5 Monivong Boulevard, P.O Box. 983, Phnom Penh, Cambodia [email protected]

Dengue viruses (DENV) infect up to 390 million individuals each year, of which 500.000 cases require hospitalization (1/S. Bhatt et al., Nature 496, 2013, 2/WHO: Dengue: Guidelines for Diagnosis, treatment, prevention and control, 2009). Since 2012, dengue is the most important vector-borne viral disease of humans and likely more important than malaria globally in terms of morbidity and economic impact (D. J. Gubler, The American journal of tropical medicine and hygiene 86, 2012). The mosquito vectors, Aedes aegypti and Aedes albopictus both thrive well in populated urbanized areas, contributing to the spread of DENV. Costs for dengue treatment are substantially and include both medical care and non-medical costs such as lost working days and preventive measurements and can be estimated to 15-20 USD per person per year. In Cambodia, during the 2006-2008 epidemic, total costs reached between 0.03-0.17 percent of the Gross Domestic Product and was mainly carried by patients themselves (J. Beaute, S. Vong, BMC public health 10, 2010).

DENV is a member of the family Flaviviridae, and consists out of 4 related serotypes (DENV-1 to DENV-4) (L. C. Katzelnick et al., Science 349, 2015). Dengue virus infection results in a range of clinical outcomes, from asymptomatic infection, to classic dengue fever (DF), to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Dengue shock syndrome is characterized by significant loss of intravascular plasma volume leading to hypovolemic shock, and is the most common life-threatening complication of dengue (WHO: Dengue: Guidelines for Diagnosis, treatment, prevention and control, 2009).

Most primary infections are mild and probably provide lifelong protection against the infecting serotype. In contrast, secondary infection with a heterologous DENV serotype can result in more severe dengue, suggesting that primary DENV infection triggers a host memory immune response that can result in either protection or enhancement of subsequent infection (B. R. Murphy, S. S. Whitehead, Annual review of immunology 29, 2011). It remains to be investigated to what extend these mechanisms contribute to human pathogenesis. Due to the incomplete understanding of the relevant adaptive immune responses leading to protection or enhancement of disease in secondary infection and the absence of conclusive biomarkers for protection, vaccine development has been hampered (S. R. Hadinegoro et al., N Engl J Med 373, 2015).

Our research is driven by the hypothesis that proper control of B cell responses such as antigen presentation and regulatory functions are needed for a favorable clinical outcome of secondary flavivirus infection in humans. A deregulated B cell response can lead to exacerbation of disease.

A. Investigation of adaptive immune responses in asymptomatic dengue-infected individuals

Most DENV infections cause few or no symptoms. Asymptomatic DENV-infected patients provide a unique opportunity to decipher the host immune responses leading to virus elimination without negative impact on the individual’s health. We used an integrated approach of transcriptional profiling and immunological analysis comparing a Cambodian population of strictly asymptomatic viremic individuals with clinical dengue patients. Whereas inflammatory pathways and innate immune responses were similar between asymptomatic individuals and clinical dengue patients, expression of proteins related to antigen presentation and subsequent T and B cell activation pathways were differentially regulated, independent of viral load or previous DENV infection.

Feedback mechanisms controlled the immune response in asymptomatic viremic individuals as demonstrated by increased activation of T cell apoptosis-related pathways and FcγRIIB signaling associated with decreased anti-DENV specific antibody concentrations. Taken together, our data

Graphical abstract of Simon-Loriere, Duong et al, Scie Transl Med 2017

illustrate that symptom-free DENV infection in children is determined by increased activation of the adaptive immune compartment and proper control mechanisms leading to elimination of viral infection without excessive immune activation, having implications for novel vaccine development strategies.

A follow up study is planned in 2018, where we will identify asymptomatic cases in the ECOMORE II project. Children will be monitored for school absences and diagnosed for DENV using point-of-care test. classmates will be screened for DENV-infection and followed-up for clinical symptoms. The field study will be done in collaboration with the Epidemiology and Public Health Unit, IPC and dengue diagnostics will be performed by the Virology Unit, IPC. Using these samples, we will confirm and extend our previous data. We will investigate B cell functionality more in detail and characterize antigen-specific B cells.

 

Simon-Loriere E, Duong V et al, Scie Transl Med, 2017

Collaborators:

  • Virology Unit, Institut Pasteur Cambodia
  • Epidemiology and Public Health Unit, Institut Pasteur Cambodia
  • Functional genetics of infectious diseases Unit, Institut Pasteur, Paris, France

B. Molecular and functional characterization of B cell responses during DENV infection

In the blood, DENV is tropic for monocytes and dendritic cells (K. Jessie, et al., The Journal of infectious diseases 189, 2004). In addition, we and others have shown that B cells can support viral replication, helping the spread of the virus (A. Srikiatkhachorn et al., PloS one 7, 2012).

The consequence of B cell infection by DENV on different B cell functionalities remains unknown. Next to precursors of antibody-secreting plasma cells, B cells perform many other functions, such as antigen presentation or cytokine production, and important immunomodulatory functions (T. Cantaert et al., Arthritis and rheumatism 64, 2012), all of which could be altered due to direct DENV infection. This hypothesis is corroborated by reports showing that the B cell compartment in DENV infection is highly disturbed in vivo. An unpreceded increase in the plasmablast population as early as 4 days after onset of symptoms, compared to non-dengue febrile controls has been observed by us and others. Remarkably, plasmablast frequencies reach up to 50% of circulating B cells and are even higher compared to frequencies observed during recall influenza vaccination (J. Wrammert et al., Journal of virology 86,2012). It remains to be determined if this is due to direct infection by DENV and how this influences antibody production.

Figure: B cells are susceptible to DENV infection in vivo. (A) B cells and monocytes were purified from DENV- fever control patients or DENV+ cases and stained with anti-CD20 (green) and anti-Envelope (red) (B) Quantification of percentage positive cells. C. FACS staining of peripheral blood mononuclear cells isolated from DENV+ cases with anti-NS3.

Collaborators:

  • Virology Unit, Institut Pasteur Cambodia
  • University of Groningen, the Netherlands
  • Erlangen University, Germany
  • Rockefeller University, USA

C. Biomarker discovery during flavivirus infection

Dengue virus infection results in a range of clinical outcomes, going from classic dengue fever (DF), to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Dengue shock syndrome is characterized by significant loss of intravascular plasma volume leading to hypovolemic shock, usually between the 4th and 6th day of fever onset, and is the most common life-threatening complication of dengue (WHO: Dengue: Guidelines for Diagnosis, treatment, prevention and control, 2009). Whereas there is no anti-viral therapy available, case-incidence of DSS can be significantly reduced via careful monitoring and fluid administration maintaining a correct intravascular composition. The earlier in disease course treatment of severe cases or cases-at-risk starts, the higher the chance of survival (WHO: Dengue: Guidelines for Diagnosis, treatment, prevention and control, 2009). In addition, it is likely that the therapeutic window of antiviral therapy under development lies within the first 48-72 hours after onset of symptoms. The identification of severe dengue is a challenging prognosis to make for a primary health clinician. Rapid diagnostic tests can determine the presence of dengue non-structural protein 1 (NS-1) in the blood, together with anti-DENV IgM and IgG antibodies, confirming DENV infection. In most cases, severe dengue is diagnosed at the moment of rapid fluid loss and hypovolemic shock.  Hence, physicians need better tools to assist in prognosis of disease severity, before the onset of shock. Therefore, We aim to identify a new biomarker predictive for the development of severe dengue within 96 hours after onset of symptoms in a clinically relevant setting.

Collaborators:

  • Kantha Bopha children Hosptital, Phnom Penh
  • Virology Unit, Institut Pasteur Cambodia
  • Institut Pasteur Italy/Fondazione Cenci Bolognetti
  • University of Groningen, the Netherlands
  • NAMRU-2 Phnom Penh, Cambodia