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1.
ACS central science ; 8(5):527-545, 2022.
Article in English | EuropePMC | ID: covidwho-1871009

ABSTRACT

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS–Spike protein–ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19. Heparan sulfate (HS) has emerged as a SARS-CoV-2 coreceptor. Pixatimod (PG545), an HS mimetic, inhibits infectivity of multiple variants offering a novel therapeutic approach against COVID-19.

2.
Vaccines (Basel) ; 10(5)2022 Apr 28.
Article in English | MEDLINE | ID: covidwho-1820432

ABSTRACT

The COVID-19 pandemic is the biggest public health threat facing the world today. Multiple vaccines have been approved; however, the emergence of viral variants such as the recent Omicron raises the possibility of booster doses to achieve adequate protection. In Brazil, the CoronaVac (Sinovac, Beijing, China) vaccine was used; however, it is important to assess the immune response to this vaccine over time. This study aimed to monitor the anti-SARS-CoV-2 antibody responses in those immunized with CoronaVac and SARS-CoV-2 infected individuals. Samples were collected between August 2020 and August 2021. Within the vaccinated cohort, some individuals had a history of infection by SARS-CoV-2 prior to immunization, while others did not. We analyzed RBD-specific and neutralizing-antibodies. Anti-RBD antibodies were detected in both cohorts, with a peak between 45-90 days post infection or vaccination, followed by a steady decline over time. In those with a previous history of COVID-19, a higher, longer, more persistent response was observed. This trend was mirrored in the neutralization assays, where infection, followed by immunization, resulted in higher, longer lasting responses which were conditioned on the presence of levels of RBD antibodies right before the vaccination. This supports the necessity of booster doses of CoronaVac in due course to prevent serious disease.

3.
ACS Cent Sci ; 8(5): 527-545, 2022 May 25.
Article in English | MEDLINE | ID: covidwho-1795854

ABSTRACT

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.

4.
Vaccines (Basel) ; 10(4)2022 Apr 08.
Article in English | MEDLINE | ID: covidwho-1786092

ABSTRACT

The ongoing coronavirus disease 2019 (COVID-19) pandemic continues to disrupt essential health services in 90 percent of countries today. The spike (S) protein found on the surface of the causative agent, the SARS-CoV-2 virus, has been the prime target for current vaccine research since antibodies directed against the S protein were found to neutralize the virus. However, as new variants emerge, mutations within the spike protein have given rise to potential immune evasion of the response generated by the current generation of SARS-CoV-2 vaccines. In this study, a modified, HexaPro S protein subunit vaccine, delivered using a needle-free high-density microarray patch (HD-MAP), was investigated for its immunogenicity and virus-neutralizing abilities. Mice given two doses of the vaccine candidate generated potent antibody responses capable of neutralizing the parental SARS-CoV-2 virus as well as the variants of concern, Alpha and Delta. These results demonstrate that this alternative vaccination strategy has the potential to mitigate the effect of emerging viral variants.

5.
Pharmaceutics ; 14(4)2022 Apr 13.
Article in English | MEDLINE | ID: covidwho-1785876

ABSTRACT

The SARS-CoV-2 virus has caused a global crisis, resulting in 0.5 billion infections and over 6 million deaths as of March 2022. Fortunately, infection and hospitalization rates were curbed due to the rollout of DNA and mRNA vaccines. However, the efficacy of these vaccines significantly drops a few months post immunization, from 88% down to 47% in the case of the Pfizer BNT162 vaccine. The emergence of variant strains, especially delta and omicron, have also significantly reduced vaccine efficacy. We propose peptide vaccines as a potential solution to address the inadequacies of the current vaccines. Peptide vaccines can be easily modified to target emerging strains, have greater stability, and do not require cold-chain storage. We screened five peptide fragments (B1-B5) derived from the SARS-CoV-2 spike protein to identify neutralizing B-cell peptide antigens. We then investigated adjuvant systems for efficient stimulation of immune responses against the most promising peptide antigens, including liposomal formulations of polyleucine (L10) and polymethylacrylate (PMA), as well as classical adjuvants (CFA and MF59). Immune efficacy of formulations was evaluated using competitive ELISA, pseudovirion neutralization, and live virus neutralization assays. Unfortunately, peptide conjugation to L10 and PMA dramatically altered the secondary structure, resulting in low antibody neutralization efficacy. Of the peptides tested, only B3 administered with CFA or MF59 was highly immunogenic. Thus, a peptide vaccine relying on B3 may provide an attractive alternative to currently marketed vaccines.

6.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-329859

ABSTRACT

The COVID-19 pandemic is the biggest public health threat facing the globe today. Multiple vaccines have been approved, however the emergence of viral variants such as the recent Omicron, raises the possibility of booster doses to achieve adequate protection. In Brazil, the CoronaVac (Sinovac) vaccine was used, however it's important to assess the immune response to this vaccine over time. This study aimed to monitor the anti-SARS-CoV-2 antibody responses in those immunized with CoronaVac and SARS-CoV-2 infected individuals. Samples were collected between August 2020 and August 2021. Within the vaccinated cohort, some individuals had history of infection by SARS-CoV-2 prior to immunization and others not. We analyzed RBD-specific and neutralizing- antibodies. Anti-RBD antibodies were detected in both cohorts, with a peak between 45-90 days post infection or vaccination, followed by a steady decline over time. In those with previous history of COVID-19, a higher, longer, more persistent response was observed. This trend was mirrored in the neutralization assays, where infection followed by immunization resulted in higher, longer lasting responses which were conditioned on the presence of levels of RBD antibodies right before the vaccination. This supports the necessity of booster doses of CoronaVac in due course to prevent serious disease.

7.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-323849

ABSTRACT

Efforts to develop and deploy effective vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continue at pace. Here we describe rational antigen design through to manufacturability and vaccine efficacy, of a prefusion-stabilised Spike (S) protein, Sclamp. This strategy uses an orthogonal stabilisation approach compared to canonical vaccines, in combination with the licensed adjuvant MF59 (Seqirus). In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease, and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. The Sclamp vaccine candidate is currently completing Phase 1 clinical evaluation, in parallel with large-scale commercial manufacture for pivotal efficacy trials and potential widespread distribution.Funding: This work was funded by CEPI.Conflict of Interest: K.J.C., D.W. and P.R.Y. are inventors of the “Molecular Clamp” patent, US 2020/0040042.

8.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-323848

ABSTRACT

Efforts to develop and deploy effective vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continue at pace with more than 30 candidate vaccines now in clinical evaluation. Here we describe the preclinical development of an adjuvanted, prefusion-stabilised Spike (S) protein “Sclamp” subunit vaccine, from rational antigen design through to assessing manufacturability and vaccine efficacy. In mice, the vaccine candidate elicits high levels of neutralising antibodies to epitopes both within and outside the receptor binding domain (RBD) of S, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells. We also show protection in Syrian hamsters, which has emerged as a robust animal model for pulmonary SARS-CoV-2 infection. No evidence of vaccine enhanced disease was observed in animal challenge studies and pre-clinical safety was further demonstrated in a GLP toxicology study in rats. The Sclamp vaccine candidate is currently progressing rapidly through clinical evaluation in parallel with large-scale manufacture for pivotal efficacy trials and potential widespread distribution.

9.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-321184

ABSTRACT

This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin-converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include: 1) serum heat treatment to prevent non-specific interactions, 2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, 3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and 4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 hours for >30 serum samples;this includes the 7.5 hours of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the ‘gold standard’ assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious, time-consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R 2 =0.975, n=25), demonstrating high sensitivity.

10.
Lancet Infect Dis ; 21(10): 1383-1394, 2021 10.
Article in English | MEDLINE | ID: covidwho-1621119

ABSTRACT

BACKGROUND: Given the scale of the ongoing COVID-19 pandemic, the development of vaccines based on different platforms is essential, particularly in light of emerging viral variants, the absence of information on vaccine-induced immune durability, and potential paediatric use. We aimed to assess the safety and immunogenicity of an MF59-adjuvanted subunit vaccine for COVID-19 based on recombinant SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a novel molecular clamp (spike glycoprotein-clamp [sclamp]). METHODS: We did a phase 1, double-blind, placebo-controlled, block-randomised trial of the sclamp subunit vaccine in a single clinical trial site in Brisbane, QLD, Australia. Healthy adults (aged ≥18 to ≤55 years) who had tested negative for SARS-CoV-2, reported no close contact with anyone with active or previous SARS-CoV-2 infection, and tested negative for pre-existing SARS-CoV-2 immunity were included. Participants were randomly assigned to one of five treatment groups and received two doses via intramuscular injection 28 days apart of either placebo, sclamp vaccine at 5 µg, 15 µg, or 45 µg, or one dose of sclamp vaccine at 45 µg followed by placebo. Participants and study personnel, except the dose administration personnel, were masked to treatment. The primary safety endpoints included solicited local and systemic adverse events in the 7 days after each dose and unsolicited adverse events up to 12 months after dosing. Here, data are reported up until day 57. Primary immunogenicity endpoints were antigen-specific IgG ELISA and SARS-CoV-2 microneutralisation assays assessed at 28 days after each dose. The study is ongoing and registered with ClinicalTrials.gov, NCT04495933. FINDINGS: Between June 23, 2020, and Aug 17, 2020, of 314 healthy volunteers screened, 120 were randomly assigned (n=24 per group), and 114 (95%) completed the study up to day 57 (mean age 32·5 years [SD 10·4], 65 [54%] male, 55 [46%] female). Severe solicited reactions were infrequent and occurred at similar rates in participants receiving placebo (two [8%] of 24) and the SARS-CoV-2 sclamp vaccine at any dose (three [3%] of 96). Both solicited reactions and unsolicited adverse events occurred at a similar frequency in participants receiving placebo and the SARS-CoV-2 sclamp vaccine. Solicited reactions occurred in 19 (79%) of 24 participants receiving placebo and 86 (90%) of 96 receiving the SARS-CoV-2 sclamp vaccine at any dose. Unsolicited adverse events occurred in seven (29%) of 24 participants receiving placebo and 35 (36%) of 96 participants receiving the SARS-CoV-2 sclamp vaccine at any dose. Vaccination with SARS-CoV-2 sclamp elicited a similar antigen-specific response irrespective of dose: 4 weeks after the initial dose (day 29) with 5 µg dose (geometric mean titre [GMT] 6400, 95% CI 3683-11 122), with 15 µg dose (7492, 4959-11 319), and the two 45 µg dose cohorts (8770, 5526-13 920 in the two-dose 45 µg cohort; 8793, 5570-13 881 in the single-dose 45 µg cohort); 4 weeks after the second dose (day 57) with two 5 µg doses (102 400, 64 857-161 676), with two 15 µg doses (74 725, 51 300-108 847), with two 45 µg doses (79 586, 55 430-114 268), only a single 45 µg dose (4795, 2858-8043). At day 57, 67 (99%) of 68 participants who received two doses of sclamp vaccine at any concentration produced a neutralising immune response, compared with six (25%) of 24 who received a single 45 µg dose and none of 22 who received placebo. Participants receiving two doses of sclamp vaccine elicited similar neutralisation titres, irrespective of dose: two 5 µg doses (GMT 228, 95% CI 146-356), two 15 µg doses (230, 170-312), and two 45 µg doses (239, 187-307). INTERPRETATION: This first-in-human trial shows that a subunit vaccine comprising mammalian cell culture-derived, MF59-adjuvanted, molecular clamp-stabilised recombinant spike protein elicits strong immune responses with a promising safety profile. However, the glycoprotein 41 peptide present in the clamp created HIV diagnostic assay interference, a possible barrier to widespread use highlighting the criticality of potential non-spike directed immunogenicity during vaccine development. Studies are ongoing with alternative molecular clamp trimerisation domains to ameliorate this response. FUNDING: Coalition for Epidemic Preparedness Innovations, National Health and Medical Research Council, Queensland Government, and further philanthropic sources listed in the acknowledgments.


Subject(s)
Adjuvants, Immunologic/pharmacology , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Squalene/immunology , Adult , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Australia , Female , Healthy Volunteers , Humans , Male , Pandemics/prevention & control , Polysorbates , Vaccination/adverse effects , Young Adult
11.
Vaccines (Basel) ; 9(12)2021 Dec 16.
Article in English | MEDLINE | ID: covidwho-1580388

ABSTRACT

This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for >30 serum samples; this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the 'gold standard' assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R2 = 0.975, n = 25), demonstrating high sensitivity.

12.
2021.
Preprint in English | Other preprints | ID: ppcovidwho-296036

ABSTRACT

SUMMARY A recent study proposed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we applied deep (>50×) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2, and did not find the virus integrated into the genome. By examining ONT data from separate HEK293T cultivars, we completely resolved 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV) positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions via ONT sequencing. That we found no evidence of SARS-CoV-2 integration suggests such events are, at most, extremely rare in vivo , and therefore are unlikely to drive oncogenesis or explain post-recovery detection of the virus.

13.
Sci Adv ; 7(44): eabj8065, 2021 Oct 29.
Article in English | MEDLINE | ID: covidwho-1494911

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 160 million people and resulted in more than 3.3 million deaths, and despite the availability of multiple vaccines, the world still faces many challenges with their rollout. Here, we use the high-density microarray patch (HD-MAP) to deliver a SARS-CoV-2 spike subunit vaccine directly to the skin. We show that the vaccine is thermostable on the patches, with patch delivery enhancing both cellular and antibody immune responses. Elicited antibodies potently neutralize clinically relevant isolates including the Alpha and Beta variants. Last, a single dose of HD-MAP­delivered spike provided complete protection from a lethal virus challenge in an ACE2-transgenic mouse model. Collectively, these data show that HD-MAP delivery of a SARS-CoV-2 vaccine was superior to traditional needle-and-syringe vaccination and may be a significant addition to the ongoing COVID-19 (coronavirus disease 2019) pandemic.

14.
Viruses ; 13(10)2021 09 28.
Article in English | MEDLINE | ID: covidwho-1481007

ABSTRACT

Nipah virus (NiV) and respiratory syncytial virus (RSV) possess two surface glycoproteins involved in cellular attachment and membrane fusion, both of which are potential targets for vaccines. The majority of vaccine development is focused on the attachment (G) protein of NiV, which is the immunodominant target. In contrast, the fusion (F) protein of RSV is the main target in vaccine development. Despite this, neutralising epitopes have been described in NiV F and RSV G, making them alternate targets for vaccine design. Through rational design, we have developed a vaccine strategy applicable to phylogenetically divergent NiV and RSV that comprises both the F and G proteins (FxG). In a mouse immunization model, we found that NiV FxG elicited an improved immune response capable of neutralising pseudotyped NiV and a NiV mutant that is able to escape neutralisation by two known F-specific antibodies. RSV FxG elicited an immune response against both F and G and was able to neutralise RSV; however, this was inferior to the immune response of F alone. Despite this, RSV FxG elicited a response against a known protective epitope within G that is conserved across RSV A and B subgroups, which may provide additional protection in vivo. We conclude that inclusion of F and G antigens within a single design provides a streamlined subunit vaccine strategy against both emerging and established pathogens, with the potential for broader protection against NiV.


Subject(s)
Antibodies, Viral/blood , Henipavirus Infections/prevention & control , Nipah Virus/immunology , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Syncytial Virus, Human/immunology , Viral Envelope Proteins/immunology , Animals , Antibodies, Viral/immunology , Female , Humans , Mice , Mice, Inbred BALB C , Respiratory Syncytial Virus Vaccines/administration & dosage , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunology , Viral Envelope Proteins/administration & dosage , Viral Envelope Proteins/genetics , Viral Fusion Proteins/immunology
15.
Cell Rep ; 36(7): 109530, 2021 08 17.
Article in English | MEDLINE | ID: covidwho-1330686

ABSTRACT

A recent study proposed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we apply deep (>50×) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2 and do not find the virus integrated into the genome. By examining ONT data from separate HEK293T cultivars, we completely resolve 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV)-positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions by ONT sequencing. That we find no evidence of SARS-CoV-2 integration suggests that such events are, at most, extremely rare in vivo and therefore are unlikely to drive oncogenesis or explain post-recovery detection of the virus.


Subject(s)
COVID-19/virology , DNA, Viral/genetics , Genome, Human , SARS-CoV-2/genetics , Sequence Analysis, DNA , Virus Integration , Aged , Animals , COVID-19/diagnosis , Carcinoma, Hepatocellular/virology , Chlorocebus aethiops , HEK293 Cells , Hepatitis B virus/genetics , Host-Pathogen Interactions , Humans , Liver Neoplasms/virology , Long Interspersed Nucleotide Elements , Male , Nanopore Sequencing , Vero Cells
16.
PLoS Pathog ; 17(7): e1009723, 2021 07.
Article in English | MEDLINE | ID: covidwho-1295527

ABSTRACT

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Gene Expression Profiling , Lentivirus , SARS-CoV-2 , Transduction, Genetic , Angiotensin-Converting Enzyme 2/biosynthesis , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/genetics , COVID-19/metabolism , Chlorocebus aethiops , Disease Models, Animal , Humans , Mice , Mice, Knockout , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Vero Cells
17.
Nat Commun ; 12(1): 3431, 2021 06 08.
Article in English | MEDLINE | ID: covidwho-1262001

ABSTRACT

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.


Subject(s)
Reverse Genetics , SARS-CoV-2/genetics , Amino Acid Sequence , Animals , Base Sequence , Chlorocebus aethiops , Culicidae/virology , Furin/metabolism , Genome, Viral , HEK293 Cells , Humans , Mice , Mutation/genetics , NIH 3T3 Cells , Polymerase Chain Reaction , RAW 264.7 Cells , Receptors, Virus/metabolism , Vero Cells , Viral Proteins/chemistry , Virus Replication
18.
Biosaf Health ; 3(4): 202-209, 2021 Aug.
Article in English | MEDLINE | ID: covidwho-1252532

ABSTRACT

The pandemic of coronavirus disease 2019 (COVID-19) has already evoked massive influence. The global pandemic has been ravaging the whole world for a year, with the number of confirmed human infection cases over 150 million and a death toll exceeding 3 million. Although the genomic sequence of the cognate pathogen SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has been quickly determined, there are still many unknown aspects, including the virus origin and evolution trend, and the effectiveness of current vaccines and drugs against the mutating virus. This review summarizes current knowledge and advances about COVID-19, including virus origin, transmission and infection, with the aim to improve the understanding of COVID-19 and provide a new perspective for future studies.

19.
Nat Commun ; 12(1): 2037, 2021 04 01.
Article in English | MEDLINE | ID: covidwho-1164849

ABSTRACT

The hallmarks of COVID-19 are higher pathogenicity and mortality in the elderly compared to children. Examining baseline SARS-CoV-2 cross-reactive immunological responses, induced by circulating human coronaviruses (hCoVs), is needed to understand such divergent clinical outcomes. Here we show analysis of coronavirus antibody responses of pre-pandemic healthy children (n = 89), adults (n = 98), elderly (n = 57), and COVID-19 patients (n = 50) by systems serology. Moderate levels of cross-reactive, but non-neutralizing, SARS-CoV-2 antibodies are detected in pre-pandemic healthy individuals. SARS-CoV-2 antigen-specific Fcγ receptor binding accurately distinguishes COVID-19 patients from healthy individuals, suggesting that SARS-CoV-2 infection induces qualitative changes to antibody Fc, enhancing Fcγ receptor engagement. Higher cross-reactive SARS-CoV-2 IgA and IgG are observed in healthy elderly, while healthy children display elevated SARS-CoV-2 IgM, suggesting that children have fewer hCoV exposures, resulting in less-experienced but more polyreactive humoral immunity. Age-dependent analysis of COVID-19 patients, confirms elevated class-switched antibodies in elderly, while children have stronger Fc responses which we demonstrate are functionally different. These insights will inform COVID-19 vaccination strategies, improved serological diagnostics and therapeutics.


Subject(s)
Antibodies, Viral/blood , Antibodies, Viral/immunology , Antibody Formation/immunology , SARS-CoV-2/immunology , Adolescent , Adult , Aged , Aged, 80 and over , COVID-19/immunology , COVID-19 Vaccines/immunology , Child , Child, Preschool , Cross Reactions/immunology , Humans , Immunoglobulin A/blood , Immunoglobulin A/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Middle Aged , Receptors, IgG/immunology , Spike Glycoprotein, Coronavirus/immunology , Young Adult
20.
Clin Transl Immunology ; 10(4): e1269, 2021.
Article in English | MEDLINE | ID: covidwho-1162553

ABSTRACT

OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

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