DEVELOPMENT OF HYBRID HAEMAGGLUTININ PSEUDOTYPED LENTIVIRUSES TO ASSESS HETEROSUBTYPIC IMMUNITY TO INFLUENZA

The influenza virus still causes hundreds of thousands of deaths globally, on top of morbidity and associated economic burden. We are currently at the height of efforts surrounding the development and employment of 'universal' vaccines against this virus, with clinical trials commencing on the most promising candidates. Despite this, the influenza virus poses more of a threat to human life than it ever has previously, with multiple subtypes of pandemic potential circulating around the globe. The key to current efforts lies in the priming of the immune system towards generating long lasting defences against conserved epitopes and conferring heterosubtypic immunity against the surface glycoprotein haemagglutinin. While vaccine strategies have expanded rapidly over recent years with the advent of 'headless' constructs as well as those derived from consensus, mosaic or chimeric sequences, the serological techniques to test how effective these vaccines are, have advanced less rapidly. Classical serological assays have been shown to be ineffective at detecting the antibodies which modern 'universal' vaccines strife to elicit, replaced by ELISA based approaches combined with mouse models measuring in vivo protection. In this thesis, an alternative method for the detection of heterosubtypic antibodies is used in depth across multiple platforms. Influenza pseudotypes have been employed using chimeric haemagglutinin constructs in a comprehensive project aimed at dissecting head and stalk directed antibodies present in human serum. Characterised broadly neutralising monoclonal antibodies have been tested on panels of influenza pseudotypes including divergent bat influenza viruses which hitherto have not been encountered in humans. A further aspect of influenza immunity has been covered in the detection of anti neuraminidase antibodies which have an important role to play in influenza heterosubtypic immunity. Finally, influenza pseudotypes bearing the glycoproteins from the less studied influenza B virus have been assayed in a large scale project aimed at correlating pseudotype assays with classical approaches

Development of a Clinical MALDI-ToF Mass Spectrometry Assay for SARS-CoV-2: Rational Design and Multi-Disciplinary Team Work.

The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has stretched national testing capacities to breaking points in almost all countries of the world. The need to rapidly screen vast numbers of a country's population in order to control the spread of the infection is paramount. However, the logistical requirement for reagent supply (and associated cost) of RT-PCR based testing (the current front-line test) have been hugely problematic. Mass spectrometry-based methods using swab and gargle samples have been reported with promise, but have not approached the task from a systematic analysis of the entire diagnostic process. Here, the pipeline from sample processing, the biological characteristics of the pathogen in human biofluid, the downstream bio- and physical-chemistry and the all-important data processing with clinical interpretation and reporting, are carefully compiled into a single high-throughput and reproducible rapid process. Utilizing MALDI-ToF mass spectrometric detection to viral envelope glycoproteins in a systems biology-multidisciplinary team approach, we have achieved a multifaceted clinical MALDI ToF MS screening test, primarily (but not limited to) SARS-CoV-2, with direct application to other future epidemics/pandemics that may arise. The clinical information generated not only includes SARS-CoV-2 coronavirus detection-(Spike protein fragments S1, S2b, S2a peaks), but other respiratory viral infections detected as well as an assessment of generalised oral upper respiratory immune response (elevated total Ig light chain peak) and a measure of the viral immune response (elevated intensity of IgA heavy chain peak). The advantages of the method include; (1) ease of sampling, (2) speed of analysis, and much reduced cost of testing. These features reveal the diagnostic utility of MALDI-ToF mass spectrometry as a powerful and economically attractive global solution

Pseudotype-based neutralization assays for influenza: a systematic analysis

The use of vaccination against the influenza virus remains the most effective method of mitigating the significant morbidity and mortality caused by this virus. Antibodies elicited by currently licensed influenza vaccines are predominantly hemagglutination-inhibition (HI)-competent antibodies that target the globular head of HA thus inhibiting influenza virus entry into target cells. These antibodies predominantly confer homosubtypic/strain specific protection and only rarely confer heterosubtypic protection. However, recent academia or pharma-led R&D towards the production of a "universal vaccine" has centered on the elicitation of antibodies directed against the stalk of the influenza HA that has been shown to confer broad protection across a range of different subtypes (H1 to H16). The accurate and sensitive measurement of antibody responses elicited by these "next-generation" influenza vaccines is however hampered by the lack of sensitivity of the traditional influenza serological assays hemagglutinin inhibition (HI), single radial hemolysis (SRH) and microneutralization (MN). Assays utilizing pseudotypes, chimeric viruses bearing influenza glycoproteins, have been shown to be highly efficient for the measurement of homosubtypic and heterosubtypic broadly-neutralizing antibodies, making them ideal serological tools for the study of cross-protective responses against multiple influenza subtypes with pandemic potential. In this review, we will analyze and compare literature involving the production of influenza pseudotypes with particular emphasis on their use in serum antibody neutralization assays. This will enable us to establish the parameters required for optimization and propose a consensus protocol to be employed for the further deployment of these assays in influenza vaccine immunogenicity studies

An Optimised Method for the Production using PEI, Titration and Neutralization of SARS-CoV Spike Luciferase Pseudotypes

The protocol outlined represents a cost-effective, rapid and reliable method for the generation of high-titre viral pseudotype particles with the wild-type SARS-CoV spike protein on a lentiviral vector core using the widely available transfection reagent PEI. This protocol is optimized for transfection in 6-well plates; however it can be readily scaled to different production volumes according to application. This protocol has multiple benefits including the use of readily available reagents, consistent, high pseudotype virus production Relative Luminescence Units (RLU) titres and rapid generation of novel coronavirus pseudotypes for research into strain variation, tropism and immunogenicity/sero-prevalence

Evidence Report: Risk of Radiation Carcinogenesis

As noted by Durante and Cucinotta (2008), cancer risk caused by exposure to space radiation is now generally considered a main hindrance to interplanetary travel for the following reasons: large uncertainties are associated with the projected cancer risk estimates; no simple and effective countermeasures are available, and significant uncertainties prevent scientists from determining the effectiveness of countermeasures. Optimizing operational parameters such as the length of space missions, crew selection for age and sex, or applying mitigation measures such as radiation shielding or use of biological countermeasures can be used to reduce risk, but these procedures have inherent limitations and are clouded by uncertainties. Space radiation is comprised of high energy protons, neutrons and high charge (Z) and energy (E) nuclei (HZE). The ionization patterns and resulting biological insults of these particles in molecules, cells, and tissues are distinct from typical terrestrial radiation, which is largely X-rays and gamma-rays, and generally characterized as low linear energy transfer (LET) radiation. Galactic cosmic rays (GCR) are comprised mostly of highly energetic protons with a small component of high charge and energy (HZE) nuclei. Prominent HZE nuclei include He, C, O, Ne, Mg, Si, and Fe. GCR ions have median energies near 1 GeV/n, and energies as high as 10 GeV/n make important contributions to the total exposure. Ionizing radiation is a well known carcinogen on Earth (BEIR 2006). The risks of cancer from X-rays and gamma-rays have been established at doses above 50 mSv (5 rem), although there are important uncertainties and on-going scientific debate about cancer risk at lower doses and at low dose rates (<50 mSv/h). The relationship between the early biological effects of HZE nuclei and the probability of cancer in humans is poorly understood, and it is this missing knowledge that leads to significant uncertainties in projecting cancer risks during space exploration (Cucinotta and Durante 2006; Durante and Cucinotta 2008)

Correlation of Influenza B Haemagglutination Inhibiton, Single-Radial Haemolysis and Pseudotype-Based Microneutralisation Assays for Immunogenicity Testing of Seasonal Vaccines.

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

Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses.

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 for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination

Development of Lentiviral Vectors Pseudotyped With Influenza B Hemagglutinins: Application in Vaccine Immunogenicity, mAb Potency, and Sero-Surveillance Studies.

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 hemagglutinin-pseudotypes (IBV PV) using plasmid-directed transfection. To activate influenza B hemagglutinin, we have explored the use of proteases in increasing PV titers via their co-transfection during pseudotype virus production. When tested for their ability to transduce target cells, the influenza B pseudotypes produced exhibit tropism for different cell lines. The pseudotypes were evaluated as alternatives to live virus in microneutralization assays using reference sera standards, mouse and human sera collected during vaccine immunogenicity studies, surveillance sera from seals, and monoclonal antibodies (mAbs) against IBV. The influenza B pseudotype pMN was found to effectively detect neutralizing and cross-reactive responses in all assays and shows promise as an effective and versatile tool in influenza research

Paucity and discordance of neutralising antibody responses to SARS-CoV-2 VOCs in vaccinated immunodeficient patients and health-care workers in the UK.

As of June, 2021, the UK population is only partly vaccinated against COVID-19, with many people having received just one vaccination dose (either BNT162b2 [Pfizer–BioNTech]) or ChAdOx1 nCoV-19 [AZD1222; Oxford–AstraZeneca]). Tracking the spread of SARS-CoV-2 Variants of Concern (VOCs) remains important for understanding the levels of vaccine-induced immunity and for identifying the emergence of vaccine escape variants. The immune correlates of protection to SARS-CoV-2 and COVID-19 established in phase 3 clinical trials following two doses of vaccine was the titre of neutralising antibodies (NAbs) to SARS-CoV-2 in study groups, before the VOCs emerged.1 Vaccination programmes are leading to promising reductions in disease severity and mortality in vaccinated populations. However, the combined situation of ongoing transmission within communities, including in some vaccine recipients, alongside newly arising VOCs, continues to pose a serious threat to public health and the efficacy of these vaccines. As of Jan 11, 2021, in the UK, the interval between the first and second dose of vaccination was extended to 12 weeks. This extension achieved the aim of maximising population coverage by immunising the greatest possible number of individuals to prevent disease and hospital admissions. Encouragingly, a growing number of studies have reported a marked reduction in the number of individuals with moderate-to-severe clinical symptoms and a substantial decline in the number of hospitalised patients with COVID-19 in the UK, underscoring the success of this strategy