Comparative spatial proteomics of Plasmodium-infected erythrocytes.

Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized subcellular compartments to extensively remodel the host cell for its survival. The information to fully understand these compartments is likely hidden in the so far poorly characterized Plasmodium species spatial proteome. To address this question, we determined the steady-state subcellular location of more than 12,000 parasite proteins across five different species by extensive subcellular fractionation of erythrocytes infected by Plasmodium falciparum, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. This comparison of the pan-species spatial proteomes and their expression patterns indicates increasing species-specific proteins associated with the more external compartments, supporting host adaptations and post-transcriptional regulation. The spatial proteome offers comprehensive insight into the different human, simian, and rodent Plasmodium species, establishing a powerful resource for understanding species-specific host adaptation processes in the parasite.

Manufacturability and functionality assessment of different formats of T-cell engaging bispecific antibodies.

T-cell-engaging bispecific antibodies (T-bsAbs) are promising immunotherapies for cancer treatment due to their capability of redirecting T-cells toward destroying tumor cells. Numerous T-bsAb formats have been developed, each with advantages and disadvantages in terms of developability, immunogenicity, effector functions, and pharmacokinetics. Here, we systematically compared T-bsAbs produced using eight different formats, evaluating the effect of molecular design of T-bsAbs on their manufacturability and functionality. These eight T-bsAb formats were constructed using antigen-binding fragments (Fabs) and single-chain variable fragments (scFvs) of antibodies linked to the crystallizable fragment (Fc) domain of immunoglobulin G. To ensure a fair comparison of growth and production data, we used recombinase-mediated cassette exchange technology to generate the T-bsAb-producing CHO cell lines. The produced T-bsAbs were assessed for their purification profile and recovery, binding capability, and biological activities. Our findings indicated that the manufacturability of bsAbs was adversely affected with increased number of scFv building blocks, while the functionality was affected by the combination of multiple factors, including the binding affinity and avidity of targeting moieties and the flexibility and geometry of formats. These results provide valuable insights into the impact of the format design on the optimal production and function of T-bsAbs.

Strategies to enhance immunomodulatory properties and reduce heterogeneity in mesenchymal stromal cells during ex vivo expansion.

Therapies using mesenchymal stromal cells (MSCs) to treat immune and inflammatory conditions are now at an exciting stage of development, with many MSC-based products progressing to phase II and III clinical trials. However, a major bottleneck in the clinical translation of allogeneic MSC therapies is the variable immunomodulatory properties of MSC products due to differences in their tissue source, donor heterogeneity and processes involved in manufacturing and banking. This variable functionality of MSC products likely contributes to the substantial inconsistency observed in the clinical outcomes of phase III trials of MSC therapies; several trials have failed to reach the primary efficacy endpoint. In this review, we discuss various strategies to consistently maintain or enhance the immunomodulatory potency of MSCs during ex vivo expansion, which will enable the manufacture of allogeneic MSC banks that have high potency and low variability. Biophysical and biochemical priming strategies, the use of culture additives such as heparan sulfates, and genetic modification can substantially enhance the immunomodulatory properties of MSCs during in vitro expansion. Furthermore, robust donor screening, the use of biomarkers to select for potent MSC subpopulations, and rigorous quality testing to improve the release criteria for MSC banks have the potential to reduce batch-to-batch heterogeneity and enhance the clinical efficacy of the final MSC product. Machine learning approaches to develop predictive models of individual patient response can enable personalized therapies and potentially establish correlations between in vitro potency measurements and clinical outcomes in human trials.

Rapid activation of distinct members of multigene families in Plasmodium spp.

The genomes of Plasmodium spp. encode a number of different multigene families that are thought to play a critical role for survival. However, with the exception of the P. falciparum var genes, very little is known about the biological roles of any of the other multigene families. Using the recently developed Selection Linked Integration method, we have been able to activate the expression of a single member of a multigene family of our choice in Plasmodium spp. from its endogenous promoter. We demonstrate the usefulness of this approach by activating the expression of a unique var, rifin and stevor in P. falciparum as well as yir in P. yoelii. Characterization of the selected parasites reveals differences between the different families in terms of mutual exclusive control, co-regulation, and host adaptation. Our results further support the application of the approach for the study of multigene families in Plasmodium and other organisms.

Immunomic Identification of Malaria Antigens Associated With Protection in Mice.

Efforts to develop vaccines against malaria represent a major research target. The observations that 1) sterile protection can be obtained when the host is exposed to live parasites and 2) the immunity against blood stage parasite is principally mediated by protective antibodies suggest that a protective vaccine is feasible. However, only a small number of proteins have been investigated so far and most of the Plasmodium proteome has yet to be explored. To date, only few immunodominant antigens have emerged for testing in clinical trials but no formulation has led to substantial protection in humans. The nature of parasite molecules associated with protection remains elusive. Here, immunomic screening of mice immune sera with different protection efficiencies against the whole parasite proteome allowed us to identify a large repertoire of antigens validated by screening a library expressing antigens. The calculation of weighted scores reflecting the likelihood of protection of each antigen using five predictive criteria derived from immunomic and proteomic data sets, highlighted a priority list of protective antigens. Altogether, the approach sheds light on conserved antigens across Plasmodium that are amenable to targeting by the host immune system upon merozoite invasion and blood stage development. Most of these antigens have preliminary protection data but have not been widely considered as candidate for vaccine trials, opening new perspectives that overcome the limited choice of immunodominant, poorly protective vaccines currently being the focus of malaria vaccine researches.