- Infectious Diseases and Allergy group,
School of Pharmacy,
University of Kent,
Kent, ME4 4TB, UK - 01634202957
Nigel Temperton
University of Kent, Medway School of Pharmacy, Faculty Member
- University College London, Immunology and Infectious diseases, Department Memberadd
- Serology, Diagnostics, Social Media, Influenza virus, Avian Influenza, Pandemic Influenza Preparedness, and 22 moreLentiviral vector, Pseudotyped Lentivirus, Biotechnology, Molecular Biology, Immunology, Genetic Engineering, Virology, Vaccine development, Molecular virology, Viral Vaccinology, Viral Immunology, Health Sciences, Pharmaceutical Sciences, Clinical Trials, Cell Culture, Clinical and Translational Research, Immunology and Serology, Molecular Microbiology, Respiratory Medicine, Reverse genetics, Cell Based Assays, and Pseudotype Virusedit
Influenza B viruses (IBV) cause respiratory disease epidemics in humans and are therefore components of seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However,... more
Influenza B viruses (IBV) cause respiratory disease epidemics in humans and are therefore components of seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, classical methods to assess influenza vaccine immunogenicity such as the hemagglutination inhibition assay (HI) and the serial radial hemolysis assay (SRH), have been proven to have many limitations. As such, there is a need to develop innovative methods that can improve on these traditional assays and provide advantages such as ease of production and access, safety, reproducibility, and specificity. It has been previously demonstrated that the use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A hemagglutinins in microneutralization assays (pMN) is a safe and sensitive alternative to study antibody responses elicited by natural influenza infection or vaccination. Consequently, we have produced Influenza B hemaggluti...
In this University of Kent Grants Factory session delivered on 27th Feb 2019, Senior Lecturer Dr Nigel Temperton (Medway School of Pharmacy) presents a personal perspective on how he uses a range of online platforms to track, disseminate... more
In this University of Kent Grants Factory session delivered on 27th Feb 2019, Senior Lecturer Dr Nigel Temperton (Medway School of Pharmacy) presents a personal perspective on how he uses a range of online platforms to track, disseminate and measure the impact of his research. This will include ORCID, KAR (Kent repository), Google Scholar, Figshare, Scopus, ImpactStory, F1000, bioRxiv, Kudos, ResearchGate and EuropePMC. He will demonstrate to you that by populating these platforms with your research outputs, you can help drive citations of your publications and find out where your research is being used.
Slides from invited talk given at the Emerging Viruses symposium at St Hilda's college, Oxford on 9th September 2019. This talk is based on data published predominantly in Nature Microbiology... more
Slides from invited talk given at the Emerging Viruses symposium at St Hilda's college, Oxford on 9th September 2019. This talk is based on data published predominantly in Nature Microbiology https://naturemicrobiologycommunity.nature.com/users/268901-efstathios-giotis/posts/51710-exploring-the-zoonotic-potential-of-the-bat-influenza-viruses and in bioRxivhttps://www.biorxiv.org/content/10.1101/499947v2<br>
Slides from offered talk given at OptionsX for the Control of Influenza conference in Singapore SUNTEC on 1st September 2019.
Background COVID-19 vaccines have demonstrated effectiveness in reducing SARS-CoV-2 mild and severe outcomes. In vaccinated subjects with SARS-CoV-2 history, RBD-specific IgG and pseudovirus neutralization titers were rapidly recalled by... more
Background COVID-19 vaccines have demonstrated effectiveness in reducing SARS-CoV-2 mild and severe outcomes. In vaccinated subjects with SARS-CoV-2 history, RBD-specific IgG and pseudovirus neutralization titers were rapidly recalled by a single BTN162b2 vaccine dose to higher levels than those in naïve recipients after the second dose, irrespective of waning immunity. In this study, we inspected the long-term kinetic and neutralizing responses of S-specific IgG induced by two administrations of BTN162b2 vaccine in infection-naïve subjects and in subjects previously infected with SARS-CoV-2. Methods Twenty-six naïve and 9 previously SARS-CoV-2 infected subjects during the second wave of the pandemic in Italy were enrolled for this study. The two groups had comparable demographic and clinical characteristics. By means of ELISA and pseudotyped-neutralization assays, we investigated the kinetics of developed IgG-RBD and their neutralizing activity against both the ancestral D614G and ...
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RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a... more
RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a large number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients as well as vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike chimeras and mutants harbouring single amino acid substitutions within the RBD demonstrated that both spike proteins c...
Purpose: Some conventional serological assays can accurately quantify neutralising antibody responses raised against epitopes on virus glycoproteins, enabling mass vaccine evaluation and serosurveillance studies to take place. However,... more
Purpose: Some conventional serological assays can accurately quantify neutralising antibody responses raised against epitopes on virus glycoproteins, enabling mass vaccine evaluation and serosurveillance studies to take place. However, these assays often necessitate the handling of wild-type virus in expensive high biosafety laboratories, which restricts the scope of their application, particularly in resource-deprived areas. A solution to this issue is the use of lentiviral pseudotype viruses (PVs)—chimeric, replication-deficient virions that imitate the binding and entry mechanisms of their wild-type equivalents. Pseudotype virus neutralisation assays (PVNAs) bypass high biosafety requirements and yield comparable results to established assays. This study explores the potential for using lyophilisation of pseudotypes as a cost-effective, alternative means for production, distribution and storage of a PVNAbased diagnostic kit. Methods & Materials: Rabies, Marburg and H5 subtype Inf...
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Influenza B is responsible for a significant proportion of the global morbidity, mortality and economic loss caused by influenza-related disease. Two antigenically distinct lineages co-circulate worldwide, often resulting in mismatches in... more
Influenza B is responsible for a significant proportion of the global morbidity, mortality and economic loss caused by influenza-related disease. Two antigenically distinct lineages co-circulate worldwide, often resulting in mismatches in vaccine coverage when vaccine predictions fail. There are currently operational issues with gold standard serological assays for influenza B, such as lack of sensitivity and requirement for specific antigen treatment. This study encompasses the gold standard assays with the more recent Pseudotype-based Microneutralisation assay in order to study comparative serological outcomes. Haemagglutination Inhibition, Single Radial Haemolysis and Pseudotype-based Microneutralisation correlated strongly for strains in the Yamagata lineage; however, it correlated with neither gold standard assays for the Victoria lineage.
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Influenza B viruses (IBV) cause respiratory disease epidemics in humans and are therefore components of seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However,... more
Influenza B viruses (IBV) cause respiratory disease epidemics in humans and are therefore components of seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, classical methods to assess influenza vaccine immunogenicity such as the hemagglutination inhibition assay (HI) and the serial radial hemolysis assay (SRH), have been proven to have many limitations. As such, there is a need to develop innovative methods that can improve on these traditional assays and provide advantages such as ease of production and access, safety, reproducibility, and specificity. It has been previously demonstrated that the use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A hemagglutinins in microneutralization assays (pMN) is a safe and sensitive alternative to study antibody responses elicited by natural influenza infection or vaccination. Consequently, we have produced Influenza B hemaggluti...
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We have developed an influenza hemagglutinin (HA) pseudotype library encompassing Influenza A subtypes HA1-18, and Influenza B subtypes (both lineages) to be employed in influenza pseudotype microneutralization (pMN) assays. The pMN is... more
We have developed an influenza hemagglutinin (HA) pseudotype library encompassing Influenza A subtypes HA1-18, and Influenza B subtypes (both lineages) to be employed in influenza pseudotype microneutralization (pMN) assays. The pMN is highly sensitive and specific for detecting virus-specific neutralizing antibodies against influenza viruses and can be used to assess antibody functionality in vitro. Here we show the production of these viral HA pseudotypes and their employment as substitutes for wildtype viruses in influenza serological and neutralization assays. We demonstrate its utility in detecting serum response to vaccination with the ability to evaluate cross-subtype neutralizing responses elicited by specific vaccinating antigens. Our findings may inform further pre-clinical studies involving immunization dosing regimens in mice and may help in the creation and selection of better antigens for vaccine design. These HA pseudotypes can be harnessed to meet strategic objective...
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BackgroundThe rise of SARS-CoV-2 variants has made the pursuit to define correlates of protection more troublesome, despite the availability of the World Health Organisation (WHO) International Standard for anti-SARS-CoV-2 Immunoglobulin... more
BackgroundThe rise of SARS-CoV-2 variants has made the pursuit to define correlates of protection more troublesome, despite the availability of the World Health Organisation (WHO) International Standard for anti-SARS-CoV-2 Immunoglobulin sera, a key reagent used to standardise laboratory findings into an international unitage.MethodsUsing pseudotyped virus, we examine the capacity of convalescent sera, from a well-defined cohort of healthcare workers (HCW) and Patients infected during the first wave from a national critical care centre in the UK to neutralise B.1.1.298, variants of interest (VOI) B.1.617.1 (Kappa), and four VOCs, B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta), including the B.1.617.2 K417N, informally known as Delta Plus. We utilised the WHO International Standard for anti-SARS-CoV-2 Immunoglobulin to report neutralisation antibody levels in International Units per mL.FindingsOur data demonstrate a significant reduction in the ability of first wa...
SARS-CoV-2 virus was first detected in late 2019 and circulated globally, causing COVID-19, which is characterised by sub-clinical to severe disease in humans. Here, we investigate the serological antibody responses to SARS-CoV-2... more
SARS-CoV-2 virus was first detected in late 2019 and circulated globally, causing COVID-19, which is characterised by sub-clinical to severe disease in humans. Here, we investigate the serological antibody responses to SARS-CoV-2 infection during acute and convalescent infection using a cohort of (i) COVID-19 patients admitted to hospital, (ii) healthy individuals who had experienced ‘COVID-19 like-illness’, and (iii) a cohort of healthy individuals prior to the emergence of SARS-CoV-2. We compare SARS-CoV-2 specific antibody detection rates from four different serological methods, virus neutralisation test (VNT), ID Screen® SARS-CoV-2-N IgG ELISA, Whole Antigen ELISA, and lentivirus-based SARS-CoV-2 pseudotype virus neutralisation tests (pVNT). All methods were able to detect prior infection with COVID-19, albeit with different relative sensitivities. The VNT and SARS-CoV-2-N ELISA methods showed a strong correlation yet provided increased detection rates when used in combination. ...
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The virus SARS-CoV-2, responsible for the global COVID-19 pandemic, spread rapidly around the world causing high morbidity and mortality because humans have no pre-existing immunity. However, there are four known, endemic seasonal... more
The virus SARS-CoV-2, responsible for the global COVID-19 pandemic, spread rapidly around the world causing high morbidity and mortality because humans have no pre-existing immunity. However, there are four known, endemic seasonal coronaviruses in humans (HCoVs) and whether antibodies for these HCoVs play a role in severity of COVID-19 disease has generated a lot of interest. Of these seasonal viruses NL63 is of particular interest as it uses the same cell entry receptor as SARS-CoV-2.We use functional, neutralising assays to investigate cross reactive antibodies and their relationship with COVID-19 severity. We analysed neutralisation of SARS-CoV-2, NL63, HKU1, and 229E in 38 COVID-19 patients and 62 healthcare workers, and a further 182 samples to specifically study the relationship between SARS-CoV-2 and NL63.We found that although HCoV neutralisation was very common there was little evidence that these antibodies neutralised SARS-CoV-2. Despite no evidence in cross neutralisatio...
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We developed an influenza hemagglutinin (HA) pseudotype library encompassing Influenza A subtypes HA1-18 and Influenza B subtypes (both lineages) to be employed in influenza pseudotype microneutralization (pMN) assays. The pMN is highly... more
We developed an influenza hemagglutinin (HA) pseudotype library encompassing Influenza A subtypes HA1-18 and Influenza B subtypes (both lineages) to be employed in influenza pseudotype microneutralization (pMN) assays. The pMN is highly sensitive and specific for detecting virus-specific neutralizing antibodies against influenza viruses and can be used to assess antibody functionality in vitro. Here we show the production of these viral HA pseudotypes and their employment as substitutes for wildtype viruses in influenza neutralization assays. We demonstrate their utility in detecting serum responses to vaccination with the ability to evaluate cross-subtype neutralizing responses elicited by specific vaccinating antigens. Our findings may inform further preclinical studies involving immunization dosing regimens in mice and may help in the creation and selection of better antigens for vaccine design. These HA pseudotypes can be harnessed to meet strategic objectives that contribute to...
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The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19... more
The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive fo...
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SummaryIt is unclear whether prior endemic coronavirus infections affect COVID-19 severity. Here, we show that in cases of fatal COVID-19, antibody responses to the SARS-COV-2 spike are directed against epitopes shared with endemic... more
SummaryIt is unclear whether prior endemic coronavirus infections affect COVID-19 severity. Here, we show that in cases of fatal COVID-19, antibody responses to the SARS-COV-2 spike are directed against epitopes shared with endemic beta-coronaviruses in the S2 subunit of the SARS-CoV-2 spike protein. This immune response is associated with the compromised production of a de novo SARS-CoV-2 spike response among individuals with fatal COVID-19 outcomes.
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Understanding the nature of immunity after mild/asymptomatic SARS-CoV-2 infection is crucial to controlling the pandemic. We analyzed T cell and neutralizing antibody responses in 136 health care workers (HCWs) 16 to 18 weeks after the... more
Understanding the nature of immunity after mild/asymptomatic SARS-CoV-2 infection is crucial to controlling the pandemic. We analyzed T cell and neutralizing antibody responses in 136 health care workers (HCWs) 16 to 18 weeks after the start of the first U.K. lockdown, 76 of whom had mild/asymptomatic SARS-CoV-2 infection captured by serial sampling. Neutralizing antibodies (nAbs) were present in 89% of previously infected HCWs. T cell responses tended to be lower after asymptomatic infection than in those reporting case-definition symptoms of COVID-19, whereas nAb titers were maintained irrespective of symptoms. T cell and antibody responses were sometimes discordant. Eleven percent lacked nAb and had undetectable T cell responses to spike protein but had T cells reactive to other SARS-CoV-2 antigens. Our findings suggest that most individuals with mild or asymptomatic SARS-CoV-2 infection carry nAb complemented by multispecific T cell responses at 16 to 18 weeks after mild or asym...
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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... more
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 mat...
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COVID-19 continues to cause a pandemic, having infected more than 20 million people globally. Successful elimination of the SARS-CoV-2 virus will require an effective vaccine. However, the immune correlates of infection are currently... more
COVID-19 continues to cause a pandemic, having infected more than 20 million people globally. Successful elimination of the SARS-CoV-2 virus will require an effective vaccine. However, the immune correlates of infection are currently poorly understood. While neutralizing antibodies are believed to be essential for protection against infection, the contribution of the neutralizing antibody response to resolution of SARS-CoV-2 infection has not yet been defined. In this study the antibody responses to the SARS-CoV-2 spike protein and nucleocapsid proteins were investigated in a UK patient cohort, using optimised immunoassays and a retrovirus-based pseudotype entry assay. It was discovered that in severe COVID-19 infections an early antibody response to both antigens was associated with improved prognosis of infection. While not all SARS-CoV-2-reactive sera were found to possess neutralizing antibodies, neutralizing potency of sera was found to be greater in patients who went on to res...
The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2... more
The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on “pseudotyping” can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of t...
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Equine influenza is a major respiratory disease of horses that is largely controlled by vaccination in some equine populations. Virus-neutralising antibodies, the mainstay of the protective immune response, are problematic in assaying for... more
Equine influenza is a major respiratory disease of horses that is largely controlled by vaccination in some equine populations. Virus-neutralising antibodies, the mainstay of the protective immune response, are problematic in assaying for equine influenza virus, as most strains do not replicate efficiently in cell culture. Surrogate measures of protective antibody responses include the haemagglutination inhibition (HI) test and single radial haemolysis (SRH) assay. For this study, a pseudotyped virus, bearing an envelope containing the haemagglutinin (HA) from the Florida clade 2 equine influenza virus strain A/equine/Richmond/1/07 (H3N8), was generated to measure HA-specific neutralising antibodies in serum samples (n = 134) from vaccinated or experimentally-infected ponies using a pseudotyped virus neutralization test (PVNT). Overall, the results of PVNT were in good agreement with results from the SRH assay (100% sensitivity, 68.53% specificity) and HI test (99.2% sensitivity, 49...
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The diversity of subtypes within Influenza A recently expanded with identification of H17N10 and H18N11 from bats. To study the tropism and zoonotic potential of these viruses, we successfully produced lentiviral pseudotypes bearing... more
The diversity of subtypes within Influenza A recently expanded with identification of H17N10 and H18N11 from bats. To study the tropism and zoonotic potential of these viruses, we successfully produced lentiviral pseudotypes bearing haemagglutinin H17 and neuraminidase N10. We investigated a range of cell lines from different species for their susceptibility to infection by these pseudotypes and show that a number of human haematopoietic cancer cell lines and the canine kidney MDCK II (but not MDCK I) cells are susceptible. Using microarrays and qRT-PCR we show that the dog leukocyte antigen DLA-DRA mRNA is over expressed in late passaged parental MDCK and commercial MDCK II cells, compared to early passaged parental MDCK and MDCK I cells, respectively. The human orthologue HLA-DRA encodes the alpha subunit of the MHC class II HLA-DR antigen-binding heterodimer. Small interfering RNA- or neutralizing antibody-targeting HLA-DRA, drastically reduced the susceptibility of Raji B cells ...
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Objective Goal of this work is to assess the feasibility to perform COVID-19 RNA tests within hospitals and communities experiencing SARS-CoV-2 virus outbreaks, to ultimately provide recommendations for hospitals with so-called fever... more
Objective Goal of this work is to assess the feasibility to perform COVID-19 RNA tests within hospitals and communities experiencing SARS-CoV-2 virus outbreaks, to ultimately provide recommendations for hospitals with so-called fever clinics. In China, these specialised clinics within a hospital, specifically receive outpatients who have fever symptoms.Methods A team with expertise in the Exposure Analysis of Critical Control Points (EACCP) framework first identified potential infection routes during the testing for SARS-CoV-2, then constructed and tested flow diagrams, which were confirmed under actual conditions, demonstrating the feasibility to be carried out in hospitals with fever clinics. The team determined critical control points to mitigate the exposure risks at each control point.Findings The sampling and inactivation steps of clinical samples in fever clinics appeared to be associated with particularly high risk levels of exposure to SARS-CoV-2. Moderate levels of exposur...
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Bats are notorious reservoirs of diverse, potentially zoonotic viruses, exemplified by the evolutionarily distinct, influenza A-like viruses H17N10 and H18N11 (BatIVs). The surface glycoproteins [haemagglutinin (H) and neuraminidase (N)]... more
Bats are notorious reservoirs of diverse, potentially zoonotic viruses, exemplified by the evolutionarily distinct, influenza A-like viruses H17N10 and H18N11 (BatIVs). The surface glycoproteins [haemagglutinin (H) and neuraminidase (N)] of BatIVs neither bind nor cleave sialic acid receptors, which suggests that these viruses employ cell attachment and entry mechanisms that differ from those of classical influenza A viruses (IAVs). Identifying the cellular factors that mediate entry and determine susceptibility to infection will help assess the host range of BatIVs. Here, we investigated a range of cell lines from different species for their susceptibility to infection by pseudotyped viruses (PV) bearing bat H17 and/or N10 envelope glycoproteins. We show that a number of human haematopoietic cancer cell lines and the canine kidney MDCK II (but not MDCK I) cells are susceptible to H17-pseudotypes (H17-PV). We observed with microarrays and qRT-PCR that the dog leukocyte antigen DLA-D...
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Pseudotype neutralization assays are powerful tools to study functional antibody responses against viruses in low biosafety laboratories. However, protocols described in the literature differ widely with respect to material, reagents, and... more
Pseudotype neutralization assays are powerful tools to study functional antibody responses against viruses in low biosafety laboratories. However, protocols described in the literature differ widely with respect to material, reagents, and methods used to perform these assays and to analyse the raw data generated. This could result in discrepancies between the results of different laboratories even when the same pseudotypes and the same samples are analysed. Here, we describe, in detail, an experimental protocol to perform pseudotype neutralization assays using lentiviral pseudotypes bearing influenza haemagglutinin and expressing firefly luciferase. We also present the steps necessary to analyse the data and calculate the half maximal inhibitory concentration of the sera analysed. This protocol will provide support for the validation and the standardization of the pseudotype neutralization assay for influenza virus serology. Additionally, it will provide a starting point for the dev...
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Haemagglutinin and neuraminidase surface glycoproteins of the bat influenza H17N10 virus neither bind to nor cleave sialic acid receptors, indicating that this virus employs cell entry mechanisms distinct from those of classical influenza... more
Haemagglutinin and neuraminidase surface glycoproteins of the bat influenza H17N10 virus neither bind to nor cleave sialic acid receptors, indicating that this virus employs cell entry mechanisms distinct from those of classical influenza A viruses. We observed that certain human haematopoietic cancer cell lines and canine MDCK II cells are susceptible to H17-pseudotyped viruses. We identified the human HLA-DR receptor as an entry mediator for H17 pseudotypes, suggesting that H17N10 possesses zoonotic potential. A comparative transcriptional analysis identifies human HLA-DR as a factor that mediates entry of bat influenza A-like H17-pseudotyped viral particles into mammalian cells.
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Influenza pseudotypes represent an alternative to wild type virus for serological assays. To date, pseudotypes (PV) have predominantly been used as surrogates for wild type viruses in microneutralisation assays, where the surface... more
Influenza pseudotypes represent an alternative to wild type virus for serological assays. To date, pseudotypes (PV) have predominantly been used as surrogates for wild type viruses in microneutralisation assays, where the surface glycoprotein of interest and a reporter gene (such as Luciferase) are used to assess if virus entry into target cells could be inhibited by serum antibodies. The influenza neuraminidase (NA) has the ability to bud and release new virions with or without the contribution of Haemagglutinin (HA). Influenza pseudotypes expressing NA alone, or with HA, were produced to evaluate the antibody response against NA using the enzyme-linked lectin assay (ELLA). The expression of an avian HA with human NAs has enabled the detection of specific antibody reponses against the human circulating subtypes of NA. Within this study a PV-based ELLA assay has been investigated with a pilot panel of sera prepared for an international CONSISE study. Preliminary results have confirm...
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(Abstract only) The 2014–2016 Ebola outbreak in West Africa highlighted the need for improved diagnostics, surveillance and therapeutics for filoviruses. The need for high containment virus handling facilities creates a bottleneck... more
(Abstract only) The 2014–2016 Ebola outbreak in West Africa highlighted the need for improved diagnostics, surveillance and therapeutics for filoviruses. The need for high containment virus handling facilities creates a bottleneck hindering research efforts. A safe alternative to working with native viruses are pseudotyped viruses (PV) which are non-replicating particles bearing surface glycoprotein(s) that can be used for antibody detection. The aim of this study was to create a diagnostic tool to distinguish between genera and species of pathogenic filoviruses (e.g. neutralization tests and ELISA), avoiding the cross reactivity currently seen. High titre PVs bearing the receptor glycoprotein (GP) of different filovirus species, plus specific epitope chimeras, were successfully generated. Next, lyophilisation studies to assess particle stability/degradation transportation and long-term storage were conducted. Filoviruses maintained their titres for at least 1.5 years after lyophilisation when kept in temperatures of up to 4 °C, with all filovirus genera following a similar trend. At higher temperatures, PVs degraded to unworkable titres. Reconstituted PVs also performed well in neutralisation assays. A chimeric cuevavirus GP bearing ebolavirus (Zaire sp.) epitopes KZ52 and 1 H3 retained infectivity, with average titres of approximately 1×10 7 RLU ml−1, similar to wild type, indicating its structure was not compromised. These chimeras are now being assessed in neutralisation tests using specific monoclonal antibodies and incorporated into ELISA with PVs as antigens. The data suggests lyophilised PVs are amenable to long-term storage, and their GPs can be modified to create artificial antigens for diagnostics and serosurveillance.
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Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists... more
Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists can be made available. Broad-spectrum antivirals are drugs with the potential to inhibit infection by viruses from different groups or families, which may be deployed during outbreaks when specific diagnostics, vaccines or directly acting antivirals are not available. While pathogen-directed approaches are generally effective against a few closely related viruses, targeting cellular pathways used by multiple viral agents can have broad-spectrum efficacy. Virus entry, particularly clathrin-mediated endocytosis, constitutes an attractive target as it is used by many viruses. Using a phenotypic screening strategy where the inhibitory activity of small molecules was sequentially tested against different viruses, we identified 12 compounds with broad-spec...
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Influenza B virus (IBV) circulates in the human population and causes considerable disease burden worldwide, each year. Current IBV vaccines can struggle to mount an effective cross-reactive immune response, as strains become mismatched,... more
Influenza B virus (IBV) circulates in the human population and causes considerable disease burden worldwide, each year. Current IBV vaccines can struggle to mount an effective cross-reactive immune response, as strains become mismatched, due to constant antigenic changes. Additional strategies which use monoclonal antibodies, with broad reactivity, are of considerable interest, both, as diagnostics and as immunotherapeutics. Alternatives to conventional monoclonal antibodies, such as single domain antibodies (NanobodiesTM) with well-documented advantages for applications in infectious disease, have been emerging. In this study we have isolated single domain antibodies (sdAbs), specific to IBV, using alpacas immunised with recombinant hemagglutinin (HA) from two representative viruses, B/Florida/04/2006 (B/Yamagata lineage) and B/Brisbane/60/2008 (B/Victoria lineage). Using phage display, we have isolated a panel of single domain antibodies (sdAbs), with both cross-reactive and linea...
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The diversity of subtypes within the Influenza A virus genus has recently expanded with the identification of H17N10 and H18N11 from bats. In order to further study the tropism and zoonotic potential of these viruses, we have successfully... more
The diversity of subtypes within the Influenza A virus genus has recently expanded with the identification of H17N10 and H18N11 from bats. In order to further study the tropism and zoonotic potential of these viruses, we have successfully produced lentiviral pseudotypes bearing both H17 and N10. These pseudotypes were shown to be efficiently neutralized by the broadly-neutralizing monoclonal antibodies CR9114 and FI6. Our studies also confirm previous reports that H17 does not use sialic acid as its cellular receptor, as pseudotypes bearing the H17 envelope glycoprotein are released into the cell supernatant in the absence of neuraminidase. However, we demonstrate that N10 facilitates heterosubtypic (H5 and H7) influenza hemagglutinin-bearing pseudotype release in the absence of another source of neuraminidase, significantly increasing luciferase pseudotype production titres. Despite this, N10 shows no activity in the enzyme-linked lectin assay used for traditional sialidases. These...
Influenza B viruses cause respiratory disease epidemics in human populations and are included in seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, the... more
Influenza B viruses cause respiratory disease epidemics in human populations and are included in seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, the haemagglutination inhibition assay, which represents the gold standard for assessing the immunogenicity of influenza vaccines, has been shown to be relatively insensitive for the detection of antibodies against influenza B viruses. Furthermore, this assay, and the serial radial haemolysis assay are not able to detect stalk-directed cross-reactive antibodies. For these reasons there is a need to develop new assays that can overcome these limitations. The use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A haemagglutinins, in microneutralization assays is a safe and sensitive alternative to study antibody responses elicited by natural infection or vaccination. We have produced Influenza B haemagglutinin-pseudotypes using...
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The Viral Pseudotype Unit was established in 2010 to act as an interface between academia, industry and public/animal health laboratories with the purpose of translating basic virus research (on viral pseudotypes) into in vitro cell... more
The Viral Pseudotype Unit was established in 2010 to act as an interface between academia, industry and public/animal health laboratories with the purpose of translating basic virus research (on viral pseudotypes) into in vitro cell culture-based assays that can be readily employed for the immunogenicity testing of preclinical vaccine candidates, and for the functional screening of new antivirals and therapeutic antibodies. More recently the Viral Pseudotype Unit has become involved with the exploitation of pseudotypes for the development of serological standards, and with training and education via traditional and new media platforms.
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Pseudotype particle neutralization (pp‑NT) is a next-generation serological assay employed for the sensitive study of influenza antibody responses, especially haemagglutinin stalk-directed antibodies. However, to date a validation of this... more
Pseudotype particle neutralization (pp‑NT) is a next-generation serological assay employed for the sensitive study of influenza antibody responses, especially haemagglutinin stalk-directed antibodies. However, to date a validation of this assay has not been performed, and this limits its use to primarily research laboratories. To identify possible serological standards to be used in optimization and validation of the pp‑NT, we have evaluated the cross-reactivity of hyperimmune chicken reference antisera in this assay. Our findings show that the cross-reactivity detected by the pp‑NT assay is only in part explained by phylogenetic relationships and protein homology between the HA subtypes analysed; further studies are necessary to understand the origin of the cross-reactivity detected, and reference standards with higher specificity should be evaluated or generated de novo for future use in pp-NT.
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Research Interests:
Seasonal influenza viruses are a common cause of acute respiratory illness worldwide and generate a significant socioeconomic burden. Influenza viruses mutate rapidly, necessitating annual vaccine reformulation because traditional... more
Seasonal influenza viruses are a common cause of acute respiratory illness worldwide and generate a significant socioeconomic burden. Influenza viruses mutate rapidly, necessitating annual vaccine reformulation because traditional vaccines do not typically induce broad-spectrum immunity. In addition to seasonal infections, emerging pandemic influenza viruses present a continued threat to global public health. Pandemic influenza viruses have consistently higher attack rates and are typically associated with greater mortality compared with seasonal strains. Ongoing strategies to improve vaccine efficacy typically focus on providing broad-spectrum immunity; although B and T cells can mediate heterosubtypic responses, typical vaccine development will augment either humoral or cellular immunity. However, multipronged approaches that target several Ags may limit the generation of viral escape mutants. There are few vaccine platforms that can deliver multiple Ags and generate robust cellul...
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The Middle East respiratory syndrome coronavirus (MERS-CoV) has infected more than 1900 humans, since 2012. The syndrome ranges from asymptomatic and mild cases to severe pneumonia and death. The virus is believed to be circulating in... more
The Middle East respiratory syndrome coronavirus (MERS-CoV) has infected more than 1900 humans, since 2012. The syndrome ranges from asymptomatic and mild cases to severe pneumonia and death. The virus is believed to be circulating in dromedary camels without notable symptoms since the 1980s. Therefore, dromedary camels are considered the only animal source of infection. Neither antiviral drugs nor vaccines are approved for veterinary or medical use despite active research on this area. Here, we developed four vaccine candidates against MERS-CoV based on ChAdOx1 and MVA viral vectors, two candidates per vector. All vaccines contained the full-length spike gene of MERS-CoV; ChAdOx1 MERS vaccines were produced with or without the leader sequence of the human tissue plasminogen activator gene (tPA) where MVA MERS vaccines were produced with tPA, but either the mH5 or F11 promoter driving expression of the spike gene. All vaccine candidates were evaluated in a mouse model in prime only ...
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Recently chimeric influenza haemagglutinins (cHAs) have been generated as potential 'universal' vaccination antigens and as tools to identify HA stalk-directed antibodies via their use as antigens in ELISA, and virus or... more
Recently chimeric influenza haemagglutinins (cHAs) have been generated as potential 'universal' vaccination antigens and as tools to identify HA stalk-directed antibodies via their use as antigens in ELISA, and virus or pseudotype-based neutralization assays. The original methods [1], [2] used for their generation require the amplification of regions of interest (head and stalk) using primers containing SapI sites and subsequent cloning into pDZ plasmid. This requires precise primer design, checking for the absence of SapI sites in the sequence of interest, and multi-segment ligation. As an alternative strategy we have developed and optimized a new protocol for assembling the cHA by exploiting Gibson Assembly. •This method also requires precise primer design, but it is rapid and methodologically simple to perform. We have evaluated that using this method it is possible to construct a cHA encoding DNA in less than a week.•Additional weeks are however necessary to optimize the...
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Pseudotyped viruses (PVs) produced by co-transfecting cells with plasmids expressing lentiviral core proteins and viral envelope proteins are potentially powerful tools for studying various aspects of equine influenza virus (EIV) biology.... more
Pseudotyped viruses (PVs) produced by co-transfecting cells with plasmids expressing lentiviral core proteins and viral envelope proteins are potentially powerful tools for studying various aspects of equine influenza virus (EIV) biology. The aim of this study was to optimise production of equine influenza PVs. Co-transfection of the HAT protease to activate the haemagglutinin (HA) yielded a higher titre PV than TMPRSS2 with the HA from A/equine/Richmond/1/2007 (H3N8), whereas for A/equine/Newmarket/79 (H3N8), both proteases resulted in equivalent titres. TMPRSS4 was ineffective with the HA of either strain. There was also an inverse relationship between the amount of protease-expression plasmids and the PV titre obtained. Interestingly, the PV titre obtained by co-transfection of a plasmid encoding the cognate N8 NA was not as high as that generated by the addition of exogenous neuraminidase (NA) from Clostridium perfringens to allow the release of nascent PV particles. Finally, i...
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Research Interests:
The continuous rapid genetic and antigenic evolution of H5 subtype influenza viruses has major implications for the sensitivity of serological assays and can limit the efficacy of pre-pandemic human vaccines and the ability to undertake... more
The continuous rapid genetic and antigenic evolution of H5 subtype influenza viruses has major implications for the sensitivity of serological assays and can limit the efficacy of pre-pandemic human vaccines and the ability to undertake effective sero-surveillance in susceptible populations. A panel of serum samples collected from the Italian population between 1992 and 2007 and previously found to be positive for antibodies against H5N1 as determined by Serial Radial Hemolysis (SRH), were evaluated using pseudotype based neutralisation assays (PPN) in combination with haemagglutination-inhibition (HI) assays using a clade 1 and a clade 2 H5N1 serological antigen. From the results obtained it can be concluded that the pseudotype assay can efficiently measure cross-reactive antibody responses that are not detected by the HI assay. It is postulated that these responses are directed against epitopes on the HA2 stalk. All three serological assays (PPN, SRH, HI) measure antibodies with different (functional), overlapping specificities, contributing to a comprehensive analysis of humoral immunity to influenza viruses.
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Design and construction of pseudotypes
Pseudotypes as serological antigens
Immunogenicity testing usung pseudotypes
Pseudotypes as serological antigens
Immunogenicity testing usung pseudotypes
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The availability of in-vitro cell culture based assays that can be readily employed for the efficacy testing of vaccines, antivirals and therapeutic antibodies are key components for effective pandemic preparedness. The exploitation of... more
The availability of in-vitro cell culture based assays that can be readily employed for the efficacy testing of vaccines, antivirals and therapeutic antibodies are key components for effective pandemic preparedness. The exploitation of retroviral vectors pseudotyped with foreign heterologous envelope glycoproteins for the development of such assays will be discussed with particular empahasis on emerging influenza viruses.