Biology subjects

Griffiths, D. T.

Publications and source records attributed to Griffiths, D. T..

2 recordsLinked to original sources

bioRxiv2021

In vitro discovery and optimization of a human monoclonal antibody that neutralizes neurotoxicity and lethality of cobra snake venom

The monocled cobra (Naja kaouthia) is one of the most feared snakes in Southeast Asia. It is a highly dangerous species with a potent venom deriving its toxicity predominantly from abundant long-chain -neurotoxins. The only specific treatment for snakebite envenoming is antivenom, which is based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines over the past 120 years, and their ongoing role in combating snakebite disease, major limitations in safety, supply consistency, and efficacy creates a need for a new generation of improved treatments based on modern biotechnological techniques. Here, we describe the initial discovery and subsequent optimization of a recombinant human monoclonal immunoglobin G (IgG) antibody against -cobratoxin using phage display technology. Affinity maturation of the parental antibody by light chain-shuffling resulted in an 8-fold increase in affinity, translating to a significant increase in in vitro neutralization potency and in vivo efficacy. While the parental antibody prolonged survival of mice challenged with purified -cobratoxin, the optimized antibody prevented lethality when incubated with N. kaouthia whole venom prior to intravenous injection. This study is the first to demonstrate neutralization of whole snake venom by a single recombinant monoclonal antibody. Importantly, this suggests that for venoms whose toxicity relies on a single predominant toxin group, such as that of N. kaouthia, as little as one monoclonal antibody may be sufficient to prevent lethality, thus providing a tantalizing prospect of bringing recombinant antivenoms based on human monoclonal or oligoclonal antibodies to the clinic. One Sentence SummaryA recombinant human monoclonal immunoglobulin G antibody, discovered and optimized using in vitro methods, was demonstrated to neutralize the lethal effect of whole venom from the monocled cobra in mice via abrogation of -neurotoxin-mediated neurotoxicity.

molecular biology↗

bioRxiv2018

MinION Nanopore Sequencing of Multiple Displacement Amplified Mycobacteria DNA Direct from Sputum

Sequencing of pathogen DNA directly from clinical samples offers the possibilities of rapid diagnosis, faster antimicrobial resistance prediction and enhanced outbreak investigation. The approach is especially advantageous for infections caused by species which grow very slowly in culture, such as Mycobacteria tuberculosis. Since the pathogen of interest may represent as little as 0.01% of the total DNA, enrichment of the input material for target sequences by specific amplification and, or depletion of non-target DNA (human, other bacteria) is essential for success. Here, we investigated the potential of isothermal multiple displacement amplification by Phi29 polymerase. We directed the amplification reaction towards Mycobacteria DNA in sputum samples by exploiting in our oligonucleotide primer design, their high GC content (approximately 65%) relative to human DNA. Amplified DNA was then sequenced using the Oxford Nanopore Technology MinION. In addition, a model system comprising standardised mock clinical samples was designed. Pooled infection negative human sputum samples were spiked with enumerated Mycobacterium bovis (BCG) Pasteur strain at concentrations spanning the typical range at which Mycobacterium tuberculosis is found in human sputum samples (106 - 101 BCG cells/ml). To assess the amount of BCG sequence enrichment achieved, sample DNA was sequenced both before, and after amplification. Reads from amplified samples, which mapped to a BCG reference genome, comprised short repeated sequences - apparently transcribed multiple times from the same fragment of BCG DNA. Therefore post-amplification, the samples were enriched for BCG sequences relative to unamplified sequences (8,101 BCG reference mapped reads, increasing to 28,617 at 106 BCG cells/ml sample), but BCG genome coverage declined markedly (for example 89.4% to 4.1%). In summary, the use of standardised mock clinical samples allowed direct comparison of data from different Mycobacteria enrichment experiments and sequencing runs. However, optimal conditions for multiple displacement amplification of minority Mycobacteria DNAs, remain to be identified.

microbiology↗