Immunology

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RESEARCH

The Immunology Unit researches the human host immune response to infectious diseases of high prevalence in Cambodia. Our mission is to provide insight into both the innate and adaptive immune response to various viral infections for novel vaccine and therapeutics design. We aim to improve vaccination strategies for vaccine-preventable diseases and identify novel biomarkers of severity or treatment response.

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Immunology

INTERNSHIP

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Dengue Viruses: Adaptive Immune Responses During Flavivirus

Dengue viruses (DENV) infect up to 390 million individuals each year, of which 500,000 cases require hospitalization.Since 2012, dengue is the most important vector-borne viral disease of humans and likely more important thanmalaria globally in terms of morbidity and economic impact. The mosquito vectors, Aedes aegypti and Aedesalbopictus both thrive well in populated urbanized areas, contributing to the spread of DENV. DENV is a member ofthe family Flaviviridae, and consists out of 4 related serotypes (DENV-1 to DENV-4). Dengue virus infection resultsin a range of clinical outcomes, from asymptomatic infection, to classic dengue fever (DF), to dengue hemorrhagicfever/dengue shock syndrome (DHF/DSS). Most primary infections are mild and probably provide lifelong protectionagainst the infecting serotype. In contrast, secondary infection with a heterologous DENV serotype can result inmore severe dengue, suggesting that primary DENV infection triggers a host memory immune response thatcan result in either protection or enhancement of subsequent infection. Due to the incomplete understanding ofthe relevant adaptive immune responses leading to protection or enhancement of disease in secondary infectionand the absence of conclusive biomarkers for protection, vaccine development has been severely hampered.Our previous work comparing the immune response in asymptomatic acute infected individuals with hospitalizedpatients revealed profound differences in the adaptive immune response profile associated with a different clinicaloutcome to infection (Simon-Loriere et al, Scie Transl Med, 2017).

Determination of antibody-independent b-cell functions during acute DENV infection

Antibodies are produced by terminally differentiated B cells, plasmablasts and plasma cells. However, besidesantibody production, B cells have diverse functions. For example, B cells with regulatory functions, termed Bregs,have important roles in maintenance of tolerance and homeostasis through the production of immunosuppressivecytokines IL-10 and TGF-β. In the context of DENV infection, not much is known about the antibody-independent Bcell responses. We detected decreased frequencies of Bregs during acute DENV infection in patients with severedengue compared to patients with mild disease, which was associated to decrease CD40L plasma concentrationsand decreased platelet counts in these patients. B cells from dengue patients were refractory to toll-like receptors(TLR) stimulation in vitro resulting in decreased B-cell specific IL-10 cytokine production, which is paralleled by anincreased expression of inhibitory FcγR in vivo in DENV-infected patients. Collectively, our results indicate that adefective B cell response in dengue patients may contribute to the pathogenesis of dengue during the early phaseof infection (Upasani et al, Frontiers Immunol 2019).

Discovery of novel biomarkers for severity at hospital admittance

Early detection of severe cases will help to identify patients that benefit from intensive therapy. Currently, noprognostic marker has been identified and early diagnosis relies on multi-parameter interpretation by the healthcare provider. We aim to identify novel biomarkers predictive for the development of severe dengue within 72hours after onset of symptoms in a clinically relevant setting. We have an ongoing collaboration with KanthaBopha Hospital, Phnom Penh. Blood samples of DENV-suspected pediatric cases are sent to the Virology Unit atIP Cambodia for dengue diagnostics. Currently, we have included 430 well-characterized DENV-infected children.After careful transcriptomic profile analysis, we have identified an 18-gene RNA signature that can detect severecases among secondary-infected dengue patients at hospital presence. This signature can yield new pointers intothe underlying pathogenesis of severe disease (Nikolayeva et al, J Infect Dis, 2018). In a separate study, we couldshow that enhanced TLR2 expression on circulating monocytes is associated with severe disease at the earlystages of disease development. In addition, increased protein expression of TLR2, an innate sentinel, generallyassociated with bacterial infections, leads to an increase in DENV-infected cell mass. In addition, our functionalanalyses uncover the ability of TLR2 to sense DENV infection and to fuel infection-mediated inflammatory responsesleading to activation of the human vascular endothelium (Aguilar-Briseno et al, submitted).

Novel vaccine development for DENV prevention

In humans, both CD4+ and CD8+ T cells contribute to protection against DENV with CD8+ T cells preferentiallytargeting non-structural proteins NS3, NS4B and NS5. Hence, a minimal DENV antigen has been designed fromconserved and highly antigenic T cell epitopes (DENV1-NS). DNA immunization with a plasmid encoding DENV1-NS in mice expressing different human HLA class I molecules confirmed the induction of a strong CD8 T cellresponse against peptides derived from these NS regions. Using this strategy, and with the intention to develop aneffective T cell-based vaccine, we show that a prime-boost immunization of human HLA class I transgenic mice withlow dose of a modified mRNA encoding DENV1-NS induces a strong T cell immunity, with a significant protectionagainst DENV1 infection, after transient blockade of the IFN type I receptor in the absence of neutralizing or subneutralizinganti-DENV antibodies (Roth et al, Frontiers Immunol 2019).

Innate Responses to HIV/TB Co-Infection

ANRS No12358: “MicroRNA (miRNAs) as prediction and/or prognostic markers of IRIS (Immune Reconstitution Inflammatory Syndrome) in TB/HIV co-infected patient (Mirbio)”

MiRNAs are reported as powerful regulators of post-translational gene expression and can act as biomarkers in several infectious diseases. Host miRNAs target certain HIV genes, affecting HIV replication thus thereby
participating in viral control. In HIV elite controllers, a set of expressed miRNA can characterize this clinical phenotype. Several studies reported the characterization of miRNA expression profile in tuberculosis (TB) patients,
but evaluation of miRNA expression in co-infections such as TB/HIV are lacking. Hence, we aim to evaluate by flow cytometry whether a circulating miRNA pattern might be used as potential biomarkers in HIV/TB coinfection and to correlate the miRNA expression profile of 28 selected miRNAs with the clinical evolution and the occurrence of IRIS. Interestingly, we identified two miRNA candidates (miR-150 and miR-145) that could discriminate between
HIV-TB co-infection from HIV, TB mono infection and healthy controls. As a next step, we will analyze exomiR, miRNA in the plasma-secreted exosome. The statistical analysis and manuscript preparation will be done in 2020.

Immune Responses to Rabies Virus (RABV) Vaccination

The international health authorities are backing an effort to eliminate canine-mediated rabies in humans by 2030.
This will require improving access to adequate and timely post-exposure rabies prophylaxis. All individuals were referred to the rabies prevention clinic at IP Cambodia and received two-dose intradermal PEP at days 0, 3, 7 and
28. We showed that all individuals demonstrated rabies virus neutralizing antibody titers considered protective (≥ 0.5 IU/ml) at day 28, immediately before the last injection. Protective titers were reached notwithstanding eRIG
use, age, sex, nutrition status or dog infective status (Cantaert T, Borand L et al, Lancet Infect dis 2019). Hence, we provide evidence that rabies PEP can be abridged to a two-dose, three-sessions, one week (D0, D3, D7 regimen.
Based on these results the WHO endorsed changes in its April 2018 guidelines. This “IPC protocol” is the first one week PEP regimen to be recommended and the shortest and most vaccine-sparing rabies PEP protocol endorsed.

Support to National Authorities

The following summarizes key support to Cambodian National Authorities during 2019 as part of our ongoing programs and projects.

- Tineke Cantaert and Pean Polidy are members of the steering committee for the international master’s degree in infectiousdisease and coordinator of immunology module in master’s year 1 (University of Health Sciences, Phnom Penh, Cambodia and Universite Paris-Saclay, Paris, France).

- Based on the results discussed in IP Cambodia’s Vaccine responses to rabies Post-Exposure Prophylaxis research, WHO endorsed changes in its April 2018 guidelines. This IPC protocol is the first one-week PEP regimen to be recommended and theshortest and most vaccine-sparing rabies PEP protocol This PEP-vaccination scheme has been implemented at the rabies vaccination centers of Institute Pasteur du Cambodge.

Teaching & Training

Drs. Tineke Cantaert and Polidy Pean also undertook the following:

- Ten 10 hours/year each of teaching in the immunology module within the master’s degree program in medical biology, UHS;
- Served as members of the steering committee for the international master’s degree in infectious disease, and coordinators of the immunology module.

PhD student:

- Vinit UPASANI was supported by a Calmette-Yersin grant from IPIN. The student has been enrolled at the University of Groningen, the Netherlands since 2017.
- Sotheary SANN is enrolled at the University of Hasselt, Belgium in 2019 Visits to UHasselt are covered by a BOF/BILA grant of the Flemish government.

The list below includes internship thesis students:

- Axelle VANDERLINDEN, University of Antwerp, Belgium: Master’s thesis in biomedical sciences: November 2018-June 2019
- David GUERRERO GOMEZ, University of Antwerp, Belgium: Master’s thesis biomedical sciences: November 2018-June 2019’
- Sokchea LAY,University of Health Sciences, Phnom Penh, Cambodia: Master’s thesis biomedical sciences: October 2018-May 2019
- Sara VAN DE KERKHOVE, University of Antwerp, Belgium: Master’s thesis biomedical sciences: November 2019-June 2020
- Mao Sowathiro, Bart University, UK: Bachelor Biological science: June-September 2019