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1.
Frontiers in immunology ; 12, 2021.
Article in English | EuropePMC | ID: covidwho-1695160

ABSTRACT

The tuberculosis vaccine, Bacille Calmette-Guerin (BCG), also affords protection against non-tuberculous diseases attributable to heterologous immune mechanisms such as trained innate immunity, activation of non-conventional T-cells, and cross-reactive adaptive immunity. Aerosol vaccine delivery can target immune responses toward the primary site of infection for a respiratory pathogen. Therefore, we hypothesised that aerosol delivery of BCG would enhance cross-protective action against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and be a deployable intervention against coronavirus disease 2019 (COVID-19). Immune parameters were monitored in vaccinated and unvaccinated rhesus macaques for 28 days following aerosol BCG vaccination. High-dose SARS-CoV-2 challenge was applied by intranasal and intrabronchial instillation and animals culled 6–8 days later for assessment of viral, disease, and immunological parameters. Mycobacteria-specific cell-mediated immune responses were detected following aerosol BCG vaccination, but SARS-CoV-2-specific cellular- and antibody-mediated immunity was only measured following challenge. Early secretion of cytokine and chemokine markers associated with the innate cellular and adaptive antiviral immune response was detected following SARS-CoV-2 challenge in vaccinated animals, at concentrations that exceeded titres measured in unvaccinated macaques. Classical CD14+ monocytes and Vδ2 γδ T-cells quantified by whole-blood immunophenotyping increased rapidly in vaccinated animals following SARS-CoV-2 challenge, indicating a priming of innate immune cells and non-conventional T-cell populations. However, viral RNA quantified in nasal and pharyngeal swabs, bronchoalveolar lavage (BAL), and tissue samples collected at necropsy was equivalent in vaccinated and unvaccinated animals, and in-life CT imaging and histopathology scoring applied to pulmonary tissue sections indicated that the disease induced by SARS-CoV-2 challenge was comparable between vaccinated and unvaccinated groups. Hence, aerosol BCG vaccination did not induce, or enhance the induction of, SARS-CoV-2 cross-reactive adaptive cellular or humoral immunity, although an influence of BCG vaccination on the subsequent immune response to SARS-CoV-2 challenge was apparent in immune signatures indicative of trained innate immune mechanisms and primed unconventional T-cell populations. Nevertheless, aerosol BCG vaccination did not enhance the initial clearance of virus, nor reduce the occurrence of early disease pathology after high dose SARS-CoV-2 challenge. However, the heterologous immune mechanisms primed by BCG vaccination could contribute to the moderation of COVID-19 disease severity in more susceptible species following natural infection.

2.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-318753

ABSTRACT

Background: Age is the major risk factor for mortality after SARS-CoV-2 infection and older people have received priority consideration for COVID-19 vaccination. However vaccine responses are often suboptimal in this age group and few people over the age of 80 years were included in vaccine registration trials.Methods: We determined the serological and cellular response to spike protein in 100 people aged 80-96 years at 2 weeks after second vaccination with the Pfizer BNT162b2 mRNA vaccine.Findings: Antibody responses were seen in every donor with high titres in 98%. Spike-specific cellular immune responses were detectable in only 63% and correlated with humoral response. Previous SARS-CoV-2 infection substantially increased antibody responses after one vaccine and antibody and cellular responses remained 28-fold and 3-fold higher respectively after dual vaccination. Post-vaccine sera mediated strong neutralisation of live Victoria (Wuhan-like prototype) infection and although neutralisation titres were reduced 14-fold against the P.1 variant first discovered in Brazil they remained largely effective.Interpretation: These data demonstrate that the mRNA vaccine platform delivers strong humoral immunity in people up to 96 years of age and retains broad efficacy against the P.1 Variant of Concern.Funding: This work was supported by the UK Coronavirus Immunology Consortium (UK-CIC) funded by DHSC/UKRI and the National Core Studies Immunity programme. Declaration of Interest: None to declare. Ethical Approval: The work was performed under the CIA UPH IRAS approval (REC 20W\0240) and conducted according to the Declaration of Helsinki and good clinical practice.

3.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-314835

ABSTRACT

Vaccines against SARS-CoV-2 are urgently required. Here we report detailed immune profiling after ChAdOx1 nCoV-19 (AZD1222) and subsequent challenge in two animal models of SARS-CoV-2 mediated disease. We demonstrate in rhesus macaques the lung pathology caused by SARS-CoV-2 mediated pneumonia is reduced by prior vaccination with ChAdOx1 nCoV-19 which induced neutralising antibody responses after a single intramuscular administration. In a second animal model, ferrets, ChAdOx1 nCoV-19 reduced both virus shedding and lung pathology. Antibody titers were boosted by a second dose. Data from these challenge models and the detailed immune profiling, support the continued clinical evaluation of ChAdOx1 nCoV-19.

4.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-293860

ABSTRACT

There are as yet no licenced therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric Spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2 (ACE2), initiating conformational changes that drive membrane fusion. We find that monoclonal antibody CR3022 binds the RBD tightly, neutralising SARS-CoV-2 and report the crystal structure at 2.4 A of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilising, CR3022 epitope is inaccessible in the prefusion Spike, suggesting that CR3022 binding would facilitate conversion to the fusion-incompetent post-fusion state. Cryo-EM analysis confirms that incubation of Spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope may be useful therapeutically, possibly in synergy with an antibody blocking receptor attachment.<br><br>Funding: This work was supported by a grant from the CAMS-Oxford Institute to D.I.S. E.E.F and J.Ren are supported by the Wellcome Trust (101122/Z/13/Z), Y.Z. by Cancer Research UK (C375/A17721) and D.I.S. and E.E.F. by the UK Medical Research Council (MR/N00065X/1). J.H. is supported by a grant from the EPA Cephalosporin Fund. PPUK is funded by the Rosalind Franklin Institute EPSRC Grant no. EP/S025243/1. The National Institute for Health Research Biomedical Research Centre Funding Scheme supports G.R.S. together with the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant number: 2018-I2M-2-002), which also supports D.I.S. G.R.S. is also supported as a Wellcome Trust Senior Investigator (grant 095541/A/11/Z). T.M. is supported by Cancer Research UK grants C20724/A14414 and C20724/A26752 to Christian Siebold. This is a contribution from the UK Instruct-ERIC Centre. The Wellcome Centre for Human Genetics is supported by the Wellcome Trust (grant 090532/Z/09/Z). Virus used for the neutralisation assays was a gift from Julian Druce, Doherty Centre, Melbourne, Australia. <br><br>Conflict of Interest: The authors declare no competing interests.

5.
Elife ; 102021 09 29.
Article in English | MEDLINE | ID: covidwho-1468709

ABSTRACT

Age is the major risk factor for mortality after SARS-CoV-2 infection and older people have received priority consideration for COVID-19 vaccination. However, vaccine responses are often suboptimal in this age group and few people over the age of 80 years were included in vaccine registration trials. We determined the serological and cellular response to spike protein in 100 people aged 80-96 years at 2 weeks after the second vaccination with the Pfizer BNT162b2 mRNA vaccine. Antibody responses were seen in every donor with high titers in 98%. Spike-specific cellular immune responses were detectable in only 63% and correlated with humoral response. Previous SARS-CoV-2 infection substantially increased antibody responses after one vaccine and antibody and cellular responses remained 28-fold and 3-fold higher, respectively, after dual vaccination. Post-vaccine sera mediated strong neutralization of live Victoria infection and although neutralization titers were reduced 14-fold against the P.1 variant first discovered in Brazil they remained largely effective. These data demonstrate that the mRNA vaccine platform delivers strong humoral immunity in people up to 96 years of age and retains broad efficacy against the P.1 variant of concern.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , RNA, Messenger/immunology , SARS-CoV-2/immunology , Age Factors , Aged, 80 and over , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19/epidemiology , COVID-19/metabolism , COVID-19/prevention & control , COVID-19 Vaccines/administration & dosage , Female , Humans , Immunity, Cellular , Immunity, Humoral/immunology , Male , Spike Glycoprotein, Coronavirus/immunology , Vaccination/methods
6.
Sci Adv ; 7(37): eabg7996, 2021 Sep 10.
Article in English | MEDLINE | ID: covidwho-1405212

ABSTRACT

There is an urgent requirement for safe and effective vaccines to prevent COVID-19. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferrets and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen 7 days after infection in ferrets. This increased lung pathology was observed at day 7 but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity.

7.
NPJ Vaccines ; 6(1): 83, 2021 Jun 17.
Article in English | MEDLINE | ID: covidwho-1387359

ABSTRACT

An array of SARS-CoV-2 virus variants have been isolated, propagated and used in in vitro assays, in vivo animal studies and human clinical trials. Observations of working stocks of SARS-CoV-2 suggest that sequential propagation in Vero cells leads to critical changes in the region of the furin cleavage site, which significantly reduce the value of the working stock for critical research studies. Serially propagating SARS-CoV-2 in Vero E6 cells leads to rapid increases in genetic variants while propagation in other cell lines (e.g. Vero/hSLAM) appears to mitigate this risk thereby improving the overall genetic stability of working stocks. From these observations, investigators are urged to monitor genetic variants carefully when propagating SARS-CoV-2 in Vero cells.

9.
Commun Biol ; 4(1): 915, 2021 07 26.
Article in English | MEDLINE | ID: covidwho-1327224

ABSTRACT

Vaccines against SARS-CoV-2 are urgently required, but early development of vaccines against SARS-CoV-1 resulted in enhanced disease after vaccination. Careful assessment of this phenomena is warranted for vaccine development against SARS CoV-2. Here we report detailed immune profiling after ChAdOx1 nCoV-19 (AZD1222) and subsequent high dose challenge in two animal models of SARS-CoV-2 mediated disease. We demonstrate in rhesus macaques the lung pathology caused by SARS-CoV-2 mediated pneumonia is reduced by prior vaccination with ChAdOx1 nCoV-19 which induced neutralising antibody responses after a single intramuscular administration. In a second animal model, ferrets, ChAdOx1 nCoV-19 reduced both virus shedding and lung pathology. Antibody titre were boosted by a second dose. Data from these challenge models on the absence of enhanced disease and the detailed immune profiling, support the continued clinical evaluation of ChAdOx1 nCoV-19.


Subject(s)
COVID-19 Vaccines/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Neutralizing/immunology , Ferrets , Macaca mulatta
10.
Nat Protoc ; 16(6): 3114-3140, 2021 06.
Article in English | MEDLINE | ID: covidwho-1203437

ABSTRACT

Virus neutralization assays measure neutralizing antibodies in serum and plasma, and the plaque reduction neutralization test (PRNT) is considered the gold standard for measuring levels of these antibodies for many viral diseases. We have developed procedures for the standard PRNT, microneutralization assay (MNA) and pseudotyped virus neutralization assay (PNA) for severe acute respiratory syndrome coronavirus 2. The MNA offers advantages over the PRNT by reducing assay time, allowing increased throughput and reducing operator workload while remaining dependent upon the use of wild-type virus. This ensures that all severe acute respiratory syndrome coronavirus 2 antigens are present, but Biosafety Level 3 facilities are required. In addition to the advantages of MNA, PNA can be performed with lower biocontainment (Biosafety Level 2 facilities) and allows for further increases in throughput. For each new vaccine, it is critical to ensure good correlation of the neutralizing activity measured using PNA against the PRNT or MNA. These assays have been used in the development and licensure of the ChAdOx1 nCoV-19 (AstraZeneca; Oxford University) and Ad26.COV2.S (Janssen) coronavirus disease 2019 vaccines and are critical for demonstrating bioequivalence of future vaccines.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Neutralization Tests/methods , SARS-CoV-2/immunology , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/blood , COVID-19/prevention & control , COVID-19 Vaccines/immunology , COVID-19 Vaccines/therapeutic use , Humans , Neutralization Tests/economics , Time Factors
11.
Nat Commun ; 12(1): 1260, 2021 02 24.
Article in English | MEDLINE | ID: covidwho-1101645

ABSTRACT

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks.


Subject(s)
COVID-19/immunology , COVID-19/virology , Lung/pathology , Lung/virology , Animals , Disease Models, Animal , Female , Immunity, Cellular/physiology , Interferon-gamma/metabolism , Macaca fascicularis , Macaca mulatta , Male , Pandemics , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity
12.
Cell ; 184(9): 2348-2361.e6, 2021 04 29.
Article in English | MEDLINE | ID: covidwho-1095900

ABSTRACT

The race to produce vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began when the first sequence was published, and this forms the basis for vaccines currently deployed globally. Independent lineages of SARS-CoV-2 have recently been reported: UK, B.1.1.7; South Africa, B.1.351; and Brazil, P.1. These variants have multiple changes in the immunodominant spike protein that facilitates viral cell entry via the angiotensin-converting enzyme-2 (ACE2) receptor. Mutations in the receptor recognition site on the spike are of great concern for their potential for immune escape. Here, we describe a structure-function analysis of B.1.351 using a large cohort of convalescent and vaccinee serum samples. The receptor-binding domain mutations provide tighter ACE2 binding and widespread escape from monoclonal antibody neutralization largely driven by E484K, although K417N and N501Y act together against some important antibody classes. In a number of cases, it would appear that convalescent and some vaccine serum offers limited protection against this variant.


Subject(s)
COVID-19 Vaccines/blood , COVID-19 Vaccines/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/immunology , COVID-19/immunology , COVID-19/therapy , COVID-19/virology , Chlorocebus aethiops , Clinical Trials as Topic , HEK293 Cells , Humans , Immunization, Passive , Models, Molecular , Mutation/genetics , Neutralization Tests , Protein Binding , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Vero Cells
13.
Cell ; 184(8): 2201-2211.e7, 2021 04 15.
Article in English | MEDLINE | ID: covidwho-1086820

ABSTRACT

SARS-CoV-2 has caused over 2 million deaths in little over a year. Vaccines are being deployed at scale, aiming to generate responses against the virus spike. The scale of the pandemic and error-prone virus replication is leading to the appearance of mutant viruses and potentially escape from antibody responses. Variant B.1.1.7, now dominant in the UK, with increased transmission, harbors 9 amino acid changes in the spike, including N501Y in the ACE2 interacting surface. We examine the ability of B.1.1.7 to evade antibody responses elicited by natural SARS-CoV-2 infection or vaccination. We map the impact of N501Y by structure/function analysis of a large panel of well-characterized monoclonal antibodies. B.1.1.7 is harder to neutralize than parental virus, compromising neutralization by some members of a major class of public antibodies through light-chain contacts with residue 501. However, widespread escape from monoclonal antibodies or antibody responses generated by natural infection or vaccination was not observed.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , CHO Cells , COVID-19/epidemiology , Chlorocebus aethiops , Cricetulus , HEK293 Cells , Humans , Pandemics , Protein Binding , Structure-Activity Relationship , Vero Cells
14.
Viruses ; 13(1)2021 Jan 15.
Article in English | MEDLINE | ID: covidwho-1067778

ABSTRACT

Ferrets were experimentally inoculated with SARS-CoV-2 (severe acute respiratory syndrome (SARS)-related coronavirus 2) to assess infection dynamics and host response. During the resulting subclinical infection, viral RNA was monitored between 2 and 21 days post-inoculation (dpi), and reached a peak in the upper respiratory cavity between 4 and 6 dpi. Viral genomic sequence analysis in samples from three animals identified the Y453F nucleotide substitution relative to the inoculum. Viral RNA was also detected in environmental samples, specifically in swabs of ferret fur. Microscopy analysis revealed viral protein and RNA in upper respiratory tract tissues, notably in cells of the respiratory and olfactory mucosae of the nasal turbinates, including olfactory neuronal cells. Antibody responses to the spike and nucleoprotein were detected from 21 dpi, but virus-neutralizing activity was low. A second intranasal inoculation (re-exposure) of two ferrets after a 17-day interval did not produce re-initiation of viral RNA shedding, but did amplify the humoral response in one animal. Therefore, ferrets can be experimentally infected with SARS-CoV-2 to model human asymptomatic infection.


Subject(s)
Asymptomatic Diseases , COVID-19/virology , Disease Models, Animal , SARS-CoV-2/physiology , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/diagnosis , COVID-19/pathology , COVID-19/transmission , Female , Ferrets , Genome, Viral/genetics , Mutation , Nasal Mucosa/virology , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Viral Load , Virus Shedding
15.
Viruses ; 13(1)2021 Jan 15.
Article in English | MEDLINE | ID: covidwho-1031172

ABSTRACT

Ferrets were experimentally inoculated with SARS-CoV-2 (severe acute respiratory syndrome (SARS)-related coronavirus 2) to assess infection dynamics and host response. During the resulting subclinical infection, viral RNA was monitored between 2 and 21 days post-inoculation (dpi), and reached a peak in the upper respiratory cavity between 4 and 6 dpi. Viral genomic sequence analysis in samples from three animals identified the Y453F nucleotide substitution relative to the inoculum. Viral RNA was also detected in environmental samples, specifically in swabs of ferret fur. Microscopy analysis revealed viral protein and RNA in upper respiratory tract tissues, notably in cells of the respiratory and olfactory mucosae of the nasal turbinates, including olfactory neuronal cells. Antibody responses to the spike and nucleoprotein were detected from 21 dpi, but virus-neutralizing activity was low. A second intranasal inoculation (re-exposure) of two ferrets after a 17-day interval did not produce re-initiation of viral RNA shedding, but did amplify the humoral response in one animal. Therefore, ferrets can be experimentally infected with SARS-CoV-2 to model human asymptomatic infection.


Subject(s)
Asymptomatic Diseases , COVID-19/virology , Disease Models, Animal , SARS-CoV-2/physiology , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/diagnosis , COVID-19/pathology , COVID-19/transmission , Female , Ferrets , Genome, Viral/genetics , Mutation , Nasal Mucosa/virology , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Viral Load , Virus Shedding
16.
Nat Commun ; 12(1): 81, 2021 01 04.
Article in English | MEDLINE | ID: covidwho-1007628

ABSTRACT

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease.


Subject(s)
COVID-19/immunology , Disease Models, Animal , Ferrets/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19 Vaccines/pharmacology , Dose-Response Relationship, Drug , Female , Lung/immunology , Lung/pathology , RNA, Viral/isolation & purification , SARS-CoV-2/genetics , Virus Replication/drug effects , Virus Replication/immunology , Virus Shedding/drug effects , Virus Shedding/immunology
17.
Sci Rep ; 10(1): 21894, 2020 12 14.
Article in English | MEDLINE | ID: covidwho-977275

ABSTRACT

The rapid emergence of SARS-CoV-2, the causative agent of COVID-19, and its dissemination globally has caused an unprecedented strain on public health. Animal models are urgently being developed for SARS-CoV-2 to aid rational design of vaccines and therapeutics. Immunohistochemistry and in situ hybridisation techniques that facilitate reliable and reproducible detection of SARS-CoV and SARS-CoV-2 viral products in formalin-fixed paraffin-embedded (FFPE) specimens would be of great utility. A selection of commercial antibodies generated against SARS-CoV spike protein and nucleoprotein, double stranded RNA, and RNA probe for spike genes were evaluated for the ability to detect FFPE infected cells. We also tested both heat- and enzymatic-mediated virus antigen retrieval methods to determine the optimal virus antigen recovery as well as identifying alternative retrieval methods to enable flexibility of IHC methods. In addition to using native virus infected cells as positive control material, the evaluation of non-infected cells expressing coronavirus (SARS, MERS) spike as a biosecure alternative to assays involving live virus was undertaken. Optimized protocols were successfully applied to experimental animal-derived tissues. The diverse techniques for virus detection and control material generation demonstrated in this study can be applied to investigations of coronavirus pathogenesis and therapeutic research in animal models.


Subject(s)
Antigens, Viral/immunology , COVID-19 Testing , COVID-19 , Immunohistochemistry , SARS-CoV-2/isolation & purification , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , COVID-19/diagnosis , COVID-19/virology , Chlorocebus aethiops , Ferrets , In Situ Hybridization , RNA Probes/immunology , SARS Virus/isolation & purification , Vero Cells
18.
EBioMedicine ; 63: 103153, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-956065

ABSTRACT

BACKGROUND: The novel human coronavirus SARS-CoV-2 is a major ongoing global threat with huge economic burden. Like all respiratory viruses, SARS-CoV-2 initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. METHODS: SARS-CoV-2 Victoria/01/2020 was passaged in Vero/hSLAM cells and virus titre determined by plaque assay. Challenge virus was delivered by intranasal instillation to female ferrets at 5.0 × 106 pfu/ml. Treatment groups received intranasal INNA-051, developed by Ena Respiratory. SARS-CoV-2 RNA was detected using the 2019-nCoV CDC RUO Kit and QuantStudio™ 7 Flex Real-Time PCR System. Histopathological analysis was performed using cut tissues stained with haematoxylin and eosin (H&E). FINDINGS: We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. After 5 days post-exposure to SARS-CoV-2, INNA-051 significantly reduced virus in throat swabs (p=<0.0001) by up to a 24 fold (96% reduction) and in nasal wash (p=0.0107) up to a 15 fold (93% reduction) in comparison to untreated animals. INTERPRETATION: The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT, to reduce SARS-CoV-2 transmission and provide protection against COVID-19. FUNDING: This work was funded by Ena Respiratory, Melbourne, Australia.


Subject(s)
Lipopeptides/administration & dosage , Respiratory System/virology , SARS-CoV-2/pathogenicity , Toll-Like Receptor 2/agonists , Toll-Like Receptor 6/agonists , Virus Shedding , Administration, Intranasal , Animals , COVID-19/drug therapy , COVID-19/pathology , Disease Models, Animal , Female , Ferrets , Immunity, Innate , Lipopeptides/chemistry , Lipopeptides/pharmacology , Nasal Cavity/pathology , Nasal Cavity/virology , Pharynx/pathology , Pharynx/virology , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction , Respiratory System/pathology , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Load/drug effects
19.
Viruses ; 12(10)2020 10 14.
Article in English | MEDLINE | ID: covidwho-905965

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Pneumonia, Viral/virology , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , COVID-19 Vaccines , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , DNA, Complementary/analysis , DNA, Complementary/genetics , DNA, Viral/analysis , DNA, Viral/genetics , Genome, Viral , High-Throughput Nucleotide Sequencing/methods , Humans , Molecular Epidemiology , Multiplex Polymerase Chain Reaction , Pandemics , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , SARS-CoV-2 , Sequence Analysis
20.
Viruses ; 12(10):1164, 2020.
Article in English | MDPI | ID: covidwho-855539

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

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