Diverse outcomes of controlled human malaria infection originate from
host-intrinsic immune variation and not var gene switching
Kathryn Milne,
1
* Alasdair Ivens,
1,2
* Adam J. Reid,
3
* Magda E. Lotkowska,
3
Áine O’Toole,
2,4
Geetha
Sankaranarayanan,
3
Diana Muñoz Sandoval,
1
Wiebke Nahrendorf,
1
Clement Regnault,
5,6
Nick J.
Edwards,
7
Sarah E. Silk,
7
Ruth O. Payne,
7
Angela M. Minassian,
7
Navin Venkatraman,
7
Mandy
Sanders,
3
Adrian V.S. Hill,
7
Michael P. Barrett,
5,6
Matthew Berriman,
3
Simon J. Draper,
7
J. Alexandra
Rowe,
1,2
† Philip J. Spence
1,2
†
1
Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, UK.
2
Centre
for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK.
3
Wellcome Sanger
Institute, Cambridge, UK.
4
Institute of Evolutionary Biology, University of Edinburgh, Edinburgh,
UK.
5
Wellcome Centre for Integrative Parasitology, University of Glasgow, Glasgow, UK.
6
Glasgow
Polyomics, University of Glasgow, Glasgow, UK.
7
The Jenner Institute, University of Oxford, Oxford,
UK.
*these authors contributed equally
†joint senior authors
email alex.rowe@ed.ac.uk or philip.spence@ed.ac.uk
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2Milne et al 2020
Abstract
Falciparum malaria is clinically heterogeneous and the relative contribution of parasite and host in
shaping disease severity remains unclear. We explored the interaction between inammation and
parasite variant surface antigen (VSA) expression, asking whether this relationship underpins the
variation observed in controlled human malaria infection (CHMI). We uncovered marked heterogeneity
in the response of naive hosts to blood challenge; some volunteers remained quiescent, others
triggered interferon-stimulated inammation and some showed transcriptional evidence of myeloid
cell suppression. Signicantly, only inammatory volunteers experienced hallmark symptoms of
malaria. When we tracked temporal changes in parasite VSA expression to ask whether variants
associated with severe disease preferentially expand in naive hosts (as predicted by current theory)
we found that var gene proles were unchanged after 10-days of infection. The diverse outcomes
of CHMI therefore depend upon human immune variation and there is no evidence for switching or
selection of var genes in naive hosts.
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3Milne et al 2020
Introduction
There is enormous diversity in the human response to identical immune challenge. This variation
is currently being interrogated in large cohorts of healthy volunteers to pinpoint the genetic and
non-genetic factors that dictate human immune decision-making (1-6). Controlled human malaria
infection (CHMI) provides a well-established challenge model in which to examine immune variation
in vivo, and heterogeneity in the host response to Plasmodium falciparum is well-recognised (7,
8). Prior work suggests a dichotomy in immune decision-making with some individuals triggering
an inammatory response, which is characterised by high circulating levels of IFNγ, whilst others
mount an alternative response distinguished by the early release of TGFβ and suppression of IFNγ
signaling (9-12). The precise mechanisms underpinning these divergent responses remain unknown.
Genome-wide transcriptional proling has the potential to map immune decision-making in
unprecedented detail and previous CHMI studies have revealed an upregulation of genes associated
with pathogen detection, NFκB activation and IFNγ signaling consistent with a systemic inammatory
response (13-15). A transcriptional signature of an alternative immune response has not been
described in naive hosts. Nevertheless, these prior studies were primarily designed to measure
shared or common signatures of infection, potentially masking variation between volunteers. Only
one study (16) has specically investigated transcriptional variation in CHMI and it reported two
distinct outcomes based on host microRNA proles; notably, the authors were able to correlate the
induction of 3 specic miRNAs with a lower parasite burden suggesting that events early in infection
may have important downstream consequences. Identifying the sources of immune variation may
therefore reveal some of the factors that underpin the clinical heterogeneity of falciparum malaria. In
this context, direct intravenous challenge with blood-stage parasites provides a unique opportunity to
assess the contribution of host-intrinsic mechanisms of variation as it ensures all volunteers receive
an identical immune challenge (17).
The potential role of parasite factors in inuencing human immune decision-making has not yet
been explored in CHMI. Expression of a subset of parasite variant surface antigens (VSA) known as
group A and DC8 var genes is associated with severe malaria in both children and adults (18-23).
These virulence-associated genes encode PfEMP1 adhesion molecules that mediate sequestration
of infected red cells in the microvasculature, contributing to pathology. Group A/DC8-expressing
parasites are thought to be more pathogenic than those expressing other var types (group B and C)
and can bind the endothelium with high afnity in sensitive sites, such as brain (24). It is unknown
whether expression of these variants can inuence the immune response, for example by causing
dysregulated activation that leads to widespread collateral tissue damage. Or conversely, whether
inammation might preferentially support the survival and expansion of these variants by upregulating
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4Milne et al 2020
their binding sites on the endothelium.
It has been observed in previous CHMI studies that parasites transcribe a broad array of var genes
in naive hosts, with predominant expression of group B variants and no marked differences between
volunteers (25, 26). Indeed, we have shown that mosquitoes reset malaria parasites to ensure
diverse expression of their VSA repertoire at the start of the blood cycle (27). It therefore remains
unclear how group A and DC8 var genes seemingly come to dominate the PfEMP1 landscape in
severe malaria. Antibody-mediated clearance of parasites expressing group B variants is a possible
explanation, but is unlikely because severe malaria develops in hosts with low or absent immunity
during their rst few infections of life (28). It has instead been suggested that parasites expressing
group A/DC8 var genes preferentially expand in naive hosts because they sequester and avoid
host clearance more effectively than parasites expressing group B or C var genes (29-31). This
explanation is widely accepted but is not based on substantial evidence.
In this study, we have used a blood challenge model to investigate host-intrinsic variation in the
immune response to falciparum malaria and examined the interplay between parasite and host
factors in shaping outcome of infection. This model has the advantage that an identical immune
challenge is given to all volunteers and furthermore the frequency of each parasite variant can easily
be measured at the start and end of infection to track changes through time. We set out to test the
specic hypotheses that a, expression of group A and DC8 var genes would preferentially increase
as the infection progressed and b, there would be a measurable relationship between inammation
and parasite VSA expression, which would shape the clinical outcome of infection.
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5Milne et al 2020
Results
Immune decision-making in falciparum malaria
To explore host-intrinsic variation in the immune response to P. falciparum a cohort of 14 malaria-
naive volunteers were infected with an equal number of blood-stage parasites (clone 3D7) by direct
intravenous inoculation (Table S1). This enabled the pathogenic blood cycle to be initiated in every
volunteer within a 30-minute window (32). Systemic changes in the host response were then captured
through time by transcriptionally proling whole blood every 48-hours from the day before infection
until the day of diagnosis (the point of drug treatment). This time-point varied between volunteers,
occurring 7.5 to 10.5 days post-infection when two out of three diagnostic criteria were met (positive
thick blood smear and/or parasite density > 500 parasites ml
-1
by qPCR and/or symptoms consistent
with malaria). To reveal the diversity of responses within our cohort each volunteer’s time-course
was analysed independently by tracking their dynamic changes in gene expression as the infection
progressed. Importantly, this approach does not assume shared features between individuals, nor
does it bias against uncommon (or rare) responses.
As a rst step, we measured the variance of every gene through time and visualised the 100 protein-
coding genes with highest variance in each volunteer (Fig. S1). To control for baseline variation in
gene expression, uninfected control volunteers were also analysed. We found that variance in one
third of infected volunteers (4/14) was comparable to the uninfected control group, suggesting these
four volunteers did not respond to infection (Table S2). To explore this further, we merged the top 100
gene lists from all infected volunteers to produce a single non-redundant list of the most dynamically
expressed protein-coding genes across the cohort (n = 517 unique genes, henceforth called the 517-
gene superset); we could then directly compare the transcriptional response between individuals.
Accordingly, we performed a principal component analysis (PCA) of these genes through time, and
for each volunteer we determined the Euclidean distance travelled to quantify the magnitude of
their response (Fig. S2 and Table S2). Using the variance and distance travelled metrics we then
set two thresholds to identify volunteers that trigger a measurable response to blood challenge
(see methods); four volunteers failed to cross both thresholds (v018, v012, v208 & v020) and were
therefore grouped and labelled unresponsive. A pairwise comparison between their diagnosis and
pre-infection samples conrmed that there were zero differentially expressed genes in this group
(adj p < 0.05). Immune quiescence is therefore a common outcome of controlled human malaria
infection.
In contrast, variance (Fig. S1) and distance travelled (Fig. S2) both suggested that the remaining 10
volunteers made a robust response to infection (Table S2). And when we clustered and visualised
the 517-gene superset expression data we found that these volunteers could separate easily into
. CC-BY 4.0 International licenseIt is made available under a
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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint
The copyright holder for thisthis version posted September 7, 2020. ; https://doi.org/10.1101/2020.09.04.20188144doi: medRxiv preprint