Biology subjects

Shattock, R.

Publications and source records attributed to Shattock, R..

3 recordsLinked to original sources

bioRxiv2022

Biophysical characterisation of the structure of a SARS-CoV-2 self-amplifying - RNA (saRNA) vaccine

The current SARS-Covid-2 pandemic has led to an acceleration of messenger - ribonucleic acid (mRNA) vaccine technology. The development of production processes for these large mRNA molecules, especially self-amplifying mRNA (saRNA) has required concomitant development of analytical characterisation techniques. Characterising the purity, shape and structure of these biomolecules is key to their successful performance as drug products. This paper describes the biophysical characterisation of the Imperial College London Self-amplifying viral RNA vaccine (IMP-1) developed for SARS-CoV-2. A variety of analytical techniques have been used to characterise the IMP-1 RNA molecule. In this paper we use UV spectroscopy, dynamic light scattering (DLS), size-exclusion chromatography small angle scattering (SEC-SAXS) and circular dichroism (CD) to determine key biophysical attributes of IMP-1. Each technique provides important information about the concentration, size, shape, structure and purity of the molecule. Statement of significanceThis paper is highly significant as it provides a prescient biophysical characterisation of an efficacious Sars-Cov-2 vaccine self-amplifying (sa)RNA molecule. RNA vaccines have been a major scientific breakthrough of the Covid-19 pandemic. saRNA is a further development of conventional mRNA vaccines, amplifying the RNA of interest in the cell, allowing the vaccine to be administered at lower dosages. These new biologics are distinct from previous biologics and have required distinct analytical characterisation. The analytics described herein provide detailed information on the size, shape, and structure of the RNA molecule. This paper is therefore an important step in characterising large saRNA biological relevant molecules.

biophysics↗

bioRxiv2021

Persistent Immunogenicity of Integrase Defective Lentiviral Vectors delivering membrane tethered Native-Like HIV-1 Envelope Trimers

Integrase Defective Lentiviral Vectors (IDLVs) represent an attractive vaccine platform for delivering HIV-1 antigens, given their ability to induce specific and persistent immune responses in both mice and non-human primates (NHPs). Recent advances in HIV-1 immunogen design demonstrated that native-like HIV-1 Envelope (Env) trimers that mimic the structure of virion-associated Env induce neutralization breadth in rabbits and macaques. Here, we describe the development of an IDLV-based HIV-1 vaccine expressing either soluble ConSOSL.UFO.664 or membrane-tethered ConSOSL.UFO.750 native-like Env immunogens with enhanced bNAb epitopes exposure. We show that IDLV can be pseudotyped with properly folded membrane-tethered native-like UFO.750 trimers. After a single IDLV injection in BALB/c mice, IDLV-UFO.750 induced a faster humoral kinetic as well as higher levels of anti-Env IgG compared to IDLV-UFO.664. IDLV-UFO.750 vaccinated cynomolgus macaques developed unusually long-lasting anti-Env IgG antibodies, as underlined by their remarkable half-life both after priming and boost with IDLV. After boosting with recombinant ConM SOSIP.v7 protein, two animals developed neutralization activity against the autologous tier 1B ConS virus mediated by V1/V2 and V3 glycan sites responses. By combining the possibility to display stabilized trimeric Env on the vector particles with the ability to induce sustained humoral responses, IDLVs represent an appropriate strategy for delivering rationally designed antigens to progress towards an effective HIV-1 vaccine.

immunology↗

bioRxiv2021

Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens

Chemical cross-linking is used to stabilise protein structure with additional benefits of pathogen and toxin inactivation for vaccine use, but its use is restricted by potential induction of local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for the purposes of understanding function, or for inducing a biological outcome such as elicitation of antibodies to conformationally-sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its functional analysis and use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent EDC to cross-link two soluble Env trimers, selected well-folded trimers using an antibody affinity column, and transferred this process to good manufacturing practice (GMP) for clinical trial use. Cross-linking enhanced GMP trimer stability to biophysical and enzyme attack, and had broadly beneficial effects on morphology, antigenicity and immunogenicity. Cryo-EM analysis revealed that cross-linking essentially completely retained overall structure with RMSDs between unmodified and cross-linked Env trimers of 0.4-0.5 [A]. Despite this negligible distortion of global trimer structure we identified individual inter-subunit, intra-subunit and intra-protomer cross-links. Thus, EDC cross-linking maintains protein folding, improves stability, and is readily transferred to GMP, consistent with use of this approach in probing protein structure/function relationships and in the design of vaccines.

molecular biology↗