Immunity to liver-stage Plasmodium falciparum in peripheral and tissue-resident immune cells

(Refer to protocol Section 1)

Malaria caused by the parasite Plasmodium falciparum poses a huge burden to
public health in endemic regions, particularly among children and pregnant
women. The parasite life cycle begins with infectious mosquito bites, injecting
sporozoite-stage parasites that migrate to the liver and develop within
hepatocytes for ~6-7 days. This liver-stage is asymptomatic. Mounting sterile
immunity to liver-stage malaria parasites results in abrogation of further
pathology, and has therefore served as the basis for many vaccine candidates.
However, conventional vaccine strategies to generate liver-stage immunity have
not been highly successful to date and a better understanding of how immunity
takes place in the liver will be required to improve the design of future
vaccines.

We have recently developed an in vitro model of P. falciparum liver-stage
parasite development using freshly isolated human hepatocytes obtained from
liver sections of patients undergoing medically-indicated surgery. This model
has been successfully used by us to study aspects of liver-stage biology. We
have moreover been able to isolate and study the phenotype and function of
liver-resident immune cells from these same liver sections.

In murine and human studies, cytolytic CD8+ T cells are important for sterile
immunity. One of the natural targets of cytolytic immunity to the liver-stage
is the circumsporozoite protein (CSP). We have developed an HLA-A2-restricted
CSP-specific cytolytic T cell clonal line capable of lysing cells which have
been artificially loaded with CSP, though this has not been demonstrated in P.
falciparum-infected hepatocytes. Demonstrating functional activity of this T
cell line against infected hepatocytes would permit investigation of a wide
range of other questions highly relevant to malaria immunity. The contribution
of other cells to liver antimalarial immunity is even less well understood. The
liver microenvironment is enriched in innate immune cells, particularly natural
killer (NK) and innate-like gd T cells. Both the cell types have been indicated
in murine studies to have a role in antimalarial protection. It is unclear how
these cells affect liver-stage immunity.

In this study we will use the in vitro fresh human hepatocyte model of P.
falciparum liver-stage infection in combination with both the CSP-specific
cytolytic T cell clone and donors* own lymphocytes from peripheral blood and
liver to investigate whether human immune cells are able to mount functional
responses to liver-resident P. falciparum parasites.

(Refer to Protocol Section 2)

Primary Objectives
• To establish an in vitro assay to study recognition and killing of P.
falciparum-infected hepatocytes by:
o CSP-specific cytolytic CD8+ T cells
o hepatic and peripheral innate/innate-like lymphocytes
Secondary Objectives:
• To assess recognition and killing of P. falciparum-infected hepatocytes by
CSP-specific cytolytic CD8+ T cell line
• To assess the differences in recognition and killing of P.
falciparum-infected hepatocytes between liver-resident and peripheral
lymphocytes
• To identify the individual lymphocyte (sub-)populations which contribute to
recognition and killing of P. falciparum-infected hepatocytes
Exploratory Objectives:
• To assess at which time point during intra-hepatocytic development (early,
middle or late) P. falciparum-infected hepatocytes are most optimally
recognised and killed
• To determine difference in recognition and killing of P. falciparum-infected
hepatocytes between parasite strains
• To compare recognition and killing of P. falciparum-infected hepatocytes in
different zonal hepatocyte types
• To characterise immunological pathways involved in recognition and killing of
P. falciparum-infected hepatocytes by lymphocyte (sub-)populations

(Refer to Protocol Section 3)

This is a single-centre investigator-initiated exploratory study. Participants
will be recruited building upon an existing collaboration with the Department
of Surgery, through which we routinely receive anonymized liver tissue which
would otherwise be considered medical waste, for in vitro P. falciparum culture
from patients undergoing medically-indicated partial liver-resection for
underlying disease. For the current study, upon initial scheduling for surgery,
written informed consent will be obtained to draw 6mL of blood to determine if
the participant has the HLA-A2 phenotype compatible with the cytolytic T cell
line. Immediately prior to surgery, 24mL of blood will be drawn, where possible
via an existing intravenous or arterial line for isolation of peripheral blood
mononuclear cells (PBMCs). No study procedures will interfere with routine
clinical care for the participants* underlying disease.
Hepatocyes will be isolated from part of the available liver tissue for in
vitro P. falciparum culture and innate/innate-like lymphocytes will be isolated
from the remaining liver tissue and PBMCs. In participants who express HLA-A2,
in vitro hepatocyte cultures will be used to assess recognition and killing of
intra-hepatocytic parasites. In all subjects with sufficient material, we will
assess recognition and killing by liver-resident and peripheral innate(-like)
lymphocytes. Read-out will be by variety of immunological techniques including,
immunofluorescence microscopy, flow cytometry, qPCR and ELISA/multiplex bead
array.

(Refer to Protocol Section 11)

There is no direct benefit to study participants. Malaria poses a significant
risk to global health and a vaccine is urgently needed to combat the burden of
disease. Development of a vaccine against the liver stage would prevent
malaria-related morbidity and mortality entirely. Unfortunately, very little is
known about liver-stage immunity. An in vitro liver stage platform to
investigate immunity to P. falciparum in the liver would advance the field
significantly by enabling more in-depth studies of the correlates of protection
and factors which can modify the host immune response.

In the proposed study, adult patients scheduled for medically-indicated partial
liver resection for underlying disease will undergo one 6mL blood draw to
determine HLA-A2 phenotype and another 24mL blood draw on the day of surgery,
where possible through an existing intravenous or arterial line. The risks
associated therewith are minimal. The liver tissue obtained for this study
would otherwise be discarded as medical waste and thus represent no additional
risk to participants.