Following a 7-month visit to the group last year as part of his doctoral thesis, a paper describing Jonas Mertens' project characterising the variability of an assay that is central in malaria research has now been published in the journal Vaccine.
The growth inhibition assay (GIA) is a fundamental tool for assessing the functionality of anti-malarial monoclonal antibodies and polyclonal immune responses on parasite invasion, and thus is vital for the characterisation and development of blood-stage malaria vaccines. Within our group this assay is routinely used to help understand why some immune responses are better than others, and how future vaccines can be optimised to take advantage of this.
The complexity of the GIA means that it can be subject to assay-to-assay variation. Our collaborator Dr Kazatoyo Miura (NIAID/NIH) has previously analysed the precision of the Plasmodium falciparum assay using some of our anti-PfRH5 antibodies. For Jonas’ project, and again in collaboration with Dr Miura, the aim was to investigate the variability within the P. knowlesi GIA. This assay is crucial for the development of a vaccine against malaria caused by P. vivax, the most widely distributed of all the malaria parasite species. Owing to difficulties culturing this strain in the lab, a genetically modified form of the P. knowlesi parasite is used instead (provided by our collaborator Prof Rob Moon at the LSHTM) which expresses the P. vivax form of a key protein, Duffy-binding protein (PvDBP), that is integral to the invasion of the parasite into red blood cells (RBCs). Using a panel of well-characterised anti-PvDBP monoclonal antibodies, across 10 GIA set-ups and 8 batches of RBCs, the average inhibition of parasite invasion was determined for each test antibody. From there differences in antibody performance in individual assays could be compared to this average. It was shown that performing separate GIAs with different batches of RBCs and on different days introduced a significant level of variation, irrespective of the sample that was being tested and at what concentration. This result was not just limited to monoclonal antibodies but was also apparent following analysis of human polyclonal antibodies. Repeating the GIA on multiple occasions and taking an average of the results gives a more precise picture of the true activity of the test sample, and narrows the range that we can be confident that this activity falls within.
In an ideal world, with unlimited time and resources, samples would be independently tested over and over again to provide the most robust dataset. However, in a busy lab with a constant stream of samples, this isn’t practical! Homing in on the number of repeats needed to generate resilient data for comparison is an important part of screening campaigns and clinical sample analysis. While choosing a single figure would be simplistic, tailoring this number depending on the needs of the investigation is sensible and this recent investigation suggests that any more than 4 or 5 repeats may be obsolete. Identification of the source, or sources, of this variation is yet to come, nonetheless this information will no doubt help to inform future research approaches within the P. vivax arena.
Publication Information:
Evaluation of Precision of the Plasmodium knowlesi Growth Inhibition Assay for Plasmodium vivax Duffy-Binding Protein-based Malaria Vaccine Development
Jonas E. Mertens, Cassandra A. Rigby, Martino Bardelli, Doris Quinkert, Mimi M. Hou, Ababacar Diouf, Sarah E. Silk, Chetan E. Chitnis, Angela M. Minassian, Robert W. Moon, Carole A. Long, Simon J. Draper, Kazutoyo Miura
DOI: https://doi.org/10.1016/j.vaccine.2024.04.073
P. falciparum analysis:
Assessment of precision in growth inhibition assay (GIA) using human anti-PfRH5 antibodies
Miura K, Diouf A, Fay MP, Barrett JR, Payne RO, Olotu AI, Minassian AM, Silk SE, Draper SJ, Long CA
DOI: https://doi.org/10.1186/s12936-023-04591-6
