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Page 8 of 15 Cheng et al. Vessel Plus 22020;4:17 I http://dx.doi.org/10.20517/2574-1209.2020.08

combination of these existing biomarkers with newly discovered EV-associated markers could significantly
improve both the specificity and sensitivity of testing.

As a consequence of their biogenesis, EVs harbour a peculiar set of proteins, nucleic acids, and lipids that
can be transferred from a parent to recipient cells, rendering these sub-micron structures unique sources
and vehicles of biomarkers. Various analytical approaches including proteomics, transcriptomics, and
metabolomics, although mostly focused on cancer-related conditions, are currently employed to study
[94]
the content of EVs derived from different cell types and bodily fluids . Nonetheless, the cargo of EVs
is now becoming an important research topic in severe malaria allowing us to both understand disease
pathogenesis and identify novel biomarkers, with proteins and microRNA (miRNA) being the most studied
components of this cargo.

EV-associated proteins can typically be studied using either untargeted proteomics, to characterise the
whole protein content, or through a hypothesis-driven targeted approach, to investigate individual proteins
or a selected set of proteins based on previous evidence. Compared to other parasitic diseases, high-
throughput untargeted proteomics - the leading technique for the discovery of new protein markers -
has not been widely applied to investigate malaria-associated EVs yet, but has been explored in the last
couple of years. The first report dates back to 2011 when exosomes from P. yoelii-infected BALB/c mice
[40]
were analysed and revealed to contain both classical exosomal markers as well as parasite proteins .
Interestingly, 30 parasite proteins belonging to two major classes, proteins associated with RBCs membrane
and proteins involved in parasite invasion into RBCs, were identified within iRBCs exosomes. Then, the
presence of Plasmodium proteins within EVs from human and mice malaria infection was confirmed by
a number of proteomics-based studies [40,51,58,82,95] . Although the majority of these studies did not have as
their main objective the identification of biomarkers, they all contributed to prove the presence of parasite-
derived proteins with antigenic and immunomodulatory properties, or as potential virulence factors within
EVs that, in the future, might be found useful for the development of novel diagnostic and prognostic tests.

Only a few studies have focused on EVs as a novel source of markers for severe malaria. In our group,
we used high-throughput proteomics to characterise the protein cargo of MVs released during ECM in
[96]
P. berghei infected mice . The vast majority of identified proteins were host-derived and only a couple
were from P. berghei. The protein content of MVs released during severe disease was significantly altered
compared to that released upon early infection or in uninfected mice. Network analysis showed that
proteins with altered abundance during ECM were associated with CM pathogenesis. Two of these proteins,
carbonic anhydrase I and S100A8 were verified to be associated with CM MV in both murine and clinical
samples, highlighting the importance of MV protein content to understand the role of EVs both in severe
[96]
malaria and as a source of protein markers . The protein cargo of MVs obtained from P. falciparum-
[97]
infected individuals was later investigated by Antwi-Baffour and colleagues , although cases with severe
malaria were not investigated. The study identified several different host-derived proteins in infected and
non-infected human subjects, as well as parasite-derived proteins in infected samples. Nonetheless, the
results remained primarily descriptive and no diagnostic marker was actually proposed. More recently,
[98]
proteomics was applied to identify novel potential biomarkers of P. vivax liver stage infection . By taking
advantage of a human liver-chimeric mouse model, plasma EVs obtained after P. vivax infection were
studied to identify potential liver-stage expressed parasite proteins that could be indicative of infection.
Among mouse and human proteins, they also identified parasite proteins showing variable distribution in
abundance over different time points post-infection, indicating that parasite proteins contained within EVs
[98]
vary with parasite developmental stages, supporting their potential role as a source of biomarkers . In
mice and human studies, there has been a consistent indication of EVs’ importance in the role of malaria
pathogenesis and their potential as markers for disease severity; however, more research is required to
confirm the potential of these EVs derived protein as biomarkers for severe malaria.