Animal Models of Dengue Virus Infection

The development of animal models of dengue virus (DENV) infection and disease has been challenging, as epidemic DENV does not naturally infect non-human species. Non-human primates (NHPs) can sustain viral replication in relevant cell types and develop a robust immune response, but they do not develop overt disease. In contrast, certain immunodeficient mouse models infected with mouse-adapted DENV strains show signs of severe disease similar to the ‘vascular-leak’ syndrome seen in severe dengue in humans. Humanized mouse models can sustain DENV replication and show some signs of disease, but further development is needed to validate the immune response. Classically, immunocompetent mice infected with DENV do not manifest disease or else develop paralysis when inoculated intracranially; however, a new model using high doses of DENV has recently been shown to develop hemorrhagic signs after infection. Overall, each model has its advantages and disadvantages and is differentially suited for studies of dengue pathogenesis and immunopathogenesis and/or pre-clinical testing of antiviral drugs and vaccines

NKG2D triggers cytotoxicity in mouse NK cells lacking DAP12 or Syk family kinases

In activated mouse natural killer (NK) cells, the NKG2D receptor associates with two intracellular adaptors, DAP10 and DAP12, which trigger phosphatidyl inositol 3 kinase (PI3K) and Syk family protein tyrosine kinases, respectively. Here we show that cytotoxicity, but not cytokine production, is triggered by NKG2D in activated NK cells lacking either DAP12 or the Syk family members Syk and ZAP70. Inhibition of PI3K blocks this cytotoxicity, suggesting that the DAP10-PI3K pathway is sufficient to initiate NKG2D-mediated killing of target cells. Our results highlight signaling divergence in the effector functions of NKG2D and indicate that alternative associations between a receptor and its adaptors may provide a single receptor with a dual 'on-switch', giving mouse NK cells more choices through which to trigger cytotoxicity

Protective Immune Response to Dengue Virus Infection and Vaccines: perspectives from the field to the bench

Dengue is the most important mosquito-transmitted viral disease in humans. Half of the world population is at risk of infection, mostly in tropical and sub-tropical areas. The World Health Organization (WHO) estimates that 50 to 100 million infections occur yearly, with 50,000 to 100,000 deaths related to dengue, mainly in children. Recent estimates show higher numbers, up to three times more, with 390 million estimated dengue infections per year, among which 96 million apparent infections (Bhatt et al. 2013). Initially localized to South-East Asia, dengue virus (DENV) started its spread in Latin America in the 80s. Little is known about DENV spread in Africa, but multiple seroprevalence surveys over several years are now clearly showing endemic areas in East and West Africa (Brady et al. 2013). Finally, due to global warming and intense traveling there is a risk of global spread towards more temperate regions, and both US Key islands (FL) and southern Europe recently faced DENV outbreaks. There are currently no specific treatments or vaccines available. Even though several dengue vaccines are in the pipeline, clear correlates of protection are still lacking. The recent failure of the live-attenuated Sanofi vaccine Phase 2b trial (Sabchareon et al. 2013) and the lack of correlation between clinical protection and in vitro neutralization assays, clearly underlines the necessity to better understand the role of the different components of the immune system in protection against dengue virus infection and the requirement for the development of additional and/or improved predictive assays. The aim of this research topic is to provide novel data, opinions and literature reviews on the best immune correlates of protection and recent advances in the immune response to DENV infection that can allow rapid progress of dengue vaccines. Authors can choose to submit original research papers, reviews or opinions on pre-clinical or clinical observations that will help unify the field, with perspectives from epidemiology, virology, immunology and vaccine developers. This research topic will discuss different aspects of the protective immune response to DENV that can influence vaccine development. It will include a review of epidemiological data generated in the field, which will address spatio-temporal diversity of DENV epidemics, the importance of cross-reactive protection and of the time-interval between infections as a predictor of disease. It will further include a review of the role of both the innate and adaptive immunity in DENV infection control, and discuss the usefulness of new improved animal models in dissecting the role of each immunological compartment, which will help define new correlate of immune protection. New data concerning the DENV structure and anti-dengue antibody structure will address the necessity of improved neutralization assays. The ultimate test to prove vaccine efficacy and study immune correlates of protection in humans before large trials will open up the discussion on human DENV challenges using controlled attenuated viral strains. Finally, the role of vaccines, administered in flavi-immune populations, in the modification of future epidemics will also be approached and will include novel studies on mosquitoes infection thresholds

Anatomy of a murder (dissecting signal transduction pathways upon activation of murine Natural killer cells)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

VALEUR DIAGNOSTIQUE DU REARRANGEMENT GENIQUE DES LYMPHOCYTES ALVEOLAIRES PAR PCR AU COURS DES SUSPICIONS DE LYMPHOME PULMONAIRE (ETUDE PROSPECTIVE CHEZ 116 F.)

PARIS-BIUM (751062103) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocSudocFranceF

Dominant Cross-Reactive B Cell Response during Secondary Acute Dengue Virus Infection in Humans

The four serotypes of dengue virus (DENV) cause dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Severe disease has been associated with heterotypic secondary DENV infection, mediated by crossreactive antibodies (Abs) and/or cross-reactive T cells. The role of cross-reactive immunity in mediating enhanced disease versus cross-protection against secondary heterotypic DENV infection is not well defined. A better understanding of the cross-reactive immune response in natural infections is critical for development of safe and effective tetravalent vaccines. We studied the B cell phenotype of circulating B cells in the blood of pediatric patients suspected of dengue during the 2010–2011 dengue season in Managua, Nicaragua (n = 216), which was dominated by the DENV-3 serotype. We found a markedly larger percentage of plasmablast/plasma cells (PB/PCs) circulating in DENV-positive patients as compared to patients with Other Febrile Illnesses (OFIs). The percentage of DENV-specific PB/PCs against DENV-3 represented 10 % of the circulating antibody-producing cells (ASCs) in secondary DENV-3 infections. Importantly, the cross-reactive DENV-specific B cell response was higher against a heterotypic serotype, with 46 % of circulating PB/PCs specific to DENV-2 and 10 % specific to DENV-3 during acute infection. We also observed a higher cross-reactive DENV-specific IgG serum avidity directed against DENV-2 as compared to DENV-3 during acute infection. The neutralization capacity of the serum was broadly cross-reactive against the four DENV serotypes both during the acute phase and at 3 months post-onset of symptoms. Overall, the crossreactiv

Characteristics of patients enrolled in the hospital-based study during the 2010–2011 dengue season, Managua, Nicaragua.

*<p>Out of 216 participants enrolled, 1 participant dropped out and 11 participants were excluded for indeterminate dengue diagnostic results.</p

Flow cytometry staining of B cell subsets in peripheral blood of DENV-infected patients.

<p><b>A.</b> Phenotype of circulating B cells in patients suspected of DENV infection. Whole blood was collected during the first 3 days of hospitalization and stained directly with anti-CD138 or anti-HLA-DR FITC, anti-CD20 PECy7, anti-CD27 PE, and anti-CD38 PECy-5. Cells were gated on the lymphocyte population, and B cell sub-populations were discriminated using anti-CD20 and anti-CD27 MAbs. Naïve B cells are CD20⁺CD27⁻, memory B cells are CD20⁺CD27⁺, and PB/PCs are CD20^lowCD27^high. Histograms represent levels of CD38, CD138 and HLA-DR expression on PB/PCs; dark grey line, positive stained sample; light grey line, negative unstained control sample. One representative flow cytometry staining (from day 5 post-onset of symptoms) is shown out of 22 OFI and 38 DENV-positive cases processed by flow cytometry. <b>B.</b> Phenotype of MZ B cells in patients suspected of DENV infection. For MZ B cell analysis, cells were processed as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001568#pntd-0001568-g001" target="_blank">Figure 1A</a> and stained with anti-IgD FITC, anti-CD20 PECy7, anti-CD27 PE, and anti-IgM PECy-5. Cells were gated on the lymphocyte population, and B cell sub-populations were identified using anti-CD20 and anti-CD27 MAbs. MZ B cells are CD20⁺CD27⁺IgD⁺IgM⁺. One representative flow cytometry staining (from day 2 post-onset of symptoms) is shown out of 23 OFIs and 31 DENV-positive cases processed by flow cytometry.</p

Cross-reactive DENV-specific serum neutralization during secondary DENV infections.

<p><b>A.</b> DENV-specific neutralization of the serum at 3 months post-onset of symptoms in 2° DENV-3 infections. Longitudinal serum samples, obtained 3 months post-onset of symptoms from a subset (n = 20) of 2° DENV-3 infections, were analyzed in a flow cytometry-based neutralization assay. Briefly, serial dilutions of the serum samples were incubated with RVPs containing the GFP gene and C-prM/M-E from the 4 different DENV serotypes for one h, and then incubated with Raji-DC-SIGN-R cells for 48 h. The percentage of infection, as defined by expression of GFP in the infected cells, was detected by flow cytometry. The NT₅₀, calculated using Prism software (see Material and Methods), represents the serum dilution at which 50% neutralization of infection is achieved. Statistical analysis was performed by Wilcoxon Rank Sum test to compare the NT₅₀ of each heterotypic serotype (DENV-1, DENV-2 and DENV-4) to the infecting serotype (DENV-3). No significant difference was found. <b>B.</b> DENV-specific neutralization of serum at day 6 post-onset of symptoms in 2° DENV-3 infections. Samples obtained at day 6 post-onset of symptoms from a subset (n = 20) of 2° DENV-3 infections were analyzed in a flow cytometry-based neutralization assay to measure the NT₅₀ as described in the legend to <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001568#pntd-0001568-g005" target="_blank">Figure 5A</a>. Statistical analysis was performed by Wilcoxon Rank Sum test to compare the NT₅₀ of each heterotypic serotype (DENV-1, DENV-2 and DENV-4) to the infecting serotype (DENV-3). No significant difference was found.</p

Increased percentage of PB/PCs in peripheral blood of DENV-infected patients.

<p><b>A.</b> Percentage of PB/PCs circulating in the blood of patients suspected of DENV infection. Mean and SE of the percentage of PB/PCs among total lymphocytes plotted according to day post-onset of symptoms. As the same patient may have had up to 3 samples processed, the patient may be represented more than once over time. The number of samples processed is shown below the graph. The percentage of PB/PCs increased over time in DENV-positive samples and peaked at day 5 post-onset of symptoms. Statistical analysis was performed using the Mann-Whitney test, and a significant difference in percentage of PB/PC between OFIs and DENV-positive cases was found at day 5 post-onset of symptoms. The p-value is shown below the symbol legend. <b>B.</b> Percentage of PB/PCs at day 5 post-onset of symptoms in 1° and 2° DENV infections. Statistical analysis was performed using the Mann-Whitney test, and no significant difference in percentage of PB/PCs was found at day 5 post-onset of symptoms between 1° and 2° DENV infections. n = 14 for 1° DENV infections; n = 8 for 2° DENV infections. <b>C.</b> Percentage of memory B cells circulating in the blood of patients suspected of DENV infection. Mean and SE of the percentage of memory B cells plotted according to day post-onset of symptoms. As the same patient may have had up to 3 samples processed, the patient may be represented more than once over time. The number of samples processed is shown below the graph. The evolution over time of the percentage of memory B cells in OFIs and DENV-positive cases is similar. Statistical analysis was performed using the Mann-Whitney test, and a significant difference between OFIs and DENV-positive cases was found on day 4 post-onset of symptoms. The p-value is shown below the symbol legend. <b>D.</b> Percentage of naïve B cells circulating in the blood of patients suspected of DENV infection. Mean and SE of the percentage of naïve B cells plotted according to day post-onset of symptoms. As the same patient may have had up to 3 samples processed, the patient may be represented more than once over time. The number of samples processed is shown below the graph. The evolution over time of the percentage of naïve B cells in OFIs and DENV-positive cases is similar. Statistical analysis was performed using the Mann-Whitney test, and a significant difference between OFIs and DENV-positive cases was found on day 3 post-onset of symptoms. The p-value is shown below the symbol legend. <b>E.</b> Percentage of MZ B cells circulating in the blood of patients suspected of DENV infection. Mean and SE of the percentage of MZ B cells plotted according to day post-onset of symptoms. As the same patient may have had up to 3 samples processed, the patient may be represented more than once over time. The number of samples processed is shown below the graph. The evolution over time of the percentage of MZ B cells in OFIs and DENV-positive cases is similar. Statistical analysis was performed using the Mann-Whitney test, and a significant difference between OFIs and DENV-positive cases was found on day 2 post-onset of symptoms. The p-value is shown below the symbol legend.</p