Development of a highly sensitive serological assay for Covid-19 based on the use of virus-like particles and protein-based nanoparticles.
Lead Research Organisation:
London School of Hygiene & Tropical Medicine
Department Name: Infectious and Tropical Diseases
Abstract
We propose to use the baculovirus expression system to generate Virus-Like Particles (VLPs) mimicking the recently emergent SARS-CoV-2. We have previously co-expressed three essential structural proteins (S, M, and E) of SARS-CoV using a single baculovirus expression vector developed in our lab and demonstrated that when co-expressed in Sf9 cells, these three proteins were able to spontaneously adopt a conformation mimicking the full virus (1). Subsequent mouse studies showed that vaccination with these elicited a strong neutralising antibody response, equivalent to that seen in a convalescent human. Recently we used these pre-existing reagents as a basis to produce SARS-CoV-2 VLPs, substituting the S, M, and E proteins into the previously generated Baculovirus constructs, and preliminary data shows that all three SARS-CoV-2 proteins are expressed. We propose to characterise thoroughly the expressed proteins, the formation of VLPs, and the immune response in mice to validate their authenticity as virus mimics. VLPs will be ultimately used to develop an antibody detection assay.
In parallel, we will express the SARS-CoV-2 Fc-tagged S protein alone to be externally encapsulated with the lumazine synthase protein-based nanoparticles (PNPs) that we have already prepared. We have shown that similar nanoparticles for a receptor binding protein of a different virus enhanced binding affinity and avidity. These PNPs self-assemble into hollow monodisperse spherical structures, which stabilises the trimeric S protein and enhances antigenicity (2).
Both SARS-CoV-2 VLPs and PNPs are excellent candidates for the development of highly sensitive diagnostic assay that will be used to detect COVID-19 infected human sera.
In parallel, we will express the SARS-CoV-2 Fc-tagged S protein alone to be externally encapsulated with the lumazine synthase protein-based nanoparticles (PNPs) that we have already prepared. We have shown that similar nanoparticles for a receptor binding protein of a different virus enhanced binding affinity and avidity. These PNPs self-assemble into hollow monodisperse spherical structures, which stabilises the trimeric S protein and enhances antigenicity (2).
Both SARS-CoV-2 VLPs and PNPs are excellent candidates for the development of highly sensitive diagnostic assay that will be used to detect COVID-19 infected human sera.
People |
ORCID iD |
Polly Roy (Principal Investigator) |
Publications
Jones I
(2021)
Sputnik V COVID-19 vaccine candidate appears safe and effective.
in Lancet (London, England)
Description | Virus like particles are macromolecular structures that closely resemble virus particles. They are formed by expression of a minimum number of virus structural proteins required for virus assembly, but can also incorporate additional viral proteins if they are compatible with the minimal assembly process. VLPs are non-infectious as no genome is present and can serve as models for the assembly process or as surrogates for authentic viruses for antibody binding or antibody generation. Importantly, VLP mimic the structure of the live virus, meaning that the individual proteins are present in a more natural context then when expressed alone. We have developed a system in insect cells, using a baculovirus system, to produce VLPs which mimic the SARS-CoV-2 particle. Co-expression of the SAR-CoV-2 membrane and envelope proteins (the minimum requirements for assembly) with the spike protein, leads to the formation of hollow VLPs with the classical coronavirus structure, decorated with Spike protein protrusions. Our VLPs bound a human monoclonal antibody specific for the receptor binding site of S and demonstrated superior sensitivity on a weight for weight basis with S alone when used as a diagnostic antigen in standard ELISA format. |
Exploitation Route | By mimicking the whole virus structure, we believe that our VLPs present the viral proteins in a more typical context, allowing them to serve as improved viral substitutes in our on-going studies of SARS-CoV2. Our VLPs can be used in diagnostic work in place of individually expressed proteins in the detection of antibody. Or could serve as a vaccine platform. Additionally our VLPs can be used as alternative for working with live virus in further SARS-CoV-2 research, particularly in structural studies. |
Sectors | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Description | Development of a highly sensitive serological assay for Covid-19 based on the use of virus-like particles and protein-based nanoparticles. |
Amount | £380,486 (GBP) |
Funding ID | BB/V006584/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 07/2020 |
End | 01/2022 |
Title | CoV19 Virus Like Particles expressed in baculovirus system |
Description | Virus-like-particles (VLPs) are macromolecular assemblies that resemble virus particles. They are formed by heterologous expression of the minimum number of virus structural proteins required for virus assembly but can also incorporate additional viral proteins if they are compatible with the assembly process. We have developed a baculovirus system to allow large scale expression of SAR-CoV-2 VLPs. Co-expression of the SARS-CoV-2 M and E proteins is sufficient to cause the generation of VLPs, however the additional expression of both the S and N proteins results in a more stable structure. |
Type Of Material | Technology assay or reagent |
Year Produced | 2020 |
Provided To Others? | No |
Impact | When examined by EM, SARS-CoV-2 VLPs appeared as the classical Coronavirus morphology, with the double corona. In addition VLPs bound a human monoclonal antibody specific for the receptor binding site of the S protein. |
Title | Evaluation of vaccine efficacy |
Description | Replication-deficient vaccine strains were tested in animals as monovalent or in multivalent combinations and the protection analysed by Elisa and serum neutralisation assays. |
Type Of Material | Technology assay or reagent |
Provided To Others? | No |
Impact | Testing these virus vaccine strains is essential to support them for commercial development |
Title | Expression of soluble spike protein using baculovirus system |
Description | The transmembrane domain and C-terminal tail of S protein was deleted, and fused with a C-terminal Fc tag and strep II tag. Furin cleavage site mutation and two proline substitutions in S2 were also introduced to increase the expression level of full length S protein. The sequence of S protein was codon optimised for expression in recombinant baculovirus infected insect cells. After 48h postinfection, the supernatant containing soluble S protein was collected and the S protein was purified using strep-tactin beads. Highly purified full length S protein with a concentration of 0.5mg/ml was detected in the first elution. Purified S-Fc tag protein were confirmed to form trimer by SDS-PAGE under denaturing and non-denaturing conditions with coomassie staining and western blot using an commercial mouse anti-SARS2 S antibody. |
Type Of Material | Technology assay or reagent |
Year Produced | 2021 |
Provided To Others? | No |
Impact | S protein bound to nanoparticle showed significantly increased binding to purified ACE2 protein in an ELISA test, meaning a multivalent presentation of S protein on the nanoparticles (PNP). Both S protein and S protein on PNPs, showed good sensitivity and specificity to commercial mouse anti-S antibody, however, the PNPs interfere with antibody detection in human sera. |
Title | Sequences of SARS-CoV2 S, M, E genes |
Description | DNA sequences of SARS-CoV2 S, M, E genes for construction of VLP and individual S protein |
Type Of Material | Database/Collection of data |
Year Produced | 2021 |
Provided To Others? | No |
Impact | N/A |
Description | Ian M Jones group baculovirus expression system |
Organisation | University of Reading |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We design and produce the baculovirus constructs and perform the major part of work in this project. |
Collaborator Contribution | Contributed in designing project and helped to perform the baculovirus based protein expression. Also performed some immunology analysis. |
Impact | A manuscript has been submitted to Journal of Virology. Preprint: BIORXIV/2021/470349 TITLE: SARS-CoV-2 Virus like Particles produced by a single recombinant baculovirus generate potent neutralizing antibody that protects against variant challenge |
Start Year | 2020 |
Description | Jeremy Brown group for Covid patients ELISA test |
Organisation | University College Hospital |
Country | United Kingdom |
Sector | Hospitals |
PI Contribution | We design the project, prepare the SARS-cov2 VLPs sample for the patients sera ELISA test. |
Collaborator Contribution | Providing patients serum samples |
Impact | A manuscript has been submitted to Journal of Virology. Preprint: BIORXIV/2021/470349 TITLE: SARS-CoV-2 Virus like Particles produced by a single recombinant baculovirus generate potent neutralizing antibody that protects against variant challenge |
Start Year | 2020 |
Description | Neil Almond group for animal trial in NIBSC |
Organisation | National Institute for Biological Standards and Control (NIBSC) |
Country | United Kingdom |
Sector | Public |
PI Contribution | We design the project, prepare the SARS-cov2 VLPs sample for the animal trial. |
Collaborator Contribution | Perform animal immunisation, antibody testing, challenging and neutralising assay. |
Impact | A manuscript has been submitted to Journal of Virology. Preprint: BIORXIV/2021/470349 TITLE: SARS-CoV-2 Virus like Particles produced by a single recombinant baculovirus generate potent neutralizing antibody that protects against variant challenge |
Start Year | 2020 |
Description | Nicole Robb group for single molecule analysis |
Organisation | University of Warwick |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We design the project, produce and prepare the materials for analysis. |
Collaborator Contribution | Co-design the project and perform analysis. |
Impact | Some preliminary data has been obtained. This collaborator also helped us to obtain EM images used for this following manuscript which is now submitted to Journal of Virology: BIORXIV/2021/470349 TITLE: SARS-CoV-2 Virus like Particles produced by a single recombinant baculovirus generate potent neutralizing antibody that protects against variant challenge. |
Start Year | 2018 |
Description | Coronavirus vaccine trials |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | The purpose was to inform the general public regarding vaccine research. |
Year(s) Of Engagement Activity | 2021 |
Description | Coronavirus vaccines |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Interview to the General Public on BBC4 Radio, |
Year(s) Of Engagement Activity | 2021 |
Description | Coronavirus: infection, new strains and vaccines, BBC News |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Interview to BBC regarding Coronavirus updates. |
Year(s) Of Engagement Activity | 2021 |
Description | Microbiology Society Conference, 2021, UK |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Talk about the research outcome: Sialic acid binding sites in VP2 of bluetongue virus and their use, Weining Wu and Polly Roy |
Year(s) Of Engagement Activity | 2021 |
Description | Russia's Sputnik V vaccine has 92% efficacy in trial |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | The aim of these interviews were to inform the general public regarding the advances in research for Covid-19 vaccines. They were held by different media channels, in different countries: BBC News, The Wall Street Journal, Financial Times, Voice of America, AP News, Reuters, France24, |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.bbc.co.uk/news/health-55900622 |