LB-3350
Profiling of T cell Responses in Recent African Immigrants Identifies a Cluster of
Antigens Associated with Clinical Malaria Episodes.
LeeAnn Talarico1, Zheng Yan1, Aula Alami1, Noelle B. Patterson2,3, Emily C. Smith2,3, Joao Aguiar2,4, Martha Sedegah2,
Thomas L. Richie2, Eileen F. Villasante2, Tobias Guennel5, Scott Marshall5, Jessica Baker Flechtner1 and Jean-Luc Bodmer1.
1Genocea
Biosciences, Inc. Cambridge MA, USA.2Naval Medical Research Center, Silver Spring MD, USA. 3Henry M. Jackson Foundation for the
Advancement of Military Medicine, Inc., Bethesda MD, USA. 4Camris International, Bethesda MD, USA. 5Biostat Solutions, Inc. Frederick MD, USA
Abstract
Memory T cell responses induced as a result of Plasmodium falciparum
(Pf) exposure are difficult to measure, and a paucity of information exists
on the correlation between responses to specific antigens and clinical
outcomes due to exposure. We hypothesized that profiling T cell
responses from donors who are recent immigrants from Pf-endemic areas,
and correlating the antigen specificity of responses with reported episodes
of clinical malaria, we could identify natural biomarkers of protection or
clinical disease. We have developed and optimized a T cell antigen
discovery platform, ATLASTM, based on expressed proteomic libraries of
medically relevant pathogens. With this technology, the full or partial
proteome of a pathogen is expressed as individual clones in E. coli which
can be processed by any donor’s antigen presenting cells and presented
as peptide epitopes in the context of MHC class I or II molecules. When
autologous CD4+ or CD8+ T cells are added that are specific for the clone
in a given well, a functional readout of activation can be measured such as
IFNg. We constructed an expression library containing 735 full-length
Plasmodium falciparum 3D7 (Pf) proteins predicted to be expressed during
the pre-erythrocytic stage of the Pf life cycle. This library was used to
interrogate memory CD8+ T cell responses from of a cohort of ~100
donors who had recently (18 months or less) emigrated from sub-Saharan
Africa to the United States. Multivariate clustering of the screening data
revealed a discrete set of 15 antigens that were correlated 14:1 to a prior
clinical episode of malaria when compared to those with no prior clinical
malaria episode. In summary, the ATLASTM technology identified the
antigen specificities of memory T cell responses that were generated
during natural Pf exposure, a subset of which correlated to a history of
clinical disease. We have identified biomarkers of clinical malaria exposure
and work is ongoing to determine if these antigens are also recognized by
T cells of protected vaccinated individuals.
Results
Study Design
This study was designed to use the ATLASTM platform to interrogate CD8+ T cell
memory responses to individual recombinantly expressed full-length Plasmodium
falciparum 3D7 antigens. Cohorts were composed of recent immigrants to the United
States from sub-Saharan Africa. Blood was collected by African Services Committee
in Harlem, NY and donors were assigned to 1 of 5 cohorts based upon their responses
to a questionnaire.
Table 1: Cohort Definition and Recruitment.
Cohort
Definition
Endemic
Return
Prophylactic
Donors who have lived in a malaria
endemic area for 18yrs or greater and
have migrated out of the malaria
endemic area within the last 18
months. Divided into two sub-cohorts:
1) reported clinical malaria; 2) did not
report clinical malaria
Donors who migrated out of a
malaria endemic area and
returned for a period of 6
months or greater without
prophylaxis and did not illustrate
symptoms of malaria including
high fevers or chills within the
last year
Prophylactically treated donor with
previous exposure to a malaria
endemic area for 6 months or
greater, with a negative history of
malaria
40
20
20
N
Naive
Donors who have never traveled
outside of the United States and
have a negative malaria history
20
Countries
Represented
Figure 1: Plasmodium falciparum 3D7 library construction. 1193 ORFs were chosen for inclusion in the
pre-erythrocytic screening library based upon transcriptome analysis1,2, P. yoelli and P. berghei antigens
identified through mouse screens (NMRC), and/or the presence of the PEXEL motif3. 901 full length
ORFs were successfully amplified from genomic DNA or cDNA. Clones were recombinantly expressed in
E. coli and full-length protein expression was validated using a cell-based assay (data not shown),
resulting in 735 full length proteins in the final screening library.
A.
Endemic v. Naive
Return v. Naive
Prophylaxis v. Naive
Endemic 1 v. Endemic 2
Conclusions
•An expression library containing 735 full-length P. falciparum
3D7 proteins predicted to be expressed during the liver-stage of
the parasite was constructed to interrogate CD8+ T cell memory
in donors from malaria-endemic areas.
B.
•Whole blood samples were collected from four distinct cohorts
of volunteers upon their immigration into the United States at the
African Services Committee in Harlem, NY. These cohorts
represent different patterns of natural exposure to Plasmodium
falciparum in endemic areas.
•The ATLASTM technology platform was used to screen the
donors’ CD8+ T cells against each individual full length protein in
the library and identified multiple protein targets of recall T cell
responses
•Cluster analysis revealed 15 antigens, T cell responses to which
predicted clinical malaria exposure.
•Experiments are currently underway to confirm malaria
exposure by Indirect Fluorescent Antibody (IFA) testing at the
NMRC
•Additional analysis to identify antigens that segregate with
protection from clinical malaria is ongoing.
1Kaiser
et al., 2004; 51:1221-32
2Tarun et al., 2008; 105:305-10
3Marti et al., 2004; 306:1930-3
Figure 2: PBMCs were isolated by Ficoll density gradient from whole blood collected from cohorts of
interest. Each donor’s T cells were non-specifically expanded for screening, and their autologous
monocytes were matured to dendritic cells (APC). On the day of screening, the donor’s APC were
incubated with individual E. coli clones expressing P. falciparum proteins then fixed, washed, and
incubated with their expanded CD8+ T cells for 24hr. Cell-free supernatants were evaluated in duplicate
by IFNγ ELISA to identify recall T cell responses to library protein antigens.
Figure 3: Genocea’s ATLASTM technology
expresses individual full-length proteins in E. coli,
with or without a co-expressed cytoplasmic variant
of Listeriolysin O (cLLO). Recombinant E. coli are
phagocytosed by each subjects’ monocyte-derived
dendritic cells, where the cLLO facilitates the
release of the contents of the phagolysosome into
the cytosol, where the proteins can be processed
through the proteosome and presented in the
context of MHC class I molecules to CD8+ T cells.
In the absence of cLLO, MHC class II presentation
to CD4+ T cells is facilitated.
Figure 4. Screening Results. A.
Supervised clustering analysis did
not reveal clusters of genes that
distinguished naturally exposed
cohorts from the naïve cohort. Upon
further analysis of the Endemic
cohort, unsupervised clustering
revealed a cluster of 15 antigens
that distinguished between endemic
donors who had reported cases of
clinical malaria and those who had
not. These genes could be useful as
biomarkers of malaria exposure. B.
Raw IFN-g ELISA values for each
plate were corrected by subtracting
values of an irrelevant antigen (GFP)
screened in multiple wells within
each plate. Corrected values were
then compared across donors within
the same cohort as well as across
cohorts. Hit distribution graphs
displaying the cumulative responses
to all clones screened show an even
distribution of responses across
clones.
We would like to thank the African Services Committee in New York, NY for collection of blood for this study. We would also like to thank the
Telemedicine and Advanced Technology Research Center (TATRC) for funding this project.
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