resemblances to humans. However, a paucity of reagents and standardized procedures has hampered research progress, especially for studying cell-mediated immunity. The approaches described here-leukocyte isolation from whole blood and secondary lymphoid tissues-are generalizable, highly reproducible, and deliver single cell suspensions with excellent cell viability. Importantly, we have now developed assays to quantify key cellular components and antigen-specific T cell responses at the single cell level from multiple tissue compartments following influenza infection in ferrets. Collectively, these methods were instrumental in flow cytometry studies that revealed alterations in immune cell composition and distribution across lymphoid tissues following viral infection. Furthermore, sorting of T cell populations and peptide restimulation ex vivo in cytokine ELISpot assays has provided novel insight into the influenza-specific CD4 and CD8 T cell repertoire. The detailed procedures for these techniques are described in this chapter and can likely be adapted for the analyses of responses to many respiratory pathogens.
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http://dx.doi.org/10.1007/978-1-4939-8678-1_24DOI Listing
April 2019

Assessment of Influenza Virus Hemagglutinin Stalk-Specific Antibody Responses.

Methods Mol Biol 2018 ;1836:487-511

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Animal models are essential to examine the pathogenesis and transmission of influenza viruses and for preclinical evaluation of influenza virus vaccines. Among the animal models used in influenza virus research, the domestic ferret (Mustela putorius furo) is the gold standard. As seen in humans, infection with influenza virus or immunization with an influenza virus vaccine induces humoral and cellular immunity in ferrets that provides protection against infection by an antigenically similar influenza virus. Antibodies against the globular head domain of the influenza hemagglutinin can provide sterilizing immunity against virus infection by blocking receptor binding. However, antibodies that bind the stalk region of the hemagglutinin also confer protection by several mechanisms including antibody-dependent cellular cytotoxicity or phagocytosis. Recently, the antigenically and structurally conserved hemagglutinin stalk has become an attractive target for the development of universal influenza virus vaccines that hold the promise to provide protection against influenza epidemics and pandemics. Herein, in vivo and in vitro assays, including optimization of assay conditions to examine hemagglutinin stalk-specific antibody responses in small animal models, are described.
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http://dx.doi.org/10.1007/978-1-4939-8678-1_23DOI Listing
April 2019

Transcription Elongation Can Affect Genome 3D Structure.

Cell 2018 09 23;174(6):1522-1536.e22. Epub 2018 Aug 23.

Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0640, USA. Electronic address:

How transcription affects genome 3D organization is not well understood. We found that during influenza A (IAV) infection, rampant transcription rapidly reorganizes host cell chromatin interactions. These changes occur at the ends of highly transcribed genes, where global inhibition of transcription termination by IAV NS1 protein causes readthrough transcription for hundreds of kilobases. In these readthrough regions, elongating RNA polymerase II disrupts chromatin interactions by inducing cohesin displacement from CTCF sites, leading to locus decompaction. Readthrough transcription into heterochromatin regions switches them from the inert (B) to the permissive (A) chromatin compartment and enables transcription factor binding. Data from non-viral transcription stimuli show that transcription similarly affects cohesin-mediated chromatin contacts within gene bodies. Conversely, inhibition of transcription elongation allows cohesin to accumulate at previously transcribed intragenic CTCF sites and to mediate chromatin looping and compaction. Our data indicate that transcription elongation by RNA polymerase II remodels genome 3D architecture.
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http://dx.doi.org/10.1016/j.cell.2018.07.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130916PMC
September 2018

A Live-Attenuated Prime, Inactivated Boost Vaccination Strategy with Chimeric Hemagglutinin-Based Universal Influenza Virus Vaccines Provides Protection in Ferrets: A Confirmatory Study.

Vaccines (Basel) 2018 Jul 25;6(3). Epub 2018 Jul 25.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Influenza viruses cause severe diseases and mortality in humans on an annual basis. The current influenza virus vaccines can confer protection when they are well-matched with the circulating strains. However, due to constant changes of the virus surface glycoproteins, the vaccine efficacy can drop substantially in some seasons. In addition, the current seasonal influenza virus vaccines do not protect from avian influenza viruses of human pandemic potential. Novel influenza virus vaccines that aim to elicit antibodies against conserved epitopes like the hemagglutinin stalk could not only reduce the burden of drifted seasonal viruses but potentially also protect humans from infection with zoonotic and emerging pandemic influenza viruses. In this paper, we generated influenza virus vaccine constructs that express chimeric hemagglutinins consisting of exotic, avian head domains and a consistent stalk domain of a seasonal virus. Using such viruses in a sequential immunization regimen can redirect the immune response towards conserved epitopes. In this study, male ferrets received a live-attenuated vaccine virus based on the A/Ann Arbor/6/60 strain expressing a chimeric H8/1 (cH8/1) hemagglutinin, which was followed by a heterologous booster vaccination with a cH5/1N1 formalin inactivated non-adjuvanted whole virus. This group was compared to a second group that received a cH8/1N1 inactivated vaccine followed by a cH5/1N1 inactivated vaccine. Both groups showed a reduction in viral titers in the upper respiratory tract after the A(H1N1)pdm09 virus challenge. Animals that received the live-attenuated vaccine had low or undetectable titers in the lower respiratory tract. The results support the further development of chimeric hemagglutinin-based vaccination strategies. The outcome of this study confirms and corroborates findings from female ferrets primed with a A/Leningrad/134/17/57-based live attenuated cH8/1N1 vaccine followed by vaccination with an AS03-adjuvanted cH5/1N1 split virus vaccine 10.
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http://dx.doi.org/10.3390/vaccines6030047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161119PMC
July 2018

Moving Forward: Recent Developments for the Ferret Biomedical Research Model.

mBio 2018 07 17;9(4). Epub 2018 Jul 17.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

Since the initial report in 1911, the domestic ferret has become an invaluable biomedical research model. While widely recognized for its utility in influenza virus research, ferrets are used for a variety of infectious and noninfectious disease models due to the anatomical, metabolic, and physiological features they share with humans and their susceptibility to many human pathogens. However, there are limitations to the model that must be overcome for maximal utility for the scientific community. Here, we describe important recent advances that will accelerate biomedical research with this animal model.
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http://dx.doi.org/10.1128/mBio.01113-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050969PMC
July 2018

A multifunctional human monoclonal neutralizing antibody that targets a unique conserved epitope on influenza HA.

Nat Commun 2018 07 10;9(1):2669. Epub 2018 Jul 10.

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

The high rate of antigenic drift in seasonal influenza viruses necessitates frequent changes in vaccine composition. Recent seasonal H3 vaccines do not protect against swine-origin H3N2 variant (H3N2v) strains that recently have caused severe human infections. Here, we report a human V1-69 gene-encoded monoclonal antibody (mAb) designated H3v-47 that exhibits potent cross-reactive neutralization activity against human and swine H3N2 viruses that circulated since 1989. The crystal structure and electron microscopy reconstruction of H3v-47 Fab with the H3N2v hemagglutinin (HA) identify a unique epitope spanning the vestigial esterase and receptor-binding subdomains that is distinct from that of any known neutralizing antibody for influenza A H3 viruses. MAb H3v-47 functions largely by blocking viral egress from infected cells. Interestingly, H3v-47 also engages Fcγ receptor and mediates antibody dependent cellular cytotoxicity (ADCC). This newly identified conserved epitope can be used in design of novel immunogens for development of broadly protective H3 vaccines.
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http://dx.doi.org/10.1038/s41467-018-04704-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039445PMC
July 2018