Glycan masking of a non-neutralising epitope enhances neutralising antibodies targeting the RBD of SARS-CoV-2 and its variants.

The accelerated development of the first generation COVID-19 vaccines has saved millions of lives, and potentially more from the long-term sequelae of SARS-CoV-2 infection. The most successful vaccine candidates have used the full-length SARS-CoV-2 spike protein as an immunogen. As expected of RNA viruses, new variants have evolved and quickly replaced the original wild-type SARS-CoV-2, leading to escape from natural infection or vaccine induced immunity provided by the original SARS-CoV-2 spike sequence. Next generation vaccines that confer specific and targeted immunity to broadly neutralising epitopes on the SARS-CoV-2 spike protein against different variants of concern (VOC) offer an advance on current booster shots of previously used vaccines. Here, we present a targeted approach to elicit antibodies that neutralise both the ancestral SARS-CoV-2, and the VOCs, by introducing a specific glycosylation site on a non-neutralising epitope of the RBD. The addition of a specific glycosylation site in the RBD based vaccine candidate focused the immune response towards other broadly neutralising epitopes on the RBD. We further observed enhanced cross-neutralisation and cross-binding using a DNA-MVA CR19 prime-boost regime, thus demonstrating the superiority of the glycan engineered RBD vaccine candidate across two platforms and a promising candidate as a broad variant booster vaccine.

Pseudotyped Viruses for Influenza.

We have developed an influenza hemagglutinin (HA) pseudotype (PV) library encompassing all influenza A (IAV) subtypes from HA1-HA18, influenza B (IBV) subtypes (both lineages), representative influenza C (ICV), and influenza D (IDV) viruses. These influenza HA (or hemagglutinin-esterase fusion (HEF) for ICV and IDV) pseudotypes have been used in a pseudotype microneutralization assay (pMN), an optimized luciferase reporter assay, that is highly sensitive and specific for detecting neutralizing antibodies against influenza viruses. This has been an invaluable tool in detecting the humoral immune response against specific hemagglutinin or hemagglutinin-esterase fusion proteins for IAV to IDV in serum samples and for screening antibodies for their neutralizing abilities. Additionally, we have also produced influenza neuraminidase (NA) pseudotypes for IAV N1-N9 subtypes and IBV lineages. We have utilized these NA-PV as surrogate antigens in in vitro assays to assess vaccine immunogenicity. These NA PV have been employed as the source of neuraminidase enzyme activity in a pseudotype enzyme-linked lectin assay (pELLA) that is able to measure neuraminidase inhibition (NI) titers of reference antisera, monoclonal antibodies, and postvaccination sera. Here we show the production of influenza HA, HEF, and NA PV and their employment as substitutes for wild-type viruses in influenza serological and neutralization assays. We also introduce AutoPlate, an easily accessible web app that can analyze data from pMN and pELLA quickly and efficiently, plotting inhibition curves and calculating half-maximal concentration (IC50) neutralizing antibody titers. These serological techniques coupled with user-friendly analysis tools are faster, safer, inexpensive alternatives to classical influenza assays while also offering the reliability and reproducibility to advance influenza research and make it more accessible to laboratories around the world.

Influenza A (N1-N9) and Influenza B (B/Victoria and B/Yamagata) Neuraminidase Pseudotypes as Tools for Pandemic Preparedness and Improved Influenza Vaccine Design.

To better understand how inhibition of the influenza neuraminidase (NA) protein contributes to protection against influenza, we produced lentiviral vectors pseudotyped with an avian H11 hemagglutinin (HA) and the NA of all influenza A (N1-N9) subtypes and influenza B (B/Victoria and B/Yamagata). These NA viral pseudotypes (PV) possess stable NA activity and can be utilized as target antigens in in vitro assays to assess vaccine immunogenicity. Employing these NA PV, we developed an enzyme-linked lectin assay (pELLA) for routine serology to measure neuraminidase inhibition (NI) titers of reference antisera, monoclonal antibodies and post-vaccination sera with various influenza antigens. We also show that the pELLA is more sensitive than the commercially available NA-Fluor™ in detecting NA inhibition in these samples. Our studies may lead to establishing the protective NA titer that contributes to NA-based immunity. This will aid in the design of superior, longer lasting and more broadly protective vaccines that can be employed together with HA-targeted vaccines in a pre-pandemic approach.

Exploiting Pan Influenza A and Pan Influenza B Pseudotype Libraries for Efficient Vaccine Antigen Selection.

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 the strengthening of global influenza surveillance, expansion of seasonal influenza prevention and control policies, and strengthening pandemic preparedness and response.

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.

AutoPlate: Rapid Dose-Response Curve Analysis for Biological Assays.

The emergence of COVID-19 has emphasised that biological assay data must be analysed quickly to develop safe, effective and timely vaccines/therapeutics. For viruses such as SARS-CoV-2, the primary way of measuring immune correlates of protection is through assays such as the pseudotype microneutralisation (pMN) assay, thanks to its safety and versatility. However, despite the presence of existing tools for data analysis such as PRISM and R the analysis of these assays remains cumbersome and time-consuming. We introduce an open-source R Shiny web application and R library (AutoPlate) to accelerate data analysis of dose-response curve immunoassays. Using example data from influenza studies, we show that AutoPlate improves on available analysis software in terms of ease of use, flexibility and speed. AutoPlate (https://philpalmer.shinyapps.io/AutoPlate/) is a tool for the use of laboratories and wider scientific community to accelerate the analysis of biological assays in the development of viral vaccines and therapeutics.

Protection From Influenza by Intramuscular Gene Vector Delivery of a Broadly Neutralizing Nanobody Does Not Depend on Antibody Dependent Cellular Cytotoxicity.

Cross-subtype neutralizing single domain antibodies against influenza present new opportunities for immunoprophylaxis and pandemic preparedness. Their simple modular structure and single open reading frame format are highly amenable to gene therapy-mediated delivery. We have previously described R1a-B6, an alpaca-derived single domain antibody (nanobody), that is capable of potent cross-subtype neutralization in vitro of H1N1, H5N1, H2N2, and H9N2 influenza viruses, through binding to a highly conserved epitope in the influenza hemagglutinin stem region. To evaluate the potential of R1a-B6 for immunoprophylaxis, we have reformatted it as an Fc fusion for adeno-associated viral (AAV) vector delivery. Our findings demonstrate that a single intramuscular injection in mice of AAV encoding R1a-B6 fused to Fc fragments of different isotypes equipped either, with or without antibody dependent cellular cytotoxicity (ADCC) activity, was able to drive sustained high-level expression (0.5-1.1 mg/mL) in sera with no evidence of reduction for up to 6 months. R1a-B6-Fc fusions of both isotypes gave complete protection against lethal challenge with both pandemic A/California/07/2009 (H1N1)pdm09 and avian influenza A/Vietnam/1194/2004 (H5N1). This data suggests that R1a-B6 is capable of cross-subtype protection and ADCC was not essential for R1a-B6 efficacy. Our findings demonstrate AAV delivery of cross-subtype neutralizing nanobodies may be an effective strategy to prevent influenza infection and provide long-term protection independent of a host induced immune response.

Combinations of chloroquine with tigecycline and telithromycin induce early onset of apoptosis in Plasmodium falciparum in vitro

OBJECTIVE: This study aimed to demonstrate that apoptosis in Plasmodium falciparum can be measured using kits originally designed for mammalian cells. The antimalarial chloroquine and antibiotics tigecycline and telithromycin were used to show the performance of the assays. METHODS: Nuclear stain DAPI fluorescence was used to estimate cytotoxicity. Apoptotic assays used were: CaspaTag™ for caspase activation, acridine orange for nuclear condensation, and TUNEL for DNA fragmentation. RESULTS: The IC50 values (95% confidence interval) for telithromycin (TL), tigecycline (TG) and chloroquine (CQ) were found to be 1.00 (0.47-1.53) µM, 4.56 (2.32-6.80) µM, and 0.019 (0.0089-0.029) µM, respectively. Activated caspase-like molecules seemed to be present in all erythrocytic stages, appearing to rise and fall with cell cycle progression with drug exposure appearing to dysregulate this pattern. Nuclear condensation and DNA fragmentation occurred late in the untreated erythrocytic life cycle of the parasite but were advanced by drug exposure. CONCLUSION: The study shows that drug-induced apoptosis can be measured in Plasmodium falciparum using the methods. These assays could be used for drug discovery, in particular, using high throughput flow cytometry.