Biology subjects

Dalby, P. A.

Publications and source records attributed to Dalby, P. A..

4 recordsLinked to original sources

bioRxiv2025

Design and development of a SARS and MERS Combination Vaccine

This work was conducted during the COVID-19 pandemic prior to the licensing of any vaccines against COVID-19. Although several COVID-19 vaccines are now commercially available, this research on the development of a combination Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV), SARS-coronavirus 2 (SARS-CoV-2), and Middle Eastern Respiratory Syndrome (MERS) is still relevant and shows how a combination vaccine can be designed, produced and rapidly tested in the laboratory. We present the development of a combination vaccine designed to provide immunity against Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV), SARS-coronavirus 2 (SARS-CoV-2), and Middle Eastern Respiratory Syndrome (MERS). The primary objective of this vaccine design is twofold: to mitigate the burden of coronavirus and to address the specific vulnerability of regions prone to recurrent MERS outbreaks. Our combination vaccine incorporates antigenic components from zoonotic sources, specifically SARS-CoV, SARS-CoV-2, and MERS. We assess the impact of combining different Spike proteins S1 subunit antigens, due to its recognised immunogenic potential, and adjuvants on serum antibody titres, virus neutralizing capabilities, and inter-antigen immune responses. We report a robust and broad antibody response against SARS-CoV-2 and related coronaviruses, which was amplified by different adjuvant formulations, including alum, MPLA, CpG, and Squalene-in-Water Emulsion.

immunology↗

bioRxiv2025

Fully functional AAV viral vectors with highly altered structural cores and subunit interfaces using ProteinMPNN

Adeno-associated viruses (AAV) have emerged as a viable vector for gene therapy, with several clinical approvals and a growing pipeline in clinical trials. These vectors have several challenges that need to be addressed to widen their use, including improving tropisms, reducing manufacturing costs, increasing storage stability, minimising their immunogenicity, or evasion of existing AAV immunity in which neutralising antibodies lead to loss of potency. Vector engineering, particularly capsid protein engineering, offers a potential route to generating new capsids that can selectively target desired cell types or evade pre-existing immunity, while also ensuring that they are manufacturable at higher titres, more stable, and have reduced immunogenicity. Extensive protein redesign is emerging as a viable option, through generative AI approaches, for engineering many types of protein. Here we explored the potential of ProteinMPNN, to extensively redesign AAV2 and yet still form stable and functional capsids. We targeted 52% of AAV2 residues for redesign by ProteinMPNN, such that only the buried protein core and subunit interfaces would be varied, leaving the capsid external and internal surface features unchanged. The aim was to significantly modify the structure responsible for assembly and capsid integrity, while maximising the probability of maintaining the wild-type DNA packaging and transduction capabilities. The final designs were between 14% and 30% mutated overall, and yet were capable of forming functional and intact capsids, with the transduction efficiency of wild type retained for some variants. The designs generally led to lower titres from cell culture, yet some designs had either improved capsid packaging efficiency or transduction efficiency. In particular, our "Pentamer" design had the best transduction efficiency, while our "Chimera" design had a packaging efficiency that was 2.5x higher than for the WT AAV2. These results demonstrate the potential to use generative AI tools in vector capsid redesign for novel core assembly features, and now pave the way for expanding this approach into selectively re-engineering their surface properties to influence tropism, immunogenicity and transduction efficiency.

bioengineering↗

bioRxiv2020

Biophysical characterization of the inactivation of E. coli transketolase by aqueous co-solvents

Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic and then aromatic substrates. This has ultimately led to the poor water solubility of new substrates, as a potential bottleneck to further exploitation of this enzyme in biocatalysis. Here we used a range of biophysical studies to characterise the response of both E. coli apo- and holo-TK activity and structure to a range of commonly used polar organic co-solvents: acetonitrile (MeCN), n- butanol (nBuOH), ethyl acetate (EToAc), isopropanol (iPrOH), and tetrahydrofuran (THF). The mechanism of enzyme deactivation was found to be predominantly via solvent-induced local unfolding. Holo-TK is thermodynamically more stable than apo-TK and yet for four of the five co-solvents it retained less activity than apo-TK after exposure to organic solvents, indicating that solvent tolerance was not correlated to global conformational stability. The co-solvent concentrations required for complete enzyme inactivation was inversely proportional to co-solvent log(P), while the unfolding rate was directly proportional, indicating that the solvents interact with and partially unfold the enzyme through hydrophobic contacts. Aggregation was not found to be the driving mechanism of enzyme inactivation, but was in some cases an additional impact of solvent-induced local or global unfolding. TK was found to be tolerant to 15% (v/v) iPrOH, 10% (v/v) MeCN, or 6% (v/v) nBuOH over 3 hours. This work indicates that future attempts to engineer the enzyme to better tolerate co-solvents should focus on increasing the stability of the protein to local unfolding, particularly in and around the cofactor-binding loops.

biophysics↗

bioRxiv2019

Simultaneous optimization of donor and acceptor substrate specificity for transketolase by a small but smart library

A narrow substrate range is a major limitation in exploiting enzymes more widely as catalysts in synthetic organic chemistry. For enzymes using two substrates, the simultaneous optimization of both substrate specificities, is also required for the rapid expansion of accepted substrates. Transketolase catalyses the reversible transfer of a C2-ketol unit from a donor substrate to an aldehyde acceptor and suffers the limitation of narrow substrate scope for widely industrial applications. Herein, transketolase from E. coli was engineered to simultaneously accept both pyruvate as a novel donor substrate, and unnatural acceptor aldehydes, including propanal, pentanal, hexanal and 3-formylbenzoic acid. Twenty single-mutant variants were firstly designed and characterized experimentally. Beneficial mutations were then recombined to construct a small but smart library. Screening of this library identified the best variant with a 9.2-fold improvement in the yield towards pyruvate and propionaldehyde, relative to WT. Pentanal and hexanal were used as acceptors to determine stereoselectivities of the reactions, which were found to be higher than 98% ee for the S configuration. Three variants were identified to be active for the reaction between pyruvate and 3-formylbenzoic acid. The best variant was able to convert 47% of substrate into product within 24 h, whereas no conversion was observed for WT. Docking experiments suggested a cooperation between the mutations responsible for donor and acceptor acceptances, that would promote the activity towards both the acceptor and donor. The variants obtained have the potential to be used for developing catalytic pathways to a diverse range of high-value products.

bioengineering↗