Biology subjects

Tham, W.-H.

Publications and source records attributed to Tham, W.-H..

22 records · Page 2Linked to original sources

bioRxiv2020

Nanobody generation and structural characterization of Plasmodium falciparum 6-cysteine protein Pf12p

Surface-associated proteins play critical roles in the Plasmodium parasite life cycle and are major targets for vaccine development. The 6-cysteine (6-cys) protein family is expressed in a stage-specific manner throughout Plasmodium falciparum life cycle and characterized by the presence of 6-cys domains, which are {beta}-sandwich domains with conserved sets of disulfide bonds. Although several 6-cys family members have been implicated to play a role in sexual stages, mosquito transmission, evasion of the host immune response and host cell invasion, the precise function of many family members is still unknown and structural information is only available for four 6-cys proteins. Here, we present to the best of our knowledge, the first crystal structure of the 6-cys protein Pf12p determined at 2.8 [A] resolution. The monomeric molecule folds into two domains, D1 and D2, both of which adopt the canonical 6-cys domain fold. Although the structural fold is similar to that of Pf12, its paralog in P. falciparum, we show that Pf12p does not complex with Pf41, which is a known interaction partner of Pf12. We generated ten distinct Pf12p-specific nanobodies which map into two separate epitope groups; one group which binds within the D2 domain, while several members of the second group bind at the interface of the D1 and D2 domain of Pf12p. Characterization of the structural features of the 6-cys family and their associated nanobodies provide a framework for generating new tools to study the diverse functions of the 6-cys protein family in the Plasmodium life cycle.

microbiology↗

bioRxiv2020

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against P. vivax antigens in a multiplexed bead-based assay using Luminex(R) technology (Bio-Plex(R)200 or MAGPIX(R))

Multiplexed bead-based assays that use Luminex xMAP(R) technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data is lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex(R) 100/200 or Bio-Plex(R) 200), magnetic beads can be run on both these and the newer MAGPIX(R) instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex(R) 200, ii) magnetic beads run on the Bio-Plex(R) 200 versus MAGPIX(R) and iii) non-magnetic beads run on a Bio-Plex(R) 200 versus magnetic beads run on the MAGPIX(R). We also performed an external validation of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were strongly correlated in all three of our comparisons, however higher amounts of protein were required for coupling to magnetic beads. Our external validation indicated that results generated in different laboratories using the same coupled beads are also highly comparable, particularly if a reference standard curve is used.

immunology↗

bioRxiv2020

Naturally acquired blocking human monoclonal antibodies to Plasmodium vivax reticulocyte binding protein 2b

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolated and characterized anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies blocked PvRBP2b binding to reticulocytes and inhibited complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.

microbiology↗

bioRxiv2018

Development and validation of serological markers for detecting recent exposure to Plasmodium vivax infection

In order to accelerate towards malaria elimination, improved targeting of limited resources is essential. A major gap in our elimination toolkit for Plasmodium vivax malaria is the identification of individuals carrying arrested liver stages, called hypnozoites. These clinically silent but frequently relapsing hypnozoites are key to P. vivax persistence. Whilst hypnozoites cannot be directly detected, individuals who have had recent exposure to P. vivax and have not been treated are likely to harbor these parasites. By measuring IgG antibody responses to over 300 P. vivax proteins, a panel of serological markers capable of detecting exposure to P. vivax infections in the prior 9-month period was identified and validated. Using antibody responses to 8 P. vivax proteins, 80% sensitivity and specificity for detecting recent infections were achieved in three independent studies conducted in Thailand, Brazil and the Solomon Islands. As these individuals have a high likelihood of harboring hypnozoites, the suite of these 8 antibody responses can serve as biomarkers for the identification of individuals who should be targeted for treatment with liver-stage drugs such as primaquine and tafenoquine in mass drug administration programs aimed at controlling and eliminating P. vivax malaria.\n\nOne Sentence SummaryThe manuscript describes identification and validation of a novel panel of P. vivax proteins that can be used to detect recent exposure to P. vivax infections within the prior 9 months.

microbiology↗