Carolina Scagnolari

ACIP 2021

Dengue virus-mosquito-host interactions: assessing type I interferon mediated immune processes and immunological effects of mosquito salivary glands on Dengue infection (ACIP N°: 221-2019)

Acronym: DeSiReS: Dengue Severe Immune Response Studies

Carolina Scagnolari (PI)1 , Darragh Duffy2 , Tineke Cantaert3 , Zanakoungo Ibrahim Coulibaly4 1. IP Rome, 2. IP Paris, 3. IP-Cambodia, 4. IP- Côte d'Ivoire

Main results obtained during the project according to each WP(n=3) are reported below:

WP1: Identification of blood IFN-I signatures in hospitalized cambodian children with mild or severe outcome of DENV infection A comprehensive analysis of type I interferon (IFN-I) responses during the earliest phase of DENV disease, within 96h of onset of fever in a cohort of Cambodian children was conducted. In particular, we performed an integrated analysis of expression of genes related to IFN-I signaling in PBMC, determination of plasma IFNα/β by ultrasensitive methods and exhaustive dendritic cells (DC) subset phenotyping in patients stratified for DENV infection history and disease severity. In total, we included 115 dengue-positive paediatric patients admitted at Kantha Bopha Children’s hospital, Phnom Penh (Cambodia). In addition, 43 age and sex-matched healthy donors were recruited from a cluster-based investigation in Kampong Cham province. Since, it remains controversial as to whether IFN-I responses are protective or contribute to immunopathogenesis during DENV infection (McNab F et al., Nature reviews. Immunology 2018), we analyzed protein concentrations of IFNα and IFNβ in plasma from DENV-infected patients with variable disease severity and healthy donors by ultra-sensitive singlemolecule array (Simoa) digital ELISA technology. In addition, we performed a comprehensive gene expression analysis of a set of IFN-I related genes [IFNα, IFNβ, IFN-I receptor subunits (IFNAR1 and IFNAR2), IRF7 and FOXO3] in PBMC isolated from the same cohort. Increased transcription of IFNAR2 and IRF7 mRNA were observed in DENV positive patients (IFNAR2: p=0.0006; IRF7: p <0.0001) compared to age and gender matched healthy donors. In parallel, we observed enhanced protein concentrations of IFNα and IFNβ in acute DENV-infected patients versus healthy donors (IFNα: p=<0.0001 and IFNβ: p=0.0138) by ultra-sensitive single-molecule array (Simoa) digital ELISA technology. Whereas IFNβ protein concentrations were correlated with IFNβ mRNA (r=0.35; p<0.01), no such correlation was observed for IFNα (r=0.10; p=0.42) during acute DENV infection. DENV viral load positively correlated with levels of plasma IFNα/β (r=0.28; p<0.01 and r=0.50; p<0.0001), IFNAR1-mRNA (r=0.30; p<0.01),

and IFNAR2-mRNA (r=0.31; p<0.01). Furthermore, when patients were stratified based on disease severity, DENV viral load correlated positively with plasma IFNα/β in patients with DF (r=0.28; p=0.02 and r=0.52; p<0.0001) and DHF/DSS (r=0.47; p=0.04 and r=0.57; p<0.01). Hence, we compared levels of IFN-I related genes, IFNα/β protein concentrations and DC subsets between primary infected DF patients and secondary infected DF patients. Here, a decrease in FOXO3-mRNA levels in patients with secondary infection compared to primary infection was observed whereas levels of other IFN-I related genes were similar in primary infected DF patients compared to secondary infected DF patients. Individuals undergoing a secondary DF infection showed decreased IFNα protein concentrations compared to primary infected patients (p=0.016). Also, we compared mRNA levels of IFN-I genes in DENV infected children who developed classical DF with patients classified as DHF or DSS). Reduced expression of IFNα (p=0.0008), IFNβ (p<0.0001), IRF-7 (p<0.0001) and FOXO3 (p=0.0004) transcripts were observed in patients with severe dengue (DHF/DSS patients) than patients with nonsevere manifestations (DF patients). In accordance to the gene expression data, serum protein concentrations of IFNα (p=0.09) and IFNβ (p=0.013) were lower in DSS/DHF patients compared to DF patients. Taken together, these data show that an elevated viral load is correlated to an increase in IFNα/β protein synthesis and downstream induction of expression of IFN-I related genes (IFNAR2, IRF7) during acute DENV infection. Moreover, these data show that the type I IFN response is modulated by the DENV infection history of the patients.

WP2: Exploration of the cross-talk between specific immune cells (i.e. DC cell subsets and B-cell) and IFN-I response underlying key mechanisms of severe DENV infection Given, pDC and virus-infected cells are the major sources of IFN-I and considering that DENV is tropic for dendritic cells of the myeloid lineage in blood, we evaluated if changes in circulating frequencies of mDC and pDC could account for the changes in IFN-I observed during acute DENV-infection. The percentages of CD11c+ mDCs were found to be significantly decreased in dengue patients compared to healthy controls (median: 5.4% versus 44.1%; p<0.0001) whereas no difference was observed in the relative proportions of pDCs between dengue patients and healthy donors. There was no correlation between the percentages of CD11c+ mDCs or CD123+ pDCs respectively with viral load in all patients or patients stratified for disease severity. However, of major interest, increased percentages of circulating CD123+ pDC positively correlated to increased IFNα plasma protein concentrations in DENV-infected patients (r=0.43; p<0.001) whereas CD11c+ mDC frequencies were not correlated with IFNα plasma concentrations. No correlation was observed between CD11c+ mDC or CD123+ pDC frequencies and IFNβ. Moreover, the percentages of circulating CD11c+ mDCs and CD123+ pDC were not different between primary and secondary DF. As frequencies of circulating pDCs are associated to IFNα protein concentration during DENV infection, we examined the influence of dengue disease severity in patients with secondary infection on the distribution of dendritic cell subsets. A significant decrease in the

relative proportions of pDCs (median: 17.2 % versus 9.1 %; p<0.05), but not mDC was observed in secondary DHF/DSS compared to secondary DF. A decrease in platelet counts is one of the hallmarks of severe DENV infection and is used in the WHO classification criteria. In accordance with the observation of a decreased IFN-I response in severe disease, we observed that lower platelet counts are correlated to lower IFNα protein plasma concentrations in DENV-infected patients (p<0.05, r = 0.27). As IFN-I play an important role in the induction of the humoral immune response during infection or vaccination, we sought to determine the relationship between type I IFN and development of anti-DENV antibodies in infected patients. As previous infection history has a major impact on the developing antibody titers during the acute phase of DENV infection, we included only secondary infected patients for this analysis. Functional neutralizing antibodies were measured by the focis reduction neutralization test (FRNT90). Even though their presence does not always correlate with protection from severe disease, the assay remains the gold standard for measuring humoral protection during DENV infection. Of interest, lower concentrations of IFNα and IFNβ proteins correlated with higher total anti-dengue antibodies measured at hospital discharge by hemagglutinin inhibition (HI) assay (p<0.0001, r=-0.57 and p<0.0001, r =-0.54). In parallel, lower IFNα (r=-0.37; p<0.01), IFNβ (r =-0.34; p <0.01), IFNAR1 (r=-0.27; p<0.05), and IRF7 (r=-0.28; p<0.05) transcripts measured at hospital admittance correlated with higher FRNT90 titers at hospital discharge. Moreover, these correlations were independent of the amount of days between hospital admittance and discharge. As type I IFN correlates with viral load and is dependent on disease severity, we also analyzed the correlation of neutralizing antibodies to viral load. Here, neutralizing antibody titers did not correlate to viral load in DF or DHF/DSS patients. Taken together, we found that a decreased pDC frequencies and an associated decreased IFN-I response are observed in patients developing severe disease during secondary DENV infection, indicating that an early IFN-I response mediated by pDCs is beneficial for the host. Also, we observed strong negative correlations between IFN-I response measured at hospital admittance and the development of anti-DENV antibody titers and the absence of autoantibodies to IFN in our cohort of acute DENV-infected Cambodian children. WP3. Determination of the ability of mosquitoes’ salivary proteins inoculated during blood feeding to modulate IFN-I response and DENV replication The entomological surveys were carried out in Anyama residential (5°50N ; 4°06’W, nonendemic area), Cocody-Vallon (5°35N ; 3°98’W, endemic area) and National Park of Banco (5°21’N ; 4°50’’W; Forest) The aedes mosquitoes obtained from the different type of collections has been identified using mosquito’s indication keys of Ethiopian region of Edwards, 1941; Rueda, 2002 and Huang, 2004 (Zoo taxa 700) and dissected to isolates the salivary glandes using standard methods performed recently published by Bio Protoc (Schmid and al. 2017; www.bio-protocol.org/e2407). A pool of salivary glandes forms endemic area (Cocody), non-endemic area (Anyama) and National Parc of banco per species. In particular, from December 2020 to November 2021; a total of 4272 mosquitoes

(3,102 females and 1170 males) with (n=1197 specimens, i.e. 28.02% of Ae. aegypti and n=1515 specimens, i.e. 35.46% of Cx. quinquefasciatus) were collected by all methods (CDC light trap, BG Trap, Traps nests, double net trap and Electric Aspirators), of which 1654 (n=213 specimens, i.e. 12. 87% Ae. aegypti) at Anyama (non-endemic area), 1709 (n= 726 specimens, i.e. 42.48% Ae. aegypti) endemic area (Cocody) and 810 (n=258 specimens, i.e. 31.85% de Ae. aegypti) in the National Parc of Banco (Forest). Of the total Ae. Aegypti female (71.17%, n=852 mosquitoes) collected, 449 (52.69%) were by double nets as against 197 (23.12%), by Traps nests, 166 (19.48%) by BG sentent Trap, 19(2.23%) by CDC light Trap and 21 (2.46%) by Electric aspirators. A total of 570 salivary glands were extracted from females of putative vectors, 312 of which were extracted in Cocody, 108 in Wanyama and 150 in Banco National Park. These extracted glands were mainly from Ae. aegypti (531 glands) and other arbovirus vectors such as Ae africanus, Ae. vittatus, Ae. apicoargenteus, Ae. luteocephalus and Ae. opok. A total of 162 monospecific pool of 1 to 40 glands were constituted per trap type per site and per month of which 44 (108 glands) in Anyama, 70 (150 glands) in Cocody and 48 (312 glands) in Banco National Park. These glands were preserved in PBS at -20°C and will be transferred to IP of Italy. Côte d'Ivoire Taken together, although there are some deviations in the research activities performed in this WP mostly due to the COVID-19 crisis, these results show that Ae. aegypti (major vector of Dengue virus) is present in all collection areas and is most abundant in the endemic area (Cocody-vallon). However, it shows unexpectedly high prevalence of Cx quinquefasciatus (putative vector of Rift vallee fever, West Nile virus, etc.). An abundance of Ae aegypti in the sylvatic environment (Banco) is also reported. Moreover, our preliminary data also indicated that mosquito SGE alters the immune response to virus infection, providing new insights into the mechanisms of host manipulation used by DENV vectors and contributing towards a more comprehensive characterization of the main mechanisms underlying the complex interactions between Aedes mosquito and the early host antiviral immunity

Publications

Vinit Upasani*, Carolina Scagnolari*, Federica Frasca, Nikaïa Smith, Vincent Bondet, Axelle Vanderlinden, Sokchea Lay, Heidi Auerswald, Sothy Heng, Denis Laurent, Sowath Ly, Veasna Duong, Guido Antonelli, Philippe Dussart, Darragh Duffy, Tineke Cantaert. Decreased Type I Interferon Production by Plasmacytoid Dendritic Cells Contributes to Severe Dengue. Front Immunol. 2020 Dec 17; 11: 605087 *VU and CS contributed equally to this work. IF=6.42